Annual Review of Immunology Volume 1 1983

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Annual Reviews

Annual Reviews

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Ant~ Rev. ImmunoL 1983. 1.’1-32 Copyright©1983by .4nnualReviewsInc. All rights reserved

GETTING STARTED 50 YEARS AGOmEXPERIENCES, PERSPECTIVES, AND PROBLEMSOF THE FIRST 21 YEARS Elvin A. Kabat Departmentsof Microbiologyand HumanGenetics and Development,and the CancerCenter/Institute for CancerResearch, ColumbiaUniversity College of Physicians and Surgeons, NewYork, NewYork10032; and the National Institute of Allergyand Infectious Diseases,NationalInstitutes of Health, Bethesda, Maryland20205

This first volumeof the Annual Review of Immunologywill appear just 50 years after I began working in immunochemistryin Michael Heidelberger’s laboratory at ColumbiaUniversity’s College of Physicians and Surgeons. The differences between then and nowin the path for a graduate student, postdoctoral, and beginning independent investigator are striking, and I thought for this prefatory chapter I wouldrecount someof the experiences of myfirst 21 years in the field, considering not only the workbut also the outside economicand political influences on mycareer, as well as World War II and its aftermath of loyalty and security investigations and the Senator Joseph McCarthy period. On January 1, 1933, I began working in Michael Heidelberger’s laboratory as a laboratory helper. Thedefinitive paper on the quantitative precipitin method by Heidelberger and Forrest E. Kendall had appeared in the Journal of Experimental Medicine in 1929 and the laboratory was well on its way to providing analytical chemical methodsfor measurementof antigens and antibodies. This paper furnished the. key to modernstructural immunologyand immunochemistry, which, not without strong resistance from the then classical immunologists, changed our way of thinking. 1 0732-0582/83/0410-0001 $02.00

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I had received a BSdegree in September, 1932, from the City College of NewYork and had majored in chemistry. I was 18 years old. The country was in the depths of the depression and myfamily had suffered acutely. I had been walking the length and breadth of NewYork visiting universities and hospitals looking for a job, literally walking,since I rarely had a nickel to take the subwayand if I had only a nickel I used it for lunch. Mymother had started selling dresses in our apartment; by chance Mrs. Nina Heidelberger had becomeone of her customers and she suggested I go up to see Michael. At the end of October or early Novemberof 1932, he offered me a job at a salary of $90 a monthto begin on the first of January, and the possibility of a future for me and for myfamily began. Michaelhad hesitated about giving me the job since I had been to college and the previous incumbenthad only been a high school graduate. I assured him that I would do the routine, which involved makingsolutions, keeping the laboratory clean, and washing glassware when MaryO’Neill, our halftime glassware worker, was ill, etc, provided he would let me do as much technical work and research as I was capable of doing. Hans T. Clarke, the Chairman of the Biochemistry Department at P and $, had interviewed me in connection with myapplication to do graduate work and had accepted me as a PhDcandidate. I had explained that I would be unable to begin until I found a job, but this was the norm during the depression. Many graduate students had part- or full-time jobs; somewere teaching at City College or elsewhere and were doing their graduate studies part-time at Columbia. Of the $90 a month, I had to give myparents $50 toward paying the rent and I had to pay the Columbia tuition, then $10 a point per semester, for mycourses, plus registration and laboratory fees. Myhours were 8:30 A.~. to 5:00 P.~., and beginning in February, 1933, I took an evening course in experimental physical chemistry with Professor Charles O. Beckman.Mygraduate courses were taken in the evening or late afternoon, or during the summersession, except for those given at P and S. During the summerof 1933 I took the course in medical bacteriology given by Calvin B. Coulter. Webecame good friends and he, Florence Stone, and I collaborated in a study of the ultraviolet absorption spectra of a numberof proteins I had prepared in Michael’s laboratory. Throughhim I met Arthur Shapiro, then a medical student, whowas full of ideas. We becamevery close friends; he was one of the very first to see the potentialities of bacterial genetics and several years later Sol Spiegelmanworkedwith him. One of the required courses, which was given only in the daytime at the downtowncampus, was quantitative organic analysis and students had to spend lots of extra time in the lab. Myjob madethis ditficult, so Hans Clarke, whohad written the classical text in the field, gave me 18 compounds to identify and I did these in Michael’s lab.

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The Department of Biochemistry was at its peak in reputation and productivity with Michael, Rudolf Schoenheimer, Karl Meyer, Erwin Chargaff, Erwin Brand, SamGurin, David Rittenberg, and SamGraft. Graduate students workedin a large laboratory on the fifth floor and included Joe Fruton, Lew Engel, David Shemin, De Witt Stetten Jr, Sarah Ratner, Konrad Bloch, William H. Stein, Abe Mazur, and Ernest Borek, and somewhat later SeymourCohen and David Sprinson. I got to know W. E. van Heyningen,whocameto us as a postdoctoral fellow. Since I workeddirectly in Michael’s lab mycontacts with the other students were muchless close. Myinterests also led me to be close to the Bacteriology Department, of which Frederick P. Gay was Chairman and whose members included Beatrice Seegal, Theodor Rosebury, James T. Culbertson, Claus Jungeblut, MaximSteinbach, Calvin Coulter, Florence Stone, Sidney J. Klein, and Rose R. Feiner. I attended both the weekly biochemistry and microbiology seminars. Professor Gay, whohad been closely associated with Jules Bordet and was still waging the Paul Ehrlich-Jules Bordet war of antibodies as substances rather than as vague properties of immuneserum, was strongly anti-immunochemical and became very impatient with my questions at seminars. This view came across clearly to the medical students and Edward H. Reisner, P and S ’39, wrote the following little ditty, which I learned about from Oscar Ratnoff. PasteurinspiredMetchnikoff andMetchnikoff wasnuts, Hehada pupil namedBordetwhohated Ehrlich’sguts, Now Ehrlichhad the goodsyouknow,but this wedare not say, For wedescendfromMetchnikoff throughBordetout of Gay. The Heidelberger laboratory in 1933-37 consisted of Forrest E. Kendall, who was working on pneumococcal polysaccharides and on the mechanism of the precipitin reaction, Arthur E. O. Menzel, who was studying the proteins and polysaccharides of the tubercle bacillus, Check M. Soo Hoo, our bacteriologist, myself, and MaryO’Neill. Several visiting scientists, among whomwere Henry W. Scherp, Torsten Teorell, D. L. Shrivastava, Alfred J. Weil, and Maurice Stacey, camefor varying periods, the longest being 1 year. WhenForrest left in 1936 to go to Goldwater Memorial Hospital with David Seegal, Henry P. Treffers, whohad just completed his PhD with Louis P. Hammettat Columbia, replaced him. Forrest Kendall worked opposite me and taught me the micro-Kjeldahl method, which in those days was the basic tool in the laboratory since all quantitative precipitin assays on the washedprecipitates were finally analyzed for total nitrogen. Digestions were carried out in 100-mlflasks. The workingrange was between 0.1 and 1.0 mgof N. I generally was responsible for doing all of the digestions for Michaeland Forrest, and after I acquired

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sufficient skill I also carriedout the distillations andtitrations for Michael. It wasnot unusualto run 40 distillations a day. I also ran manyquantitative iodine determinations on thyroglobulin, using the methoddevelopedby Professor G. L. Foster in the BiochemistryDepartment,until Herbert E. Stokinger becamea graduate student with Michael and was given the thyroglobulin problem. Thelaboratorywasa very exciting place in whichto workandI generally kept Forrest busyaskinghimquestions, no doubtinterfering often with his train of thought. Hewasvery patient and helpful, but Michaeloncesuggested that I had better put someof myquestions to him rather than to Forrest. I wasalwaysable to get answersor wastold whereto find them. After a few months,as Michaelhas described (2), I suggestedthat one might use a well-washedsuspension of heat-killed bacteria of knownN content to removeantibody and measureantibodyN as agglutinin. Michael said that I could do this so I started with type I pneumococci. Themethod wasvery successful, provided one took care in growingthe organismsto avoid autolysis. Whathappenswhenthis procedureis not strictly adhered to has already beenpublished(3). Thecombinedapplication of the quantitative precipitin and quantitative agglutinin methodspermittedus to establish that the antibodyto the typespecific polysaccharideof pneumococci was the samewhethermeasuredas precipitin or as agglutinin. Naturally, the bacterial suspensionwasable to removeantiprotein, and this wasmeasuredindependentlywith a suspension of rough unencapsulatedpneumococci.In those days wewere only beginning to be awareof antibodyheterogeneity,that our type-specific antibody was a complexmixture of antibodies, someof which were non- or coprecipitating, and that all of these werebeing measuredas agglutinin or as precipitin. MichaelandI also studiedthe courseof the quantitativeagglutinin reaction and showedthat it followed the empirical equation he and Forrest had described for the quantitative precipitin reaction, and which they later derived from the Lawof MassAction. Thequantitative agglutinin workwassupposedto be myPhDdissertation; the first two paperson the methodand on the identity of agglutinin and precipitin had beenpublishedin the Journalof ExperimentalMedicine in 1934and 1936. These,as reprints, and the third part on the mechanism of agglutination were submittedearly in 1937. However,the University’s rules had been changedso that the student was required to be the first author on any publication and Michaelhadbeenthe first author on the two papers. I couldof coursehaveused the third part as the thesis, but I had also been studying the quantitative precipitin reaction betweenR-saltazobiphenylazo-serum albuminand rabbit antibody to serumalbumin.This systemhadcertain unsuspecteduniqueaspects since the introductionof the

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haptenic group had not altered the reactivity with anti-serum albumin. Michaelsuggestedthat I write this up and use it as myPhDdissertation, whichI did in about 3 or 4 weeks. It created a minorsensation at the Faculty of Pure Science office whenI broughtdownthe copies of the new dissertation and took backthe other. ArneTiselius had suggestedsometime earlier that it wouldbe nice to have someonefrom Michael’s laboratory to study the physicoehemical properties of purified antibodiesat Uppsalaand that the RockefellerFoundation might provide a Fellowship. He had just developed the moving boundarymethodof electrophoresis with the rectangular cell, whichfirst madepossibleprecise quantitativeestimatesof purity andpermittedresolution of mixtures of proteins. Michaelhad spent two summersin Uppsala workingwith KaiO. Pedersenand with Tiselius and had suggestedme. This wasan extraordinaryopportunityto learn the newermethodsof fractionating and characterizing macromolecules in the Svedberglaboratory. Frank Blair Hansoncameto interview mefromthe Foundationand I wasawarded the Fellowship. The Fellowshippaid $125per month.Onthe original application there wasa questionas to whetherone hadany special financial obligations and I had noted that I gavemyparents $50a month.WhenI received the award, it specified $125per month.I wentto the Foundation’soffices to discuss whetherI could get along on $75 per month.Theylooked at myoriginal application and assured methat $50 wouldbe sent to myparents and that I wouldreceive the full $125.I madesure that I did not spendmuchof my first month’sstipenduntil several weekslater whena letter frommymother arrived(this wasbeforetransatlantic airmail)sayingthat the first checkhad beenreceivedpromptlyon September 1, 1937.After that I wasable to live quite comfortably. TheRockefellerFoundationalso paid for cabin class transportation from NewYorkto Uppsala.Since I wasentitled to a monthvacation for mywork with Michael,I wantedto spend it in Europeand especially to visit the SovietUnion,to whichat that time I wasvery favorablyinclined, predominantly becauseof the great economicsuffering of myfamily during the depressionand also becauseof Russia’s UnitedFront policy in opposition to Hitler andtheir supportof the Spanishloyalists. TheRockefellerFoundation agreed to give mewhat it wouldcost themto send medirectly in cabinclass and I could go anywhere I chose. In those days if one purchased a tourist class ticket on the QueenMary,onecould go by rail secondclass anywhere in Europefor $10 extra. I choseLeningrad,spendingseveral days in Paris and Warsaw.For $50 one could spend 10 days in Leningradand Moscow with all hotels, meals, travel betweencities, and guidedtours to places of interest included.In ParAsI visited the Institut Pasteur, sawthe

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Pasteur Museum,and met Gaston Ramon, who invited me to lunch at his home where I met Andr6 Boivin and Lydia Mesrobeanu. In the Soviet UnionI was unable to see any scientists and so I was essentially a tourist visiting museums,factories, etc, on standard guided tours. I then spent a day or two in Helsinki, took the boat to Stockholmand the train to Uppsala, and arrived at the Institute early in September. Before leaving and in anticipation of mybeing able to go to Uppsala, Michael had suggested that we immunize several different species with suspensions of pneumococci, so Soo Hoo and I injected two pigs and a monkey.These immunizations were less traumatic than those we had done earlier, injecting two goats (4). Rabbit and horse antisera were available the lab and a cow was immunized at Sharpe and Dohme.Torsten Teorell with Forrest and Michael had found that a given quantity of pneumococcal polysaccharide precipitated less antibody in 15 %salt than in physiological saline, due to a shift in the combiningproportions at equilibrium whenthe reaction was carried out in high salt. Michael and Forrest then used this finding to purify antibodies to polysaccharidesby precipitating the antibody from a large volumeof antiserum with polysaccharide under physiological conditions of 0°C, washing repeatedly with cold saline, and extracting the washedprecipitate with 15%NaCI at 37°C to dissociate a portion of the antibody. With individual antisera as much as 15-30%purified antibody could be obtained and over 90%was precipitable by polysaccharide. This was the first time substantial amounts,tens of milligrams, of antibody could be prepared. Michael and I also extended the methodby using a suspension of bacteria to remove the antibody followed by washing and elution with 15 %salt. Thesepurified antibodies plus various antisera and antigens were shipped to Uppsala and were available when I arrived. The laboratory was at a peak in its productivity with Professor Svedberg popping in and out, with Arne Tiselius, then a docent, and with Kai O. Pedersen and Ole Lamm.Numerous visitors came for various periods, including Basil Record from Haworth’s laboratory, Frank L. Horsfall from the Rockefeller Institute, whoseinterests were similar to mine, J. B. Sumner with crystalline urease and concanavalin A, G. Bressler from Leningrad, G. S. Adair from Cambridge, and Gerhard Schramm from Germany. Professor Svedberg was very anxious to test his new separation cell, which had a membranedividing the cell into an upper and a lower compartment. Using some of myhorse antipneumococcal serum, he centrifuged it until the 18S peak had gone below the membrane.I tested both proteins and found all of the antibody in the lower compartment. I had also brought someof the crystalline horse serum albumin used in myPhDthesis. Tiselius was very surprised whenwe examinedit by electrophoresis at a concentration of 0.5 or 1%and saw only a single peak. He

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said it wasthe first time he had seen a protein that wasa single component electrophoretically. Althoughnewtechniques provide moreand often better criteria for establishing purity, they frequently only showthat not enoughcare wastakenin purifyingthe material. Theneedfor such care was most forcefully demonstrated years later whenKnight (8) examined paper chromatography Emil Fischer’s collection of 34 synthetic peptides madehalf a century earlier, whichhis son Hermann had broughtto Berkeley. All but three, whichcontaineda trace of oneof the constituent amino acids, gavea single spot. I got to workright awaywith the help of Kai Pedersenlearning to run the oil turbine ultracentrifuge and the diffusion apparatusto measurethe molecularweightsof the antibodies I had brought. Surprisingly, the horse antibodies werequite polydisperse. Wewereat a loss to understandthis. Fortunately, the State SerumInstitute had a horse that had been under immunizationfor a short time, and on purifying its antibody by the salt dissociation methodwefounda single 18Speak. All of the other antibodies gave multiple peaks, as did a second samplefrom the same horse after further immunization.Therapeuticuse of antipneumococcal horse sera in Swedenhad not been very successful, so I was able to get serum from humansrecovering from lobar pneumoniawhohad not received antibody, throughthe courtesyof Jan Waldcnstrtim.I tested the serumof quite a few convalescents, found one with about 1 mgof antibody protein per ml, purified the antibodyfrom about 40 ml of serum, and measuredits molecular weight. ArneTiselius andI studied the electrophoretieproperties of antibodies. Oneof the rabbit hyperimmune anti-ovalbumin sera I had brought had 36.4%precipitable antibody. Weexaminedits electrophoretic pattern before andafter removalof the antibodyand found, by the decrease, 37.2%, in area of the gamma globulin peak, that wehad removedthe sameproportion of the total serumprotein. This definitely establishedthat these antibodies were gamma globulins [nowimmunoglobulin (Ig) G, one of the five majorimmunoglobulin classes]. Theelectrophoretic patterns wepublished appear in most textbooks of immunology. I also spent a considerableamountof time studyingspecific precipitates of ovalbuminrabbit anti-ovalbumin and horse serumalbumin-rabbit antiserumalbumindissolvedin excessantigen both by ultracentrifugation and by electrophoresis, calculating the compositionof the soluble complexes. This was all done by the Lamm scale method,whichwas very tedious and especially hard on the eyes. Beforemyreturn to the USI left a manuscript with ArneTiselius, which, becauseof the war, never waspublished, althoughhe cited our workand an electrophoretic pattern showingthe soluble complexesas a schlieren band migrating behind the ovalbuminband

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appears in Tiselius’ HarveyLecture (10). It remainedfor JonathanSinger and DanCampbellto do the definitive study. Bythat time, the automated Schlieren methodhad replaced the laborious scale methodand work was mucheasier. I have already described myexperiences at the Nobel Ceremonyin December1937 (6). The terms of the Rockefeller FoundationFellowshiprequired that the sponsorpermit the Fellowto return to a position in the laboratory from whichhe cameand Michael had assured the Foundationhe would do so. However,he told mehe woulddo his best to find mea moreindependent position, and somemonthsafter I had arrived in UppsalaI receiveda letter from Jacob Furth offering me a position as Instructor in Pathology at Cornell University MedicalCollegeat $2400per year beginningSeptember 1, 1938, to work on viruses causing tumors and leukemias in chickens. Michaeladvised meto accept and I did. I believed it wasimportantfor meto visit certain laboratories before returning to the USand discussedthis with HarryMiller of the Rockefeller Foundation.Theytended to discouragesuch travel but finally agreed that I could leave Uppsalaabout July 15 to visit Linderstrom-Lang and the State SerumInstitute in Copenhagen;J. D. Bernal, I. Fankuchen,and John Marrack in London; F. G. Hopkins in Cambridge; W. N. Haworth and MauriceStacey in Birmingham; HansKrebs in Shettield, whereI spent 4 days learning to do someenzymereactions in the Warburgapparatus; Gorter in Leyden;and den Doorende Jong in Amsterdam, finally going to Zurichto attend the International PhysiologicalCongressbefore returning to the US.All of these visits established lasting friendships andcontacts with junior as well as senior investigators, andin myreport to the Rockefeller FoundationI emphasized the desirability of providingsimilar opportunities to their other fellows. In Zurichin August,1938,everyonewasvery disturbed about the situation in Spain wherethe Franco forces were about to or had already cut Catalonia from the rest of loyalist Spain. Manyof the membersof the Loyalist government were physiologists, including Juan Negrin, the Prime Minister, and Cabrera, the ForeignMinister, and a good-sizeddelegation attended the Congress.Agroupof us felt weshoulddo somethingto express our support and organized a dinner at the Congress,whichwasvery well attendedand raised a considerablesum.I wasanxiousto go to Spainto find out whatpeople in the UScould do to help, and it wasarrangedfor meand LewEngel to go to Spainfor a few days before sailing for the US.MyUS passport wasnot valid for travel to Spainso Dr. Cabrerawroteon a sheet of paper that all border patrols shouldpermit meto enter and leave Spain without makingany marks in mypassport. Wetook a train from Zurich

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to Perpignan,France,andto Cerb~re,the usual crossing point, thinkingit wouldbe easy to get across. I very soonhada Frenchsoldier with a bayonet proddingmealong until I got on the train backto Perpignan. Wondering whatto do wedecided to visit the Spanishconsul in Perpignan. Whenhe sawthe Cabreranote, he told us to go out andget pictures taken, andwhen wereturned he at~xed themto a SpanishCarte d’Identit6 on the back of whichhe had written the contents of the Cabreraletter (Figure 1), summonedhis car and chauffeur, andhad us driven fromPerpignanto the Hotel Majesticin Barcelona.TheHotel Majesticwasthe center for foreign correspondents and there I met Herbert L. Matthews, the NewYork Times correspondent.Foodwasvery scarce and I washungryall the time, most especiallyafter the rationedmeals.Indeed,whenI left a Frenchdiningcar, on mywayback, i emptiedthe bread tray into mypockets. Mostof mytime was spent visiting hospitals wherethe woundedAmericansof the Lincoln Brigadeand other International Brigadevolunteers werebeing treated. I vividly remember speakingto one wounded anti-fascist Germansoldier who felt his problemswerenot being understoodbecausehe spokeonly German; I wasable to translate his wishesinto Englishto oneof the doctors. Onreturning to NewYork, I immediatelybeganto workat Cornell with Jacob Furth. EugeneOpie, the Chairmanof the Pathology Department, had built up an important departmentwith Jacob, MurrayAngevine,Robert A. Moore,and Richard Linton, whohad been at Columbiaand with whoseworkin India on cholera antigens I had becomefamiliar. Vincent du Vigneaudwas Chairmanof Biochemistry and permitted me to attend their weeklyseminars. I becameclose friends with W.H. Summerson and soon collaborated in studies with DeanBurk, Otto K. Behrens, Herbert Sprince, and Fritz Lipmann,whohad left Germanyand arrived shortly thereafter from Denmark.Cornell MedicalSchoolwasnot anxious to have too manyJewson its staff and du Vigneaud,whohad madea definite offer to Lipmann,indicated that he wouldresign if the appointmentdid not go through. At Cornell the mainproblemJacob Furth wishedmeto workon was to purify the virus of oneof his chickenstrains that causedleukosisif injected intravenouslyandtumorslike the Roussarcomaif injected subcutaneously. Theapproachwasessentially to centrifuge crude extracts in the Pickels air-drivenultracentrifuge,followingthe biologicalactivity by assaysin baby chicks, as well as bynitrogento estimatethe extent of purification. I wanted to buy a micro-Kjeldahlapparatus, but a previouspostdoctoral fellow with Jacob had purchaseda Dumasapparatus. Jacob, considering mea chemist and wishing to save money,wantedto get the Dumasworking. I had to insist that this wasnot a suitable methodif one hadto run manydeterminations and he finally consentedto buy a micro-Kjeldahlapparatus; he was

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Annual Reviews GETTING STARTED 50 YEARS AGO

Figure 1 SpanishCarte d’Identit6, with the Foreign Minister’s letter.

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very happywhenhe saw 40 to 50 analyses a day comingout immediately and told methat they had tried to use the Dumasfor a wholeyear without a single successfulresult. Albert Claudehad essentially done a study with Rous sarcomavirus similar to whatwewere planningand had foundthat high-speedsedimentable materialswerepresentin normaltissues. This wasall in the daysbefore subcellular organdies, mitochondria,microsomes, etc, and it took mea long time to convinceJacobthat onecouldnot get a purevirus just by centrifugation of tumorextracts. Among our interesting findings at that time werethe demonstrationthat alkaline phosphatase and the Forssmanand Wassermannantigens wereassociated with these high-molecular-weight particles. Wealso preparedrabbit antisera, whichweused to try to characterizethese materials. Jacob also suggestedthat I try to workout somehistochemicalmethods for localizing enzymesin tissues. I wasjust getting started whenGeorge Gomoripublishedhis elegant methodfor localizing alkaline phosphatasein paratfin sections of alcohol-fixedtissues. Weadoptedit andcarried out a study of the distribution of alkaline phosphatasein normaland neoplastic tissues. Thestained sections werevery impressive;coloredlantern slides werejust cominginto use and Jacobthoughtweshouldhavea coloredplate for our paper in the American Journalof Pathology,whichwouldcost $400. This wasconsideredtoo expensive.It wasfinally arrangedthat the Departmentof Pathologywouldpay $100, the research fund wouldpay $200, and Jacob and I would each pay $50. It was a very worthwhile investment becauseit stimulated muchworkon the subject. In EuropeI had hopedto visit KSgl’s laboratory at Utrecht; he had startled the world by announcingthat malignanttumorshad large amounts of D-aminoacids instead of L-aminoacids. Muchof his data could be accounted for by racemization, but from one tumor several grams of t~-glutamic acid had been isolated. Naturally, everyonebeganto try to checkthis and Jacobgot mesometumors, thinking that after I established that I could isolate D-aminoacids I could then workon leukemiccells, whichwere available in muchsmaller amounts. However,I workedup a numberof tumors but only found L-glutamic acid. Twoconfirmations of KiSgl’s workwere soonreported and it seemedclear that I wasnot a very goodchemist. Theproblemwas solved by the genius of Fritz Lipmann,who suggested that after hydrolysis, one could add D-aminoacid oxidase and estimate D-aminoacid concentrations by using the Warburgapparatus. He, DeanBurk,Otto Behrens,and I soonshowedthat there wereno significant amountsof D-aminoacids. DavidRittenberg at Columbiaapplied the isotope dilution methodand also failed to find D-amino acids. This turned out to be oneof the instancesof falsification of data, someone havingapparently

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added o-glutamic acid to K/Jgl’s hydrolysates. It led to mycoining an aphorism:"Everyincorrect discoveryhas alwaysbeenindependentlyverified." In our efforts to purify the tumorviruses, wefrequentlyfoundthat saline extracts of tumors were extremelyviscous, and from one chicken with a tumor,wewereable to get a considerablequantityof this very viscousfluid. The viscosity reminded me of the pneumococcalpolysaccharides I had prepared. I addedsomesodiumacetate to the fluid and then two volumes of ethanolandgot an extraordinarilystringy precipitate, almostall of which adheredto the stirring rod and could be removed.Karl Meyerat P and S had reported the isolation of hyaluronicacid as behavingsimilarly and I said to Jacob, whowaswatchingmeprecipitate the polysaccharide,that it looked like hyaluronic acid. Hebecamesomewhatupset that I could make sucha statementfromso little evidence,but whenI addeda little hyaluronidase providedby Karl Meyer,the Viscosity disappeared.Welearned it was essential to treat our tumorextracts with hyaluronidasebefore ultracentrifugation. DavidShemin,whohad been at City College with me, had received his PhDin biochemistryat Columbia,and had taken a job there in the Departmentof Pathologywith JamesJobling, the Chairman,working on Roussarcoma,had similar findings. DeanBurkwasalso very interested in tumormetabolismand he, Jacob, Herbert Sprince, Janet Spangler,Albert Claude,andI carried out studies in the Warburgon chicken tumors. On the international scene mystay at Cornell was the period of the Munichagreement, the conquest of Czechoslovakiaby Hitler, the NaziSoviet pact, the partition of PolandbetweenGermany and Russia, and the Finnish-Sovietwar. Theseevents shookmeand I began to worryabout my political views. Aboutthe spring of 1940,TracyJ. Putnam,Director of the Neurological Institute at Columbia, wantedan immunochemistto work on multiple sclerosis and asked MichaelHeidelbergerto suggest someone.Michaelgave hima choice, assuring himthat if he picked mehe ffould have a strong personality on his hands. Heinvited mefor an interview and said that he understood that I had lots of myownideas and that I could work on whateverI wishedexcept he hopedI wouldnot discriminate’againstneurological problems. The salary was $3600 per year and Hans Clarke had agreed that I wouldhave an appointmentas ResearchAssociate in Biochemistry(assigned to Neurology).Fundswereavailable for a technician; two laboratories were available in the NeurologicalInstitute, whichhad formerlybeenintern’s bedrooms,plus somespace sharedwith the clinical laboratory. It took little thoughton mypart to decideto accept. RobertF. Loeb, for whomI had the greatest admiration and affection, was then

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Associate Director of the Neurological Institute and was a great attraction for me. I was especially thrilled when I met him at WoodsHole in the summerof 1940 and he came over to me saying, "Elvin, we need you, we can hardly wait until you get to Neuro." I arranged to leave Cornell at the end of May, 1941, taking June as my terminal vacation. I started at P and S and the Neurological Institute on June 1, 1941, and took myvacation later in the summer. Mylaboratories were temporarily occupied by NormanWeissman, a friend who had gotten his degree in biochemistry, and Murray Glusman, with whomI was to collaborate years later when he returned from a Japanese prison camp. Harold Landow,a resident in neurology, was interested in collaborating. Wedecided to look at the electrophoretic patterns of cerebrospinal fluid in the Tiselius electrophoresis apparatus. Dan H. Moore, who had come from Lederle laboratories where he had studied horse antisera electrophoretically, had set up a laboratory for electrophoresis and had an air-driven ultracentrifuge. For the Tiselius electrophoresis, ¯ the cerebrospinal fluids had to be concentrated by pressure dialysis to a small volumeand had to be run in a 2-ml micro cell. Large amountsof fluid were available from pneumoencephalograms and we arranged to obtain these. Wereadily obtained good patterns on concentrated cerebrospinal fluid and showedthat patients with multiple sclerosis often had substantial increases in the gamma globulin fraction, as did patients with neurosyphilis, and that positive colloidal gold tests correlated with increased gamma globulin. This led naturally to a study of serum proteins with Franklin M. Hanger, which showedhigh levels of gammaglobulin to be responsible for positive cephalin flocculation tests in various sera. Thesefindings provided someinsight into the mechanismof these two clinical diagnostic tests. At the same time, Harold Landowand I began a quantitative study of passive anaphylaxis in the guinea pig. Oddlyenough, although the antibody N content of rabbit antisera could be determined by quantitative precipitin analysis, nobodyhad measuredhowlittle antibody was required to sensitize a guinea pig so that fatal anaphylaxis wouldresult on subsequent administration of antibody. Our data showed, with anti-ovalbumin and antipneumococcalserum that 30/.tg of antibody N sensitized 250-g guinea pigs, so that fatal anaphylaxis wouldresult if 1 mgof ovalbuminor of pneumococcal polysaccharide were given 48 hr later. This was the beginning of a series of studies on quantitative aspects of allergic reactions. In the summer of 1940, while still at Cornell, MaryLoveless and I had workedat Woods Hole, trying to measure uptake of skin-sensitizing antibody (later recognized as IgE) in sera of ragweed-sensitive patients, using a suspension of formalinized pollen with negative results. The skin sensitizing powerof the sera was removed,but no increase in N in the washedpollen was detectable.

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As we nowknowfrom the work of the Ishizakas and Bennich, IgE is so muchmoreactive per unit weightthan rabbit IgGthat the methodwasnot suitable. For similar reasonsI also obtainednegativeresults trying to measure uptakeof bacteriophageby suspensionsof bacteria. Also while at Cornell Professor du Vigneaudhad given mepermission to use the Tiselius electrophoresisapparatushe hadjust purchased.I examined numerous leukemicsera for changesin protein pattern, but significant changeswere not seen. However,a BenceJones protein he also provided gave a beautiful homogeneous peak. Duringthe first weeksafter myreturn to Columbiain June, 1941, I stopped in to see Alexanderand Ethel Gutman,whoselaboratories were opposite Michael’sand from whomI had received advice while with Michael. They were studying myelomasera in the Tiselius apparatus and showedmetheir patterns with their sharp peaks movingbetween/32and gamma globulin, the mobilityof the peaksdiffering amongindividual sera. I asked themwhythey were not studying BenceJones proteins since the mobility of mypeakhad also beenbetweenthat of B2and gamma globulins. AI took out a pattern of a BenceJones protein he had sent to someone several years earlier. Therewasa beautiful sharp peaklike the one I had observed--it was labeled "boundary disturbance." Wedecided to add BeneeJones proteins from a myeloma patient to normalserumand compare the pattern with that of the patient’s serumby itself; wefoundwecould reproducethe patient’s serumpattern in a numberof instances by using BenceJones proteins of different mobility. Wethen used a rabbit antiserum to urinary BenceJones protein from a myelomapatient to measurethe levels in the serumof the samepatient. I also continued the histochemicallocalization of enzymes,studying normaland neoplastic tissues of the nervous system with Harold Landow and WilliamNewman, whocameto us as a technician, later studied medicine, and becameProfessor of Pathologyat GeorgeWashington University. AbnerWolf, whowasProfessor of Neuropathology,also wasinterested in this field. Wecollaborated closely on histochemicalstudies on acid and alkaline phosphatases and especially on producing and studying disseminatedencephalomyelitisin monkeys by injection of brain tissue emulsified in Freundadjuvants, until the grant for this workwassummarily terminated in 1953 during the hysteria generated by Senator Joseph McCarthy. Onthe eveningof June22, 1941,I wasat a party--everyonewasdiscussing the rumorsthat Germany wasgoing to attack Russia. I arguedvigorously that Hitler wouldnot, andwhenI hadjust about convincedeveryone, weturned on the radio. This markedmyretirement as a political prognosticator. It wasfar moretragic for mankindthat Stalin wastaken in than

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that I was. I joined with manyothers to set up a group for Russian War Relief at the MedicalCenter. The doubts generated by the Nazi-Soviet pact werestilled. On Sunday, December7, 1941, I was working in the laboratory and heard the news about Pearl Harbor. No one knew what was happening but we decided to move acids and hazardous chemicals to a storeroom in the basement. It was clear that medical scientists, immunologists, and immunochemists were going to make important contributions to the war effort. Michael Heidelberger, whoI saw very frequently since myreturn to Columbia, had been working for the PneumoniaCommissionof the Board for the Control of Epidemic Diseases, USArmy, on immunization against lobar pneumonia, using pneumococcalpolysacchaddes. I proposed that the type I (now group A) meningococcal polysaccharide, which had been purified by Geoffrey Rake and Henry W. Scherp, might be used for immunization in man. Meningitis had been a serious problem during recruiting in World WarI. The Meningitis Commissionunder the Chairmanship of J. J. Phair was centered at Johns Hopkins and gave me an initial subcontract for $1000, renewedfor a year at $2500, to purify type I meningococcalpolysaccharide and study its antigenicity in man. I needed to order someequipment, which I did before going on a trip. WhenI returned I found that the order had not been processed. I phoned Dean Willard C. Rappleye, who said they were having a problem about whether my subcontract would receive overhead. I asked how much the overhead was and when he said $40, I shrieked into the. telephone, "Andfor forty dollars you are holding up my work on immunization against meningitis with a war going on!" There was a momentof deathly silence and I expected to hear that I was no longer employedby Columbia.Instead, he said, "I’m sorry. I guess you are right. We’ll order it right away." Fromtoday’s perspective it is easy to see howthe Universities’ attitudes on getting the incredible amounts of moneythey demandin indirect costs developed, to understand what happensto the reputation of the investigator who cannot bring in what they consider his share, and to marvel at the unanimity administrators exhibit in lobbying against any proposed reductions whenthey can agree on almost nothing else. I hired Hilda Kaiser, whose husband, Samuel Kaiser, had been dismissed from Brooklyn College as a consequence of the NewYork State Legislature’s Rapp Coudert Committee and later suggested to Michael that he hire Samfor the pneumoniawork, which he did. All of the individuals fired from the City Colleges during that period were reinstated with apologies 40 years later, manyposthumously. Webegan to immunize medical student volunteers with type I meningococcal polysaccharide following Michael’s schedule for pneumococcal

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polysaccharides. In these and all mylater studies on blood group substances, dextrans, levans, etc, I generally injected myself first with any new materials unless they were expected to cross-react with antibodies to antigens I had already received. Weused Michael’s precipitin assay with about 4 ml of serum per test and with Hilda Kaiser and Helen Sikorski were able clearly to demonstrate that precipitins were formed; C. Philip Miller and Alice Foster at the University of Chicago assayed the sera for protective antibody and we demonstrated that the polysacchadde was antigenic. Only 4 of 38 individuals, however,produced a significant antibody response and this seemed poor by comparison with the pneumococcal polysaccharides, so immunization was not considered promising. Whenimmunization with meningococcal polysaccharides was taken up years later, radioimmunoassay was available and detection of an antibody response becamethousands of times more sensitive. Wetoo would have found detectable antibody by radioimmunoassayin a substantial proportion of the 38 subjects. Mylaboratory was involved in two other projects during the war: one with the Ottice of Scientific Research and Developmentand the Committee on MedicalResearchon false-positive serological tests for syphilis; and the other for the National Defense Research Committee on the plant toxin dcin, a possible chemical-biological warfare agent, with a view toward developing methodsof detecting it and protecting against its toxic effects. Our experiences in buying and immunizingtwo horses as part of this work have already been described (5). Bernard D. Davis was assigned to the laboratory by the USPublic Health Service to work on serological tests for syphilis. With Ad Harris and Dan Moore we studied the anticomplementary action of humangammaglobulin and succeeded in purifying Wassermannantibody by absorption on and elution from lipid floccules. For the studies on ricin, which were classified secret, Michael Heidelberger and I joined forces as co-responsible investigators. Ada E. Bezer cameto work in mylaboratory on this problem and we began an association that was to last over 20 years until she went to work with WHO in Ibadan, Nigeria. Wewere able to demonstrate by immunochemicalmethods that the toxic and hemagglutinating properties of dcin were due to different substances. This was later confirmed by Moses L. Kunitz and R. Keith Carman,whoindependently succeeded in crystallizing ricin. One very puzzling observation was that the protective powerof anti-dcin sera could be assayed readily in animals but did not correlate with the amountof precipitable antibody. Wenowknowof course that ricin has a combiningsite for carbohydrates and therefore was reacting with and precipitating non-antibody gammaglobulin and probably other serum glycoproteins, as well as antibody.

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During the war years Manfred Mayer had replaced me in Michael’s laboratory. He continued the study of the cross-reaction of types III and VIII antipneumococcal antibodies Michael, D. L. Shrivastava, and I had begun earlier. Webecamevery close friends. The war years had seen important changes in mypersonal life. Harold Landow,a brilliant neurologist and close friend, had committed suicide in 1940 because of his parent’s objections to his marrying outside of his religion. In 1940 at a literary meeting I had been introduced to a young Canadian named Sally Lennick. She had come to NewYork from Toronto to study painting. Wemet again a year later. Then in the summer of 1942 we met at a party, began seeing one another frequently, and were married on November28, 1942; our oldest son, Jonathan, was born on June 5, 1944. It had becomeclear at this time that a book outlining the thinking and methodology of quantitative immunochemistry was sorely needed. Most workers using the quantitative precipitin methodhad learned it directly from Michael or from someone who had learned it from Michael and this waslimiting growthof the field. I had been invited to write a review entitled "Immunochemistry of the Proteins" for the Journal of Immunology by Alfred J. Weil; it appeared in December,1943, and I was astonished at the hundreds of requests for reprints. Manfred Mayerand I decided to write a text called Experimental Immunochemistry, which would not only give the methodsin detail but wouldalso outline principles and concepts so that it would be useful to students and workers who might think immunochemistry of value in attacking their problems.It wouldalso include the preparation of materials needed for work in immunochemistry. Weprepared an outline and submitted it to John Wiley, who did a survey and assured us that it would never sell even a thousand copies. Fortunately, Charles C Thomascameto visit Michael and immediatelyagreed to take it and invited Michael to write a preface. Weset right to work by dividing up the chapters and beginning to write. Weusually met on Saturday or Sunday at one or another’s apartment and read aloud what had been written, correcting, revising, and reordering sections. Our wives were left to entertain each other or to be bored by listening to what we had written. The book was sent to the publisher at the end of 1945, but because of paper shortages after the war, and various other delays, it did not appear until 1948. Fortunately, we were aware of many studies during the war years that were just being prepared for publication and insisted on doing extensive revisions in proof so that ~the bookwas up to date. It had a substantial influence on the field and the first edition went through four printings, the last being in 1958. The second edition, which appeared in 1961, also went through four printings. After Pearl Harbor, a group of us at Columbiawho were membersof the

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AmericanAssociationof Scientific Workersdevoteda considerable amount of time to consideringwhatcould be doneto aid the wareffort, especially with respect to defense and improvingmethodsof immunization.Theuse of bacterial andviral warfareagentsby the Naziswasconsidereda possibility and TheodorRoseburyand I undertookto prepare from the literature a report of the agents that mightbe used; wewereassisted by MartinH. Boldt, then a medical student. Wespent several monthspreparing the review, and AlphonseDochez,whowasan adviser to the Secretary of War, gaveit to him.Naturally,it waswithheldfrompublicationby us voluntarily during the war. The Armywas also very concerned about the possible military use of infectious agents and set up CampDetrick near Frederick, Maryland,as a large-scale military research facility. TedRoseburystarted workingthere full time and I becamea consultant, spendingseveral days there each month.Ourreport was amongthe first items newpersonnel at Fort Detrick were given to read. At the end of the war, whenthe SmytheReport on the AtomicBomb was published, wefelt it was in the public interest that our report on bacterial warfareshould also be publishedand werequestedclearance from the WarDepartment.Several incidents arose. In our original request for clearance westated in a footnote that wehad withheld the report from publication during the war but that it wouldnowbe published with the approval of the WarDepartment.In their letter stating that wecould submitthe reviewfor publication, they requestedthat the wordswith the approval of the WarDepartmentbe changedto "in view of the removalof war time restrictions" (Figure 2). Wenaturally complied.TheJournal of Immunology indicated it wouldconsider publishing it. In accordancewith Universitypolicy wethen gavethe report for approvalto Dr. Dochez,who wasChairmanof the BacteriologyDepartment.Althoughhe had originally takenthe report to the Secretaryof War,he said it hadnonscientificimplications and that he wouldhave to take it up with DeanRappleye.Rosebury and I werecalled to the Dean’sot~ce andweretold the report mightoffend religious groupsandthat if weinsisted on publishingit weshouldwrite our resignationson the spot. Sincewewerein no position to do this, and since our appeals about Freedomof Speechandthe Press wereunavailing, weput the report in a drawer. Wealso wentto see Osmond K. Frankel, a leading civil liberties attorney, whotold us essentially that wehadunlimitedfreedomto publish but havingdone so wedid not have the right to workfor ColumbiaUniversity. About6 monthslater, DeanRappleyecameto Ted Rosebury’soffice and said that he had madea mistake, that the University did not intend to limit our freedomto publish, and wecould submitthe report for publication. Wedid, andit waspublishedin the May,1947,issue of Journal of Immunology (9), whichalso arranged to sell reprints. The report created a world-widesensation (Figure 3).

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~l~ti~

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9e~art~t oF Bacteriology 6:~ ~est l~thStreet Dea~ I have looked over the revi~

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~Little,

in The Nashville Tennessean

Figure3 Political cartoon(by TomLittle, in The Tennessean)resulting fromthe published bacterial warfarearticle. (Reprintedwith permission.)

Whenit appeared, Timemagazineimplied that wewere procommunist and interpreted the statementin the footnote to indicate that wehad not obtainedclearance but had publishedit on our own"in viewof the removal of wartimerestrictions." MichaelHeidelbergerwrotea letter supporting our loyalty, whichthey published. At the sametime HowardJ. Mueller, Professor of Bacteriologyand Immunology at Harvard, whohad also been a consultant at CampDetrick, wrote DeanRappleyesuggesting that Rosebury and Kabatshould be fired becausethey had timed the publication of their report to appear whenCongresswas considering the WarDepartment’s budgetso that they wouldget moremoneyfor biological warfare, although the war was over. Dean Rappleye knewthat if any one had

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determined if and whenthe report would appear it was he and not Rosebury and Kabat. However, we learned that our clearances for CampDetrick were cancelled just after the report was published. The FBI was assigned to investigate us and the following episode with mylandlord was told to me. Sally and I were leaving for Europe on June 20, 1947, to attend the International Cytology Congress in Stockholm and the International Microbiology Congress in Copenhagen. I wrote mylandlord, giving him the dates we would be away and enclosing two rent checks dated July 1 and August 1. Weleft our two children, Jonathan, age 3, and Geoffrey, born May11, 1946, with myparents. Shortly after our departure, an FBI agent visited the landlord. The conversation as recounted to me went about as follows. Did you know the Kabats were leaving for Europe? Do you think they are ever comingback? He showedthem the letter and the checks. The questions continued. If you were plann’ing to leave the country wouldn’t this be a good way to hide your intentions? Mylandlord naturally was very surprised when we returned on August 20. In Stockholmat the CytologyCongress, there was substantial interest in the report on bacterial warfare and a group of Swedish microbiologists invited Sally and meto an elegant lunch at the Operkellaren; several of the guests were evidently assigned to entertain Sally while the others turned the topic of conversation to myreport. Since Roseburyand I had been involved in research subsequent to our report, all of whichwas still secret, it would have been almost impossible for meto discuss anything without creating the impressionthat it represented the report plus additional classified information. Fortunately, suspecting what the lunch was about, ! had put a reprint of the report in mypocket and in reply to each specific question, I merely read the pertinent sections written in 1941 and finally gave the reprint to one of the microbiologists sitting next to me. At the end of the war, I took up two major lines of investigation. One, on the immunochemistryof blood group A and B substances, was to continue to the present day. The backgroundand details of work in this area and the personal aspects have been described by merecently (7). The second was an attempt, together with AbnerWolf and Ada Bezer, to produce acute disseminated encephalomyelitis in monkeysbecause of its possible relation to multiple sclerosis. The use of the Pasteur treatment for rabies had been knownto result in what were termed paralytic accidents, which involved disseminated demyelinating lesions in the brain and spinal cord. This had been shown by TomRivers and Francis Schwenkter to be due to the nervous tissue and they succeeded in producing the disease in monkeysby injecting suspensions of spinal cord tissue. Manyinjections were neededand the disease did not appear for a long period. Tracy Putnamhad always been most interested in multiple sclerosis and was delighted when we got back

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to this problem. Jules Freund had shownthat one could get a very enhanced and protracted antibody response, as well as delayed type hypersensitivity, by incorporating antigens with mineral oil and killed mycobactedumby using an emulsifying agent, and I was anxious to see if this procedurecould be used to produce disseminated encephalomyelitis in monkeysmore rapidly and reproducibly. Wefound that after only one to three injections of brain tissue emulsified in the Freund adjuvant, the monkeysdeveloped the disease and we sent a short note to Science early in 1946. While having dinner with Isabel Morganduring the Federation meetings I learned that she had made similar observations in monkeysshe had been injecting with spinal cord tissue containing poliomyelitis virus with Freund adjuvants and then had omitted the virus with similiar results. She was to present her work at the Society of American Bacteriologists meetings in May. Wedecided that we wouldwrite up our detailed papers completely independently, then send themto one another to criticize and request that they be published side by side. Since her paper arrived a few days before ours had been typed, I gave the unopened envelope to Robert Loeb for safekeeping until I had mailed our manuscript. The results were completely concordant and both papers appeared together in the Journal of Experimental Medicine in January 1947. The National Multiple Sclerosis Society had just been founded and sometime after our paper appeared I received a phone call from them saying they wantedme to apply for a grant. I said that I really was not interested in applying and didn’t need the money.Theyreplied that I "just have to" since they had received an anonymouscontribution on condition that they used it to support our work. WhenI inquired as to the amountof the contribution and was told it was $10,000, I said that this was not really sufficient to makethe more intensive effort they wished. I drew up an application for $64,350 for 3 years of support, which they showed the anonymousdonor whogenerously provided the entire sum. This was the first grant madeby the National Multiple Sclerosis Society. I was able to increase the size of my monkeycolony from about 10 or 12 to 40. In 1950, the Public Health Service gave mea grant to continue this work, but it was cancelled in 1953. By the end of the war mysalary had increased to $4500per year, but with the inflation following the removal of price controls, plus my growing family, I had to take a part-time job teaching elementary chemistry at City College. The multiple sclerosis grant provided an equivalent sum and made it possible for meto give up this outside work. Wespent the summer of 1948 in WoodsHole where we first met Fred and Sally Karush and their children. This was the beginning of a life-long friendship between the two families and Fred spent 6 months in mylaboratory in 1950. The laboratory was then so crowded that he had to work a second shift beginning in the late afternoon.

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Towardthe end of that summerI saw a note on the bulletin board at the MBLthat a house was for sale by the Professor of Microbiology at Washington University, Jacques Bronfenbrenner, whomI knewquite well. It took only a few minutes to arrange to buy the house. Our children becamevery attached to it and we spent part of each summerthere during the years when they were growingup, except whenI was on sabbatical. I wouldeither work in the library or rent a laboratory bench. In 1949, I rented a laboratory bench, which then cost $50 for the summer, so that I could prepare an enzymefrom snail hepatopancreas that split blood group substances. The business office at Columbiarefused to pay the bill, saying that grants stipulated that the institution wasto provide laboratory facilities. I assured them that the $50 entitled me to order and receive snails collected by the MBL boats and that if they wouldput snails on mydesk in 48 hr I woulddo the work in NewYork--they paid "the $50. WoodsHole was, of course, a marvelous place for makingscientific contacts. In 1949, ShlomoHestrin of the Hebrew University had the laboratory bench opposite me and we becamevery close friends. Around this time a problem developed in myrelationship to the Biochemistry Department at Columbia. For several years Tracy Putnam, the Professor of Neurology, had been asking Hans Clarke to promote me to Assistant Professor, but to no avail. This was in no way directed towards me personally, nor did it reflect any doubts about mywork. Hans Clarke was a very fine person, but he was completely unaware of the need to promote people. He did not consider persons in other departments with titles in biochemistry as real membersof his department, although he did not promote the regular memberseither. A substantial numberof Research Associates had been in these positions for years, requests of the chairmen of the departments in which they workedbeing unavailing. Indeed, Walter W. Palmer, the Professor of Medicine, told me he had asked Hans Clarke to promote Michael Heidelberger to full Professor for 17 years before he agreed. I broke the log jam of Research Associates by transferring to the Department of Bacteriology. The Chairman, Dr. Dochez, was very anxious to introduce immunochemistryinto the medical teaching. In discussing my situation he suggested that he would be willing to give me an Assistant Professorship. The appointmentwas delayed for a year by the objections of Hans Clarke, whoevidently didn’t want to promote me or lose me, but it finally took effect on July 1, 1946. Louis Levin, whofor about 10 years had been Research Associate in Biochemistry assigned to Anatomy,an appointment similar to mine, had left a year earlier for the University of Chicago because the AnatomyDepartment could not arrange his promotion. He was brought back as Assistant Professor of Anatomyand later went to the Office of Naval Research and the National Science Foundation.

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After this, Hans Clarke agreed to several promotions of other Research Associates. As Assistant Professor of Bacteriology, I was entitled to have graduate students and my first was SamM. Beiser, who had been in the Navy and came to Columbia under the GI Bill. He was recommended by Arthur Shapiro. His PhDdissertation was on blood group substances from bovine gastric mucosa. The Academyof Allergy was just setting up a fellowship program(1). Even though Beiser was not working in allergy, at mysuggestion that the fellowship might stimulate his interest in allergy, he received it and later devoted sometime to work in this area. After two postdoctoral years with Bernard Davis, he returned to Columbia,rising to Professor and was Acting Chairmanat the time of his death from cancer in 1972. Among his most important contributions were studies on antibodies to nucleotides with Bernard F. Erlanger. His bovine blood group glycoproteins have remained important standards and Michael and I are still using the pneumococcal C-polysaccharide he prepared. Myposition in the Department of Bacteriology, the title of which was changed to Microbiology, and in the Neurology Department was eminently satisfactory over all these years. Tracy Putnamleft in 1946 and was followed by EdwinG. Zabriskie, and in 1948 by H. HoustonMerritt. Professor Dochez retired, and Beatrice Seegal became Acting Chairman of the Department of Bacteriology until Harry Rose was appointed in 1951. Drs. Zabriskie and Dochez had recommended my promotion to Associate Professor in 1947 and I was promoted in 1948. Harry Rose was also very supportive. Indeed, when he was offered the position as Chairmanof Microbiology in 1951 he laid downtwo conditions--that I be promotedto full Professor and that mysalary, which until then had come from grants, be paid out of departmental funds. This took effect on July 1, 1952. By 1947, the laboratory had three major lines of investigation immunochemistry of blood group substances (see 7), acute disseminated encephalomyelitis, and quantitative studies on allergic reactions. EdwardE. Fischel, who was in the Department of Medicine, and I measured the amounts of antibody needed to produce Arthus reactions passively in the rabbit, and GrangeCoffin, a medical student, and David M. Smith, a dental student, continued studies on passive anaphylaxis. Baruj Benacerraf came to see mein 1946, having completed medical school and a 1-year internship before serving in the Armyand wanting to spend a postdoctoral year in the laboratory. There were nb funds, but he told me he had independent means and came, as he later put it, on the "Benacerraf Fellowship." He was a very intense and dedicated worker and we studied quantitative aspects of the latent period in passive anaphylaxis and also the passive Arthus reactions in the guinea pig. John H. Vaughanspent two postdoctoral years in the

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laboratorystudyingthe skin-sensitizing properties of rabbit antibodies to ovalbuminand conalbuminin humanskin. Wehad foundincreases in the gamma globulin in the cerebrospinalfluid of patients withmultiplesclerosis by electrophoresis,but this wasimpractical for diagnostic purposes. MurrayGlusman,whohad returned to the NeurologicalInstitute after the war, VestaKnaub,and I decidedto measure the amountsof gamma globulin by using a microquantitative precipitin reaction. Wealso measuredalbuminimmunochemically at the same time. It becameclear that the methodwouldgive moreinformative results than the colloidal gold tests, and it becamepossible to followgamma globulin levels in cerebrospinalfluid in patients over prolongedperiods. DavidA. Freedman, Jean Murray, and Vesta Knaubmeasured the albumin and gamma globulinlevels in 100 cases of multiple sclerosis and found85 with elevatedgamma globulinlevels. In carryingout sucha studyit wasessential not to report our findings until the diagnosis hadbeenmadein the absence of these data, otherwiseclinicians whoavidly grasp for objectivedata would soongain a clinical impressionof the valueof the test andwouldnot make their diagnosisuntil the test wasin the patient’s chart. Subsequent studies were carried out with MelvinD. Yahrand Sidney S. Goldensohn,and the quantitative precipitin methodfor determininggamma globulin in the cerebrospinal fluid of patients at the NeurologicalInstitute becameone of my routine responsibilities for 30 years, until it wassupersededby moresensitive and automatedimmunochemical methods. I had manyclose friends and other colleaguesat the NeurologicalInstitute during this period, ineluding Saul R. Korey, Harry Grundfest, and David Nachmansohn. At the end of the war AbnerWolf had becomean attending consultant in Neuropathologyat the BronxVeterans Administration Hospital. The VeteransHospitals wereencouragingresearch and weresetting up substantial laboratory facilities. WilliamNewman had received his MDand had gone there, as had another MD,Irwin Feigen, and Abnersuggested that wecontinuestudies on histochemicallocalization of enzymesthere. I was appointedattending consultant and spent one afternoon a weekthere for severalyears. WhileI was at the VA, President Trumanissued an Executive Order initiating loyalty andsecurity investigationsof Federalemployees with the followingcriterion: "Reasonablegroundsmustexist for the belief that one is disloyal to the UnitedStates." JamesB. Sumner,with whomI had been very friendly at Uppsalain 1937-1938and whoI had later met briefly on only one occasionin the US,wentto the FBIto tell themthat I had been a communist while in Uppsala.This initiated a series of FBIinvestigations on the basis of whichI was presented with charges about the various organizationsto whichI hadbelongedor contributedduringthe late 1930s.

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I had a hearing before the VAloyalty board, whichdismissed me. I appealedfinally to the Presidential LoyaltyReview Board,whichreversedthe decision and reinstated me. I returned to the VA,but it wasobviousthat there werepressures to reducefurther the rigidity of the criteria andthe quality of the evidenceuponwhichan individual could be dismissed, so I decidedto resign. This essentially led to mygiving up workon the histochemicallocalization of enzymesin tissues. ThePresidential LoyaltyReviewBoardwasabolishedby President Eisenhower,respondingto pressures that it had beentoo lenient. Indeed, it wasthe only board whosemembers were not Federal employeesand thus was not subject to pressures from Congressand fromwithin the Executivebranch. I wasvery pleased that I had carried out all myappeals without a lawyer. The Bronx VAHospital Loyalty Board that dismissed me had also written a letter to the passport office telling themthat I should not be allowedto travel, and so mypassport wascancelled; it was not returned whenI was reinstated. I had been invited to address the First International Congressof Allergists in Zurich in the summer of 1950but was unable to obtain a passport. I wentto the passport office to discuss the matter and was told that if I gave the namesof anyoneI knewor thought to be a communistI could get a passport. Needlessto say I was unable to travel until the decision of the USSupremeCourt in 1955that every Americancitizen had an unlimited right to travel, whenI attended the International Congressof Allcrgologyin Pctropolis, Brazil. Baruj Bcnacerraf broughtmysituation to the attention of the Zurich Congressin his address. I received the invitation to the AllergologyCongresswhile in Woods Hole. It provided$1000for travel expenses.I wrotea letter sayingthat I could not accept becauseI could not get a passport. I walkedinto townand droppedthe letter in the boxandwalkedacross the street to the drug store to buy the NewYork Times. The headline announced the US Supreme Court Decision. I rushed to the Post Office and was able to retrieve my letter. Sanford Elberg, with whomI becamefriendly at CampDerrick, invited meto teach two coursesat the Universityof California at Berkeleyduring the summerof 1950.Sally, Jonathan,Geoffrey,and I droveacross country, stoppingin Springfield,Illinois, to see the Lincolniana,in Denver,in Salt LakeCity, and at Boulder(nowHoover)Dam.I had a very enjoyable time teaching--amongthe students or auditors were A. A. Benedict, MelHerzberg, Fred Aladjem, and Keith Smart. I met and becamefriends with Ed Adelberg, RogerStanier, MikeDoudoroffat Berkeley, and K. F. Meyer, Professor of Bacteriologyat San Francisco.Whilein BerkeleyI wasinvited to speakat the NavalBiologicalLaboratory,the Navy’sequivalentof Camp Derrick. In this highly classified institution andat a time of considerable

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hysteria, Keith Smart, in trying to shorten an obituary-like, overly long introduction, brought downthe house by saying, "Dr. Kabatis a member of a large numberof organizations, whichI had better not mention." Of coursehe then felt obligatedto list them. In 1952Congressappropriated $I00,000for research in immunochemistry in the budgetof the Office of NavalResearch(ONR).To evaluate what problems were worthy of support, William V. Consolazio, whomI had never met, and Louis Levincalled a conferenceat P and S. About10 or 15 people were present, including myself, Michael Heidelberger, and Dan Campbell.Everyonearound the table indicated what problemshe thought were important. I stressed use of immunochemical criteria of purity of proteins and polysacch.arides. The meetinglasted until lunch time. When it wasover andeveryonewasleaving, Bill Consolaziosaid to me, "Youare staying here," and then asked, "To whomwould you give moneyon the basis of the suggestions?" F’outlined the programsI thought should be supported. Hethen said, "Youhave to take somemoney."I responded,"I don’t need any money,I’m loaded with money."He gave mea yellow pad, saying, "Youare not leaving here until you write out a proposal. Youcan hold on to the moneyand you can activate the contract any time within the next five years." I wrote out a title, "Immunochemical Criteria of Purity of Proteins and Polysaccharides," myname, the University’s nameand address, a short abstract, and a budgetthat providedfor a postdoctoral fellow and supplies. WhenI cameto overheadI asked, "Whatare you going to do about overhead?"At that time ONR was havinga battle with Columbia-they wouldonly pay 10%overhead, believing that the investigator wasinterested in doing the workanyway.TheDeanwasvery dissatisfied with 10%and had threatened to throw ONRout completely. Consolazio replied, "Oh, put down25%overhead--youdon’t want the moneyand I had to twist your arm." Several weekslater two Navycontract negotiators cameto see DeanRappleyewith the piece of paper containing myalmost illegible handwritingsaying, "We’vecometo give you this contract." The Deansaid, "Whatcontract?"Theyreplied, "Its all written on this piece of yellowpaper." I wasin the processof havingit typed. TheDeanthen said, "Whatabout overhead?"Theyreplied, "Twenty-fiv~percent." Mystock at Columbiarose enormously.I only activated the contract a year later when the Public Health Service cancelled mygrants at the height of the McCarthyhysteria. It was like having moneyin the bank. ONR supported mefor 17 years. Consolazioand Levin left ONR whenthe National Science Foundation wasset up andBill suggestedthat I applyfor a grant. I received oneof the first awards-S60,000 for 3 years, appropriatedout of the first year’s molecularbiologybudget;it representedabout 8%of the total. This replaced myblood group grant, whichthe Public Health Service cancelled in 1953. NSFhas been the mainsupport of mylaboratory since then.

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In 19511 wasaskedto serve on the NationalResearchCouncil’sSubcommittee on Shock,whichwasconcernedabout the severe allergic reactions being found on administration of dextran, whichhad beendevelopedas a plasmaexpanderin Sweden.It wasgenerally believed that the reactions weredue to contamination with bacterial protein. I naturally suggestedthat the dextran itself, like the pneumococcal polysaccharides,mightbe antigenic in man.Althoughclinicians werepreparedto inject 30 g of dextran intravenously, no one on the committeewouldinject 1 mgto see if it was antigenic. I did an initial skin test, had a bloodsampletaken, and gave myselftwo injections of 0.5 mgof dextran a day apart. A secondskin test 3 weeks later showeda typical wheal and erythema, and quantitative precipitin assays on the pre- and post-immunizationsera showedthat my antidextran level had risen from1.5 to 25/.tN/ml. At the next meetingof the Subcommittee on Shock, I demonstratedmyprecipitates and also did a skin test on myselfand on DougLawrason,the Secretary of the Committee, as a control. DeborahBergand I studied the quantitative precipitin reaction of dextran and antidextran producedin medicalstudent volunteers. I suggested to the NationalResearchCouncilthat Paul Maurerbe asked to do a study to confirmour findings, whichhe did. To prove that the antibodies were indeedantidextranweused biosyntheticallylabeled [~4C]dextran to precipitate the antibody, and 14Canalyses by DavidRittenberg, LauraPontecorvo at P and S, and LeonHellmanand MaxwellEidinoff at Sloan-Kettering established that the dextran wasprecipitated by the antibody. Theantigenicity of dextranmadepossible studies probingthe size of the antibody combiningsite. Oneof the dextrans, B512, developedat the NorthernRegionalResearchLaboratoryof the Departmentof Agriculture at Peoria, Illinois, wasbuilt of 96%c~1~6-and 4%ctl~3-1inkedglucoses andso had very long stretches of ctl-~6-1inkedglucoses. AlleneJeanesat Peoria and Turveyand Whelanin England were isolating the series of al-~6-1inked isomaltose oligosaccharides. Thesystem, al-~6 dextran and humanantidextran and the c~1~6oligosaccharides, essentially provideda molecularruler, since they permitted one to comparethe potency on a molarbasis of the various oligosaccharidesin inhibiting precipitation of antidextranby dextran. This becamea fourth area of interest of the laboratory, whichcontinuesto the present day. In the summerof 1952,Sally and I again droveacross country with our three sons (David, born August7, 1951). I wishedto learn morecarbohydrate chemistry and decided to workwith HermanO. L. Fischer at Berkeley, havingbecomevery attracted to the BayArea frommyearlier visit. Hermanasked me what I wished to do and whenI said somethingabout learning methylation of sugars, he took out a sampleof galactinol, an

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a-galactoside of inositol, and said that methylation and hydrolysis would show where galactose was linked to the inositol. Clinton E. Ballou and Donald L. MacDonald collaborated on the problem and taught me the technics; fortunately, the methylated compound crystallized. The sabbatical was a wonderful experience and we were able to renew old friendships and make manynew ones that.have lasted. One unexpected consequence of the methylation study was that galactinol turned out to be one of the best inhibitors of the blood group B-anti-B reaction until the disacchadde DGal al-~3DGal and larger oligosaccharides were isolated. Mysabbatical ended in February, 1953, and we drove back to NewYork via the southern route, visiting various friends at universities and doing some sightseeing, arriving in NewYork early in March. It was hard to adjust to the rest of the NewYork winter after Berkeley. The laboratory was thriving; the allergic encephalitis, spinal fluid gammaglobulin, blood group substance, and dextran problems were all going well. I had been cleared by the top Presidential Loyalty Review Board and presumably could carry on with my activities normally. However, rumors of grants being cancelled were becomingmore frequent. Linus Pauling had his Public Health Service Grants cancelled and had to appoint others as Responsible Investigator so that the work could continue. My3-year grant for the monkeystudies was running out and I had naturally applied for a renewal. I received a letter from Frederick L. Stone, Chief, Extramural Programs, National Institute of Neurological Diseases and Blindness, dated December 14, 1953, which read, DearDr. Kabat: Your application for a research grant, identified as B-9(C4), and entitled "Immunochemical Studies on Acute Disseminated Encephalomyelitis and on Multiple Sclerosis" has nowhad a final review. I regret to report that this request falls in the group of applications for which grants cannot be made.

This was followed by a visit to Houston Merritt saying that this was because of the political climate, that they didn’t knowexactly why,etc. He then madethe suggestion that the work could go on if someoneelse’s name was substituted as Responsible Investigator. Althoughmanyothers in this situation complied, I refused, responded with the appropriate four-letter words, and began a boycott of the US Public Health Service, which had imposed the policy on NIH. Anyonewishing to work in mylaboratory had to agree not to accept any funds from any unit of the USPHS for the period he was working with me, nor could any employee of the USPHSset foot in my laboratory unless he was coming for some unrelated purpose. I received muchsupport from scientists at NIH, but the top administration did nothing.

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The reactions of my colleagues were varied. Most expressed support. Columbia University through Dean Rappleye, Houston Merritt, and Harry Rosesupported meunequivocally. The scientific societies fought the policy vigorously. Michael and manyothers refused to review grant applications. Unfortunately, not all actions were of this type: One person, whomI had considered one of myclosest friends, avoided me and on one occasion even hid in the stacks of the WoodsHole library when he saw me comingtoward him. Abner Wolf, Ada Bezer, and I had to kill off the only~monkeycolony in the world then being devoted to the multiple sclerosis problem. Shortly thereafter I was informed that the tenth committedyear of myBlood Group Grant was cancelled. I shall have to leave the story at this point. The suspense will not be dreadful. Youall knowthat I survived and continued to work actively, that I spent a year at NIHas a Fogarty Scholar, and that I have been spending 2 days a weekat NIHfor the past 7 years. The rest of the story must wait for another occasion. ACKNOWLEDGMENT

I thank Daniel M. Singer and Harvey N. Bernstein of Fried, Frank, Harris, Shriver, and Kampelman,Washington,D. C., for their advice, assistance,and direction in obtaining various government files about me under the Freedom of Information/Privacy Act. Literature Cited 1. Cohen, S. D. 1979. The American Academyof Allergy--An historical review. J. Allergy Clin. Immunol. 64:332466 2. Heidelberger, M. 1979. A "pure" organic chemist’s downwardpath: Chapter 2--The years at P .and S. Ann. Rev. Biochem. 48:1-21 3. Kabat, E. A. 1978. Life in the laboratory. Trends tliochem. Sci. 3:N87 4. Kabat, E. A. 1978. Close encounters of the odoriferous kind. Trends Biochem. Sc£ 3:N256 5. Kabat, E. A. 1979. Horse sense? Trends Biochem. ScL 4:N21M 6. Kabat, E. A. 1980. Wrongdoor at the

7.

8. 9. 10.

Nobel banquet. Trends Biochem. Sci. 6:VI Kabat, E. A. 1982. Contributions of quantitative immunochemistry to knowledge of blood group A, B, H, Le, I, and i antigens. Am. J. Clin. Pathol. 78:281-92 Knight, C. A. 1951. Paper chromatography of some lower peptides. J. Biol. Chem. 190:753-62 Rosebury, T., Kabat, E. A. 1947. Bacterial warfare. £ lmmunol. 56:7-96 Tiselius, A. 1939-1940. Electrophoretic analysis and the constitution of native fluids. Harvey Lectures 35:37-70

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CELLULAR MECHANISMS OF IMMUNOLOGIC TOLERANCE G. J. V. Nossal TheWalterandEliza Hall Institute of MedicalResearch,Post Office,Royal Melbourne Hospital,Victoria3050,Australia INTRODUCTION Immunologictolerance, the phenomenon wherebyantigen interacts with the lymphoid systemto impairits later capacity to respondto that antigen, remainsone of the most fascinating problemsin cellular immunology. The capacity of individuals to discriminate"self" from"not self" has assumed a newdimensionsince the realization that T lymphocytesonly see foreign antigens in the context of "self" moleculesof the majorhistocompatibility complex(MHC)(131), and so the newer field of MHC restriction becomeintertwined with immunologic tolerance and indeedwith all aspects of immunoregulation.Althoughthe central interest of students of immunologictolerance lies in the establishment and maintenanceof selfrecognition, experimentalrealities mandatethat mostworkon tolerance in fact is performedwith modelsystemswhereforeign antigens, rather than autologousconstituents, are presented to the lymphoidsystemto induce a state of non-reactivity. It then becomesa matterof considerabledit~culty to judge whetherthe effects observedmimicthe physiologicalsituation of lymphocytescomingto grips with the need to avoid horrorautotoxicus, or whether the modelillustrates somefacet of immunoregulation neededto limit the cascadeof immunoproliferation that follows the introduction of antigen. Facedwith this constraint, investigators of immunologic tolerance fall into three groups.Thefirst, numericallythe largest, describespartienlar modelsystems and refrains from speculation as to howmajor a role the cdlular mechanism revealed by the study plays in physiologicalself-recognition. Somemembersof this group form a "tolerance is dead" subgroup, whobelieve nothing is all that special about self antigens, and that the avoidance of autoimmunity depends on myriad immunoregulatoryand 33 0732-0582/83/0410-0033502.00

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34 NOSSAL feedbackmechanisms, none of whichis uniquely importantto self-recognition and all of whichmayapply in particular circumstancesto foreign antigens as well. Thesecondgroup considers suppressorT lymphocytesas central to immunologictolerance. Onthis view, the repertoire of immunocytes is perfectly competentto recognizeself antigens, but the capacity to threaten health by actually switching on these immunocytesis thwartedby a powerfulactivation of the appropriatesuppressors.Thethird groupsees the mostimportantbulwarkagainst auto-reactivity as lying in somepurging of the immunologicdictionary, effector precursors with receptors for self being either destroyedor inactivated in someway. Whatis not revealedin mostarticles on toleranceis that these three major approachesare not mutuallyexclusive, nor are the conceptualdemarcations betweenthemas rigid as mayfirst appear. Giventhe importanceof avoiding uncontrolledautoimmunity,mayit not makesense to buttress a repertoirepurgingmechanism with a suppressorsystemthat acts as a fail-safe device? Giventhe immunopathological consequencesthat can result from continued antibody productionin the face of persisting antigen, could one not envisagethe bodyutilizing a trick developedprimarilyfor self-recognition to limit the immuneattack against foreign antigens, such as intestinal commensal organisms?Is the thought that an anti-idiotypic suppressor T cell stops a particular responseso far fromthe thoughtthat the idiotype is silenced by clonal purgingof the cell bearing it? Thetemptationto place tolerance phenomena into tight compartmentsmustbe resisted for another reasonas well. Aselective immune system, gearedto react to the unknown, must be degenerate and redundant(47), otherwise the repertoire of unispecific cellular elementswouldhaveto be infinitely large. A necessary consequence of this fact is that the at~nity of an.tibodiesvaries greatly, and that cross-reactivities amongunrelated antigens and antibodies are common.This beingso, andthe numberanddiversity of self antigensbeing very large, clonal deletion of all cells with any reactivity .to anyself antigen cannotbe absolutelycorrect, becausethere wouldbe a real risk of deleting the total repertoire! Thusany thought of clonal purgingas the mechanism of tolerance must immediatelybe qualified by exemptionsfor sequestered antigens, antigenspresentin lowconcentrationsin extracellular fluids, and (for antigens present at intermediateconcentrations)perhapswith respect to antibody alfinity thresholds, belowwhichthe purging mechanism could not be expectedto operate. Bythe sametoken, a limited amountof autoantibodyproduction,e.g. to heart muscleantigensafter a myocardialinfarction, neither transgresses the rules of tolerance nor causesany harm. It appears, therefore, that immunologic tolerance cannotbe describedor explainedsimplistically or in isolation fromimmune responsesas a whole. Fromthe viewpointof this review, it is fortunate that twoother chapters

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IMMUNOLOGIC TOLERANCE 35 in this volumedeal with idiotypic regulation (100) andsuppressorpathways (53a), whichthereforereceiveless emphasishere. Theplan of this review as follows. A brief historical introduction describes the mainstreamsof thinking in the field. Themajor section of the reviewdeals with the B lymphocyte,becausemoredetailed knowledge about its receptors for antigenand moreadvanced technologyfor its studyat the single cell level have given us a clearer picture than is available for T cells of howit can be signaled followingencounterwith antigen. Present knowledge of howactivation takes place is presentedto contrast with the various mechanisms of negativesignaling. Akeyfindingthat emergesis that not all B cells react equally to a given signal. The more complexproblem of T lymphocyte tolerance is then addressed, with special emphasison tolerance to MHC antigens. HISTORICAL

PERSPECTIVES

Thewordtolerance wasfirst applied to this field by Owen(89, 90), in his classical studies on binovular twin cattle sharing a common placenta. He noted that "erythrocyte precursors fromeach twin fetus had becomeestablished in the other and had conferred on their newhost a tolerance towards.... foreign ceils that lasted throughoutlife" (89). Evenearlier, Traub(122) had noted that miceinfected in utero with lymphocyticchoriomeningitisvirus carried the virus in their bloodandtissues throughoutlife, without an apparentimmune response, whereasmice first infected in adult life madea brisk, typical antibody response. Onthe basis of these two findings, Burnet& Fenner(19) predicted that an antigen introducedinto the bodyduring embryoniclife, before the immuneapparatus had developed, wouldbe mistakenfor self, and that it wouldnot evoke antibody formationthenor if reencountered later in life. It is of interest to recall that the somewhat awkwardself-marker theory of antibody production proposedin that workin fact postulatedself-recognitionunits in antibodyproducingcells that gavethemthe capacityto recognizeself. Thepostulated characteristics of these receptors are somewhat similar to those popular today for self-MHC recognition by proponentsof the two-receptortheory of MHC restriction. Burnet’s ownattempts to provide experimental evidencefor the theory wereunsuccessful(20), but experimentalinduction tolerance wasachievedsoonafter by Billinghamet al (9), whousedliving cells as the sourceof MHC antigens andskin graft rejection as the readout system. Theseauthors coined the phrase "actively acquired immunological tolerance." This study usheredin an avalancheof research on the subject. It wasperhapsfortunate that mostearly workerswhosoughtto validate the conceptof tolerance by using non-livingantigens, and antibodyproduc-

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36 NOSSAL tion as a readout, usedxenogeneicserumproteins as the test toleragen(24, 32, 56, 109, 110), as had they followedTraub’slead and used microorganisms, the results wouldhavebeenless successful(20, 82). In the event, the conclusionthat antigens introducedin the perinatal period preferentially caused tolerance quickly becamewidespread.Toleranceinducedin adult animalsby lethal doses of X-irradiationand allo- or xenografting(69) was explicable on the postulate that a repopulating lymphoidsystemrecapitulates the (unknown)events of early ontogeny.Thefinding of Felton (44) that small but supraimmunogenicamountsof pneumococcalpolysaccharide could cause nonreactivity even in normal, adult mice was generally placedinto a separate conceptualbasket until muchlater. A major stride towardsa frameworkfor the understandingof tolerance wasthe developmentof Burner’s (18) clonal selection theory. If immune recognition were indeed encodedin a population of immunocytes with one specificity per cell, then deletion of self-reactive clones could elegantly accountfor self-tolerance. Thenotion wasfurther elaboratedby Lederberg (65), whopostulated that lymphocytespassed throughan obligatorily paralyzable state early in their ontogeny,during whichantigenic encounters wouldsilence or delete them;if, this milepostwerepassedwithoutantigenic encounter,the cell wouldbecomeinducible,i.e. susceptibleto activation by clonal selection processes.Thediscoverythat onecell alwaysmadeonly one antibodyspecificity gavean early boost to clonal selection (83), and the discoveryof great heterogeneityin antigen binding amgnglymphocyteslent strong support (80). It wasnot until the late 1960s:thatclonal selection cameto be almostuniversally accepted, at least for the B lymphocyte.In the meantime,the notion of somethinguniqueabout the newbornstate that facilitated tolerance induction had received somerude shocks. In 1962, Dresser found that if care was taken to free preparations of serumproteins of all aggregatedmaterial, intravenousinjections of minute amountsinto normaladult animals could induce tolerance (37). This was an importantdiscovery, becauseit raised the possibility that evenmature immunocytes could receive negative signals from antigen. Graduallythe perception dawnedthat unprocessedantigen, acting alone, wasinsut~cient to trigger an immune response, but that someaccessorystimulusor characteristic wasrequired. Theterm adjuvanticity wascoined. Thebelief grew that direct encounters betweenlymphocytesand soluble antigens lacking this characteristic werelikely to deliver a negativestimulus(46). Yetthe nature of this signal remainedentirely unclear. Until the middle1960s, writers on tolerance considered phenomena involvingpredominantly T cells (e.g. graft rejection) andthose depending reducedantibody responsesmoreor less together. Thencametwin revolutions in rapid succession, namelythe clear separation of lymphocytes into

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two great families, T and B cells, and the realization that these families had to collaborate in antibody formation (reviewed in 73). The impact on research into tolerance was immediate and profound. Weigle’s group (23, 127-129) soon reached the view that humangammaglobulin injected into adult mice produced tolerance in the T cell compartmentmore rapidly and at a muchlower dose of antigen than for the B cells. The greater ease of tolerance induction in T cells was confirmedin several other studies (74, 76, 99, 116). Nevertheless, one probable cause at workin these experimentsstill remained to be uncovered by independent work. The discovery of suppressor T cells by McCullagh(70) and t3ershon & Kondo(49) gave new thrust to tolerance research. It is in someways remarkablethat suppressor T cells had been missed until then. In retrospect, the ultra-low dose tolerance caused by femtogramamountsof flagellar antigens in our laboratory (107) could hardly have had any other explanation. In any event, once discovered, suppressor T cells were found to be active in manysituations. As early as 1973, a review by Droege(38) cited no fewer than 81 references on suppressor T cells, and the pace of research has certainly not slackened since. The key role of regulatory T cells in both antibody formation and T cell-mediated immunereactions raised another issue of great importance to the tolerance field. Giventhat the precursors of effector cells, particularly B cells, might not be tolerant of certain self components,the possibility was raised that autoimmunization might follow if a self molecule came into association with a foreign molecule that could act as a carrier, thus being capable of inaugurating T cell help (3, 127). A similar mechanismcan invoked for breakdown of experimentally induced tolerance in some models. Thoughthe emphasis of research in the early 1970s swungheavily towards regulatory T cells, the concept that B cells might be negatively signaled through direct contact with antigen was kept alive. Animportant experimental contribution was that of Borel’s group (1 l, 50), whoshowed that haptenic antigens coupled to autologous immunoglobulin acted as powerful, direct toleragens for adult B cells. A seminal theoretical work was the articulation by Bretscher &Cohn (17) of their two-signal theory lymphocyteactivation. This states that lymphocytesrequire two inductive signals to becomeactivated. One is provided by antigen occupying the receptor for antigen; the other is provided by the action of a helper T cell. If the first signal acts alone, in the absence of the second, the result is a negative signal or tolerance. In manyways, this formulation still provides a useful framework. By the mid-1970s,the last of the crucial issues mentionedin the introduction, namelythat of MHC restriction, entered the arena, profoundly affecting all our concepts of the T cell and therefore also of tolerance. AsDoherty

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NOSSAL

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& Benninkhaveput it (33), the tolerance problemcould no longer be seen simplyas a needto distinguish betweenself and non-self, but rather as a requirementthat the T cell be capableof distinguishingself fromself plus other. Theintriguing possibility emergesthat within the thymuscells with a high affinity for self-MHC components are eliminatedby a clonal abortion mechanism(10, 33), whereasthose with low affinity for self-MHCare positively selected. Theextensiveliterature on MHC restriction is, however, outsidethe scopeof this review. SIGNALS INVOLVED TRIGGERING

IN

B LYMPHOCYTE

Before considering the possible mechanismsof induction of tolerance amongB lymphocytes,it is essential to summarize whatis knownabout the induction of immunityin this. population. Studies with purified B cells culturedat lowdensity(40, 58) or singly (97, 114,124, 130)havematerially enlargedour understanding.It is nowclear that a cross-linkingof the cell’s membrane immunoglobulin (mIg) receptors alone, whetherby anti-Ig antibodiesor by a multivalent,T-independent antigen, doesnot suffice to trigger the cell. Additionalsignals are required. For someB cells, a T-independent antigenandoneor moreB cell growthanddifferentiation factors(s) initiate proliferation and subsequentantibodyproduction. For others, an antigenspecific, MHC-restricted, T cell-derivedhelper signal is also needed(4, 81). Furthermore,the stimulatory co-factors able to movethe B cell from a resting Gostate to active blastogenesis maynot be identical to those requiredfor continuedcycles of multiplication,and these, in turn, maydiffer from factors that promotedifferentiation to antibody-producingstatus. Someevidencesuggests that macrophage-derived, interleukin l-like molecules are neededearly in the cascadeandat least twotypes of T cell-derived factors are neededsomewhat later. Thenature of these various co-stimulatory factors is under active investigation at the moment.Frommyviewpoint, the most important point is that this recent workvalidates the requirementfor somethingother than Signal 1 alone, openingthe door to the possibility that Signal1 withoutthis complexof factors that constitute Signal 2 indeedmayexert a negativeeffect on the cell. Somefurther interesting points warrant mentionwith respect to Signal 1. Althoughit appearsthat receptorcross-linkingis an essential elementin immuneinduction, not every cross-linker behavesequally. Dintzis’ group (30, 31), by using size-fractionated linear polymersof acrylamidesubstituted with hapten, reached the conclusion that a minimumnumberof receptors (about 12 to 16) mustbe connectedas a spatially compactcluster before an immunogenic signal is delivered. Polymerssubstituted with fewer

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IMMUNOLOGIC TOLERANCE 39 haptenmoleculesexert an inhibitory effect. Theimportanceof somecritical degreeof epitope spacingin immune signaling has also beennoted in other systems(42, 43). Weshouldtherefore be awarethat subtle differences may exist betweenthe Signal1 that combineswith Signal 2 in immune induction, and the Signal 1 only, whichmaybe operative in someformsof tolerance. Anotherimportantquestion in the creation of an appropriate micropatch on the B lymphocytesurface involves the cell Fc receptor. This must concernus, particularly as so manyof the toleragens used in current research feature Ig as the carrier molecule.Isolated Fc portions of humanor murine gammaglobulin are capable of polyclonally activating murine splenic B ceils to proliferation and antibodyproduction(8), mimicking the effects of lipopolysaccharides(LPS). Althoughthe effect wasclaimed involve only Fc-receptor-positiveB cells and not macrophages or T cells, the point is difficult to provein high-densitycultures. Multivalentantigens attached to the B cell surface could easily engagethe Fc receptors in a micropatch through capturing secreted antibody. Furthermore, antigen attached to dendritic follicle cells is complexedto natural or acquired antibody and so could lead to the formation of a mixedmlg-Fcreceptor patch on the lymphocytesurface. Weneed to knowmuchmore about the characteristics of the macromolecular interactions that constitute Signal1, which maybe more complexthan we currently imagine.

NEGATIVE SIGNALS TRANSDUCEDVIA THE B LYMPHOCYTEMIG RECEPTOR Manystudies showthat an engagement of the B cell mlgreceptors can also lead to negativesignaling. This section reviewsevidenceof this important processin immunologic tolerance. EveryB cell with mIgis susceptible to this negativesignal, but a dominantfeature that will engageour attention is the fact that cells at different maturationstages in B cell development displaymarkedly differing sensitivities to this influence.B lymphocytes are derivedfromproliferating pre-Bprecursor cells and exit fromthe mitotic cycles that generate themas small, non-dividinglymphocyteslacking mlg (88). Theythen developmlg, first IgMand later IgMplus IgD, during non-mitoticmaturationphase. I arguethat cells encounteringantigenas the first receptors emergeare exquisitelysensitive to negativesignaling. This phaseis whena potentially anti-self B cell wouldfirst meetantigen. Immature B cells already possessingmIgMcan be isolated from newbornspleen or adult bone marrow,and can be confrontedwith antigen in vitro. Note that this is subtly different fromallowingthe mlgreceptor coat to develop on the cell surface in the constant presenceof antigen. AnimmatureB cell is less susceptible to negative signals than one caught in the pre-Bto B

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transition, but it is muchmoresusceptible than is a mature mIgMplus mIgDopositive B cell. Finally, evenan activated B cell, engagedin largescale antibodysynthesis, can be switchedoff by antigen, but only at high concentration. AsI reviewthe data on whichthese assertions are made,the picture that emergesis not a dramatic,all-or-nonedifference conferringuniqueproperties on the immaturecell, but rather a carefully programmed quantitative difference geared to help self-recognition and also to limit appropriate immuneresponses. Negative Signaling During the Pre-B to B Transition The first evidence that receptor cross-linking could have profoundand long-lasting effects on B lymphocytedifferentiation was producedby an elegant series of studies by Cooper’sgroup(reviewedin 64). Whenantibodies to IgMare introducedinto an animalbefore B cells havedeveloped,B cell maturation can be prevented, and an agammaglobulinemic, B celldeprivedindividualresults. However, such animalsstill possesspre-Bcells. Whensuch lymphocytesare cultured in the absence of further anti-/.t antibody, mIg-positive B cells can emerge. Thesefindings could be explained by postulating that as soonas the first few mIgM receptors appear on the maturingB cell, contactwith anti-kt immediately initiates the patching-capping-endocytosis cycle and thus the cell never really developsinto a B cell. This viewgainedfurther credenceby two independentstudies (98, 108), whichshowedthat maturingB cells were muchmore susceptible to modulationby anti-Ig than were mature B cells. Furthermore,although mlgreceptor deprivationin matureB cells wasreadily reversible, a considerable proportionof immatureB cells failed to recovertheir receptor coat in tissue culture followinganti-Ig treatment.It seemslikely that suchcells woulddie in vivo without ever becomingmlg-positive, especially in the continuingpresenceof the modeluniversal toleragen, anti-Ig. Wewishedto take this line of investigationfurther. In somerecentstudies (93), Pike et al set out to determinewhetheror not concentrationsof anti/.t chain antibodiesinsufficient to modulatethe totality of the mlgM receptor coat couldneverthelessexert functionaleffects on B cells. In particular, what wouldhappenif very low concentrationsof anti-/.t were present as immaturesmall lymphocyteschangedfrom being mlgM-negativeto mIgMpositive? Theydevelopedan anti-bt chain monoclonalantibody, termedE4. Thefluorescence-activated cell sorter (FACS)wasused to select small, mIgM-negative cells from adult murinebone marrowor spleen. Thesewere cultured for 1-2 days, and in the absenceof E4, a populationconsisting of 30-40%mlg-positiveB cells resulted. Aspredicted, E4 wasable to inhibit this pre-Bto B transition. At a concentrationof 10/.tg/ml,virtually no cells with high mlgdensitydeveloped.At 1/.tg/ml, a slight inhibition of receptor

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IMMUNOLOGICTOLERANCE 41 emergencewas noted. At concentrations of 0.1 #g/ml or below, the E4 failed to affect mIgappearance. With these low concentrations, FACS analysispost-cultureshowed the mIgstatus of the cells to be exactlyequivalent to that of controls. Accordingly, Pike et al (93) allowedB cell maturation fromthe pre-Bpool to proceedin the presenceof 0.1 #g/mland still lowerconcentrationsof E4. Following1 or 2 days of such preculture, the resulting B cells weresubjected to functional tests. Theywerestimulated with the potent mixtureof polyclonal B cell activators, LP$plus dextran sulfate. Thecapacityof single B cells to giverise to proliferating clusters wasstudied, as wastheir capacity to produceIg, measuredby the protein Areverse plaque technique. It wasfound that 0.1 #g of E4 present during maturation completelyabrogated response capacity. With10-2/xg/ml, 97100%inhibition wasinduced, and even with 10-4 #g/ml, only half as many B cells respondedin the donal cultures as in controls with no E4 present duringmaturation.Yet, in all these groups,the number of B ceils appearing duringpreculture, andthe averagenumberof mIgreceptors per B cell, were perfectly normal. Thus, the B cells must have received and stored some negative signal, which,however,did not impair the insertion of mlginto the plasmamembrane of the cell. If this experimentaldesignis a modelfor whatmaybe occurringin vivo duringthe induction of self-tolerance, it oughtto be possible to produce similar effects on antigen-specificB cells. Wehaveexamined this possibility in vivoby introducingtolerageninto the tissues of fetal micevia the maternal placenta(94), at a time beforeanyB cells werepresentin the fetus. The hapten fluorescein (FLU)linked to the carder humangammaglobulin (HGG)waschosen. Isotope tracer studies indicated free passage into the fetus and helpedto establish tissue concentrations.Togaugethe effects on the emergingB cell pool in a strictly quantitative manner,Pike et al (94) onceagainmadeuse of B cell cloningtechniques,on this occasionusing the T-independent antigen FLU-polymerized flagellin to trigger the B cells. The results led to very similar conclusionsto those describedabove.It wasfound that with a fetal serumconcentrationof around80 pg/ml(8 X 10-5 ptg/ml) or 5 X 10-13 M, half the FLU-responsiveB cells wererendered incapable of responding,indicatingan extraordinarysusceptibility to negativesignaling. Oneinteresting feature of this studywasthat fully tolerant mice,given muchlarger doses of toleragen in utero, possessedin their spleens mlgnegative pre-B cells on the verge of receptor emergence.Whenthese were placed in vitro in a highly stimulatory environment,they acquired their receptors and, then confrontedwith Signal 1 plus Signal 2, reacted normally. This is at least onereasonwhyantigen mustpersist for tolerance to be maintained,and it soundsa warningfor studies in whichlarge numbers of cells are adoptivelytransferred to environments full of Signal 2. Thecapacityof suchvery lowconcentrationsof antigento affect the cells

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functionally suggests that some mechanismother than modulation of the mIgreceptor coat must be at work. It is therefore of considerable interest to ask whether the antigen, despite its low concentration, delivers some death blowto the maturingantigen-specific cell, or whetherthe functionally impaired cell can persist for sometime. Nossal &Pike studied this question in vivo by inducing tolerance via the placental route and subsequently searching for antigen-binding cells (85). Whensmall doses of toleragen were used, no significant reduction in FLU-binding cell numbers was noted. Furthermore, the antigen avidity profile of tolerant and control populations was identical.

The Concept of Clonal Anergy, and Competition between Signals Takentogether, these results suggested that clonal abortion was not really an accurate description of the events induced, at least not whencritically low concentrations of antigens were used. Rather, they are more consistent with the induction of a potentially reversible anergic state in the maturing B cell. I havetherefore coined the phrase clonal anergy to denote that Signal 1 only can render a cell non-responsivewithout actually killing it. I believe this finding settles an old controversy. Tolerant cells do seemto exist, and this allows one to pose the question of whether or not somereversal of this intracellular anergy might form part of the spectrum of autoimmunity. Important evidence exists that even at the earliest phase of B cell neogenesis tolerance induction is not the only possible outcomeof an encounter with antigen. The first hint of this was the work of Metcalf &Klinman(71, 72), who used unfractionated cell sources rich in immature B cells in T-dependent B cell cloning system, the splenic microfocus assay. They found that whenthese cells were confronted with specific multivalent antigen in the absence of T cell help, the cells were rendered tolerant. However, when they saw antigen for the first time simultaneously with a strong activation of helper T cells present in the immediatevicinity, they formed antibody-forming clones. Thus even here, Signal 1 plus Signal 2 resulted in immunity.Teale et al weresutficiently interested in this result to repeat it, using FACS-fractionated mIg-negative pre-B cells from either newborn spleen or (in small numbers) adult spleen (118). In the absence of T help, hapten-HGGpresent during their maturation rendered the immature B cells tolerant. In the presence of help, a clonal response was obtained. An analogous result was obtained in experiments where fractionated pre-B cells were placed in culture simultaneously with hapten-HGGand the polyclonal B cell activator LPS (96). This mitogen has been seen by some as representing a mixture of Signal 1 and Signal 2. In this situation, about two thirds of the calls of relevant specificity wereindeed rendered tolerant,

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but one third wenton to clonal proliferation and antibodyproduction. In that substantial minority, the tolerance signal hadbeenoverridden.Asomewhatsimilar intervention of a polyclonalB cell activator duringtolerance induction had been reported previously (68). Negative Signaling of Immature B Cells If the line of reasoningpostulatinga gradationof progressivelydecreasing sensitivity to negativesignalingas the B cell maturesis correct, interest attaches to the behaviorof B cells with mlgreceptors that maystill not be fully mature.Alikely sourcefor suchcells is the mlg-positivecell pool from newbornspleen or adult marrow,whereimmaturecells predominate. To study suchceils, onecan either resort againto the modeluniversal toleragen, anti-/~ antibody,or seek to identify, isolate, andstudy the immature B cells of a givenspecificity. Nossalet al (84, 85, 87, 92-96)havedoneboth and havedocumented that such B cells do represent an intermediatestage in sensitivity to toleranceinductionbetween the exquisitesensitivity of cells in the pre-Bto B transition state and matureB cells. Let us consider,first, the behaviorof B cells fromnewborn spleenfollowing a brief encounterwith anti-~ chain antibody(87). Thesewerefound be about 30-fold moresusceptible to negative signaling than wereadult B cells similarly treated. To achieve60%inhibition of clonal proliferation followingmitogenstimulation, 0.1 /~g of anti-/~ antibodyper ml wasrequired, acting for 24 h on the cells, whereas3 #g/ml only reducedthe proliferative potential of maturecells by 40%.A similar difference was noted for antigenic stimulation and antibody formation as the readout. Note, however,that the threshold concentration delivering an effective negativesignal is about1000-foldhigherthan for the pre-Bto B transition. As immatureB cells, thus defined, possess adequateamountsof mlg, it is possible to prepareantigen-specificimmature B cells by the sameantigenaffinity fractionationproceduresas for maturecells (55). Onecan then react such cells with multivalent antigen, e.g. for 24 h, and subsequentlycan challenge with antigen in immunogenic form. Their analysis (84) showed that with an oligovalent FLU-HGG that has 3.6 haptens per molof cartier, hapten-specific newbornB cells could be tolerized with 1000-foldlower antigenconcentrationsthan couldadult 13 cells, the thresholdbeing around 0.1 /zg/ml. In other words,the sensitivity is muchlowerthan that of cells that first see antigenbeforeachievingfull receptorstatus, but muchhigher than that of fully maturecells. Pike et al haveperformedan extensiveexplorationof negative signaling amongthis intermediatepopulationto determinethe importanceof molecular arrangementof haptens and of the cartier molecule(92). Spleencells from newbornor adult mice were incubated for 24 h with FLUcoupled to

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HGG,the F(abl)2 fragment of HGG,or bovine serum albumin (BSA). Conjugates were prepared that possessed a meanof 2, 5, 8, or 12 FLU residues per moleculeof protein. After this putative tolerance-inducing regime,the cells wereplacedinto a limit dilution cloningassaywith FLUPOL(polymerized ttagellin) as the antigenicstimulus.It wasfoundthat with all conjugates tested, the newborncells were moresensitive to negative signaling than were adult cells. However,the gap betweenimmatureand maturecell, in terms of sensitivity, varied widelyfromconjugateto conjugate. For all three carriers, the higher the haptendensity, the lowerthe molarity required for tolerance induction. TheFc piece appearedto play somerole in makingHGG the mosteffective of the three carriers. At a given hapten substitution rate, FLUconjugatedto the F (abe)2 fraction of HGG (FLU-FAB)was less toleragenic than was FLU-HGG. This difference could be overcome by crowding on more haptens. FLU-BSA was less toleragenic than either FLU-HGG or FLU-FAB. Dependingon the conjugate, the sensitivity difference factor betweenimmatureand maturecells varied from6 to 53. Withhighly multivalentconjugatesit is easy to see how susceptibility differencescouldhavebeenmissedby other investigators, as the differences are less pronounced than with oligovalent antigen. Thoughfew authors havesoughtto dissect the pre-Bto B transition from slightly later stages of ontogeny,the sensitivity of the immature B cell to negative signaling has beenwidelyconfirmed(21, 39, 71, 72, 105). Nevertheless, the adult B cell is not completelyrefractory, and wemust now examinemlgas a transducerof negativesignals in morematurecells of the Bseries. Negative Signaling of Mature B Cells and their Progeny I havealready madereference to the workof Borel (11, 50), to studies Nossalet al (see above), and to workby Weigle’sgroup (35, 36, 91, 128), whichamplydemonstratethat a sufficient (and by no meansunphysiologically high) concentrationof antigen can deliver a negative signal evento matureB cells. Conjugateswith Ig carriers are particularly effective, which brings us backto the questionof the role of the Fc receptor. This has been addressedin an interesting series of studies by Taylor’s group(117, 119121). Theypreparedstable covalent antigen-antibodycomplexes,for example linking haptendirectly to antibody, or hapten-proteinsto anti-hapten antibody. The hapten-antibody conjugates were profoundly suppressive whenT-independentanti-hapten responses were later measured.Evidence suggesteda direct effect on B cells rather than a suppressorT cell effect, and the Fc piece of the antibody in the conjugatewasrequired. This was closely analogousto earlier studies with non-covalentflagellin-antibody complexes(43). However,whenthe animalhad beenpre-primedagainst the

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rabbit antibody in the conjugate, the end result was an enhancedresponse, suggesting that the conjoint action of (normally toleragenic) conjugate and active T cell help was immunogenic. Whenribonuclease was linked to antibody in this fashion, this normally poor immunogeninduced priming or immunologicmemory,but this immunogenicitywas lost if recipient mice had been previously madetolerant to the rabbit IgG carrier. In that case the complexeswere frequently toleragenic. Again, the suggestion is strong that an engagementof the Fc receptor within the micropatch on the B cell surface somehowcauses the generation of a more effective Signal 1, which requires the Signal 2 of T cell help to becomeimmunogenicand in its absence acts to produce tolerance. It wouldbe of considerable interest to quantitate the effects of these stable antigen-antibody conjugates on immature B cells. The regulatory role of the mlg receptors as signal transducers does not end at the momentof lymphocyteactivation. In fact, activated B blasts and early antibody-forming cells including manyplasma cells still bear mlg. Presumablythis indicates that such cells are. still under the regulatory influence of antigen. Certainly premature removalof antigen halts expansion of immunocyteclones (95). There may be circumstances, however, such as the existence of immunecomplexes in the circulation, where it might be wise to halt the ongoing immuneproliferation, or even antibody formation at the single cell level. Someyears ago, Schrader & Nossal discovered that an interaction of a single antibody-forming cell with multivalent, specific antigen can result in profound inhibition of that cell’s secretory rate (104). This represents an unexpected late down-regulatory signal mediated via mlg, which may represent, though at a much less sensitive level, a mechanismanalogous to the tolerance induction just described. Independent confirmation ~ was provided by Klaus (62), whose group also soon showed that antibody production in monoclonal plasmacytomacell cultures could be similarly inhibited (1). Antigen-antibody complexeswere particularly effective in this regard (2). Mylaboratory has recently sought to characterize this phenomenon, termed effector cell blockade, more fully. As a first step (12), a mouse hybridoma cell line that secretes IgMspecific for FLUwas developed. Treatment of these cells with highly multivalent FLUconjugates (e.g. FLU20-gelatin) resulted in inhibition of hemolytic plaque formation. Biosynthetic labeling studies indicated this was a result of reducedIg synthesis. Total protein synthesis and cell proliferation were not affected, documenting the specificity of the inhibitory effect. Fluorescencestudies showedthat FLUconjugates capable of causing blockade aggregated on the cell surface, and clearance of cell-associated antigen correlated with recovery from blockade. Reversibility has also been documentedin other systems (78).

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In later studies, Boyd&Schraderhaveprovidedfurther valuableinsights (14, 15). Colchicinereverses effector cell blockade, providedexogenous antigen wasremoved.In the presenceof colchicine, cappingoccurs, presumablyaiding antigen removal. However,in the continued presence of exogenousantigen, colchicine wasnot able to reverse the negative signal, arguingagainst the notionthat colchicine-sensitivestructures, suchas microtubules, are essential for signal transmission. It wasalso found that antibody-forming cells (AFC)fromthe spleens of 10-day-oldmiceare more susceptible to blockade than are adult AFC.Furthermore, blockade in neonatal AFCcould not be completely reversed by enzymaticremovalof surface antigen. This increased susceptibility of neonatal AFCis reminiscent of the greater sensitivity of newborn splenic B cells to negativesignaling by antigen. It supportsthe notionof someintrinsic extra susceptibility in mlg-transducedsignaling in the neonatalstate. Anothermodelof a relatively mature B cell is a B lymphoma, such as the cell line WEHI 231, whichresemblesa B cell in bearing large amounts of IgMon its surface but not in secreting Ig. It wasfound(13) that anti/.t chain antibodies, including E4, profoundlysuppressedthe growthof WEHI 231 in both soft agar and liquid culture. Other antibodies binding to non-Igcell surface antigens lackedthis effect, and LPS,whichinduces antibody formation in normalWEHI 231 cells, could neither prevent nor reversethe growthinhibition. This negativesignal differs fromthat involved in effector cell blockadein affecting cellular life span. It provokesthe thought of whetheror not receipt of the anergic signal in normalB cells mayaid their early demise. Thepicture that emerges,then, is a graduallydiminishingsusceptibility to negative signaling as the B cell matures. Themonoclonalanti-/~ chain antibody, E4, has allowedsomequantitative comparisonsin this regard (93). It takes approximately 5.5 log~omoreE4to causeeffector cell blockade in matureantibody-forming cells than it raises to cause clonal anergyduring the pre-B to B transition. Operationally, this could well meanthat many exogenousantigens never reach the concentrations that might impair the B cell responseto them. CONSTRAINTS ON THE UNIVERSAL APPLICABILITY OF CLONAL ANERGY IN SELF-TOLERANCE Althougha strong case can be madefor ~he plausibility of the clonal anergy modelas beingrelevantto self-tolerance, particularlyfor antigens(e.g. cell surface molecules)present in multivalentform, it is importantto mention someof the constraints on the universality of the mechanism. Perhapsthe

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most obvious of these is that monovalent antigens do not induce clonal anergy in vitro. Most of the evidence suggests that the negative signal requires mlg receptor cross-linking. One could imagine in vivo matrixgenerating mechanisms,e.g. self antigens associating with macrophagesurfaces, or even one B cell presenting toleragenic antigen to another. Given the extreme sensitivity during the pre-B to B transition, even someselfassociation through protein-protein interactions at the B cell surface could not be entirely excluded. It is also of interest that despite the enormous amount of work on autoimmunediseases in both experimental animals and man, no one has yet reported an autoimmunedisease that involves antibody production to autologous albumin, transferrin, complementcomponents, or other soluble monomericserum proteins present in high concentration (rheumatoid factors are an interesting exception). If tolerance to albumin did not exist within the B cell compartment,it is hard to see howantibody production could be avoided on occasions of abnormal induction (e.g. endotoxins present in septicemia). There are no albumin-binding cells in humanperipheral blood (5), though B cells capable of binding autoantigens present in low concentrations in serumdo exist (5, 41, 102). This suggests that for antigens present in high concentration, someclonal purging mechanism may be at work in vivo, perhaps one we have not yet been able to modelin the artificial in vitro models. Nevertheless, the possibility must be faced that someself antigens cannot evoke the clonal anergy/clonal abortion pathway, but cause tolerance through other mechanisms.From that viewpoint, recent studies from Dienet’s groupare of interest (29, 125, 126). The sensitivity of the fetal immune system to tolerance induction in vivo was investigated by injecting various antigens into the maternal circulation. Transplacental passage was monitored, as was persistence of antigen in serum and lymphoidtissues. Despite adequate exposure, the fetuses failed to becometolerant in most instances. Of seven antigens, only two gammaglobulins (HGGand bovine) induced significant tolerance. Furthermore, whenhaptens were introduced multivalently onto the proteins, the various non-tolerageniccarriers failed to induce hapten-specific tolerance. This was despite fetal concentrations of up to 10-6 M. In other studies it was found that some carbohydrates, but not others, can act as a toleragenic carrier for hapten-specific B cell tolerance, and there appeared to be no clear correlation betweenthe capacity to induce tolerance and either the molecular weight of the carrier or its epitope density. Indeed, rather subtle chemical changes were found to abolish an antigen’s tolerag~nic potential. Thesesomewhatsurprising findings are interpreted as suggesting that the toleragenie potential of certain extdnisic antigens reflects someobscure carrier-related property. To account for the results, Diener’s group has

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postulated (28, 125) a dual recognition event in self-tolerance induction that bears somesimilarities to the dual recognition mechanismresponsible for immuneinduction in T cells. Consider an organ-specific cell membraneself antigen, S, and a ubiquitous, polymorphicself marker or interaction structure, Z. The latter is postulated to be present on all cells including lymphocytes. Whenan anti-S lymphocyteadzes in ontogeny its anti-S receptor meets S, and its self-marker Z meets the Z present on the S-bearing cell This interaction is postulated to cause clonal abortion. Extrinsic, non-self antigens are seen as incapable of inserting into cell membranesin such a way as to allow correct dual recognition, with the exception of somemolecules with special properties. This novel speculation, however, does not account for mechanismsby which animals become tolerant of self components not associated with cell surfaces, e.g. serumproteins. Preliminary studies in our laboratory (B. L. Pike, G. J. V. Nossal, unpublished data) have sought to use transplacental FLUoBSA in vivo as an agent to induce clonal anergy in FLU-specific B cells emergingin fetal and early postnatal life. Even large doses achieved only moderate reduction in the numbersof clonable anti-FLU B cells. This contrasts with the relatively poor but still definite potential of FLU-BSA to give a negative signal to immatureB cells in vitro (92). The result confirms that the carrier molecule is not a neutral entity in tolerance induction. Obviouslythis constraint will require muchfurther investigation.

B CELL TOLERANCE AND AUTHENTIC SELF ANTIGENS There would obviously be many advantages if students of immunologic tolerance, rather than introducing foreign modeltoleragens into animals or cultures, could investigate interactions betweenauthentic autologous antigens and the lymphoid system of the body. However,surprisingly little workhas been done in this way, probablybecause of the practical diti~culties posed by omnipresent antigen, e.g. through neutralizing any antibody that may be formed or through blockading immunocytereceptors. I have already mentioned some of the studies (5, 41, 102) seeking autoantigenbinding B cells, whichshowedpresence of B cells for self antigens occurring at low molarity but absence for those in serum at high concentration. This finding seems consistent with the rules of clonal anergy/clonal abortion. Twoearly studies that are frequently quoted are those of Triplett (123), who claimed that extirpating an organ early in ontogenyled to a loss of tolerance to the organ-specific antigens so that later transplantation led to rejection, and the hemoglobinstudies of Reichlein (101), whoconcluded that rabbits could only form antibodies to such determinants of hemoglobinchains as

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IMMUNOLOGICTOLERANCE 49 are not represented in rabbit hemoglobin.In this section, three recent studies are reviewed,two of whichargue against, while one argues for, clonal anergy/clonal abortion as an important mechanism. The first study comesfrom Borel’s group (57) and exploits a pair congenicstrains of mice, one of whichlacks the fifth component of complement, C5, a serumprotein normallypresent at a concentration of 50-85 /.tg/ml. Lymphoid cells fromC5-deficientmicewereadoptivelytransferred into X-irradiatedhosts of the normalstrain andvice versa. C5-deficientcells were not tolerant to C5. They formedantibody whentransferred to C5sufficient (normal)irradiated hosts. Cells fromnormalmiceweretolerant, failing to form antibody to C5on primary or secondaryimmunizationof the C5-deficient hosts with C5. T and B cell mixingexperimentsdemonstrated that the tolerance appearedto reside in the T cell compartment of the CS-sufficient(normal)lymphoid cells. T cells fromCS-deficient(nontolerant) donorscould collaborate either with B cells fromnontolerant (C5deficient) donorsor fromtolerant (normal)donors.This arguesthat anti-C5 B cells had not beeneliminatedin normalmice, nor werethey functionally silenced. This thought-provokingstudy exhibits two features that are somewhat worrying.First, the adoptivehosts receivedonly 760--780rads of irradiation in a split dose. In myexperience,this wouldallow considerableendogenous reconstitution. Second,70 million cells weretransferred, a very large number, whichraises the possibility that T cell sourcesmightbe contaminated by significant numbersof B cells and vice versa. Takenat face value, the results provide another exampleof a monovalentantigen not leading to B cell tolerance. Asa general point, caution mustbe taken in adoptivetransfer studies to excludethe possibility that effects attributed to B cells in fact are not ascribableto mlg-negative pre-Bcells or evenearlier precursors.It is clear that such occur in spleen and bone marrowin substantial numbers,and if they maturein the concomitantpresence of antigen and T cell help, they will immediately be triggered. Of course, pre-Bcells that lack mlghavenot yet beensubjectedto self-censorship. Thesecondstudies concernan alloantigen, the cytoplasmicliver protein F. This moleculeof 40,000 mol wt occurs in two forms in mice, F1 and F2. Serumconcentrationsare around10-8 to 10-9 M.It has long beenknown that in certain strains of micealloimmunization can lead to an autoantibody responsedirected against determinantscommon to F~ and F2, implyingthat self-tolerance is normallymaintainedby T cells (59). This lack of B cell tolerance is not too worrying,given the relatively lowserumconcentration. Detailedinvestigationof cellular interactions in this systemare nowpossible becauseof the recent development of an in vitro assay(113). Thesystem

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measuresthe proliferation of F-primedT cells exposedto F-pulsedsplenic adherentcells. SyngeneicF antigen fails to activate undercircumstances whereallo-F workswell. It has beensuggested(27) that this is dueto vivoeliminationof self-F reactiveT cells. If so, this wouldbe oneinteresting exampleof clonal deletion affecting T cells but not B cells, an interesting counterpointto the usual explanationsinvolvingsuppressorT cells. Thethird exampleinvolves tolerance to antigenic determinantson mouse cytochrome c (60). This study fromKlinman’sgroupis of particular interest, not only becauseit supports clonal anergy/clonalabortion modelsof tolerance,but also becauseit dependson the careful, quantitativeanalytical technologyso vital in tolerance research. To understandthe study, it is necessaryagain to recall that pre-B cells can form antibody-forming cell clonesif artificially transplantedinto a milieuwheretheymatureinto B cells in the concomitantpresenceof antigen andT cell help (Signal 1 plus Signal 2 overridingthe toleragenicpotential of Signal 1 alone). It is possible to prepare pre-B cell-rich cell populations, e.g. by using bone-marrow depletedof mlg-positivecells. Onecan then contrast this pre-Bcell repertoire of clonotypes with that of mature splenic B ceils. If clonal anergy or analogousmethodsof repertoire purging are importantin self-tolerance, oneshouldfind autoreactiveB cells presentin the pre-Bcell repertoire but absent fromthe matureB cell pool. Jemmerson et al (60) synthesizedtwo peptides from mousecytochromec and probed the T-dependent immune response against each in the clonal splenic microfocusassay. Withone peptide it wasfoundthat the pre-Bcell pool containedmanyclonal precursors against the self component,the numbersbeing analogousto those for a foreignpeptideof equivalentsize. MatureB cells showed far fewerprecursors, indicating an in vivo repertoire purgingduringthe pre-Bto B transition. Withthe other peptide, whichincluded an evolutionarily highly conservedregion at the N-terminalend of cytochrome c, neither cell population madean anti-self response.Thisraises the fascinatingpossibility that V genes whoseproducts mighthave reacted with this region mayhave been eliminated fromthe specificity repertoire by evolutionarymeans. In somerespects, wefind this examplemoreilluminating than the other two. Wehavealready stated that clonal abortion/clonal anergycannot be absolutelytrue for everyself antigen, for fear of purgingthe wholerepertoire throughcross-reactivity. Thedegreeof the contributionof this mechanism to self tolerance therefore becomesa quantitative question. The existenceof someundetermined numberof anti-self B cells, as there appear to be in the C5and F antigen examples,cannot invalidate clonal silencing as an importantquantitative contribution to the immune balance. Whatone needsto knowis whetheror not there are feweranti-self C5or anti-self F antigen B cells than there wouldbe for a foreign antigen of comparable size

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TOLERANCE PHENOMENAWITHIN THE T CELL COMPARTMENT The subject of tolerance within the T cell compartmentis muchmore difficult to addressat either the cellular or the molecular level. Westill know relatively little about the T cell receptor for antigen. Wehaveno method of enumeratingeffector T cells that can comparewith the Jerne plaque technique.Aboveall, wehaveto face the complexitythat whereasthe B cell can encounterand bind antigen free in solution, and evidently can receive signals from such an event, the T cell appears to be programmed to see antigenonly at cell surfaces. Furthermore,both the triggering of resting T cells into activation andthe deliveryby the effector progenyof someeffect (cytotoxickilling, help, suppression)requireboth a unionof T cell receptor with foreign antigen and reeogrtition by the T cell of certain self-MHC determinants, the nature of that "self-hood" having been somatically learned by the T cell pool. Theonly exceptionto these rules appearsto be the effector cell engagedin mediatingsuppression, whichcan adhere to antigen-coated surfaces that lack any MHC molecules. Cell interactions involvingproductsof the I-J genesare probablyactive in getting resting suppressor precursors moving,so evenfor this category of cells, signal inductioninvolveslinked recognition. Conceptually,this extra complexityposes no major dilemmafor clonal anergy/clonalabortion theories. Onewouldsimplyhave to postulate that for those self antigens not already on a cell surface, mechanisms exist for presentingthemin cell-associatedformto the maturingT cell, e.g. by means of accessorycells in the thymus.Onthe other hand,a large bodyof opinion prefers to attribute self-recognition within the T cell compartment to the induction and maintenance of an anti-self-suppressor T cell population.If this special kind of immune responseis placedat a post-thymicstage of T cell development, it is hard to see howthe peripheralpool of (not clonally purged)T cells couldoperationallydistinguishself fromnot self, activating exclusivelyor predominantly a suppressorresponseonly against the former. If, on the other hand, one were to postulate an intrathymic preferential genesis of suppressorsshouldthe maturingT cell see antigen, a formulation that wouldgive ever-presentself antigens the edgethey possesson a clonal abortion model, then weare left with the need to explain whyforeign antigens,in the face of a partial blood:thymus barrier, can so readily gener-

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ate suppressor cells under somecircumstances, even in adult animals. For these reasons, I prefer to regard the generation of suppressor cells as a regulatory mechanism,an ancillary device in self-tolerance, rather than as the primary cause. The main point, however,is that the existence of a family of immunocytes knownas suppressor T cells cannot explain self-recognition. It merelyshifts the problemto another level. Furthermore, the existence of an (undefined) numberof suppressor cells in a T cell population that behaves as tolerant does not necessarily implythat these cells are the sole cause of the tolerance. Clonal silencing of effector precursors maybe present concfirrently, but they escape detection unless sought. Onceagain, only strictly quantitative methodscan address the relative contributions of the various mechanisms. Suppressor T Cells in Tolerance Measured by Antibody Formation It is surprising that someantigens that are good at negatively signaling B cells in vitro or in vivo also seemto be excellent at inducingsuppressorcells. For example, HGGhas been used in manystudies of suppression (6, 7, 35), although it has also been claimed that suppressor cells may not be the essential element in keeping the animal as a whole unresponsive (36, 91, 126). Most authors study induction of suppressor cells in adult mice, but some(66, 126) have used fetuses and the transplacental route of antigen administration and find it effective in initiating suppressor cell generation. Aninteresting recent series of studies (66, 67) documentsthat HGG can not only produce suppressor cells, but also can set up a state of suppressor T cell memory.Thus, whenthe first effective waveof active suppression has passed, animals maybe left with memorysuppressor T cells ready to spring into a secondary wave of multiplication and active suppression should antigen be encountered again. HGGin vitro can generate memorysuppressors that even 6 monthslater can be reactivated by antigen. Such cells may have been missed in previous studies, possibly explaining some examples where tolerance appeared to persist though active suppressors could not be detected. The memorysuppressor cells were antigen specific and were found to have the same phenotype as those mediating primary suppression, namelyLy - 1-23 +, Thy- 1 +, I a+. Moreover,thoughit requires morethan 100 ktg of HGGto generate primary suppression, as little as 1 ktg induces suppressor T cell memory.Exposure to very small concentrations of self antigens throughoutlife might give a continuing series of boosts to the pool of memorycells without necessarily generating detectable active suppressor activity. Anyunexpectedpulsatile rele~ase of self antigen, e.g. from organ damage, could quickly lead to emergenceof active suppressors, preventing autoimmunity. Thus, a powerful case has been made for a mechanismwith

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physiological relevance. Wecan only conclude that this co-exists with B cell-silencing mechanismsas demonstrated in our studies.

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Suppressor T Cells in MHCReactions A large literature exists on suppressor T cells in various modelsof T cell tolerance involving MHC antigens (e.g. 34, 45, 52, 53, 61, 75, 103). This is beyond the scope of this review. The view has been expressed (77) that in this case clone elimination is the prime process and suppressor cells operate as a fail-safe mechanism. Functional Clonal Deletion in T Cell Tolerance I wish nowbriefly to review some evidence supporting the view (16) that functional clonal deletion does occur amonganti-allo-MHC T lymphocytes. Given that T cells of a given specificity cannot be enumeratedby an antigenbinding technology, it is difficult to ask howmanyT cells active against a given self antigen exist in the body. However,the point can be approached by cloning technology. Wecannot visualize the specific T cells nor isolate themby antigen-affinity binding techniques as in the case of the B cell, but we can enumerate specific cells capable of yielding progeny in vitro whose effects can be measured. The best knownsuch technology is the enumeration of precursors of cytotoxic T lymphocytes(CTL-P), which develop from single cells by appropriate stimulation with irradiated allogeneic cells acting as an antigen source, and T cell growth factor as a co-stimulus, a methodology now in use in many laboratories. Nossal & Pike have applied this methodto the classical tolerance modelof newbornmice receiving semiallogeneic spleen cells (86). FUNCTIONAL CLONALDELETION OF ANTI-ALLO-MHC CYTOTOXIC T LYMPHOCYTE PRECURSORS CBA(H-2 k) mice were rendered tolerd ant to H-2 antigens by injection of (CBA× BALB/c)F 1 spleen cells on the day of birth (86). At intervals of 2 days to 12 weeks, the frequencies anti-H-2 d CTL-Pin the thymus and spleen were determined by limiting dilution cloning technology. A profound and long-lasting functional clonal deficit was noted. This wasfirst clear-cut by day 5 of life in the thymusand by day 8-10 in the spleen, as if to suggest that the clonal purging occurred in the thymus and movedto the spleen only as that organ came to be dominated by purged thymus migrants. The functional clonal deletion reduced the observed proportion of anti-H-2 d cells in adult spleens from a normallevel of about 1 in 500 spleen cells (1 in 150 T cells) to 3%of that figure. This showsthat whether or not suppression is also induced concomitantly, clonal silencing is a mostimportant feature of the tolerant state. Its rapid occurrence in the thymus makes a clonal abortion/clonal anergy mechanismhighly plausible.

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Althoughthis strict quantitative approach awaits confirmation, there is a considerable amountof indirect evidence for functional clonal deletion emergingfrom bulk culture studies. For example, Streilein’s group (54, 112) failed to detect lymphocytesreactive with tolerated H-2 alloantigens in a wide battery of tests, including mixedlymphocytereactions, graft versus host reactions, cell-mediated lympholysis, or concanavalin A-mediated, polyclonal activation of tolerant T cell populations. Suppressor cells were not found in these studies, and the inference was of an active process achieving specific clonal deletion of an early stage in the ontogenyof the alloreactive T cells. Proponents of the suppressor T cell might then postulate that suppressor T cells (anti-idiotype in nature?) might actually be responsible for this functional or actual clonal deletion. A hint in this direction was provided by Gorczynski&MacRae (52, 53). Streilein (11 l) investigated this possibility in an original way by using MHCrecombinant strains to induce tolerance. Newbornmice were rendered tolerant to F1 semiallogeneic spleen cells in such a mannerthat donor and host differed over the entire H-2 region, over a region including the K or D locus alone, or over a region embracing either K or D and the mid-I (probably IJ) region. Althoughthe first and last groups developedexcellent tolerance, groups differing at the Class I antigens alone showedlittle or no tolerance as judged by skin graft rejection. It is as if isolated K or D antigens acted as poor tolerogens, requiring some kind of activation (presumably of suppressor cells) dependent on IJ disparity to reach the final tolerant state. If the K or D antigen is regarded as a hapten, and the mid-I (presumably IJ) antigen is regarded as a carrier, then active suppression is induced only whenthe immunogen differs from the host in both hapten and carrier. Howthis activation of suppression finally achieves clonal deletion is not clear. These somewhatunexpected results prompted Nossal & Pike to investigate the effects of anti-IJ sera on tolerance induction in their modeldepending on injecting F1 spleen cells differing at the full MHC into newbornmice (86). Repeated high doses of anti-IJ injected into newborns during the period of toleragenesis partially inhibited clonal deletion, an effect, however, that proved to be quite transient. This has been a frustrating finding to follow further. Thoughthe phenomenonwas readily reproducible, it dependedon a particular batch of anti-IJ serum, which ’so far has not been reproduced with several other batches. The role of suppressor cells in functional clonal deletion therefore remains obscure. It must be remembered that the injection of semiallogeneic cells into newbornmice at birth represents an imperfect modelfor self-tolerance. The toleragen-beadng cells enter the body at a time whensmall but significant numbersof alloreactive T cells are already present in the thymicmedullaand the periphery. It could

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be that suppressor cells are moreinvolved in preventing these mature cells from getting out of hand than in clonal abortion/anergy within the thymus. FUNCTIONALCLONAL DELETION OF ANTI-HAPTEN CYTOTOXICT LYMPHOCYTE PRECURSORS Another model of T lymphocyte physiology extensively explored in recent years is the reaction of T lymphocytes to haptenic antigens presented in association with syngeneic cells (106) isolated cell membranes (25). Tolerance can be induced in such systems and is usually ascribed to suppressor T cells (e.g. 25, 115). Alerted to the fact that suppression mayco-exist with an underlying clonal deletion or anergy, Good& Nossal studied anti-hapten T cells in a model system involving adult tolerance (51). Reactive haptens were administered intravenously adult mice and doses were carefully chosen to provide convincing nonreactivity. Presumablythe operative toleragen was hapten coupled to autologous cells. The anti-hapten CTL-Pnumbers of control and tolerant animals were studied in a limiting dilution microculture system. After apprbpriate correction for anti-self activity within the population, the antihapten CTL-Pnumbers in tolerant mice were found to be reduced by 90%. In other words, functional clonal deletion was present. Kinetic studies showedthat a substantial degree of tolerance existed already at 24 h. These studies documentthat tolerance within the T lymphocyte system, even in models where suppressor T cells have been noted, maycontain an element of functional clonal deletion. Again, the relative importanceof each process in physiological self-tolerance must await the developmentof modelsthat moreclosely mimicthe real-life situation. Other Possibilities for Clonal Abortion of T Cells So far it has appearedas thoughelonal purging of the T cell repertoire must take place in the thymus. However,there are somehints that bone marrow precursors of thymic cells already bear receptors for antigen (26, 63) and are susceptible to toleragenesis by self antigens. Althoughthis woulddestroy the elegant notion of the thymusas the chief generator of diversity amongT cells, it wouldnot offend the central tenets of clonal abortion/clonal anergy. Anintriguing recent study (79) involves radiation chimeras in which bone marrowcells repopulated a thymus graft syngeneic to them, but within the body of a lethally irradiated semiallogeneic thymectomized host. The intrathymic lymphocytes, proven to be of bone marrowgenotype, were nevertheless tolerant not only to themselves, but also to the peripheral alloantigens. Noalloantigenic material was detected intrathymically, nor were suppressor cells found. Althoughthe results were interpreted as favoring pre-thymic functional inactivation of pre-T cells, the presence of sub-

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detectable but still toleragenic alloantigen in the thymus cannot be excluded. An interesting example of what appears to be clonal abortion/anergy of T cells, as judged by a limiting dilution assay, has been reported by ChiecoBianchi et al (22). Mice inoculated at birth with Moloneymurine leukemia virus were challenged as adults with Moloneymurine sarcoma virus. Although other adults (regressors) rejected the resulting sarcomas, these dually infected mice (progressors) died with rapidly growing sarcoma. Progressor mice were found to lack virus-specific CTL,and this was not due to suppressor cells. A limit dilution study showeda severe defect of antiviral CTL-Pin the Moloneymurine leukemia virus carrier mice, as well as in the dually infected progressors. This study has reached similar conclusions to Nossal & Pike involving MHC antigens (86). On the basis of these results, it maybe concludedthat functional clonal deletion is well established as one mechanismof T lymphocytenon-responsiveness. Whetherwe are dealing with clonal abortion or clonal anergy will not be clear until knowledgeof T cell receptors increases materially.

CONCLUSIONS This review has concentrated on" recent studies supporting the notion that functional silencing of particular elements in the immunologicrepertoire is an important mechanismin immunologic tolerance for both B and T lymphocyte classes. It has stressed, however,that it is not the only mechanism, and, in particular, a substantial role is seen for suppressor T cells, not perhaps as the primary mechanism, but as an important ancillary one. For the B cell, the view has been promotedthat mIg is the vital signal transducer, and that each stage in the B cell life history that exhibits mIg displays its ownparticular sensitivity to such signals. Effective signaling may involve the aggregation of receptors into an array with particular characteristics, e.g. a critical minimalnumberbefore signaling can occur. Involvement of the Fc receptor, although not essential, mayamplify the signal. Formation of an mIg receptor micropatch is a prelude either to activation or to a down-regulatory event. The former requires conjoint activity of T cells and perhaps macrophages, with B cell growth and differentiation-promoting factors of an antigenically nonspecific, MHCunrestricted nature prominently involved. The latter is the end result when non-immunogeniccarriers with particular characteristics multivalently present antigenic determinants to the B cell in the absence of co-stimulatory signals comingfrom T cells or artificial mitogens. Thehierarchy of sensitivities to such negative signaling is as follows, in decreasingorder of susceptibility: cells in transition frompre-B to B cell status (self antigens obviously

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first interact with devdoping B cells at this stage); immature B cells; mature B calls; activated, antibody-forming B ceils. The negative signal, particularly if delivered by small concentrations of antigen, insutt~cient to modulate the mIg receptor coat, does not kill the cell but rather renders it anergic. Functional clonal deletion is also a possibility within the T lymphocyte class. This has been formally demonstrated for anti-hapten, anti-viral, and anti-allo-MHC CTL-P. Whether an actual deletion or the induction of an anergic state is involved is not known. Functional deletion can co-exist with induction of suppressor cells. Although some evidence suggests that functional deletion occurs in the thymus, a pre-thymic repertoire purging remains a possibility. The complex process of self-tolerance is probably an amalgam of repertoire purging, suppression, sequestration of self antigens, and blockade of receptors by monovalent, non-immunogenie self molecules and other regulatory loops, possibly including anti-idiotype networks. Evolutionary elimination of V genes with self-reactive potential is also conceivable, but chimeric states amply testify that muchof tolerance is somatically acquired. Carefully quantitated experiments with cloning techniques for enumeration of competent B and T cells should continue to enlarge our understanding. More experimentation on authentic self antigens is badly needed. ACKNOWLEDGMENTS Original work reviewed in this chapter was supported by the National Health and Medical Research Council, Canberra, Australia; by U.S. Public Health Service Grant AI-03958; and by specific donations to The Walter and Eliza Hall Institute. Literature Cited 1. Abbas, A. K., Klaus, G. G. B. 1977. Inhibition of antibody production in plasmacytoma cells by antigen. Eur. J. lmmunol. 7:667-74 2. Abbas, A. K., Klaus, G. G. B. 1978. Antibody-antigen complexessuppress antibody production by mouseplasmacytoma cells in vitro. Eur. J. IrnmunoL8:217-20 3. Allison, A. C. 1971. Unresponsiveness to self antigens. Lancetii:1401-3 4. Andersson, J., Melchers,F. 1981.T cell dependentactivation of resting B cells: requirementfor both nonspecifie,unrestricted and antigen-specific, Iarestricted soluble factors. Proc.Natl. Acad. ScL USA78:2497-501 5. Bankhurst,A. D., Tordgiani,G., Allison, A. C. 1973. Lymphoeytes binding humanthyroglobulin mhealthy people

andits relevanceto tolerance for autoantigens. Lanceti:226-29 6. Basten,A., Miller, J. F. A. P., Sprent, J., Cheers,C. 1974.Cell-to-cellinteraction in the immune response.X. T-celldependentsuppressionin tolerant mice. Z Exp. Med. 140:199-217 7. Benjamin,D. C. 1975. Evidencefor specific suppressionin the maintenanceof immunologictolerance. Z Exp. Med. 141:635--46 8. Berman,M. A., Weigle, W.O. 1977. B lymphocyteactivation by the Fc region of IgG. J. Exp. Meal 146:241-56 9. Billingham,R. E., Brent, L., Medawar, P. B. 1953.Activelyacquiredtolerance of foreign ceils. Nature172:603-6 10. Blanden,R. V., Ada,G. L. 1978.A dual recognitionmodelfor eytotoxie T cells basedon thymicselection of precursors

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with lowd.affinity for 7:181-90 self H-2antigens. Scand. Immunol. 11. Borel, Y., Kilimm,L. 1974. Carrierdeterminedtolerancein various strains of mice:the role of isogonicIgGin the inductionof haptenspecific tolerance. Proc. Soc. Exp. Biol. Med.145:470-74 12. Boyd, A. W., Schrader, I. W. 1980. Mechanism of effector cell blockade.I. Antigen-induced suppressionof Ig syathesis in a hybridoma cell line, andcorrelation withcell-associatedantigen. £ Exp. Med. 151:1436-51 13. Boyd,A. W., Schrader,J. W.1981. The regulationof growthanddifferentiation of a murine B cell lymphoma. II. The inhibition of WEHI231 by antiimmunoglobulln antibodies. Z Immunol. 126:2466-69 14. Boyd, A. W., Sckrader, J. W. 1983. Mechanism of effector cell blockade,II. Colchicinefails to block effector cell blockadebut enhancesits reversal. Cell Immunol.In press 15. Boyd, A. W., Schrader, j. w. 1983. Mechanism of effector cell blockade. IIL Theincreasedsensitivity of neonatad PFCto ¢ffcetor cell blockade.Cell ImmunoLIn press 16. Brent, L., Brooks, C., Lubling, N., Thomas,A. V. 1972. Attemptsto demonstrate an in vivorole for serumblock. ing factors in tolerant mice.Transplantation 14:3~2-87 17. Brgtacher,P,, Cohn,M.1970. A theory of self-nouselfdiscrimination:paralysis and inductioninvolvethe recognitionof one and two determinants on an antigen, respectively. Science169:1042-49 18. B~rnet, F, M. 1957. A modification of erne s theory of antibody production using the conceptof clonal selcetion, Aust. £ Sc~ 20:67-69 19. But’net, F. M., Fetmer, F. 1949. The Productionof Antibodies, 2nded. London: Macmillan 20. Burnet,F. M., Stone, J. D., Edney,M. 1950. Thefailure of antibodyproduction in the chick embryo,dust. d. Exp. Biol. Med. Sc£ 28:291-97 21. Cambier,L C., Vitetta, E. S., Uhr, J. W., Kettman,J. R. 1977. B cell tolerance. II. Triultrophenyl humangamma globulin-inducedtolerance in adult and neonatal mnrineB cells responsive to thymus dependent and independent formsof the samehapten, d. Exp. Med. 145:778-83 22. Chieco-Bianchi, L., Collavo,D., Biasi, G., Zanovello,P., Ronchese,F. 1983. T lymphocyte tolerance in RNAtumor VLrUSoncogenesis: a model for the

clonal abortion hypothesis. In Biochemical and Biological Markers of Neoplastic Transformation, ed. p. Chandra, In press. NewYork: Raven 23. Chiller, J. M., Habieht,G. S., Weigle, W. O. 1970. Cellular sites of immunologic unresponsiveness. Pro¢, Natl. Acad. Sc£ USA65:551-56 24. Cinader, B., Dubert, J.-M. 1955. Acquired immunetolerance to humanalbuminand the response to subsequent injections of diazo humanalbumin.Br. £ Exp. Pathol. 36:515-29 25. Claman,H. N., Miller, S. D., Sy, M.-S., Moorhead, J. W. 1980. Suppressive mechanisms involving sensitization and tolerance in contact allergy. Immunol. Rev. 50:105-32 26. Cohn,M. L., Scott, D. W21979. Functional differentiation of T cell precursors. I. Parametersof carrier-specific tolerance in murinehelper T cell preenrsors, d, Immunol.123:2083-87 27. Czitrom, A., Mitehison, N. A., Sunshine, G. H. 1981. I-l, F, and hapten conjugates:canalisationby suppression. In Immunobiolog~ of the Major ttiatocompatibility Complex,7th Intema~ Convoc.lmraunoL,pp. 243-53. Ba~l: Karger 28. Diener, E., Waters, C. A., Sin#, B. 1979.Restraints on enrrent conceptsof self-tolerance. In T andB Lymphocytes: Recognition and Function, ICN-UCIM Syrup. MolecularandCellularBioL, ed. F. Bach,B. Bonavidm E. Vitetta, C. F. Fox, 16:209-27. NewYork: Academic 29. Diner, U. E., Kunimoto, D., Diener, E. 1979. Carboxymethyl Cellulose: a nonimmuttogenie hapten carrier with toleroge~nic properti~, d.. Immunol. 122:1886-91 30. Dintzis, H. M., Dintzis, R. Z., Vogelstein, B. 1976. Moleculardetgrminants of immunogenicity: the immunon modelof immune response. Proc. Natl. Acad. Sc~ USA73:3671-75 31. Dintzis, R. Z., Vogelstein,B., Dintzis, H. M.1982. Speeitic cellular stimulation in the primary immune response: experimental a quantized model. Proc. Natl. test Acad.ofScL USA79:884--88 32. Dixon, F. J., Manrer, P. 1955. lmmtmologicunresponsivenessinducedby protein antigens. J.. Exp. Med.101: 245-57 33. Doherty,P. C., Bermink,J. R. 1980.An examinationof MHC restriction in the context of a minimalelonal abortion modelfor self tolerance. Scand.d. ImmunoL12:271-80

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IMMUNOLOGIC 34. Dorsch,S., Roser,B. 1977. Recirculating, suppressorT cells in transplantation tolerance. J. Exp. Med. 145:1144-57 35. Doyle,M.V., Parks, D. E., Weigle,W. O. 1976.Specificsuppressionof the immuneresponse by HGG-tolerantspleen cells. I. Parameters affectingthe level of suppression. J. ImmunoL116:1640-45 36. Doyle,M. V, Parks, D. E., Weigle,W. O. 1976.Specific,transient suppression of the immune response by HGG-tolerant spleencells. II. Effector cells and target cells. J. ImmunoL 117:1152-58 37. Dresser,D. W.1962.Specificinhibition of antibodyproduction.I. Protein over loading paralysis. Immunology5: 161-68 38. Droege,W.1973.Five questions on the suppressive effect of thymns-derived cells. In Curt. Titles ImmunoL, Transplant. Allergy, 1:95-134 NewYork: Pergamon 39. Elson,C. J. 1977.Tolerancein differentiating B lymphocytes. Eur. J. ImmunoL7:6-10 40. Farrar, J. J., Benjamin, W.R., Hiifiker, M., Howard,M., Farrar, W.L., FullerFarrar, J. 1982. The biochemistry, biologyandrole of interleukin2 in the inductionof cytotoxic T cell and antibody-formingB cell responses. ImmunoLRev. 63:129-66 41. Feizi, T., Wernet,P., Kunkel,H. G., Douglas, S. D. 1973. Lymphocytes formingred cell rosettes in the cold in patients chronic cold agglutinin cfisease. with Blood 42:753-62 42. Feldmann,M., Howard,J. G., Desaymard,C. 1975. Role of antigen structure in the discriminationbetweentolerance and immunityby B cells. Trans. plant. Re~.23:78-97 43. Feldmann,M., Ntr~al, G. J. V. 1972. Tolerance, enhancementand the regulation of interactionsbetween T cells, B cells and macrophages.Transplant. Rev. 13:3-34 44. Felton,L. D.1949.Signilicanceof antigen in animal tissues. J.. Immunol. 61:10%17 45. Fitch, F. W.,Engers,H. D., Cerrottini, J.-C., Brunner,K. T. 1976. Generation of cytotoxic T lymphocytesin vitro. VII. Suppressiveeffect of irradiated MLCcells on CTLresponse. J. Immunog116:716-23 46. Frei, P. C., Bcnacerraf,B., Thorbecke, G.J. 1965.Phagocytosis of the antigen, a crucial step in the inductionof the primaryresponse.Proc.Natl. Acad.Sci. USA53:20-23

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47. Gaily, J. A., Edelman,G. M. 1972.The genetic control of immunoglobulin synthesis. An~Rev. Genet. 6:1-46 48. deleted in proof 49. Gershon,R. K., Kondo,K. 1971. Infectious immunologicaltolerance. ImmunoL21:903-14 50. Golan, D. T., Borel, Y. 1971. Nonantigenicity and immunologic tolerance: the role of the carderin the inductionof tolerance to the hapten. Z Exp. Med. 134:1046-61 51. Good, M. F., Nossal, G. J. V. 1983. Characteristics of tolerance induction amongstadult hapten-spccific T lymphocyte precursors revealed by clonal analysis. Z Immuno~ In press 52. Gorczynski, R. M., MacRae,S. 1979. Suppressionof cytotoxic response to histoincompatible cells. I. Evidencefor the two types of T lymphocyte-derived suppressorsactingat different stagesin the induction of a cytotoxicresponse.Z Immunol. 122:737-46 53. Gorczynski, R. M., MacRae,S. 1979. Suppressionof cytotoxic response to histoincompatible ceils. II. Analysisof the role of two independentT suppressor pools in maintenanceof neonatally inducedallograft tolerance in mice. Z ImmunoL122:747-52 53a. Green, D. R., Flood, P. M., Gershon, R. K. 1983. ImmunoregulatoryT Cell Pathways. Ann. Rev. Immuno~ 1:439-63 54. Gruchalla,R. S., Streilein, J. W.1982. Analysis of neonatally inducedtolerance of H-2alloantigem.II. Failure to detect alloantigenspecific T lymphocyte precursors and suppressors. Immunogenetics 15:111-27 55. Haas, W., Layton, J. E. 1975. Separation of antigen-specificlymphocytes. I. Enrichment of antigen-bindingcells. J. Exp. Med. 141:1004-14 56. Hanan,R., Oyama,J. 1954. Inhibition of antibodyformationin maturerabbits by contact with the antigen at an early age. ,~. Immunol.73:49-53 57. Harris, D. E., Cairns,L., Rosen,F. $., Borel, Y. 1983. A natural modelof immunologictolerance: tolerance to mufine C5is mediatedby T cells andantigen is required to maintain unresponsiveness. Z Exp. Med.In press 58. Howard,M., Farrar, J., Hilflker, M., Johnson,B., Takatsu,K., Hamaoka, T., Paul, W.E. 1982.Identification of a T cell-derived B cell growthfactor distinct fromInterleukin 2. J. Exp. Med. 155:914-23

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59. Iverson, G. M., Lindenmann, J. 1972. Therole of a carrier determinantandT cells in the induction of liver-speciticantoantibodies in the mouse.Eur. d. Immunol. 2:195-97 60. Jemmerson,R., Morrow,P., Klinman, N. 1982. Antibodyresponses to synthetic peptides correspondingto antigenic determinants on mouse, cytochromec. Fed. Proc. 41(3):420 61. Kilshaw,P. J., Brent, L., Pinto, M. 1975. SuppressorT ceils in mice made unresponsiveto skin allografts. Nature 255:489-91 62. Klans, O. O. B. 1976.B cell tolerance inducedby polymericantigens. IV. Antigen-mediatedinhibition of antibodyforming cells. Eur. J. ImmunoL6: 200-7 63. Kubara,T., Hosono,M., Fujiwara, M. 1981.Studiesonthe resistanceto tolerance induction against humanIgG in DDD mice. IV. Transienttolerant state of T-cell precursors in bone marrow. Cell IramunoL57:377-88 64. Lawton,A. R., Cooper, M. D. 1974. Modificationof B lymphocyte differentiation by anti-immunoglobulin.Contemp. Top. ImmunobioL3:193-225 65. Lederberg,J. 1959. Genesand antibodies: doantigensbearinstructionsfor antibodyspecificity or dothey select cell lines that arise by mutation. Science 129:1649-53 66. Loblay,R. H., Fazekas,B., PritchardBriscoe, H., Basten, A. 1983. Suppressor T cell memory. II. Therole of memory suppressorT cells in tolerance to humangammaglobulin..L Exp. Meal In press 67. Loblay,R. H., Pritchard-Briscoe, H., B~ten, A. 1983. Suppressor T cell memory.L Induction and recall of HGG-specific memorysuppressor T cells andtheir role in regulationof antibodyproduction.,L Exp. MecLIn press 68. Louis,J., Chiller, J. M., Weigle,W.O. 1973. Fate of antigen-bindingcells in unresponsiveand immune mice..L Exp. Med. 137:461-69 69. Loutit, J. F. 1959. Ionizing radiation and the whole animal. Sc£ /lt~ 201:117-34 70. McCullagh,P. J. 1970. The immunological capacity of lymphocytesfrom normaldonors after their transfer to rats tolerant of sheep erythrocytes. ~lust. d. Exp. BioLMed.ScL 48:369-79 71. Metcalf,E. S., Klinman,N. R. 1976.In vitro tolerance induction of neonatal murine spleen cells. ,L Exp. Med. 143:1327-40

72. Metealf,E. S., Klinman,N. R. 1977.In vitro tolerance of bonemarrow cells: a markerfor B cell maturation, d. ImmunoL118:2111-16 73. Miller, J. F. A. P. 1972. Lymphocyte interactionsin antibodyresponses.Int. Rev. CytoL33:77-130 74. Miller, J. F. A. P., Basten,A., Sprent, J., Cheers,C. 1971.Interactionbetween lymphocytes in immune responses. Cell Immunol. 2:469-95 75. Miller, S. D., Sy, M.-S.,Claman,H. N. 1977.Theinduction of hapten-specific T cell tolerance using hapten-moditied lymphoidmembranes. II. Relative roles of suppressorT cells and doneinhibition in the tolerant state. Eur. Z lmmunoL7:165-70 76. Mitchison, N. A. 1971. The relative ability of T and B lymphocytesto see protein antigens. In Cell Interactions and Receptor/lntibodies in lmmuneResponses,ed. O. M~ikel~,A. Cross,T. U. Kostmen,pp. 249--60. NewYork: Academic 77. Mitchison,N. A. 1978.Newideas about self-tolerance and autoimmunity. Clinics RheumaticDR4:539--48 78. Moreno,C., Hale, C., Hewett,R., Esdaile, J. 1981.Inductionandpersistence of B cell tolerance to the thymnsdependent componentof the a(1-,6) glucosyl determinantof dextran. Recovery inducedby treatment with dextranase in vitro. Immunology44: 517-27 79. Morrissey, P. J., Kruisbeek, A. M., Sharrow,S. O., Singer, A. 1982.Tolerance of thymiccytotoxic T lymphocytes to allogeneic H-2determinantsencountered prethymically: Evidencefor expression of anti-H2 receptors prior to entry into the thymus.Proc.NatLAcad. ScL USA79:2003-7 80. Naor,D., Sulitzeanu,D. 1967.Bindi~.g of radioiodinated bovine serumalbumm to mousespleen cells. Nature 214: 68%88 E., Singh,B., Diener,E. 81. Nisbet-Brown, 1981. AntigenRecognitionV: Requirementfor hlstocompatibilitybetweenantigen-presentingcell and B cell in the response to a thymus-dependentantigen, andlack of allogeneicrestriction between T and B cells. £ Exp. Meg 154:676-87 82. Nossal, G. J. V. 1957. Theimmunological responseof foetal miceto influenza virus. ~lust. d. Exp. BioL Med. 35:549-58 83. Nossal, G. J. V., Lederberg,J. 1958.

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Antibodyproduction by single cells. 96. Pike, B. L., Nossal, G. J. V. 1979. Mechanisms of clonal abortion toleroNature 181:1419-20 84. Nossal, G. J. V., Pike, B. L. 1978. genesis. III. Antigenabrogates funcMechanisms of clonal abortion tolerotional maturationof surfaceIg-negative genesis. I. Responseof immature,haptadult bone marrow lymphocytes. Eur. J. lmmunol. 9:708-14 en-specific B lymphocytes.Z Exp. Med. 148:1161-70 97. Pike, B. L., Vanx,D. L., Clark-Lewis, 85. Nossal, G. J. V., Pike, B. L. 1980. I., Schrader, J. W., Nossal, G. J. V. Clonalanergy: persistence in tolerant 1983. Proliferation and differentiation mice of antigen-bindingB lymphocytes of single, hapten-specifieB lymphocytes incapable of respondingto antigen or promotedby T cell factor(s) distinct mitogen. Proc. NatL Acad, Scl. USA fromT cell growth factor. Proc. NatL Acad.Scl. USA.In press 77:1602-6 86. Nossal,G. J. V., Pike, B. L. 1981.Func- 98. Raft, M.C., Owen,J. J. T., Cooper,M. tional clonal deletion in immunological D., Lawton,A. R., Megson,M., Gathtolerance to major histocompatibllity ings, W.E. 1975.Differencesin suscepcomplex antigens.Proc. Natl. Acad.Scl. tibility of matureand immaturemouse USA78:3844-47 B lymphocytesto anti-immunoglobulln 87. Nossal,G. J. V., Pike,B. L., Battye,F. suppression in vitro. ,L Exp. Med. L. 1979. Mechanisms of ¢lonal abortion 142:1052-64 tolerogenesis. II. Clonalbehaviourof 99. Rajewsky,K. 1971. The carrier effect immatureBcells followingexposureto and cellular cooperationin the indueanti-~ chain antibody. Immunology tion of antibodies. Proc.R. Soc. London 37:203-15 Ser. B. 176:385-92 88. Osmond, D. G., Nossal, G. J. V. 1974. 100. Rajewsky,K., Takemori,T. 1983. GeDifferentiation of lymphocytes in netics, Expression,andFunctionof Idiotypes. Ann. Rev. Imraunol.1:569-606 mousebone marrow. II. Kinetics of maturationandrenewalof antiglobulln- 101. Reiehlein, M.1972. Localising antibindingcells studiedbydoublelabeling. genie determinants in humanhacmoCell. Immunol.13:132-45 globin with mutants:molecularcorrela89. Owen,R. D. 1945. Immunogenetic contions of immunologic tolerance, d. Mol. sequencesof vascular anastomosesbeBiol. 64:485-96 tweenbovine twins. Science 102:400-1 102. Roberts, I. M., Whittingham, S., 90. Owen,R. D. 1956. Erythrocyte antiMackay,I. R. 1973. Tolerance to an gens and tolerance phenomena. Proc. R. autoantigen-thyroglobulin. AntigenSoc. LondonSer. B 146:8-18 binding lymphocytes in thymus and 91. Parks, D. E., Doyle,M.V., Weigle,W. blood in health and autoimmunedisO. 1978. Induction and modeof action ease. Lancetii:936-40 of suppressor cells generatedagainst 103. Rouse,B. T., Warner,N. L. 1974. The human gammaglobulin. I. An imrole of suppressorcells in avian allomunologic unresponsivestate devoidof geneic tolerance: implications for the demonstrablesuppressorcells, d. Exp. pathogenesis of Marek’sdisease. Z Iramunol. 113:904-9 Med. 148:625-38 92. Pike, B. L., Battye,F. L., Nossal,G.J. 104. Schrader,J. W.,Nossal,G. J. V. 1974. V. 1981. Effect of haptenvalency and Effector cell blockade. A newmechacarrier compositionon the tolerogenic nism of immune hyporeactivity induced potential of hapten-proteinconjugates. by multivalent antigens. Z Exp. Med. Z Immunol. 126:89-94 139:1582-98 93. Pike, B. L., Boyd,A. W., Nossal,G. J. 105. Scott, D. W., Venkataraman,M., JanV. 1982.Clonalanergy:the universally dinski, J. J. 1979.Multiplepathwaysof anerglcB lymphocyte. Proc. Natl. Acad. B lymphocytetolerance. Iramunol.Rez Scl. USA79:2013-17 43:241-80 94. Pike, B. L., Kay,T. W.,Nossal,G.J. V. 106. Shearer, G. M. 1974. Cell-mediated 1980.Relativesensitivity of fetal and cytotoxicityto trinitrophenyl-modified newbornmice to induction of haptensyngeneic lymphocytes. Eur. ,L Immunol. 4:527-33 specific Bcell tolerance, d. Exp. Med. 152:1407-12 107. Shellam,G. R., Nossal,G. J. V. 1968. 95. Pike, B. L., Nossal,G. J. V. 1976.ReMechanismsof induction of immunoquirementfor persistent extracellular logical tolerance.IV. Theeffects of ulantigen in cultures of antigen-binding B tra-low doses of flagellln. Immunology lymphocytes,d. Exp. Med.144:568-72 14:273-84

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108. Sidman, C. L., Unanue,E. R. 1975. Receptor-mediated inactivation of early B limphocytes. Nature257:149-51 109. Smith,R. T., Bridges, R. A. 1958.Immunologicalunresponsivenessin rabbits producedby neonatal injection of defined antigens. ,L Exp. MecL108: 227-50 110. Stevens, K. M., Pietryk, H. C., Ciminera, J. L. 1958. Acquiredimmunological tolerance to a protein antigen in chickens. Br. d. Extx PathoL39:1-7 111. Streilein, I. W.1979./Neonataltolerance: towardsan immunogenetic defmition of self. ImmunoL Rev. 46:125-46 112. Streilein, J. W.,Gruchalla,R. S. 1981. Analysisof neonatally induced tolerance of H-2alloantigens. I. Adoptive transfer indicatesthat toleranceof Class I and Cla~II antigensis maintainedby distinct mechanisms.Immunogenetics 12:161-73 113. Sunshine, G. H., Cyrus, M., Winchester, G. 1982.In vitro responsesto the liver antigen F. Immunology 4:357-63 114. Sw?aJn,S. L., Wetzel,G. D., Saubiran, P./, Dutton,R. W.1982.T cell replacing factors in the Bcell responseto antigen. ImmunoLRe~. 63:111-28 115. Tagart, V. B., Thomas,W.IL, Asherson, G. L. 1978. Suppressor T cells whichblock the inductionof cytotoxic T cells in vivo. Immunology 34:1109-16 116.Taylor, R. B. 1969. Cellular cooperation in the antibody responseof mice to two serumalbumins:specific function of thymuscells. Transplan~Re~. 1:114-49 117. Taylor, R. B., Tire, J. P. 1979. Immunoregulatory effects of a covalentantigen-antibody complex.Nature 281: 488-90 118. Teale, J. M., Layton,J. E., Nossal,G. J. V. 1979. In vitro modelfor natural toleranceto self antigens, d. Exp.Med. 150:205-17 119. Tire, J. P., Morrison,C. A., Taylor,R. B. 1981. Immunoregulatory effects of covalent antigen-antibodycom.plexes. III. Enhancementor suppres~on dependingon the time of administration of complexrelative to a T-independent antigen. Immunology42:355-62 120. Tite, J. P., Morrison,C. A., Taylor,R. B. 1982. Immunoregulatory effects of covalent antigen-antibodycomplexes. IV. Primingandtolerance in T-depend-

ent responses. Immunology46:809-17 121. Tite, J. P., Taylor, R. B. 1979. Immunoregulationby covalent antigenantibody complexes.II. Suppressionof a T cell-independent anti-hapten response. Immunology38:325-31 122.Traub,E. 1938.Factors influencingthe persistenceof choriomeningitis virus in the bloodof miceafter clinical recovery. d. Exp. Med~68:229-50 123. Triplett, E. L. 1962, Onthe mechanism of immunogenic self-recognition, d. ImmunoL89:505-10 124. Vanx,D. L., Pike, B. L., Nussal,G. J. V. 1981.Antibodyproductionby single, hapten-specificB lymphocytes: an antigen-drivencloningsystemfree of filler or accessorycells. Proc NatLAcad.ScL USA78:7702-6 125. Waters, C. A., Diener, E., Singh, B. 1981. Antigen-relatedsusceptibility to toleranceinductionin utero: an objection to the current dogma.In Cellular and MolecularMechanismsof Immunological Tolerance, ed. T. Hraba, M. I-Iasek, pp. 481-91. NewYork:Dekker 126. Waters, C. A., Pilarski, L. M., Wegmann,T. G., Diener, E. 1979. Tolerance induction during ontogeny. I. Presenceof active suppressionin mice rendered tolerant to human~-globulin in utero correlates with the breakdown of the tolerant state, d. Exp. Med. 149:1134-51 127. Welgle, W.O. 1971. Recent observations and concepts in immunological unresponsiveness and autoimmunity. Cli~ Exp. ImmunoL9:437-47 128. Weigle, W. O. 1973. Immunological unresponsiveness. Adv. ImmunoL16: 61-122 129. Weigle,W.O., Chiller, J. M., Habicht, G. S. 1972. Effect of immunological unresponsiveness on different cell populations. Tmn~plan~ Rez 8:3-25 130. Wetzel, G. D., Kettman, J. R. 1981. Activation of routine B lymphocytes. III. Stimulationof B lymphocyte clonal growth with lipopolysaccharide and dextran sulfate. .L ImmunoL126: 723-28 131. Zinkernagel, R. M., Doherty, P. C. 1974.Imunologicalsurveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature 251:547-48

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An~ Rev. lmmunol 1983. 1:63-86 Copyright © 1983 by Annual Reviews Inc. All right reserved

T-CELL AND B-CELL RESPONSES TO VIRAL ANTIGENS AT THE CLONAL LEVEL L. A. Sherman, A. Vitiello,

and N. R. Klinman

Departmentof Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037

INTRODUCTION Over the years it has becomeapparent that the immunesystem can invoke a multiplicity of mechanismswith which to combatinfectious agents. These include the specific activation of both phagocytic and lymphoid cell subpopulations. Althoughthe action of antibodies in neutralizing infectious agents and facilitating opsonization is well known,in recent years the central role of cellular immunity in defense mechanismshas becomemore apparent. With respect to immunologicdefenses against viral infection per se, it has long seemedevident that even relatively low concentrations of antibodies in the serum or target organs could neutralize or immobilize virus and thus, if present, could protect against primary infection and subsequent viremic spread of the infectious agent. Under certain normal infectious circumstances, however,it appears that T-cell responses, by limiting or abolishing primary or secondary infectious foci, mayserve as the main weaponin the self-defense armamentarium(9). Since, in the course of a natural infection, it is likely that the primary presentation of viral antigens is in the context of the surface of infected cells, an understanding of immunerecognition of infected cells will be fundamentalto our understanding of both natural and prophylactically induced anti-viral responses. This review focuses on the recognition of viral determinants, particularly as they are expressed on infected cells, by both the B- and T-cell systems. Recent findings have indicated that B cells as well as T cells can recognize viral determinants that are uniquely expressed in the context of the infected cell. This finding provides a comparativeanalysis ofT-cell and B-cell recognition not heretofore available. Weattempt, wherever possible, to empha0732-0582/83/0410-0063502.00

63

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size studies carried out at the clonal level since it is probablethat an unambiguous study of recognition will ultimately dependon the relative simplicity of monoclonalresponses. Noattempt is madeto be comprehensive in termsof serologicalrecognitionof viruses per se, since the plethora of informationconcerningthis subject has already been reviewedextensively (20, 46). Rather, wetry to assess emergingconceptsof recognition to elucidaterelevant issues. ~iventhe enormousselective pressure that survival of virus infections must haveplayed in the evolution of the immune system, it is likely that both mammalianviruses and the mammalianimmunesystem, as we perceivethemtoday,in large part, are the productof these selective forces. For example,it has been shownthat encounters with the immunesystem are probablythe selective force for variations in influenzaantigens (34, 52). Similarly, since majorhistoeompatibility(MHC) antigens are so integrally involvedin immune recognition of foreign antigens expressedon cell surfaces, it is likely that the structure and function of MHC alloantigens has beeninfluenced over evolutionary time by their essential involvementin immune recognitionof infected cells. In this context, therefore, it maybe inappropriate to attempt to separate immune recognition of viral determinants per se from the recognition of such determinantsin the context of givenMHC alloantigens. Asthe studies presentedin this reviewreveal, both antigensystemsincreasinglyappearto be integrally related in terms of the structure of determinants perceived by the immunesystem. Nonetheless, elements of the immunesystem, primarily B cells, can recognize virion determinantsper se, and in the first section wereviewaspects .of that recognition that mayfurther our understandingof defense mechanisms in general andthe specific recognitionof viral determinantsin the context of MHC alloantigens, whichwill be discussed subsequently. THE RECOGNITION OF VIRION DETERMINANTS PER SE Thecapacity of serumantibodies to recognize various virion components is well established. Overthe past few years, these studies have beenadvancedmarkedlyby the advent of monoclonaltechnology(33, 48). Studies with monoclonal antibodies, derivedeither by the stimulation of individual B cells in culture (33) or by hybddoma technology(48), haveallowed detailed dissection of viral determinantsexpressed both on the isolated vidonand on infected cell surfaces. Mostimpressivein this regard, and a paradigmfor such studies, has beenthe analysis of influenzahemagglutinin (HA)by large panels of monoclonalanti-influenza antibodies (18, 31-33, 47). Thefirst studies that successfullygeneratedmonoclonal antibodyre-

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CLONAL RESPONSES TO VIRAL ANTIGENS

65

sponses to viral determinants and used such antibodies to delineate viral determinants were carried out with the fragment culture system in which isolated B cells were stimulated in vitro and the antibody product of their clonal progeny was collected over a period of several weeks (17, 18, 31, 33, 47). This technique can provide microgram quantities of monoclonal antibodies which have proven sut~cient for detailed specificity analysis. These studies also introduced the use of a large panel of influenza variants to study both the diversity of recognition of monoclonal antibodies and to delineate the determinants recognized by such antibodies. Such an analysis is shown in Table 1. This table shows a comparative analysis of monoclonal antibodies derived from primary versus secondary B cells. The antibodies were prescreened on viral strains that share only HAwith PR8 versus those that Table 1 Relative frequency of reactivity patterns in primary and secondary anti-PR8 aHAresponses Heterologousvirus W E I S S

C A M

+

+ + +

+ + -

+ +

+ +

+ -

+ -

+

-

b 7.0 4.5

1.6 5.3

.c

+

*

1.6 1.5

* *

* *

* *

* *

÷

3.1 ¯

* 3.0

* *

0.8 *

* *

0.8 *

* *

2.3 *

+

-

1.6 3.8

* 0.8

0.8 0.8

* 0.8

0.8 *

* 6.0

* *

1.6 0.8

-I-

÷

2.3 12.0

* *

2.3 5.3

* 3,0

* 0.8

* 3.0

¯ ¯

* 0.8

0.8 0.8

* *

* *

2.3 *

* 0.8 0.8 0.8

* * 2.3 1.5

0.8 0.8

0.8 0.8

0.8 0.8

* *

* *

2.3 0.8

4.7 0.8

1.6 1.5

1.6 ¯

* 0.8

* *

* 0.8

0.8 0.8

5.5 0.8

1.6 2.3

1.6 1.5

--

B E L

-

BH WSE MEL

5.5 2.3 1.6 7.8 3.1 6.2 5.5 14.0 1.5 * * * * * 12.8 23.3 a Relative frequencies are given as a percentage of total response and are based upon 129 primary and 134 secondary monoclonalanti-HA antibodies (from 18). bUppernumberrefers to primary response; lower numberrefers to secondaryresponse. c*, RPnot observed.

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SHERMAN ET AL

share other virion determinants, including neuraminidase(NA),and those that share only chickenhost component. Thetable, therefore, includes only those antibodies that presumablyrecognize determinants on the HAof PR8.This andsimilar analysesestablished several highly importantgeneral principles.First, it is clear that a panelof viral variantsis a verypowerful tool in the dissectionof availableantibodyspecificities. Second,these studies give definitive evidenceof the enormous degreeof diversity availablein the B cell repertoire. Byexpandingsuch panels (26, 84), it has nowbeen foundthat morethan 103 different recognitionpatterns are available within the Balb/cmouse.Since B cells responsiveto the influenzaHAare approximately1 in l0 s of all B cells (18), such studies haveestablished that the slowerlimit to the size of the antibodyrepertoire is of the order of 10 specificities. Third, if eachindividualreactivity pattern canbe equatedwith the recognition of a given determinant,then it maybe concludedthat the B cell repertoire is not redundantin its recognitionof mostdeterminants. Thus, for manydeterminants a fully mature mousemayhave only one or no B cell clones capable of recognition. Fourth, since subsequentstudies haveshownthat only a few limited regions of the HAmoleculeare responsible for this complex pattern of binding(32, 81), it is clear that smallnuances in the structure of a regionof a protein can be recognizedanddiscriminated by monoclonalantibodies. Althoughin someinstances such nuances may be createdby the substituted aminoacid per se it is equallylikely that in someinstances aminoacid substitutions maybe recognizedas perturbations in other regions of the molecule. Subsequentstudies with monoclonalantibodies derived from hybridomas haveconfirmedall of the abovefindings(26, 32, 35, 84). In addition, it has beenpossible, by the use of hybridoma antibodies, to select for influenzaHA variants that represent a single mutationaldifference (34, 52). Suchnewly selected variants haveservedas invaluabletools in further discriminating the B cell repertoire and further delineating regions of the HAmolecule whereinsequencevariation affects immunologic recognition (81). _ Figure1 is a schematicrepresentationof the three-dimensional structure of influenza HAas determinedby X-ray crystallography by Wyley& Wilson (81). Includedin this figure are point mutationsselected by monoclonal antibodies.It is apparentthese mutationaldifferencescluster in immunologically relevant regions of the HAmolecule(81). Thecombinedanalysis monoclonalantibodies, selection of mutants, and crystal structure represents an extraordinarily powerfulnewset of approachesfor the understanding of determinantrecognition and the powerof selective forces on both immune expressionand viral expression.Recently,several other viral systemshave beensubjected to analysis by monoclonalantibodies (49, 56, 58). Someof these studies havebeenthe subject of extensiverecent reviews, including comprehensiveanalyses of the antigenic structures of murine

Annual Reviews CLONALRESPONSES TO VIRAL ANTIGENS

67

leukemia viruses (58), the 70S glycoproteins of mudneretroviruses (56), and rabies viruses (49). In each of these instances, it has been possible the use of monoclonalantibodies to more clearly delineate structural relationships and variations amongviral strains.

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VIRAL COMPONENTS RECOGNIZED ON INFECTED CELLS It is nowclear that both T cells and B cells can function, and mayprimarily function, by recognizing viral determinants as they are expressed on infected cells. Suchrecognition is particularly relevant to viruses that repli-

HA2 N

Figure 1 Schematicdrawingof a monomer of the AICHI-1968 hemagglatininshowing locations of aminoacid substitutions on HA,.¯ Site A; ¯ site B; ¯ site C; ¯ site D (subunitinterface); <) surface; o neutral. Summary of all known alterations from1968 1977includinglaboratory-selected variants(81).

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care by budding.In terms of immune recognition of viral determinantsas they are expressedon infected cells, several factors are importantto consider. First, it is likely that determinantsthat can be recognizedon the maturevirion maynot be accessible for recognitionas they are expressed on the cell surfaceandprior to virion maturation.Second,it is likely that somevirion determinantsare accessible on the surfaceof infected cells that are not accessible on the intact virion. If suchrecognitionis importantin the immunologic defense against viral infection, then immunizationwith inactivated virion maynot be expectedto be as effective as the immunization that results frominfection (67). Finally, it is possiblethat a considerable amountof immune recognition is directed towardscomplexantigenic determinantsthat mayinvolve both virion and cell surface determinants. Thus, our understandingof immunologic defenses against viral infection maydependon a clear understandingof the recognition of viral determinants as they are expressedon cell surfaces. Viral Determinants on Infected Cells Recognized by B Cells Recentstudies have demonstratedthat serumantibodies directed to viral antigens can cause the lysis of vitally infected cells via a complementmediatedprocess (61). Suchfmdingsunderline the importanceof understandingB cell recognitionof virion determinantsas they are expressedon infected cells. Recentstudies by Gerhardand his co-workershavedemonstrated, by the use of monoelonal antibodiesdirected to the various protein componentsof the influenza virion, that although both the HAand NAcan be readily recognizedon the surfaceof infectedcells, little or no representation of the matrix(M)protein can be foundon suchcells (35, 84). Additionally, a small amountof recognition of the viral nuclear protein (NP) apparentby lowlevels of bindingof the hybridoma antibodydirected to this protein on the surfaceof infectedcells (35, 84). Therelevanceof this latter recognition remainsto be determined.Anotherviral protein that maybe present on infected cellg is non-structuralprotein 1 (NS1)(72). Recently,this laboratory has carried out an extensiveanalysis of monoclonal B cell responsesto influenza-infectedsyngeneicmurinecell lines (82, 83). Tothe extentthat the analyseshavebeencarried out, it is apparentthat vigorous monoclonalresponses can be obtained to both the HAand NAof the influenzavirion as they are expressedon infected cells. Importantly,the frequencyof such responses is somewhatlower than the frequencyof responses to the same determinantswhenthe intact virion is used as the immunogen (18). That is, wheninfected cells are used as the immunogen andantibodiesare screenedon the intact virion, the frequencyof responses by HA-specificB cells is about two thirds of that foundin responseto the HAas presented on the intact virion. Onlyapproximatelyone third of B

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cells stimulatedby NA,as expressedon the intact virion, respondwhenthe NAis presentedon the infected cell (82). To determinethe basis for this lack of responsiveness,monoclonal antibodiesderivedby stimulationby the intact virion wereassessedfor their capacity to bind either the HAor the NAas expressedon infected cells. Thefindings indicate that approximately one third of all virion-stimulatedanti-HAantibodies recognizeHAdeterminantsnot accessible on the infected cell. Similarly, two thirds of the anti-virion NA-specificmonoclonalantibodies do not bind to NAas expressed on the infected cell (82). Thus, although both HAand NAare expressedon the surfaceof infected cells, this expressionis apparentlynot inclusive of all potential responsesto these determinantsas they are expressed on the virion. Importantly, amongthe subset missing from the anti-HAresponsewhenvirus is presentedon infected cells is the majority of antibodies that are broadlycross-reactive amongclosely related viral strains of the H1 subtype.This findingwouldpredict that antibodiesderived by stimulation during the course of an infection maybe somewhatmore specific for the infecting virus than those whichmaybe derivedby immunization with high concentrationsof the intact virion. In summary,it wouldappear that someviral determinants, such as the NP,that are poorlyrevealedon the intact virion maybe recognizedon the infected call. Conversely,it is also apparentthat somedeterminantsnormallyreadily recognizedon the intact virion are not seen whenviral determinantsare presented as infected ceils. As moreinformationaccumulates concerningdifferencesin B cell recognitionof the intact virion versus the infectedcell, it is likely that a better understanding of the basis andspecificity of immune recognitionof infected cells will be achieved.

T-Cell Recognitionof Viral Determinantson lnfected Cells Preciseidentification of viral proteins responsiblefor recognitionby cytolyti c T lymphocytes(CTL)has proven difficult. Immunologistshave approachedthis task with the reasonableexpectationsthat the viral structures involved should be expressed on the target cell membrane and that the specificity of CTLshould, in general, reflect recognitionof determinants definedby the structure of viral proteins. Inherentin the latter assumption is the belief that T cell receptors recognizedeterminantson the foreign antigen.Theinability to identify a viral structure that fulfills bothof these expectationswhich,parenthetically, has beenthe basis for identification of HAas the viral protein involved in MHC-restrictedantibody recognition of influenzavirus-infectedcells (83), has hampered efforts to identify the viral proteins requiredfor T cell recognitionof influenza-infectedcells. The basic finding reported by most laboratories that have studied the specificity of influenza-specificCTLis that a CTLpopulationstimulatedin

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responseto viral infection with a particular influenzaAstrain virus crossreacts on target cells infectedwithessentiallyall other Astrain viruses(12, 23, 91). This is in markedcontrast to the specificity of antibodyfor which cross-reactivityis restricted to viral strains.of the samesubtype.Thespecificity of such CTLpopulationsis defined by the lack of recognition of B strain-infectedtargets andtargets infected with unrelatedvirus (14). Therefore, CTLdemonstratea broader range of recognition than do antisera. Suchcross-reactiverecognitionis also characteristic of T helper cells and effectorcells of delayed-type hypersensitivityspecific for influenza(1, 3, 4, 53) andhas also beendescribedin T cell recognitionof vesicularstomatitis virus (VSV)-infected cells (90). Severaltypes of evidencesuggest that subtype-specificCTLdo exist but are obscuredby a second population of highly cross-reactive CTL.Ennis andco-workers(24) describedsuch subtype-specificCTLas the only potlulation that could be observedwhenkidneycells infected with virus were used as CTLtargets as opposedto morecommonly used tumor cells. With recombinant strains of virus, it wasdemonstratedthat specific recognition required the presenceof the viral HAand not NA.It wasalso possible to demonstratesubtype-specificrecognition by .cold target competitionstudies. Whencold target inhibitors infected with heterologousAstrain virus of a different subtypewereaddedto a cross-reactiveeffector cell population, the remaininglytic activity wasgreatest on targets infected with the homologous strain of virus (14, 23, 91). In addition, it waspossible amplifythe cross-reactive CTLpopulationpreferentially by in vitro stimulation with heterologousAstrain virus (14, 23) or specific population using either UV-inactivated virus (25) or purified HAfor in vitro stimulation (13, 91). Thesedata havebeeninterpreted as evidencethat at least portion of the CTLresponse is subtype specific and is dependentupon expressionof the appropriateHAfor target recognition. However,there is reasonto believe that the precise determinantsrecognizedare not the same as those recognizedby HA-specific antisera. First, it is difl~cult to demonstrate antibodyblockingof CTLrecognition with HA-specificantisera (5, 14). This is the case evenwhensomesubtype-specificCTLare analyzedand is in contrast to the markedinhibition observedwith antisera specific for class I MHC molecules (5, 89). Second, whensubtype-specific CTLare obtained by stimulation with purified HA,such CTLdo not exhibit the samehierarchy of cross-reactive recognitionas is observedfor HA-specific antibody(13). If subtypespecificity is attributable to HArecognition,then whichprotein is responsible for cross-reactive recognition?Again, with the basic assumptionthat the specificity of CTLshouldreflect the serologically defined antigenic properties of a viral protein, it wouldbe concludedthat

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highly conserved viral proteins are the most likely candidates. This would exclude the two proteins expressed in greatest abundanceon infected cells, HAand NA. The recent demonstration that several relatively invarient viral-encoded molecules such as Mprotein and NS 1 may be expressed on the surface of infected cells place these proteins on the list of possible candidates. The only evidence that suggests recognition of Mproteins by CTLis the success of one laboratory (68) [in the face of failure by others (14, 40)] to demonstrate CTLinhibition by using anti-M protein antisera. There are no reports as yet on the possible role of NS1. There is, however, substantial evidence that HAmay not only represent the target molecule most often responsible for subtype-specific CTLrecognition but mayalso represent the major target molecule for cross-reactive CTL. First, Askonas &Webster were able to demonstrate antibody inhibition of cross-reactive CTLusing a combination of monoclonal antibodies directed against HAand H-2 (5). Second, Koszinowski and co-workers were able to construct target cells for influenza-specific CTLby Sendaimediated fusion of liposomes containing purified viral glycoproteins (HA and NA)with uninfected H-2 compatible cells (50). The resultant targets were cross-reactively lysed by CTLraised against a different A subtype. Finally, the fact that T helper cells can be stimulated in vitro with purified HA,or HAfragments obtained from a strain heterologous to that used for in vivo palming, indicates that class II MHC-restdctedT cells, which are cross-reactive, are unambiguouslyHAspecific (3). Aninteresting parallel is the case of VSVrecognition by CTL. Nonserologically cross-reactive VSV-G (glycoprotein) have been positivdy identified as the target structure for recognition by cross-reactive CTL(19, 90). Nevertheless, purified G protein as an immunogen elicits a highly specific CTLpopulation (71) muchas does purified HAin the case of influenzaspecific CTL. Therefore, for both VSVand influenza, stimulation with viral-infected cells generate both specific and cross-reactive CTL,whereas stimulation with purified viral glycoproteins results in stimulation of the specific population only. Althoughone interpretation of these data is that a second viral protein is responsible for the cross-reactive CTLpopulation, it is also possible to interpret thembased on the difference in the modeof antigen presentation. Whereasthe viral HAis expressed as a transmembrane protein on the surface of infected stimulator cells, it is unlikely membraneinsertion occurs whenpurified HAis used as antigen. It is likely that the type of association betweenH-2 and antigen is qualitatively different whenantigen is transmembranethan whenit is not. For example, it is possible that association between H-2 and HAin regions proximal to the membranemay be achieved with transmembrane antigen but not solubilized protein, thereby resulting in a greater spectrum of newdeterminants,

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including perhaps the majority of those that are cross-reactively recognized. Alternatively, the type of association between H-2 and HAmaybe significantly affected by the presence of Mprotein or someother viral protein. To summarizefindings obtained by studies of CTLpopulations, it would appear that two types of CTLspecificities exist: one appears subtype specific and is likely to be HA-directed; the other is cross-reactive and mayor may not be HA-specific. The possibility exists that neither type of population reflects recognition of determinants identical to those seen by antibody; however, this could only be resolved at the level of individual receptor specificities by studying CTLclones. CTL Clones In the past few years, several laboratories have studied the viral specificity of cloned CTLobtained either by limiting dilution of effector cell populations or cloning after multiple in vitro stimulations of a CTL-containing population. As the findings from each laboratory have contributed different information, each is reviewed separately. Lu &Askonasdescribed the specificity of a CTLline obtained by multiple in vitro stimulations (54). This line requires interleukin-2 for growthbut not stimulator cells. Insofar as clones obtained from this line react with all A strain subtypes, it represents the first demonstration that a single CTL specificity can, indeed, cross-reactively recognize cells infected with distantly related viral strains. It is not possible to estimate the proportion of the original effector cell population that wouldexpress such specificity. However, Owenet al (62) have described the specificity of CTLclones obtained by in vitro stimulation under limiting dilution conditions without prior in vitro culture. The frequency of influenza-specific CTLwithin the spleen of an in vivo primed animal is in the range of 1 in 1700-4700spleen cells. Of these clones, approximately 80%were cross-reactive on heterologous A strain-infected target cells. Therefore, the vast majority of their clones were of the cross-reactive category. Another series of CTLclones has been described by Braciale et al (15). These are long-term clones that require both stimulator cells and interleukin-2 for maintenanceof activity and growth. Braciale found three categories of specificity in examining14 such clones. These included clones that demonstratedspecificity restricted to the homologousvirus used for in vivo stimulation (43%), clones that appeared subtype specific (21%), and that were cross-reactive on all strains tested. One done demonstratedrather paradoxical recognition insofar as it did not recognize target cells infected with certain heterologous virus of the same subtype as that used for stimulation, yet it did recognize targets infected with virus representing an unrelated subtype.

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Our own studies on viral specificity of influenza-specific CTLclones indicate that such paradoxical recognition mayindeed be characteristic of a large proportion of PR8-specific CTLclones (A. Vitiello, L. A. Sherman, manuscript in preparation). It was frequently observed that PR8(HI)stimulated clones could discriminate amonga panel of target cells each infected with different H 1 A strain virus and yet wouldreact on target cells infected with an H3-type virus. Howcan these various specificities be classified with respect to antigen recognition? Recently, Bennicket al (6) performedan analysis of the specificity of a PR8-specificclone that had the general characteristics of subtype specificity and, therefore, could have been considered HA-specific insofar as the clone reacted with target cells infected with someH1strains but not heterologous H2 or H3 virus. With defined recombinant viral strains created between PR8 (H1) and Hong Kong (H3), it was found that requirement for target recognition genetically segregated not with HAbut rather with the viral polymerase, P3. Considering that intermediate levels of lysis were observed with different recombinant strains, all of which express the required P3, this wouldfurther suggest that the level of expression of the recognized structures is under multigenic control. Although these results do not rule out HAas the requisite target cell structure, they do indicate that in view of the potential for multiple genes to affect expression of the target molecules, it mayprove exceedingly difficult to identify precisely the viral proteins required for CTLrecognition. It maybe concluded that studies with CTLclones have not yet provided definitive answers with regard to determinant recognition by virus-specific CTL.Clearly, it is also necessary to have well-defined target structures. Perhaps one way this may be achieved is through the use of cloned viral genes as transfection agents. Nevertheless, clones have been quite informative with respect to the demonstrationof great diversity within the virusspecific CTLrepertoire.

MHC-RESTRICTED RECOGNITION OF VIRAL DETERMINANTS ON INFECTED CELLS The most striking feature of immunerecognition of virally infected cells, particularly by T cells, is the restricted recognition of viral determinants such that antigens are recognized only in the context of MHC-encoded determinants of the infected cell (89). Both the nature of such recognition and the lymphocyte receptor mechanism necessary to accomplish such recognition remain amongthe most controversial and provocative aspects of the immunesystem. In general, studies that have attempted to demonstrate a physical association of viral determinants and MHCcomponents

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have beenambiguousin that, in someinstances, such associations maybe present(11, 19, 30, 60, 85), whereasin others they appearnot to be, or may be of insufficient stability to permitdetection (21, 39). Mostimportant, however,is that recognition of viral determinantsin the context of given MHC alloantigens is often exclusive for one MHC determinant, and such exclusivity appears to be MHC-haplotype-dependent (22, 87). Thus, recognition wouldappearto be a function of selection of the receptor repertoire rather than a function of the stability of complexes on the cell surface. In any case, attempts to understandthe mechanism of restricted recognition of infected cells, will be a central themeof future immunologic studies.

MHC-Restricted Recognitionof Virally Infected Cells by T Cells T cells function throughtheir recognitionof cellularly presentedantigen. The strategy used by the immunesystemto restrict T-cell recognition to cells rather than antigen per se wouldappearto be the requirementthat T cells recognizecellular MHC antigens as well as foreign antigen. This is referred to as MHC-restricted recognition. For T helper cells this entails recognition of class II (I region) MHC molecules, whereas CTLhas requirementfor recognition of class I molecules(K, D, and L) (89). MHC restriction is observed as the requirement for MHC sharing betweenthe cell presentingantigenduringT cell activationandthe target cells recognizedby the activated effector cell population.Therefore,MHC determinantsas well as antigenselect the specificity of the effector cell population. However,available data supportingCTLrecognition of determinants on H-2moleculesis far morecompellingthan available data demonstrating recognition of antigen determinants. Antisera to H-2blocks target cell recognitionby CTL(5, 11, 29, 36, 37, 51, 69). In addition, subtle changes in the structure of the H-2molecule,such as point mutations,can markedly affect the ability of CTLto recognizetarget cells. Theseresults are in contrast to the relative insensitivity of CTLreactivity to blockingby antiviral antisera, or to extensivestructural changesin viral proteins(see previous section). Theprecise H-2determinantsrecognizedin restricted recognitionare not yet known.Somedata suggest there maybe relatively few H-2determinants utilized in such a manner.WhenKb-restricted CTLare tested for recognition of target cells that bear different mutationsin the Kb molecule,the hierarchy of cross-reactive recognitionfor a variety of different viruses appears to be similar. For example,Kbml-expressing targets are not recognized by Kb-restricted CTLin all viral systemsthat havebeentested (10, 63, 86). This suggests bml, whichdiffers fromwild-typeby only two amino acids, mayhavelost all viral-restricting determinantspresent on the wildtype molecule.However,that this conclusionmaybe an oversimplification

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CLONALRESPONSESTO VIRAL ANTIGENS 75 is suggestedby the recent demonstrationof the complexityof H-2determinants recognizedin conjunctionwith minorcell surface histocompatibility antigens(80). Asis the case for viral recognition,Kb-restrictedandminor antigen-specific CTLdo not recognize the gbml mutant. However,cold target competitionstudies with other Kb mutantssuggest the existence of multiple determinants on the Kb molecule, only someof whichare conservedby various Kb mutants. In addition, whereasrecognitionof the H3.1 minorantigen in association with determinantsshared by Kb and Kbm3 was observed, Kb-restricted H4.1recognition wasnot found with bm3targets. Therefore,a different pattern of restricting elementsis seen in conjunction with different minorantigens. In light of these results, a morecareful examinationof viral-restricting H-2determinantsas maybe achievedby studyingthe specificity of individualclonesis required. Thepossibility that different antigens mayuse a different array of H-2 determinantsas restricting elementspresents the interesting questionas to whetheror not different Ho2moleculesmaypresent a different mosaicof viral determinants. Wehaverecently examinedthis question by studying the fine specificity of viral recognition by CTLclones restricted to two different H-2molecules.Theseresults indicate that there is very little overlap betweenviral determinants recognized by Db-restdcted (B6) versus Dd- and Ld (B10.ASR)-restricted influenza-specific CTL(A. Vitiello, L. Sherman,manuscriptin preparation). Sharedclonotypesrepresent less than 20%of the repertoires expressed by B6and B10.ASR.Consideringthat different clonesthat share reactivity patterns do not necessarilyrecognize identical determinants, this represents a maximum estimate of determinant sharing. Thereare several possibleinterpretations of these results. First, there is considerableevidencefor physical association of H-2and virus. Physical proximityof H-2andinfluenza HAon target cells has beendemonstrated by Hackett&Askonas (39). It is possiblethat the odentiationof this associationmaybe different for different H-2molecules.If so, thendifferent restricting elementsmaylead to the presentationof different viral determinants. Theobservation that the sameanti-HAmonoclonalantibodythat is effective in inhibition of Dr-restricted CTLdoes not inhibit recognitionof Kd-restricted CTLmaybe interpreted as support for this possibility (28). Second,it is possible, that different viral proteins are associatedwith the MHC moleculesof these different murinestrains. Third, in view of the evidencethat MHC moleculesinfluenceCTLrepertoire selection (7, 41, 73, 88), it is possiblethe repertoiresare sut~cientlydifferentin these twostrains as to preclude recognition of comparableviral determinants. Comparison of the allo-specific repertoire of twoMHC congenicstrains does not necessarily support this view (73). Themajority of H-2Kb-specificclonotypes observedin H-2d mice are also foundin H-2k mice. Rather, it is the frequencyof representation of individual clonotypesthat is mostaffected by

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H-2differences. Finally, it is possible that the determinantsactually describedby these influenza-specificclonotypesare not viral structuresper se but instead represent altered H-2determinants(see below)(74). Regardless of the basis for the lack of correlation betweenthese tworepertoires with respect to fine specificity for viral antigens, the findingthat the repertoire is so dramaticallyshifted by the H-2-restrictingelementmakesit less surprising that there is so little correlation betweenviral determinantsrecognized by antibodies that are not MHC restricted and determinants recognizedby MHC-restrictedT cells. Anotherdramaticeffect that the MHC has on the anti-viral response is the ability of H-2 to determine which MHC molecule~are utilized as restricting elements.In general, two types of such immune responsedeficiencies are attributable to class I MHC molecules.In somesituations, lack of responsiveness appearsto be inherentin the restriction elementitself. For b plus influenza (22). This low example,B6miceare low respondersto H-2K responsephenotypeis reflected as a low frequencyof CTLactivated in the immune animal(2). That this type of defect is inherent in the b molecule bm3mutant responds well maybe concludedfrom the fact that the B6-H-2 to the Kbm3-infectedtargets (A. Vitiello, L. A. Sherman,manuscriptin preparation). This type of nonresponderphenotypeis also observedfor simian virus 40 (SV40)recognition whereB6respondswell in this case Kb-infected cells but the bml mutant is a nonresponder(63). Another examplehas been described for Moloneyleukemiavirus (MoLV)-infected cells (76). In this case, mutationin the Db(bml4) results in lack of D recognition by the mutantstrain. A second type of immuneresponse defect commonlyobserved maps to an H-2moleculeother than the one used as the restricting element. For example,Db-restricted and vaccinia-specific CTLcannot be obtained from mice that bear Kk, whereas B6 mice (Kb Db) are able to mounta good D b-restricted vaccinia response (22, 87). As another example, it has been observedthat Dr-restricted SV40-specifieCTLcannot be raised in mice that bear Kb or Kk antigens, but it can be elicited in Kd and Kq haplotypes (65). The observation that CTLfrom B10.D2mice that mount a vigorousDa-restrictedSV40responsecan recognizetarget cells expressing Kb and Dd suggests that the defect is probably not due to association betweenH-2andantigen on the cell surface. Similarly, it wasdemonstrated that whereasH-2b mice respond to Db in association with MoLV virus, a mutationin the Db moleculecan result in increased utilization of Kb as a restricting elementfor this virus (76). Similarobservationshavebeenmade in other viral systems(89). The mechanism responsible for these two types of nonresponderphenotypes mayor maynot be similar. Twotypes of explanations have been proposed(89). Thefirst evokeslack of associationor preferred association

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CLONALRESPONSESTO VIRAL ANTIGENS 77 betweenH-2and antigen as responsible for instances of haplotypepreference. Althoughthere is evidencefor preferred associationof viral protein and H-2moleculesin somesystems, for such a modelto explain all of the experimentalresults it wouldalso be necessary to explain whysomeH-2 moleculesassociate well enoughto act as targets but not well enoughto stimulateCTLprecursors.In addition, the fact that Kb is a poorrestricting elementfor influenza-specific CTLyet serves as the dominantrestricting elementfor H-2-restrictedantibodyalso makesthis less likely (see below) (83). Asecondmodelpredicts that the CTLrepertoire is deleted for recognition of somecombinationsof H-2andvirus becauseof eliminationof such potential specificities by self tolerance. Recentexperimentsby Mullbacher (55) supportthe notion that toleranceto an alloantigencan result in elimination of a particular MHC-restricted antigenresponse.Finally, it is possible that both types of phenomenon mayoccur in different situations. There are numerousstudies in which CTLpopulations that are H-2restricted and virus or antigen specific havebeenfoundto cross-react on alloantigen(8, 16, 27, 44, 64, 78, 79). Sincethis often representsthe specificity of a minorsubpopulation,examplesof this phenomenon at the level of individual CTLclones were sought and found (16, 44, 78, 79). It is now firmlyestablishedthat a highproportionof self plus-X-specificT-cell clones can react with alloantigen. Equallyimpressive is the general trend for specific alloantigens to be recognizedmorefrequently than others. For example,whereas10%of all CTLclones specific for Db plus HYreacted on the Dd alloantigen, no clones were found that reacted on H-2k targets (44). Similar cases of preferred alloreactivity havebeenreported for restricted antigen-specific T-cell clones (70). Anextremeexampleof this type of cross-reactivityhas beenobservedfor Kb-restrictedinfluenzarecognition. In an analysis of 22 Kb-plusinfluenza-specificclones manydemonstrated lyric activity on uninfected Kbin1° targets (A. Vitiello, L. A. Sherman,manuscriptin preparation). In addition, each of these clones was heteroclytic insofar as lyric activity on bml0wasmuchgreater than on Kb-infectedtargets. Thesetypes of data further supportthe notionthat self tolerance to H-2 antigens maybe the most common mechanism responsible for IR phenomena in manyof the examplesdescribed above. Further, the selectivity as to whichalloantigensare recognizedsuggeststhat the basis for cross-reactivity is not fortuitious but, rather, mayrepresent truly shared determinants. MHC-Restricted Recognition of Vitally-Infected by B Cells

Cells

Recently, several laboratories have demonstratedthat in someinstances B-cell stimulationandrecognition,like T-cell stimulation, can display the

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need for antigen presentation in the context of an appropriate MHC alloantigen (38, 42, 45, 57, 59, 66, 75, 77). This laboratory has carded out detailed examination of the role of such recognition by B cells responding to influenza-infected syngeneic cell lines (83). For this purpose, individual B cells were antigenically stimulated by influenza-infected cell lines of two different strains. In both instances, responses were observed wherein monoclonal antibodies recognized influenza antigens expressed on the purified vidon. However,in both the responsive H-2b B cells to influenza-infected EL-4 (H-2b) cells and H-2k B cells responsive to influenza-infected L929 (H-2k) cells, the majority of antibody-forming cell clones produced monoclonal antibodies that recognized the appropriate infected cell but not the puttied vidon or uninfected cell alone. The majority of these monoclonal antibodies were found to be dependent on the presence of the appropriate influenza HA.The recognition of HAwas relatively specific in that little cross-reactivity was observed whenthe same cell lines were infected with influenza viruses bearing other H1 variants. Most important, a majority of monoclonal antibodies (57%) that responded to influenza-infected cells recognized influenza determinants only in the context of cells expressing either the H-2K or H-2Dend antigenic determinants of the syngeneic stimulating cell lines. These findings are summarizedin Table 2. It can be seen that amongthe monoclonal antibodies derived against influenza-infected L929(H-2k) cells, the majority recognized only infected k class 1 antigen. Similarly, H-2b B cells that cell lines that bore the H-2D recognized influenza-infected EL-4 (H-2b) cells recognized primarily inb molecule. Since panel analyses fluenza-infected cells bearing the H-2K were carried out with influenza-infected primary kidney fibroblasts, it is

Table

2 MHCrestriction

of monoclonal

antibodies

specific

for PR8-infected

L929 (H-

2K) cells

Percentage of total PR8-L929 PR8-BALB.K PRS-3T3 PR8-B6 PR8--C3H.OH PRS-BI0.A(4R) monoclonal PR8 L929 (H-2 k) (H-2 k) (H-2 b) (H-2 d) (H-2K d, Dk) (H-2K k, Db) antibodies + + ÷ + ÷ + + + +

aNT NT + + + + + +

aNT, Not tested. (From 82, 83.)

NT NT NT + + -

NT NT NT + + -

NT NT NT NT NT NT + + -

NT NT NT NT NT NT + +

35 4 4 10 5 5 2 30 5

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CLONALRESPONSESTO VIRAL ANTIGENS 79 unlikdy that this recognition wasdependenton the modeof infection, productiveversus nonproductive,or other antigens expressedon the tumor cell lines. Additionalevidencethat the anti-influenza-infected EL-4reb locus wasobtained by an analysis of sponseswererestricted to the H-2K b mutants. The bindingto influenza-infectedfibroblasts obtainedfromH-2K majority of these antibodies could discriminate amonginfluenza-infected cells beating these mutants. Thus,these antibodies weresensitive to both small changes in the influenza HAas well as point mutations in the b molecule. H-2K Thefinding that the majority of antibody responsesto infected cells displaysrestricted recognitionreminiscentof T-cell recognitionhas several important implications for our understanding of immunerecognition of infected cells. First, it is dear that the dichotomypreviously observed betweenB-cell recognition and T-ceil recognition is not absolute and is basedmoreon the fact that antibodiescan be foundthat bind andneutralize virus particles per se than on the fact that B cells cannot also display restricted recognition as do T calls. Second, it is clear that maximum immune responses by both T and B ceils wouldrequire the presentation of viral antigens in the contextof syngeneicinfected cells. Third, the finding that monoclonal antibodies can so readily recognizecomplexantigen determinantshas profoundimplicationsfor the nature of T-cell receptors, since such recognitionobviouslydoesnot necessitate the existenceof twoseparate receptors. Fourth, whateverthe nature of the complex antigen, it apparently pre-exists antibodyinteractionwith the cell since the infectedcells usedfor bindingassayswereglutaraldehydefixed prior to interaction with antibody. Fifth, whateverthe mechanism of recognition of complexantigens by immunereceptors, such recognition wouldappear to be extremelyspecific both with respect to its discriminatorycapability for the viral determinants andits discriminatorycapability for the H-2antigen involved.Finally, it is of great interest that the recognitionby Bcells of influenza-infected cells, like that previously observedfor T cells, is markedlyskewedtowardsone endof the MHC locus versus the other in both strains tested. Interestingly, the skewingin both cases is opposite that previously reported for T cell recognitionof influenza-infectedcells of these haplotypes(22), an observation similar to that of Ohno,et al in a studyof anti-HYantisera (59). Thus, althoughT cells recognizinginfluenza-infectedH-2b cells are restricted to recognition of viral antigens in the context of D-endantigens, the monoclonal antibodies apparently prefer Kend. Similarly, T cells recognizing influenza-infected H-2k cells are predominantlyKend restricted, whereas the monoclonalantibodies appear to be primarily D-end restricted. Whethersuch endednessin recognition represents a phenomenon similar to the IR phenomenon attributed to T-cell recognition of one end versus

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another has yet to be determined. Nonetheless, the opposite preference of the two cell systems wouldseem to eliminate a preference of the virus to complex with one end versus the other as a major determining element in either T-cell or B-cell recognition.

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CONCLUSION:COMPARISON OF THE T- AND BCELL REPERTOIRESSPECIFIC FOR VIRALLY INFECTEDCELLS Comparisonof determinant recognition by B and T cell receptors has been an area of great interest to immunologists. Often, such comparisons have led to the conclusionthat different epitopes are recognizedby these different lymphocyte subsets. However, such results have always been interpreted with the understanding that comparison maynot be valid since the B cells that were analyzed were specific for antigen per se whereas T cells are confined to recognition of antigen in the context of MHC molecules. Therefore, the recent demonstration of an additional subset of antibody forming cells which are MHC restricted provides a unique opportunity to study Band T-cell recognition on commonground. Of course, the most important conclusion to be drawnfrom these studies is the fact that a single receptor molecule, immunoglobulin,is sufficient to display restricted recognition. This finding lends credence to the view that a single combiningsite is sufficient for dual (complex) recognition and, therefore, supports single combining-site models for T-cell recognition as well as providing evidence for H-2-antigen interaction on the cell surface. However, closer inspection of the determinants actually recognized by CTL- and MHC-restdcted antibody suggests more differences than similarities betweenthese two repertoires. Certainly, the most striking difference between MHC-restricted antibody and CTLis their choice of different H-2-restricting elements. In H-2b mice, MHC-restricted influenza-specific antibody is Kb-directed, whereas CTLare Db-directed. If tolerance is the underlying mechanismresponsible for haplotype preference, then this observation implies that T and B cells are differentially tolerized perhaps by different self-determinants, since the CTLand B cell repertoires have been deleted of different self-plus-Xspecificities. Alternatively, it is possible that the germline repertoires of B cells and CTLare very different to begin with bsuch that few T cell receptor specificities exist that can recognize H-2K plus influenza, whereas few B cell specificities exist that can recognize H-2Kb plus influenza. This again would lead us to conclude that T and B cells havedifferent specificity repertoires. Next, let us consider the nature of viral determinants recognized by MHC-restdctedT and B cells. As is the case for antibody recognition of

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CLONALRESPONSESTO VIRAL ANTIGENS 81 virus per se, MHC-restricted antibodiesdemonstrateexquisite sensitivity to subtypevariation in virus. Indeed, the MHC-restrictedcomponent of the anti-HAresponserepresents a subset that is mostsensitive to structural variations as measuredby the relative lack of cross-reactiverecognitionof H1variants. In contrast, the MHC-restricted T cell response is far less discriminatorythan antibody responsesas measuredby the broad range of cross-reactivity. With respect to MHC recognition, only 57%of cellularly restricted antibodies recognizevirus in the context of cells of the immunogenic haplotype (83). This is far less MHC discriminationthan is generally observed for CTL.In addition, the Kb determinantsdescribed by fine specificity analysis of Kb-restdctedantibodies on a mutantpanel are a very different set from those observedin allogenic recognition by CTL(83). Takentogether, these findings suggestMHC-restdcted antibodiessee both a different set of H-2determinantsthan do CTLand also see a different set of viral determinants than do CTL. Theseobservations lead us to consider the nature of the determinants actually engagedby MHC-restrictedT- and B-cell receptors. There are three possible candidates for immunogenic epitopes. First, there maybe structures that are complexdeterminantscomposedof sequenceinformation contributed by both the viral moleculeand H-2. Second,there maybe newdeterminantsthat arise on the viral moleculeas a result of conformational changeinducedthroughinteraction with H-2. Finally, there maybe newH-2 determinantsthat result fromconformationalchangesin H-2that are inducedby virus. Responsesto all three types of determinantswould fulfill the requirementsnecessaryto be considered an MHC-restdcted response. It is possible that the differences observedin the T- and B-cell repertoires maybe interpreted on the basis of different emphasisby these repertoires as to whichof these three types of determinantsare immunogenie. For example,the greater specificity of MHC-restdctedantibody (versus CTL)to slight variations in viral structure maybe interpreted a greater emphasisby B cells on direct engagementof viral structures whereasthe specificity of CTLmaybe morereadily explainedas a consequenceof T-cell receptor engagement of conformationchangesin H-2. That CTLare actually capable of recognition of conformationalchangein H-2 has beendemonstratedby studies of Kb mutants(74), and certainly, there is muchevidencethat antibody is sensitive to conformationalchangein antigens. The conceptthat the CTLrepertoire is predominantlydirected against subtle changes in H-2 allows one to explain manyexperimentalobservations. As discussed above, CTLclones specific for H-2Aplus X often cross-react on specific alloantigenH-2B.This maybe explainedby considering that H-2Aplus Xreveals an H-2determinantalready exposedon H-2B.

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Also, it provides the best possible explanation as to why a CTLrepertoire directed against the alloantigens of the species is indeed the correct choice for recognition of self plus X, since many of the new self H-2 determinants that arise through interaction with antigen are determinants normally exposed on at least some other H-2 haplotypes. In any case, the resolution of the issues confronted by a comparative assessment of the mode of recognition by B and T cells of viral determinants and virus-infected cells will prove to be central to our ultimate understanding of several major phenomena. As monoelonal B- and T-cell responses continue to be applied to the dissection of determinant recognition and individual MHCand viral antigen expression is manipulated by gene cloning and transfection technology, significant insights should accumulate concerning the mechanism of determinant recognition by the immune system, the mutual effects of viral and immunological evolutionary selective pressures, and the means to maximize prophylactic immunization. Literature Cited 1. Ada,G.L., Lcung,K. N., Ertl, H. 1981. Ananalysisof effector T cell generation and function in mice exposed to influenza A or sendai virus ImmunoL Rev. 58:5-24 2. Allouehe,M.A., Owen,J. A., Doherty, P. C. 1982. Limit-dilution analysis of weak influenza-immune T cell reb and sponses associated with H-2K H-2Db. J. ImmunoL129:689-93. 3. Anders,E. M., Katz, J. M., Brown,L. E., Jackson,D. C., White,D. O. 1981. In Genetic Variation AmongInfluenza Viruses, pp. 547-65. NewYork: Academic 4. Askonas,B. A., Mullbaeher,A., Ashman, R. B. 1982. Cytotoxic T-memory cells in virus infectionandthe specificity of helper T cells. Immunology 45:79-84 5. Askonas,B. A., Webster, R. G. 1980. Monoclonalantibodies to hemagglutinin andto H-2inhibit the cross reactive cytotoxic T cell populationinducedby influenza. Eur. J. Immunol.10:151-56 6. Bennick,J. R., Yewdell,J. W., Gerhard, W.1982. A virus polymeraseinvolvedin recognitionof influenzavirusinfectedcalls bya cytotoxicT cell clone. Nature 296:75-77 7. Bevan,M.J. 1977.In a radiation chimera, host H-2antigens determineimmuneresponsivenessof donorcytotoxic ceils Nature LondonNewBiol. 269: 417-18 8. Bevan,M.J. 1977.Killer cells reactive to altered-self antigenscan also be al-

loreactive. Proc. Natl. Acad. ScL USA 74:2094-98 9. Blanden,R. V. 1974.T cell responseto viral and bacterial infection. Transplant. Rev. 19:56-88 10. Blanden,R. V., Dunlop,M. B. C., Doherty, P. C., Kohn,H. I., McKenzie, I. F. C. 1976. Effects of four H-2Kmutations on virus-inducedantigens recognized by cytotoxic T cells, lmmunogenetics 3:541-48 11. Blank,K. J., Lilly, F. 1977. Evidence for an H-2/viral protein complexon the cell surfaceas the basis for the H-2restriction of cytotoxicity. NatureLondon NewBioL 269:808-9 12. Braciale, T. J. 1977. Immunologic recognition of influenza virus-infected cells. I. Generation of a virus-strainspecific anda cross-reactivesubpopulation of eytotoxic T cells in the responseto type Ainfluenzavirusesof different subtypes. Cell. lmmunol.33:423-36 13. Braciale,T. J. 1979.Specificityof cytotoxicity T cells directed to influenza virus hemagglutinin. J. Exp. Med. 149:856-69 14. Braciale,T. J., Ada,(3. A., Yap,K. L. 1978.Functionaland structural considerations in the recognitionof virus infected cells by cytotoxicT lymphocytes. In ConternporayTopics in Molecular Immunology,ed. R. A. Reisfeld, F. P. Inman, 7:319-64. NewYork: Plenum 15. Braciale,T. J., Andrew, M.E., Braciale, V. L. 1981.Heterogeneityand specificity of clonedlines of influenza-virus-

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specific cytotoxic T lymphocytes. cancc of alloreactivity: T cells stimu,Exp. Med.153:910-23 lated by Sendaivirus coated syngeneic 16.Braciale, T.J.,Andrew, M.E.,Braciale, cells specifically lyse allogeneietarget V. L. 1981. Simultaneousexpressionof cells. Proc. Natl. Acad, Sc~ USA75: 5154-49 H-2restricted andalloreactiverecogni28. Frankel,M.E., Effros, R. B., Doherty, tion bya clonedline of influenzavirus specific cytotoxic T lymphocytes,d.. P. C., Gerhard,W.1979. A monoclonal Exp. Med. 153:1371-76 antibody to viral glyeoprotein blocks virus-immune effector T cells operating 17. Braciale, T, J., Gerhard,W.,Klinman, a but not at H-2K d. Z lrnraunol. at H-2D N. R. 1976.Theanalysisof the in vitro 123:2438-40 humoralimmuneresponse to influenza virus, d. lmrnunol.116:1539-46 29. Gardner, I. D., Boweru,N. A., Blanden, R. V. 1974.Cell-mediatedeytotox18. Canero, M.P., Gerhard,W., Klinman, icity against ectromcliavirus-infected N. R. 1978.Thediversity of the influenza-specificprimaryB cell repertoire in target cells. II. Identificationof effector Balb/c mice. J. Exp. Med.147:776-87 cells andanalysis Eur. d. lmrnunol. 4:68--72of mechanism. 19. Ciavarra, R., Forman,J. 1981. cell membrane antigens recognizedby anti30. Geiger,B., Rosenthal,K.L., Klein, J., Zinkernagel,R.M.,Singer, S. J. 1979. viral andanti-trinitrophenyl cytotoxic T lymphocytes, lmmunol. Rev. 58; Selective and unindirectional membraneredistribution of an H-2antigen 73-94 andan antibody-clustered viral antigen: 20. Cowen,K. M. 1973. Antibodyresponse to viral antigens. Adv. lmraunol.17: Relationship to mechanismsof eytotoxic T cell interactions. Proc.Natl. 195-253. 21. Dales, S., Oldstone,M.B. A. 1982.LoAcad. Sci. USA76:4603-7 31. Gerhard, W.1977. The delineation of calization at high resolution of antibody-inducedmobilization of vaccinia antigenic determinants of the hemagglutinin of influenzaAviruses bymeans virus hema~.glutinin and the majorhisof monoclonal antibodies, Top. 1n feet. tocompatibdityantigens on the plasma membraneof infected cells. J. Exp. Di~ 3:15 Med. 156:1435-47 32. Gerhard, W. 1978. In The lnfluenza Virus Hemagglutinin,ed. W.G. Laver, 22. Doherty,P. C., Biddison,W.E., Bennink, J. R., Knowles,B. B. 1978.CytoH. Baehmayer, R. Weil, pp 15-23. toxic T cell responsesin miceinfected Wien:Springer. 259 pp, withinfluenzaandvacciniaviruses vary 33. Gerhard,W., Braciale, T. J., Klinman, in magnitude with H-2 genotype. N. R. 1975. Theanalysis of the monoExp. Med. 148:534-43 clonal immuneresponse to influenza virus. I. Productionof monoclonalan23. Efl’ros, R. B., Doherty,P. C., Gerhard, W., Bennink,J. 1977. Generation of tiviral antibodiesin vitro. Eur. £ Imbothcross-reactiveandvirus-specificT munoL5:720-25 cell populations after immunization 34. Gerhard,W., Webster,R. O. 1978. Sewith serologicallydistinct influenza A lection and characterization of antiviruses. Z Exp. Med.145:557-68 genie variants of A/PR/8/34(HONI) 24. Ennis,F. A., Martin,W.J., Verbonitz, influenza virus with monoclonalantiM. W. 1977. Hemagglutininspecific bodies. J. Exp. Med.148:383-92 cytotoxic T cell response during in35. Gerhard, W., Yewdell,J., Frankd, M. D., Lopes, A, D., Staudt, L. 1980. fluenza infection. J. Exp. Med.146: MonoclonalAntibodies against In893-98 25. Ertl, H., Ada,O. L. 1981.Rolesof influenza Virus. In MonoclonalAntibodfluenzavirus infectivity andglycosylaies, ed. R. Kennett, T. McKearu,K. tion of viral antigenfor recognitionof Bechtol, pp. 317-333. NewYork: Pletarget cells by eytolytie T lymphocytes. num Immunobiology158:239-53 36. Oermain,R., Doff, M.E., Benacerraf, 26. Fazekasde St. Groth,S. 1981.Thejoint B. 1975. Inhibition of T lymphocyteevolutionof antigensandantibodies.In mediatedtumorspecific lysis by alloanThe lmmuneSJ~stem.A Festschrift in tisera directedagainstthe H-2serologiHonorof Niels Kaj Jerne on the Occacal speeificities of the tumor.J. Exp. sion of his 70th Birthday, ed, C. M. Med. 142:1023-28 Steinberg,I. Letkovits,1:155-68.Basel: 37. Gomard,E., Duprez, K., Henin, Y., Karger Levy,J. P. 1976.H-2region productas 27. Finberg,R., Burakofl’,S., Cantor,H., determinantin immune cytolysis of synBenacerraf,B. 1978.Biologicalsignifigeneic tumorcells by anti-MSVT lym-

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phocytes. Nature LondonNewBiol. 260:707-9 38. Gorczynski, R. M., Kennedy,M. J., MacRae, S., Steele, E. J., Cunningham, A. J. 1980.Restrictionof antigenrecognition in mouseBlymphocytesby genes mappingwithin the major histocompatibility complex. J. lmmunol. 124: 590-96 39. Hackett, C. J., Askonas,B. A. 1982. H-2and viral hemagglutininexpression byinfluenzainfectedcells: Theproteins are close but do not cocap. Immunology 45:431-38 40. Hackett, C. J., Askonas,B. A., Webster, R. G., vanWyke,K. 1980.Quantiration of influenzavirus antigensoninfectedtarget cells andtheir recognition by cross reactive cytotoxicT cells. J. Exp. Med. 151:1014-25 41. Hunig,T., Bevan,M.J. 1980.Self H-2 antigensinfluencethe specificity of alloreactive cells. J. Exp. Med. 151: 1288-98 42. Ivanyi, P., Cornelis,J. M., vanMourik, P., Vlug,A., de Greeve,R. 1979. Lymphocyteantibodies producedby H-2allo-immunizationdistinguish between MuLV-positive and -negative substrains of the same haplotype. Nature 282: 843-45 43. Joseph,B. S., Oldstone,M.B. A. 1974. Antibody-induced redistribution of measlesvirus antigenson the cell surface. Z lmmunol.113:1205-9 44. Kanagawa,O., Louis, J., Cerottini, J. -C. 1982.Frequencyand cross-reactivity of cytolytic T lymphocyte precursors reacting128:2362-6 against malealloantigens. J. lmmunol. 45. Katz, D. H., Skidmore,B. J., Katz, L. R., Bogowitz, C. B. 1978. Adaptive differentiation of murinelymphocytes. I. BothT and B lymphocytesdifferentiating in Fl-parental chimerasmanifest preferentialcooperativeactivity for partner lymphocytesderived from the sameparental type correspondingto the chimerichost. J. Exp. Med.148:727-45 46. Kilbourne, E. D., ed. 1975. The Influenza Viruses and Influenza. New York: Academic 47. Klinman,N. R., Segal, G. P., Gerhard, W.,Braciale, T., Levy,R. 1977.Obtaining homogenousantibody of desired specificity fromfragmentcultures. In Antibodies in HumanDiagnosis and Therapy,ed. E. Haber, R. Krause,pp. 225~-36. NewYork: Raven 48. Kohler,G., Milstein, C. 1975.Continuousculturesof fusedcells secretinganti-

bodyof predefmedspecificity. Nature 256:495-97 49. Koprowski, H., Wiktor, T. 1980. Monoclonalantibodies against rabies virus. In Monoclonal Antibodies,ed. R. Kennet, T. McKearn,K. Bechtol, pp. 335-51. NewYork: Plenum 50. Koszinowski, U. H., Allen, H., Gething, M. J., Watertidd, M.D.,Klenk, H. D. 1980. Recognitionof viral glycoproteins by influenza A-specific cross reactive cytolytic T lymphocytes. Z Exp. Med. 151:945-58 51. Koszinowski,U., Ertl, H. 1975. Lysis mediatedby T cells and restricted by H-2antigenof target cells infectedwith vaccinia virus. Nature London New Biol. 255:552-54 52. Laver, W.G., Air, G. M., Webster,R. G., Gerhard, W., Ward, C. W., Dopheide, T. A. A. 1979.Antigenicdrift in type Ainfluenzavirus: Sequenceditferences in the hemagglutinin of Hong Kong(H3N2)variants selected with monoclonal hybddomaantibodies. Virology 98:226-37 53. Lin, Y.-L., Askonas,B. A. 1981. Biological properties of an intiuenza A virus-specifickiller T cell clone.Z Exp. Med. 154:225-34 54. Lu, L. Y., Askonas,B. A. 1980. Cross reactivity for different type Ainfluenza virusesof a clonedT-killercell line. Nature 288:164-67 55. Mullbacker,A. 1981. Neonatal tolerance to alloantigens alters majorhistocompatibility complex-restrictedresponsepatterns. Proc. Natl. Acad.ScL USA78:7689-91 56. Niman,H. L., Elder, J. H. 1982. Monoclonal antibodies as probesof protein structure: Moleculardiversity among the envelope glycoproteins (gp70s) the murineretroviruses, In Monoclonal Antibodiesand T cell Products,ed. D. H. Katz, pp. 23-51 BocaRaton, Fla: CRC 57. Nisbet-Browm, E., Singh,B., Diener,E. 1981. Antigenrecognition. V. Requirementfor histocompatibilitybetweenantigen-presentingcell andB cell in the response to a thymus-dependentantigen, and lack of allogeneierestriction betwecnT and B cells. J. Exp. MecL 154:676-87 58. Nowinski,R. C., Stone, M. R., Tam,M. R., Lostrom,M. E., Burnette, W.N., O’Donnell,P. V. 1980.Mapping of viral proteins with monoclonalantibodies. Analysisof the envelopeproteinsof mufine leukemiaviruses. In Monoclonal Antibodies, ed. R. Kennett, T.

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CLONAL RESPONSES TO VIRAL ANTIGENS McKearn,K. Bechtol, pp. 295-316. NewYork: Plenum 59. Ohno,S., Matsunaga, T., Epplen,J. T., Hozumi,T, 1980.Interaction of viruses and lymphocytes in Evolution, Differentiation and Oneogensis.In Immunology80-Progress in Immunology IV, ed. M. Fougereau,J. Dansset, pp. 577-98. London: Academic 60. Oldstone, M. B. A., Fujinami, R. S., Lampert, P. W. 1980. Membraneand cytoplasmicchangesin virus-infected cells inducedbyinteractionsof antivirai antibody with surface viral antigen. Prog. Meal.Firol 26:45-93 61. Oldstone,M. B. A., Sissons, J. G. P., Fujinami,R. S. 1980. Action of Antibody and Complementin Regulating Virus Infection. In Immunology 80-Progress in Immunology IV, ed. M. Fougereau, J. Dausett, pp. 599421. New York: Academic 62. Owen,J. A., Allouche,M., Doherty,P. C. 1982. Limitingdilution analysis of the specificity of influenza-immune cytotoxic T cells. Cellular. Immunol. 67:49-59 63. Pan, S.-H., Wettstein,P. J., Knowles, b mutationslimit the B. B. 1982. H-2K CTL response to SV40TASA.J. Immunol. 128:243-46 64. Pfizenmaier,K., Jung, H., Kurrle, R., Rollinghoff, M., Wagner,H. 1980. Anti-H-2Dd alloreactivity mediated by herpes simplexvirus specific cytotoxic H-2t T lymphocytesis associated with k. Immunogenetics10:395-404 H-2D 65. Pfizeumaier,K., Pan, S., Knowles, B. B. 1980. Preferential H-2association in cytotoxic T cell responsesto SV40tumor-associated specific antigens. Z Immunol. 124:1888-97 66. Ramos, T. Moller, G. 1978. Immune response to haptenatedsyngeneie and allogeneie lymphocytes.Scand. Z Immunol. 8:1-7 67. Reiss, C. S., Schulman, J. L. 1980.Cellular immune responsesof miceto influenza virus vaccines. J. Immunol. 125:2182-88 68. Reiss, C. S., Schulman, J. L. 1980.Inttuenza type Avirus Mprotein expression on infectedcells is responsiblefor cross-reactiverecognitionby eytotoxic thymus-derivedlymphocytes.Infect. Immun. 29:719-23 69. Schrader, J. W., Edelman,G. M. 1976. Partipatioa of the H-2antigens of tumorcells in their lysis by syngeneieT cells. J. Exp. Med.143:601-14 70. Schwartz,R. H., Sredni, B. 1982. A1loreacfivity of antigen-specificT cell

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clones. In Isolation, Characterization, andUtilization of T lymphocyteclones, ed. C. G. Fathman,F. W.Fitch, pp. 375-84. NewYork: Academic 71. Sethi, K. K., Brandis,H. 1980.Therole of vesicular stomatitis virus major glycoproteinin determiningthe specificity of virus-specificandH-2restricted cytolytic T cells. Eur. J. lmmunol. 10:268-72 72. Shaw, M. W., Lammon,E. W., Cornpans, R. W.1981. Surfaceexpressionof a non-structural antigen on influenzaA virus-infccted cells, lnfect, lmmu~ 34:1065-67 73. Sherman,L. A. 1982. Theinfluence of the major histocompatibility complex onthe repertoireof allospecificcytolytic T lymphocytes. J. Exp. Med. 155: 380-89 74. Sherman,L. A. 1982.Evidencefor recognition of conformational determinants on H-2 by cytolytie T lymphoeytes. Nature297:511-13 75. Singer, A., Hathcock,K. S., Hodes,R. J. 1981.MHC restrictions in the generation of TNP-Ficollresponses:evidence for sdf-recognition by B cells. Fed. Proc. 40:1061(Abstr.) 76. Stukart, M. J., Vos, A., Boes, J., Mdvoid, R. W.,Bailey,D. W.,Melief,C. J. M. 1982. A crucial role of the H-2 D locus in the regulation of both the Dand the K-associatedcytotoxic T lymphocyte responseagainst Moloneyleubkemiavirus demonstratedwith two D mutants. Z lmmunol. 128:1360-64 77. van Leeuwen, A., Goulmy,E., van Rood,J. J. 1979. Majorhistocompatibility complex-restricted antibodyreactivity mainly,but not exclusively,directed against cells frommaledonors. Z Exp. Med. 150:1075-83 78. yon Boehmer,H., Hengartner,H., Nabholz, M., Lenhardt,W., Schreier, M., Haas,W.1979.Finespecificity of a continuouslygrowingkiller cell clonespecific for H-Yantigen. Eur.J. Immunol. 9:592-97 79. Weiss, A., MacDonald,H. R., Cerottini, J.-C., Brunner,K.T. 1981.Inhibition of cytolytic T lymphocyteclones reactive with Moloneyleukemiavirusassociatedantigensby monoclonal antibodies: A direct approachto the study of H-2 restriction. Z lmmunoL 126:482-85 80. Wettstein, P. J. 1982. H-2effects on cell-cell interactionsin the responseto single non-H-2alloantigens. V. Effects b mutations on presentation of of H-2K

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H-4and H-3alloantigens. Z Immunol. cell mediatedcytolysis. J. Exp. Meg 143:437-43 128:2629-33 87. Zinkemagel, R. M., Althage, A,, 81. Wyley,D. C., Wilson,I, A., Skehel.J. Cooper,S., Kreeb,G., Klein,P. A., SefJ. 1981.Structuralidentificationof the ton, B., Flaherty, L, Stimptiing, J., antibody binding sites of HongKong Shretiter, D., Klein,J. 1978.Ir genesin influenza hemagglutininand their inH-2 regulate generation of antiviral volvementin antigenic variation. Nacytotoxic T cells: Mappingto K or D ture 289:373-78 and dominanceof unresponsiveness.J. 82. Wylie,D. E., Klinman,N. R. 1981. The Exp. Med. 148:592-606 mufineB cell repertoire responsiveto 88. Zinkernagel, R. M., Callahan, G. N., an influenzainfectedsyngeneiccell line. A1thage, A.,Cooper, J.,Klein, P.A,, Z ImmunoL127:194-98 Klein, J.1978. Onthethymus inthe 83. Wylie,D. E., Sherman,L. A., Klinman, differentiation ofH-2self-recognition N. R. 1982.Theparticipationof the mabyT cells: Evidence fordual rccognijor histocompatibilitycomplexin antition? Z Exp. Med. 147:882-96 bodyrecognition of viral antigens ex89. Zinkernagel, R. M., Doherty, P. C. 1979. MHC-restdeted cytotoxic T cells: pressedon infected cells. J. Exp. Med. Studies on the biological role of poly155:403-14 morphicmajortransplantation anttgcns 84. Yewdell,J. W., Gerhard,W.1981. AndeterminingT cell restriction specifictigenic Characterizationof viruses by ity, function, andresponsiveness.Adv. monoclonalantibodies. ,4n~ Rev. MiImmunol. 27:51-177 crobio~ 35:185-206 90. Zinkcrnagel, R. M., Roscnthal, K. L. 85. Zarling, D. A., Miskimen,J. A., Fan, 1981. Experimentsand speculation on D. P., Fujimoto, E. K., Smith, P. K. antiviral specificity ofTandBcells. Immunol. Rev. 58:131-55 1982. Associationof sendal virion envelope and a mousesurface membrane 91. Zwcednk,H. J., Courtn¢idge, S. A., polypeptide on newlyinfected cells: Skehel,J. J., Crumpton, M.J., Askonas, Lack of association with H-2K/Dor B. A. 1977. CytotoxicT cells kill inalteration of viral immunogenicity. Z tiuenza virus-infected cells but do not Immunol. 128:251-57 distinguish betwccnscrologically dis86. Zinkernagel,R. M. 1976. H-2compatitinct Type A viruses. Nature London bility requirementfor virus-specific T NewBiol. 267:354-56.

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Ann. Rev. Immuno£1983. 1:87-117 Copyright©1983by AnnualReviewsInc. All rights reserved

STRUCTURAL BASIS OF ANTIBODY FUNCTION David R. Davies

and Henry Metzger

NationalInstitute of Arthritis, Diabetes,Digestive, and KidneyDiseases, National Institutes of Health, Bethesda, Maryland20205

INTRODUCTION It is less than 20 years since the general architecture of antibodies was elucidated and even less time since the explicit molecularbasis of antibody specificity first becameclear. In the subsequent explosion of information about the immunesystem, two basic principles have emerged: (a) Antibodies remainthe only knownstructures whosediversity is sufficient to explain the fine specificity exhibited by the immuneresponse; and, (b) antibody function is mediated by a molecule whosestructure consists of two distinct regions---one that carries a recognition site for antigenic determinants, and a secondby whichthe antibody reacts with receptors of a variety of etfeetor systems. In this review we examine the current information on the structure of antibodies. Wedo not describe again the basic four-chain structure of immunoglobulinsnor the division into variable and constant regions, which are by now well known(e.g. 4, 90, 118). Weinstead concentrate on the higher resolution data, muchof which is still in the course of refinement. Wediscuss the Fabs, in particular with reference to the combiningsite and the specificity of binding to hapten; the Fc region with reference to the binding site of protein A of Staphylococcus aureus and of Clq; and the structure of the hinge with reference to its possible role in separating Fab and Ft. Whereasthe characterization of the structures of individual proteins and of their interactions with small molecules can nowbe carried out with some 87 0732-0582/83/0410-0087502.00

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sophistication, the interaction of two or more macromolecules still presents considerable difllculties. It is not surprising therefore that our understanding of how antibodies interact with the macromolecular receptors on effector systems (e.g. Clq in the complement pathway, Fc receptors on cell membranes) is much less advanced. Our review of this aspect of antibody structure and function therefore involves more questions than answers.

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IMMUNOGLOBULIN STRUCTURE General Comments Our knowledge of the three-dimensional structure of antibodies at atomic resolution rests mainly on X-ray diffraction investigations of fragments. Intact proteins for which X-ray analyses have been carried out consist of Kol(106), an IgGl(A) human myeloma protein, the protein Dob (161), an IgG 1( K ) human cryoglobulin, and recently the human myeloma IgG 1(A) protein, Mcg (129). In Kol, and also apparently in another immunoglobulin, Zie (%a), the crystal structure contains an unusual feature: The Fc occupies a number of different positions in the crystal that are not crystallographically related, with the result that no significant electron density occurs in this region of the crystal, there being an abrupt drop in density at the end of the hinge. In both Dob and Mcg there is a 15-amino-acid-residue deletion in the hinge (63, 169), thus, presumably, reducing the flexibility of the molecule and enabling the Fc to be located in the electron density, although in the case of Dob the crystals are disordered and do not diffract to high resolution. The structures of three Fabs have been published, Newm (148), Kol (106), and McPC603 (152), as well as the structures of a number of VL dimers and L chain dimers (4). The structure of human IgG Fc has been determined, and also its complex with protein A of Staphylococcus aureus (43). These structures have been reviewed most recently by A m e l & Poljak (4) and are not covered comprehensively in this review. Because of the deficiencies in the crystals of the intact molecules, our knowledge of the whole antibody molecule has to be a sum of its parts. The flexibility of the molecule, in particular in the region between the Fab and the Fc, may preclude for some time visualizing directly by X-ray diffraction an intact molecule with intact hinge at atomic resolution. However, the checks that can be made on this composite three-dimensional picture of the antibody molecule are reassuring. Thus, the Kol Fab in isolated form in the crystal is quite similar to the Fab of the whole molecule in its crystal form. Also, the Fc in Dob has, within the experimental error of the comparison, the same overall structure as does the Fc in the isolated Fc crystals.

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Fa6 Structure and the Antibody Combining Site McPC603 A N D THE PHOSPHOCHOLINE BINDING SITE The structure of McPC603 Fab, a mouse myeloma IgA ( K ) with phosphocholine (PC) binding capability, has been determined at 3.1-8, resolution (41, 42, 125, 152) and is being refined to 2.7 8, (Y. Satow, D. R. Davies, manuscript in preparation). The overall three-dimensional structure of the Fab is illustrated in Figure 1, which demonstrates the strong lateral association between domains of the light (L) and heavy (H) chains, together with the relatively weak longitudinal interactions along each chain. Figure 1 also shows the clustering of six of the seven hypervariable regions at the tip of the Fab, forming the complementarity-determining surface (89, 90). The variable domains have a very similar three-dimensionalstructure for both the L and H chains and across species (4, 42, 123). The constant domains CLand CH1are also very similar. Both the variable and the constant pairs of domains are related by approximately twofold (rotation about the V axis of 180" will superpose VL on VH) and the angle between the two axes has been referred to as the elbow bend of the Fab and has been observed to vary

Figure I The a-carbon backbone of McPC603 Fab. The heavy chain is represented by the thick line. The two variable domains are at the top and the constant domains are at the bottom of the figure. The complementarity-determiningresidues (CDR) are shown as 6lled circles. Two residues in each CDR loop have been labeled.

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between approximately 137° for Fab Newm(4), 135° for McPC603(152), 147° for Dob (161), and approximately 170° for Kol (106). Figure 2 shows the combining site of McPC603with PC bound. (Y. Satow, E. A. Padlan, G. H. Cohen, D. R. Davies, manuscript in preparation). The choline is attached at the bottom of a pocket located principally between the hypervariable regions H3 and L3. The phosphate is on the surface and contacts residues from the heavy chain. It is apparent that PC is a small molecule and that the greater part of the hypervariable surface is not directly in contact with it. At the front of the pocket there are two hydrogen bond donors positioned within reasonable hydrogen bonding distance of the phosphate oxygens; these are the hydroxyl group of Tyr 33H and the guanidiniumgroup of Arg 52H(152). The residues lining the inside of the pocket are Tyr 94L on the right side, Asp 91L on the left, Leu 96L at the back, (125, 146), and the side chain of Trp 100all at the top left. addition, the backboneof residues 92-94L form the lower rim of the front of the pocket. CONFORMATIONAL CHANGEOne of the mechanisms proposed for cffector function activation involves an allostcdc change uponantigen binding (110). Since crystals of immunoglobulinshave large solvent channels and can bind to haptens soaked in through these channels, crystallographic investigation offers a direct way for observing conformational changes, when they occur. WhenPCbinds to McPC603 in the crystal, no significant conformational change occurs in the protein. There is a small movementof Trp 104a away

F31 ~

Figure 2 Stereo drawing of the combining site of McPC603with phosphocholine bound. The lower residues (91-96 and F32) are from the light chain. The remaining residues belong the three complementarity-determining regions of the heavy chain. The phosphocholine has the phosphate group in front with the choline moiety buried in a pocket.

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from the pocket, but no other changeof any significance. However,there are several reasons whyit cannotbe concludedfromthis observationthat antigen-antibodyinteraction results in no conformationchange: 1. Thecrystals are grownin a concentratedammonium sulfate solution, andit has beenobservedthat in the absenceof PCthere is a peak at the phosphatebinding site interpreted to be a sulfate ion (126). A conformational changemighthavebeentriggeredby the presenceof this sulfate ion, so that no additional changewouldbe observeduponPCbinding. However, in this respect it should be noted that in Fab Newm, (148) no conformational changeoccurs uponbinding of a neutral vitamin K~derivative. 2. PCis small and the association constant (---105 -1) with McPC603 might be insut~cient to trigger a conformationalchangethat could be inducedby a larger, moretightly binding antigen. Thesameconsideration applies to Fab Newm. 3. Theonly two structures at atomicresolution of immunoglobulins with knownbinding specificity are of Fabs. Althoughimprobable, one cannot rule out the possibility that changesthat occur with the intact molecule mightnot occur with fragments(81). Thus,althoughthere is no supportfromX-raydiffraction for a conformational changeassociated with antigen binding, such a changecannot be rigorously excluded. McPC603: THE CONTACTINGRESIDUES AND THE EFFECT OF CHANGES IN THECOMBINING SITE The PC molecule is in direct contact with only a limited numberof residues. Theyinclude side chains from all three heavychain hypervariableregions and fromone (L3) light chain region. Thenext mostdistant range of contacts contain manyresidues that play a role in positioning the directly contactingresidues and changesin these mightbe expectedto influence PCbinding. Anexampleof such a side chain is Glu 35H,a residue in the interface betweenVII and VLthat makes a hydrogenbondwith the hydroxylof Tyr 94L, whichis in turn a major contacting residue with hapten. A mutant of S107, a PC-bindingmyeloma protein, has beenobservedthat has lost the ability to bind PCandalso that fails to agglutinate PC-SRBC (144). Aminoacid analysis showedthat the mutationresults in substitution of an alanine for glutamicacid in position 35H.Althougha changeof this magnitudeis likely to producea significant rearrangement of side chainsin its vicinity simplybecauseof the difference in volumeof the twoside chains, the loss of contact with Tyr 94Lreduces an importantconstraint on a residue in direct contact with hapten. Another mutant observed by Cooket al (38) is more puzzling. The mutantstill boundPC, but it boundless well than S107to PCcoupledto

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different carriers, and showeda decreasein affinity for a variety of PCcarrier conjugates. The only aminoacid changeobservedwas that of Asp -~ Ala in the fifth position of the heavychain J region. Changesin the carboxyterminal half of VLwerenot entirely ruled out, but they did not appearin a tryptic peptideanalysis. Thestrangeaspect of this mutationsite is that it is spatially well removedfromthe PCpocketso that it mightnot have been expected to be involved in antigen binding. Anothercurious feature is that diversecarderscoupledto PCwereall affected, whichimplies they all havesomecontact with this residue, or with a region influencedby it. SEQUENCE COMPARISON OF PC-BINDINGANTIBODIESSequences andbindingdata are available for a variety of PC-bindingmyeloma proteins and monoclonalantibodies. Theyhave recently been reviewedby Rudikoff (145) and are only discussed here in relation to the three-dimensional structure of McPC603. The heavy chain The sequences of 19 heavy chains of BALB/cimmunoglobulins that bind phosphocholinehave been analyzed (74, 145). Ten these are identical and employthe T 15 sequence.Theremainingnine differ by oneto eight residues fromthe T 15 sequence.Geneisolation and analysis haverevealedthat all 19 of the Vr~regionsmusthavearisen fromthe single germline T15Vn gene segment(39). M167is the most divergent protein with eight Vn substitutions. In both M167and HPCG13 the same change (Thr at position 40) occurs, but all the other substitutions are unique, occurringin only oneprotein. The PC-contactingresidues Tyr33and Arg52,together with Glu35, are present in all of these sequences.Similarily, all of the BALB/c sequences with the single exceptionof M167contain a tryptophanat 100b. There is considerablevariation in the D region for these proteins, accompanied by only relatively small changesin PCaffinity, consistent with the fact that, with the exception of Trpl00b, CDR3 of the heavy chain does not play a major role in defining the PCpocket. The light chains The light chains of PC-bindingantibodies can be represented by the three BALB/cmyelomaproteins, T15, M603,and M167(34, 35). Theselight chainsdiffer considerablyin sequence,despite the similarity of their correspondingheavychain sequences. However,in the PC-binding region, in particular with the contactingresidues Tyr94,Pro95,and Leu96, their sequencesare the same. Theyalso all employthe sameJr sequence 05).

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The invariant residues in these light and heavy chains provide strong support for the suggestion that these PC-binding antibodies all have the sameoverall combiningsite for PC(125), with differences in binding specificity being contributed by the aminoacid substitutions.

Fab NewmThe structure of the Fab of Newm,a human IgG (k) mydoma protein, has been relined to a nominal2-,~ resolution (4, 148). The elbow bend is 137° and the L chain has a seven-residue deletion that includes residues 55 and 56 of CDR2,and 56 to 62 of the frameworkregion FRIII. The combiningsite is formed by the association of the remaining five hypervariable regions. The principal feature of the site is a shallow groove 15 X 6 X 6 ~, deep, bordered by residues from the H and L chains. Fab Newm binds several haptens at this site with affinity constants ranging from 103 to 105 M-l. A derivative of vitamin K1binds with the higher affinity and a crystallographic investigation has demonstrated that the menadionering system binds in the shallow groove with the phytyl chain draped along the surface, makingcontact with a numberof residues from the light and heavy chains (5). The number of contacts provided by the phytyl chain can probably account for the difference in binding between menadioneand the vitamin K1 derivative (103 vs 1.7 × 105 M-~). As noted above, no conformational change is observed upon the hapten binding.

KoL The humanIgG h cryoglobulin Kol and its Fab both crystallize and both structures have been solved, the intact molecule at 3.5 ]k and the Fab, for which the combiningsite specificity is unknown,at 1.9/~ resolution (106). The crystals of the intact moleculedisplay an unusual form of disorder, described above, that prevents visualization of the Ft. The Fab crystallizes well and has provided a detailed high resolution structure. The L chain conformation is quite similar to that of Fab Newm, except for the presence of the seven additional residues around CDR2. However, in the H chain, CDR3is eight residues longer than Newm(and six longer than McPC603),and these additional residues fold into the combiningsite and fill it completely, thus obliterating the grooveobserved in Newm.This combiningsite is rich in aromatic side chains, being filled largely by Trp (47H, 52H, 90L and 108H), Tyr (35H, 35L and 97L), His (59H). In both the crystals of the intact molecule and the Fab, the samecontact is made between the hypervariable surface and the hinge segment, Cys221Cys230, the light chain C terminus Glu212-Ser214, and the residues 133-

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138 and 196-199 of CHI of a neighboring molecule. This contact involves 2 of the Kol considerable surface area and it has been estimated that 1314A hypervariable surface is excluded from solvent. The contact is tight and closely packed and involves hydrophobicinteractions as well as salt links and hydrogen bonds. Marquart ct al (105) suggest that this interaction could be a prototype antibody-antigen interaction and might be responsible for the cryo properties of this molecule.

ANTIBODY COMBINING SITES Data on antibody combining sites have been comprehensivelyreviewed by Givol (76). Here, we only highlight a few structural topics. What commonfeatures, if any, will the three-dimensional structures of the combiningsites share? Givol (76) notes that the sizes of these sites are comparable to those of someenzymes. Lysozyme,for example, has a groove that will accommodatea hexasacchadde, and this is believed to represent about the upper limit in size for this kind of antigen (88, 102). The binding of antidextrans can be divided into two classes: end-binders like W3129, which bind only the nonreducing end of the dextran; and middle-binders like QUPC52, which bind in the middle of the chain to runs of six glucose units. It has been suggested that the former type of antibody might have a pocketlike site, whereasthe latter might be morelikely to have a lengthy groove(33, 89). Sincd three-dimensional structures are known for but two Fabs with knownbinding specificities, only a limited picture can be obtained from direct observation. It is nevertheless suggestive that one of these structures, McPC603,has a pocket for binding PC, whereas the other, Newm,has a shallow groove where the menadionebinding site is located. Again, if we were to argue from analogy with enzymes we might expect a pocket or groove in most antibodies to provide specificity. However,specificity can also be produced by complementarity between two interacting surfaces without necessarily invoking grooves or pockets, and significant contributions to the free energy of interaction can comefrom exclusion of hydrophobic groups from contact with water. This kind of specificity, like the interactions betweensubunits in a multisubunit protein, can be quite precise and can be destroyed by single aminoacid changes. For instance, not only is it necessary to maintain complementarityof two interacting surfaces, but there also needs to be a suitable juxtaposition of oppositely charged groups forming salt bridges as well as appropriate disposition of hydrogen bond donors and receptors. Accordingly, although antibodies specific for small antigenic determinants will probably have a groove or pocket, other antibodies specific for an array of amino adds such as epitopes on the surface

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ANTIBODYFUNCTION 95 of a protein need not necessarily have these features. TheKol protein combiningsite, although similar in somerespects to Newm, has its groove partially filled with aromaticaminoacid side chainsandit is significantthat this site interacts strongly with the C-terminalportion of another Fabin what could be a prototype antibody/protein antigen complex. Theavailability of monoclonalantibodies to specific antigens through hybridoma technologyshouldprovidea significant increase in the diversity of antibodies studies by X-raydiffraction. In particular, they offer the opportunityto study interactions with ligands that are larger than simple haptens and that could completdyfill the combiningsite. Oneexampleis an anti-influenza virus neuraminidasethat has already been crystallized (37). Moreof these anti-protein antibodies needto be studied, both alone and complexedwith antigen, to obtain structural comparisonswith other studies (12, 57, 164). Othermonoclonal antibodies to polysaccharidessuch as c~ 1 ~ 6 linked dextranshouldclarify the mechanism of bindingfor linear antigens (90) and the effects of aminoacid changeson specificity.

MODEL BUILDING STUDIES OFFv Since in the last 10 years high resolution structures havebeendeterminedfor only three different Fab’s, and only two (M603and Newm) have knownbinding specificities, it appears that X-ray crystallography can only provide a small fraction of threedimensionalstructures for interesting antibodycombiningsites. Thereis also the dit~culty that only someFab’s can be inducedto crystallize. An alternative approachto direct X-rayanalysis is to utilize the knowledge available from crystallography together with knownaminoacid sequences to construct modelsof the Fv’s of interesting antibodies. Thegeneral problemof protein folding, i.e. howa polypeptidechain several hundredamino acid residues long folds into its final globular form, is being extensively investigated. However,it is mostunlikely that it will soonbe possible to predict correctly the final foldedprotein structure basedon sequence.Nevertheless, the forcesthat contributeto the stability of a proteincontinueto be studied andare being refined. Thereis also an increasedappreciationof the dynamicaspects of protein structure. Theproblemof predicting antibodycombining site structures is a special case with somefeatures that makeit an attractive candidatefor investigation by molecularmodeling.The similarity of variable domainstructures is quite remarkable(4, 120-122),whichindicates a strong conservation the three-dimensionalstructure of the frameworkpart of the variable domains.Also, the mostvariable parts of the V domainsoccur largely at one end of the domainin the hypervariableloops. As a result, a comparisonof the sequencewith that of Vdomainsof knownstructure could, in optimal

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cases, directly lead to a preliminary modelthat could then be refined by a suitable energy minimization technique. The problem becomes more complicated whenthere are aminoacid insertions and deletions that change the length of the hypervadable loops. The new loops could be copied wherever possible from similar size loops in proteins whose structures have been determined. The YL: VI~ dimers Rei and Au(60) provide an exampleof very similar three-dimensional structures associated with loops of the same length, although there are 18 amino acid differences in each domain. A different point of view would be inferred from the Lchain dimer of Meg (58) where corresponding hypervariable loops do not preserve the local twofold symmetryof the V domains but have different conformations as a result of interaction with neighboring molecules in the crystal. The earliest modeling studies involved the 2,4-dinitrophenol-binding mouse myeloma protein MOPC315.The combining site of MOPC315has been discussed (135) relative to the structure of Newm.Padlan et al (124) constructed a molecular model for MOPC315 that utilizes the framework stru.cture of M603and the hypervariable loops derived from other V regions that have CDR’sof similar length whose structure had been determined by X-ray diffraction, An extensive nuclear magnetic resonance investigation of MOPC315 (54, 55, 186) and its interaction with ligands has led to a refined modelthat is similar to the original model, but has a different orientation for the DNPbinding site (89). The crystal structure of the Fv of MoPe315 is being investigated and should ultimately provide a basis for evaluating these models (6). Subsequently, Davies & Padlan (40) constructed a model for the homogeneousrabbit antibody (BS5) to type III pneumococcalpolysaccharide. Potter et al (136) presented a model for the inulin-binding myelomaprotein EPC109.More recently, Stanford & Wu(167) have constructed a backbone model for MOPC325,and Feldmann et al (62) have proposed models for J539 and included a proposal for the binding of hexasaccharide. The crystal structure for J539 has been determined at 4.5/~ and the atomic resolution structure is under investigation so that it should soon be possible to test this model. None of the models described above has been subjected to any form of energy minimization. They may give an approximate general, low resolution picture of the combiningsites particularly wherethey illustrate some stalking insertion or deletion, as in EPC109.However,they are unlikely to be accurate to better than several angstroms for the backbone atoms, and could be quite incorrect in positioning the aminoacid side chains. Until, for at least a few cases, they are comparedwith the results of X-ray diffraction these modelsshould be regarded as being quite hypothetical and should be treated with caution. In the case of MOPe315, the structure investigation by Padlan et al (124) was consistent with the knownchemical data from

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affinity labeling anddid lead to the discoveryof an error in the sequence determination,but these correlations derive from coarse rather than fine detail in the model. Since single aminoacid changes can producelarge effects on structure, it is perhapsoptimisticat this stage to expectto define an antibodysite with reasonableprecision. Certainly, somepowerfulform of energyminimizationwill be necessaryto ensure that the modelsprodueeddo at least satisfy the basic requirementof stereo-chemistry.However, satisfactory prediction also requires a larger library of known structures of antibodiesto a greater variety of antigens. THEH: L ASSOCIATION For the combinatorial mechanismfor generating antibodydiversity to be reasonablyeffective, mostlight chains should have the ability to combinewith mostheavychains. This requirementhas been examinedboth in vitro and in vivo. The~,-L interaction has beenshownto obeysecond-orderkinetics (7, 13, -~ (13). When 22, 69) and has a high affinity with Ka> 10~° M the competitive association of autologousand heterologouspairs of chains(i.e. pairs derived or not from the same myeloma)was examined,it was discovered that there wasa preferred association with the autologouschain (46, 78, 104, 170). When heterologousVt¢wasaddedto Fd’, no significant association took place except in the presence of CK.This was not true for the autologous VK,whichdid not require the presence of Cr (96). Isolated Vr recombinedwith the autologous Vri, but heterologous pairs did not associateby the criterion of UVdifferencespectra (80). It is dear that the association of the two completechains mustbe significantly aided by the presenceof the const.’,nt domain of the light chain. Nevertheless,the preferential associationfor the autologouspair observedin these in vitro experiments does imply some form of prior selection. Whetheror not any additional sdection is neededother than that for antigen bindingper se is not clear. Clearly, if the VnandVL do not associateat all or associatevery weakly,then their effectivenessin forminga combining site will be greatly reduced. Thesource of the variability in the Vr~:Vr~association comesfromthe involvementof hypervariable residues in the Vn:V L interface, as noted previously (41, 42, 148). Figure 3 showsthe residues that makecontact across the interface between Va and VLin McPC603 (Y. Satow, D.R. Davies, manuscriptin preparation). There are manyhighly conservedresidues that interact, particularly those in FRII, such as Glu39.However,at one end of the interface a variety of interactions can be seen to occur betweenhypervariableand frameworkresidues and betweenpairs of hypervariable residues. Thesemustcontribute to the forces governingthe exact positioning of VLrelative to VI~.That this is not alwaysthe sameevenin related pairs has beendemonstrated by Marquartet al (106)for the proteins

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Kol (IgG h) and Newm(IgG1 h) where small differences (’--9 °) occur in the position of VHrelative to VLthat are outside the limit of experimental error. In vivo results from cell fusion experiments demonstrated the existence of most of the possible "artificial" hybrids, so that it was concludedthat most, if not all, H and L chain can randomly recombine to produce new immunoglobulins that do not interfere with cell viability (114). This apparent contradiction raises the possibility that the preferential association is an in vitro artifact, but this wouldappear to be ruled out by the numerousvery carefully controlled experiments. Alternatively, the difference in the at~nities between autologous and heterologous pairs of chains maybe so small as to be unobservable under cellular conditions. Whena hybrid H/L recombinant is formed, then there is the additional question of howits binding properties will relate to those of the parent molecules. This would appear to depend on how similar the parent chains are, and Sher et al (157) observed that with reconsituted H/L hybrids from

L H

36Y

D35 Q39

38Q 43P

R44 L45

44P 46L

W47 Y91 $99 T100 W100a Y100b F100c

49Y 87Y 89Q 91D 94Y

W103 A105

98F

95P 96L

100A Figure3 Theresiduesthat formthe interface between VaandVLin McPC603. Thevertical bars indicatethe complementarity-determining.residues. Theconnecting lines join thoseresiduesthat haveatomswithina distanceof 4 A of oneanother.

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ANTIBODYFUNCTION 99 three anti-PC myelomaproteins, those with common idiotypes retained specificity uponrecombination,whereasthose without common idiotypes did not. Similadly, Manjulaet al (103) showedthat all the recombinants of anti-galactan myelomaproteins they examinedshowedanti-galactan activity comparable to the parents. However, as reviewedby Rudikoff(145), these anti-galactanheavyandlight chainsare so similar to oneanotherthat it is not surprising they exhibit the samebinding properties. Recently, Kranz& Voss(97) examinedthe fluorescein binding of all the possible recombinantsfromsix monoclonalantibodies to fluorescein and foundthat no hybridhas anti-fluorescein activity; the sequenceandidiotype of these antibodies was not analyzed. The Fc ThehumanI~GFc has been crystallized and the structure has been determinedat 2.9-Aresolution, Figure4 (43, 44). Thestructure differs fromthat of a Fab in that there is no protein/protein contact betweenthe two CH2domains.Instead, the complexcarbohydrateattached to Asn297occupies the ihterface region betweenthe two domainswith weakinteractions betweenthe two carbohydratechains. The two CI~3domainsassociate in a mannerslmdar to the CH1:CLdomainsof the Fab. The two C~2domains wouldseemto be positioned by the contacts madebetweenCH2and C1~3 of the samedomainand there is evidencethat someflexibility can occur in this region. In the protein Dob,the Fc has a very similar structure to the isolated Fc (161), despite the absenceof the hinge disulfide bridge due a deletion of 15 residues in the heavychain at this point. Thestructure of Fe with a fragmentof protein A from Staphylococcus aureus attached to it has been determined(43). Twocontacts are made between the fragment B of protein A and two neighboring, symmetryrelated Fc molecules. Oneof these, involving primarily hydrophobicinteractions andbelievedto be the interaction occurringin solution, involves residues 251-254, 310-315of CH2,and 430-437of CH3.Since binding of protein Adoes not inhibit complement activation (192) and since the binding site of Clqhas beenreportedto be on C~2(195), the Clq-Fcinteraction site must be located elsewhereon Ca2(see belowand Figure 4). The Hinge Thehinge region of the heavychain is encodedfor by a separate minigene (147) and varies in length (91) fromfive residues in IgGto 62 residues humanIgG3, wherethere appears to be a clear exampleof gene quadruplication. Marquartet al (106)in the intact Kol crystal observethe structure of the hinge to be double-stranded,disulfide-linked parallel chains, with each chain adoptinga three-fold polyproline-like helical structure.

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Figure4 Stereo drawingof the a-carbonbackboneof the Fc of humanIgG boundto fragment B of proteinAfromStaphylococcus aureu~Basedon coordinatesobtainedfromthe Brookhaven ProteinDataBank(43). Thecomplex carbohydrate occupiesthe spacebetween the twoCa2domains.Theprotein Ais shown withclusters of opencircles in the region betweenCa2and Ca3. ¯ , .,, and <3 on the Fc are the o-carbonsof those residues, respectively,that havebeenproposed to bindto Clqin 138,16, and24. Klein et al (91) have investigated the effect of major deletions in the hinge on the expression of biological effector functions. Theyfound that proteins Dob and Lec, both of which have 15 amino acid deletions, leaving them with effectively no hinge peptide, could not express several effector functions. In particular, they showedno ability to bind Clq or to activate complement. However, neither displayed any difference with normal IgG1 in binding to protein A from Staphylococcus aureus. The three-dimensional structure for the Dob protein is approximately T shaped (161). In this protein the overall structure of the Fc region is very similar to that observed by Deisenhofer (43) in the crystals of the isolated Ft. Thus, there is evidence that the lack of ability to perform effector functions is due to a major quaternary change (although minor changes would not be detected). The protein A binding indicates that the junction between C~/2 and C),3 is not significantly changed. However, in the Dob protein the CL and C~2domains are quite close to each other, and in the conformation observed in the crystal, they wouldblock binding of a large molecule such as the head of Clq to the end of C),2 proximal to the Fab. The presence of a somewhatrigid two-stranded hinge, with the strands linked by disulfide bridges and with each strand adopting a polyproline-like conformation (95, 106), would produce a greater separation of the Fab and Fc. Indeed, in IgG3, if the total hinge region adopted an extended rigid polyproline conformation (106), it would be more than 150 ~ long.

MembraneImmunoglobulinson ll-L ymphocytes The unique characteristic of B-lymphocytesis the presence of membranebound immunoglobulinon their surface. The special nature of this immuno-

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aTable 1 Extra sequencesof membrane immunoglobulin Segment

I II III

Net charge

Homology

12 (~) 18

-6 to -7

0.08b c(0.78)

extracellular

0

0.65 (0.96)

intramembranous

+2 to +3

1.0 (0.71)

intracellular

26 3 (tz)

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Presumed disposition

Length

aData from IgG1, 2a, 2b, and IgM(142, 182, 194). bFraction of identical residues at positions shared by gt and 3’. c Fraction of identical residues at positions shared by %

globulin has recently been elucidated by sequencing the cloned DNAfrom Balb/c mice. In each case studied so far, the molecules differ from secreted immunoglobulins having a membrane-specific portion coded for by two exons, M1 and M2. M1 is separated from the exon coding for the carboxy terminus of the secreted immunoglobulinby 1.4-1.8 kbases and M2in turn is separated from M1by an intron from "--0.1-0.8 kbases. The amino acid sequences show features commonto other membraneproteins that have a transmembraneportion: Proceeding in the direction of C-terminus, several negatively charged residues are followed by a hydrophobie stretch that is followed by several positively charged residues (Table 1). It is likely that these represent regions disposed on the outer surface of the membrane, within the bilayer and on the cytoplasmic surface of the membrane,respectively. Tyler et al (182) have suggested that the presence of as manyas six negatively charged residues in the stretch of 18 aminoacids in the external segment of mlgGmaypreclude muchsecondary structure and thus lead to membrane hinge. It contains a cysteine in the middleof the sequence, which mayform an interchain disulfide bond (182). In mlgMthis region is five residues shorter and contains no cysteine. A notable feature is the conservation of the hydrophobie sequence of 26 amino acids in the transmembrane portion. The three T chains show only a single aminoacid difference (albeit two base changes) in this segmentand nine differences when compared to the membrane/z chains. This is much less divergence than is observed for the other constant region domainsof these heavychains. Theconservation is also striking in view of the variability seen both in the hydrophobicleader sequencesof various proteins as well as for the intramembranoussegments of other (even related) integral membrane proteins (182, 194). That is, the mererequirement that the 26 residue segments are intramembranouswould not necessarily lead to the prediction of the markedsequence homologyobserved. If the 26 hydrophobie segment is an a-helix with 1.5 ,~ per residue, its length wouldbe "~4 nm, adequate to span the membranebilayer. Moreover, this wouldcluster those residues

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that havedivergedon one"side," leavingthe other aspect virtually invariant for the four isotypes for whichinformationis available (23, 75). This raises the possibility that these mIgare interacting with anothermembrane protein (23, 75). It wouldbe interesting to test this possibility by the use of cross-linking reagents or by isolating the mIgunder conditions that appear to stabilize interactions betweenthe subunits of other membrane receptors (141). The hydrophilic segment of mIg presumedto be on the cytoplasmic aspect of the membrane is variable: For mIgM it appears to consist of only three residues (142), whereasfor yl, y2a, andy2b it is 28 residues. Among the latter, the variability increasestowardsthe C-terminus; sevenof the final 14 residues showexchanges. FUNCTIONAL

ASPECTS

OF IMMUNOGLOBULINS

General Comments CONSEQUENCES OF ANTIGENBINDINGTO ANTIBODY The combining sites of mostfunctional proteins such as enzymesreflect a pervasive evolutionary trend. Oncea structure capable of effectively cleaving the substrate has evolved,subsequentselective pressurespromoteits stability. Theresidues that contact the ligand are mostpreserved, whereasthose that providesimplythe scaffolding are less constrainedfromvarying (reviewed in 189). Withimmunoglobulins the situation is virtually the opposite. It is those residues capable of contacting the antigen that showthe greatest diversity, whereasthe supporting "framework"residues tend to be more invariant (68, 193). Whatis gained with such a systemis a diversity of combiningsites so great that at least somemembers of the repertoire are capable of binding to anygivencluster of atoms,includingepitopes that neither the individual nor evenits species are likely to haveencounteredpreviously.Whatis lost is the capacity to catalyze a covalent changein the ligand in the waythat the sites on enzymes,finely honedby evolution, can. Instead, the binding of antigen is madeproductiveby other means.In someinstances, this occurs simplyby the antibody preventing the antigen from adhering to and penetrating vulnerabletissues (107). In other instances,it is the ability of the antibodycomplexed with antigen to activate an effector systemthat is critical. For the mostpart, the effector systemsrecruited are cells. The numberof different such systemscorresponds muchmore closely to the numberof distinctive Fc regions with whichthey interact than to the numberof combiningsite specificities, but there is no simpleone-to-onecorrespondence of heavychain isotype to the

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effector systemsit can stimulate. Severaldifferent Ig isotypesmayactivate the samesystemandanysingle isotypecan initiate several diverse effector reactions. Theeffector systems are usefully divided into clonal and nonclonal classes. In clonal systems, the immunoglobulins on a single cell that play a critical role in its activation all bear the sameantigen combining site. Within this class are the central elements of the immuneresponse; the antigen receptors (membraneimmunoglobulin)on B lymphocytesare suitable example.In nonclonalsystems,an activation event can be mediated by a mixtureof immunoglobulins bearing different combiningsites. These nonelonalsystemsconstitute the principal mechanisms by whichthe organismactively rids itself of the offendingantigen. MECHANISM OF ANTIBODY ACTIONThe biochemical perturbations activated by the antigen-antibodycomplexesare many.Nevertheless, the preponderanceof evidence suggests somecommon features. Just as the evolutionary mechanism that generates the diversity of combiningsites precludesthese sites fromhavingenzymicactivity, it seemsequallyunlikely that in each case the antigen wouldbe capableof acting like a heterotropie allosteric modifier that wouldchangethe conformationof the Fc region reproducibly. Similarly, if only those antigens wouldbe effective whose epitopeshappenedto be just so spacedthat a reproducibledistortion of the semi-rigid hinge region of the immunoglobulin wouldensue, this would substantially limit the numberof productiveantigen-antibodyencounters. Thesetheoretical argumentsare supported by considerable experimental data. Thelatter provide strong evidenceagainst either an allosteric or distortive mechanism of antibody action. The data instead suggest that clustering of twoor moreFe regions is the initiating eventin most,if not all, of the effector systemsactivated by antigen-antibodycomplexes.The older data have been analyzed previously (109, 110, 190); somenewer results are discussedin the reviewof specific systemsthat follows. Clonal Systems B-LYMPHOCYTES The consequences of antigen binding to the immtmoglobulins displayedon the surface of B-lymphocytes are varied. Theyrange frommakingthe calls unresponsiveto subsequentchallenge(118a) to promotingproliferation, anddifferentiationinto cells that secrete antibodiesof different isotypes(80a). Elucidatingthose factors that are decisivein regulating the outcomeis one of the central problemsin immunology. It has becomeapparent that interaction with exogenousmoleculesand cells, as well as the state of maturationof the B call itself, critically affect the

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pathwaychosen. Despite this complexity, it is dear that perturbation of the surface immunogiobulin can provide an essential signal without which nothing happens. The most persuasive studies are those in which the perturbation is induced by anti-immunoglobulin. The latter experimental tool permits examination of a population of B-lymphocytesthat bears immunoglobulins of diverse specificity (115). Withfew exceptions (155, 156), those studies showthat the critical common feature is the clustering of the surface immunoglobulinby bivalent antibodies or antibody fragments [F(ab’2)]. Such protocols may be somewhatartificial [though see Rajewsky(139a)], but investigations with hapten conjugates have led to similar conclusions (49, 61). The latter more physiological experiments have other complexities associated with them, however. At present, it is unclear how the size or number of immunogiobulin clusters on the cell affect the eventual outcome.It is also not clear whether there are other events associated with the clustering that are common to all the pathways the stimulated B-lymphocytecan eventually follow. The answers will not comequickly. Current available assessments of the outcome involve measurementstemporally distant from the stimulus---often several days. What is sorely needed is to identify early events that presage the ultimate result. Acute changes, apparently resulting from the clustering of the surface immunoglobulin in and of itself, have been reported, e.g. mobilization of bound Ca2+ (18), association of the clustered immunoglobulin with the cytoskeletal matrix (17), and changes in the surface distribution of the mIg (177). These cannot yet be correlated with the final fate of the stimulated cells. Other perturbations reported [methylation of phospholipids, activation of a serine esterase, and generation of cytoplasmic factors (94)] appear to require additional exogenousfactors (B-cell growth factor and interleukin 2) that are knownto affect the cell’s future activity. The quantitative role of the clustering of the membraneimmunoglobulinhas not yet been analyzed in those cases. Althoughthe immunesystem as a whole is unlikely to play a significant role in general homeostasis of an organism’s internal environment, it obviously does have a variety of regulatory elements within the context of immuneresponses themselves. The mechanisms by which hormone receptors are controlled (downregulation, desensitization, amplification) are therefore likely to have their counterparts in the control of events mediated by the immunoglobulins on B-lymphocytes also, but appropriate systems for study of the latter are only nowemerging. Twoother aspects of the activation of B-lymphocytes mediated by mIg are of interest. Since the mIgare mobilein the plane of the membrane [albeit not as mobile as might be expected from theoretical considerations (56)], it is reasonable to ask why spontaneous clustering of the mIg does not activate the cell if the association is a critical event. One answeris that

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whereasit may’be a critical event, it is likely to be inadequatein the absence of accessoryfactors, as already discussed. Nevertheless,in systemswhere suchaccessoryfactors are not required, a similar questionarises (below). Asecondquestion of interest is the followingapparentparadox(86). the one hand, B cells bearingIg with higher affinities for the antigen are activatedpreferentiallywhenantigenis limiting(atfinity selection)(162); the other hand, multipoint attachment of antigen to mlg-bearing lymphocytes can be expected on theoretical grounds, to makethe amountof antigen boundbe quite insensitive to the intrinsic bindingaffinity of the combiningsites on the mlg(86). Thetwoquestionsmayin fact be related. Whatis likely to be the significant factor is the size andpersistenceof the clusters of mIg,whetherthey arise spontaneouslyor result fromthe interaction of antigen with mlgof lowor high affinity (111). This subject is discussedl~urther in the context of a simplersystem(see below). T-LYMPHOCYTES Thymus-processed lymphocytes are capable of mediating a variety of antigen-specific effects apparentlywithoutthe use of exogenousimmunoglobulins.Theuncertain nature of the T-cell antigenspecific receptor that mustbe involvedcontinuesto be a major challenge with whichimmunologists are wrestling (85). Althoughthese receptors may share common features with secreted or membrane-bound Ig, not enough is knownto maketheman appropriate subject for discussion in a review of antibodystructure and function.

NonclonaISystems: Cells BearingFc Receptors Manycells interact with the Fc region of immunoglobulins (183). In those cases wherewehavethe mostinformation,the interaction is mediatedby a plasmamembrane glycoprotein usually of molecularweight 50,000-70,000. The binding maybe more or less firm to the monomericIg and more or less specific for certain heavychainisotypesandthe speciesof origin. The nature of these "Fc receptors" (130) to whichthe Ig binds is outside the subject area of this survey; reviews on one or moreof themhave been publishedrecently (70, 112, 183). Herewefocus on two aspects only: (a) the sites on the Ig that interact with the Fc receptors, and(b) the role the immunoglobulinin mediating the functions in whichthe receptors participate. SITES ONIg TO WHICHTHE Fc RECEPTORS BIND lgG The literature about the sites in the Fc region that interact with cellular receptors is confusing(reviewedin 2). In part, this arises because various investigators havestudied immunoglobulins and cells fromdifferent

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animals. Therelationship betweenisotypes fromdifferent species is by no meansa simple one nor can one assumethat the receptors on alveolar macrophages,peritoneal macrophages,and peripheral blood monocytes will be similar evenwithina species, let alone fromdifferent species. The assays employedmayalso be important. Thosein whichmultipoint attachmentcan amplifyweakintrinsic association constants can be expectedto yield results different fromthose in whichdirect (monomeric) binding examined.Becausecertain types of fragmentsof Fc are morereadily pre¯ pared from the Ig of somespecies than of others, numerousreports deal with the binding of Ig fragmentsto cells of a heterologousspecies. Data derivedfromsuchstudies maybe useful for providingspecificity criteria for distinguishing different receptors, but it mayconfusemorethan clarify physiologicallysignificant interactions. We,therefore, focusprincipally on those studies involvingreagents fromthe sameor closely related species. Theuse of fragments, peptides, or mutant proteins even from homologous species maypresent additional problemsof interpretation becauseof sequence homologiesin the domainsof immunoglobulinsand related proteins. To cite just two examples,Ciccimarraet al (32) observedbinding activity in a decapeptidefromCT3that, however,is likely to be buried in the native structure (51). Furthermore,B2-microglobulin, the light chain the class I majorhistocompatibility antigens, wasfound by Painter et al (128) to bind to macrophage Fc receptors. In someinstances binding of isolated CT3modulesto call receptors can be observed. HumanpFc’ and CT3fragments interact weaklywith human monocytes,although not to humangranulocytes (65, 66). A weakinteraction has also beenseen in an analogouscombinationwith reagents prepared fromrabbits (73) and mice (50) but not fromguinea pigs Isolated CT2domainsare inactive but manystudies demonstratethe involvement of these regions. Cleavageof the interheavychain disulfides in the hingeregion, whichincreases the segmentalflexibility of CV2,reduces the binding activity of humanIgG1 to humangranulocytes(66, 73). Masking the interdomaincleft betweenCT2and CT3with a fragmentof protein A from Staphylococcusaureus also interferes with the binding of human IgG1 to humangranulocytes, but not to humanmonocytes(67). Using variants of the mouseMPC11 myeloma,Diamondet al (48) showedthat the receptor for IgG2bon mousemacrophagesrecognized a segment in the CT2domainand the receptor for IgG2a, sequences from both CT2and C’y3domains.Thecomplexityof these interactions is strikingly illustrated in a report on IgG producedby another variant of this myeloma. It fails to bind to the mousemacrophage receptors for IgG2aeven though the variant has completelyintact CT2and CT3regions of the 2a isotype(14). This mutanthas hybridheavychainswith the first 220 residues of the 2b isotype and the remainingresidues (221 to the carboxyterminus)

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of the 2a class. Cleavageby papainof the hingeregionof this proteinoccurs oneresidue awayfromthe normalsite of cleavage,after residue 240versus 239 for both native IgG2a and IgG2b. This showsthat this region is conformationallydifferent fromthat of the normalproteins. Thefragment derived from such cleavage of the variant protein binds to the receptors perfectly well. Theseunusualresults indicate that althoughthe principal contacts between the receptors and IgG may be in the CT2and CT3 domains,the hinge regions mayindirectly affect binding by affecting the relationship of the Fab regions to the C72domains.Althoughthe human myeloma proteins that havea deleted hinge haveso far beentested only on murine macrophages,the results of those studies suggest an analogous phenomenon (95). A study of Facb fragments ((Fab-CT2)2)in a homologous system has been reported by Johansonet al (87). Theyfound that rabbit Facb boundvery well to the Fc receptors on membranesof the yolk-sac. Notably, the reducedmolecules,whichdissociate into monomers of Fab-C~2,boundequally well, whereasneither the Fab nor the pFc’ (C’y3)fragmentswereactive. Activity of Facbfragmentsalbeit with Ig and macrophagesfrom heterologousspecies has also been observedin certain instances, but not in others (of. 8, 119). IgE Several types of cells have one or moredifferent surface receptors capable of binding IgE (26, 165). However,one of the receptors on mast cells and basophils is unique: Its at~nity for monomeric IgE is at least 100-foldgreater than that observedfor any other Fc receptor for any other immunoglobulin. In the homologoushumansystem, the Fc fragments ((C~2-C,3-C,4)2)bind well, but fragmentscomparableto Facb, i.e. (FabC,2)2, or pFc’, i.e. (C~4)2,do not (53). A report that a pentapeptide the C,2 domaininhibited binding of the intact IgE (79) could not corroborated in a detailed study (11). That in the rodent systemsuch peptide could displace boundIgE (185) is hard to reconcile with data showingthat even intact IgE cannot displace boundmolecules(187). exclusion,it has beenpostulatedthat C,3 plays a majorrole in the interaction (53). This wassupportedby indirect data concerningthe location irreversible physicochemicalchangesinducedby conditions that lead to irreversible changesin binding. Recently,the rates of tryptic cleavageof unboundIgE and IgE boundto solubilized receptors were comparedin the rodent system(132). Whereascleavageat all sites wasinhibited, the most stalking(>40-fold)inhibitionwasobservedat a site likely to be close to the C,3:C,2interdomainregion. Theseresults also point to the importanceof the C~3domains.It is interesting that by several criteria the latter domains maybe homologousto the CT2domainsof IgG (53). There is evidence against the carbohydrateon the IgE contributingsignificantly to the binding affinity (98).

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Overall, the results with IgGandIgE suggestthat in mostinstances the penultimatecarboxyterminalCH domainsplay a significant, perhaps even the most important, role in the interaction betweenimmunoglobulins and the Fc receptorson cells. MECHANISM BY WHICHCELLS BEARINGFc RECEPTORSARE ACTIVATED The Fe receptors on cells are implicated in a wide variety of immunemechanisms.Someof these can be meaningfully groupedtogether as involving immunoregulatory functions. Despite the apparent importance of this group, we do not consider it since the mechanismby whichIg mediatesthese functionsis still largely unknown. Auseful series of articles on the role of such Fc receptors in regulating the function of T cells has recently been compiled(116). Asecondfunctional groupthat can be distinguishedincludes those receptors by whichthe cell directly or indirectly disposesof antigen. Wediscuss two of such functions for whichthe role of the Ig and its mechanism of action havebeenactively investigated. EndocytosisIt is likely that engulfmentby macrophages of antigens that havesuccessfully penetratedthe organismsexternal barriers is quantitatively the most important mechanismfor the disposal of antigens. This engulfmentis markedlyenhancedif the antigen has antibody boundto it (reviewedin 160). It has beenknownvirtually from the time Fc receptors werediscoveredthat antigen-antibodycomplexesare readily distinguished by the cell from uncomplexed.IgG. This can be observed even whenthe concentrationof the latter exceedsthat of the formerby four or moreorders of magnitude(150). Theevidenceis overwhelming that the clustering of regions engenderedby the antigen-antibody interaction is necessaryand sufficient to explainthe selectivity. This has beenrigorouslyanalyzedtheoretically and experimentallyby Segal and his colleagues (150, 151, 154). These workers provide evidence that the Fc receptors cycle betweenthe plasmamembrane and the interior. Theyproposethat the multipoint attachmentthat aggregationof Fc promotescan accountfor the preferential internalization of the complexed immunoglobulin (151, 154). That is, given a movingchain of cycling Fc receptors, the complexesof Ig simply hop aboard morereadily. The importantcorollary is that the complexesneed not generateany signal. Secretion: macrophagesThoughendocytosis of immunecomplexes by macrophagescan apparently be accountedfor by a simple mechanism,this processseemsinadequateto explain the stimulation of synthesis andsecretion of various mediators by macrophageswhenchallenged with antigenantibodycomplexes (15, 27, 134, 139, 143, 176). In these instances, the cell

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clearly has beenactivated to do somethingnew.That the activation occurs on the surface of the cell is indicated by the results of Ragsdale&Arend (139)and Rouzeret al (143). Thelatter stimulatedprostaglandinsynthesis andsecretion by beadscoatedwith antibodiesin the presenceof cytochalasin D, whichprevents phagocytosis. That immunecomplexesof guinea pig IgG2 induce superoxide anion production by homologousmacrophages threefold moreefficiently than IgG1 despite their being equivalently phagocytosed (176)could be similarly interpreted. In this andother studies (134), the greater efficacy of larger complexes wasnoted. Macrophages also have Fc receptors moreor less specific for homologous IgE. Artificially dimerizedIgE was sut~cient to trigger release of lysosomalenzymesfrom the cells (47). Secretion: mastcells Mastcells havereceptors that bind IgEwith extraordinarily high at~nity. Moreover,triggering suchcells via the IgE boundto their surfacerequires nothingmorethan somethingthat will react appropriately with the IgE and (in most studies) a 1 mMpinch of 2+. These factors havepermitted a morecompleteinvestigation of this systemthan has beenpossible with any other cellular systemactivated by antibodies (113). Thecurrent consensusis that IgE-mediatedtriggering of mastcells occurs whenantigen, reacting with the cell-boundIgE, clusters the previously unassociatedreceptors for IgE. Threeprincipal observationsformthe basis for this model:(a) the receptors in situ are univalent (108, 149); (b) receptors are mobile(9, 28, 100, 149, 171); and (c) multivalentantibodies to the receptor mimiccompletelythe action of the antigen (83, 84) in the total absenceof IgE. Thefirst observationindicates that the process is intermolecularrather than intramolecular;the secondshowsthat the molecules that mediatethe reaction neednot be closely adjacentprior to triggering; and the third demonstratesthat the importantmoleculesare likely to be the receptors and not the IgE. Themechanism by whichclustering of these Fc receptors mediates degranulation is reviewedelsewhere(113). Here weindicate a few salient points only. In this system also, evidence showsthat larger and more numerousclusters provokemorecompleteor rapid activation (64, 92) but that evendimerscan providea unit signal (64, 92, 153). In this systemalso (see above),the questionarises of why,if clusteringis all that is required, ~ There is as yet no answerto this is spontaneousrelease not observed? qt could be argued that since these cells have a desensitization mechanismthat is also triggered by clustering (92), low levels of clustering are insufficient to cause release because of an unfavorable balance betweenthe activation and deactivation. If this were true, one would then anticipate spontaneous desensitization. Since this has not been observed, the dilemma remains the same.

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questionbaseduponexperimentaldata. It is possiblethat effective clustering cannotoccur becauseof an energybarrier that is only overcomeby the free energyprovidedby the bindingof antigento IgE. It is also possiblethat the lifetime of spontaneousaggregatesis too short to inducea signal; even a ligand that bindsrelatively weaklyto an individual IgEcoulddramatically increase the time the receptors remainclustered, thereby permitting the necessarymolecularchangesto take place (45). Theinteraction of mobile ligands (e.g. in a planar layer of lipids) at a limited interface with the cell-boundIgEis sufficient to cluster receptors(10, 188)andcausemediator release in this system(188), whichdemonstrates that rather indirect clustering is all that is required. Other Althoughits functional role is undefined, isolation from human leukemic cells of an Fc receptor has been reported by Suzuki and his colleagues(173). Onsodiumdodecylsulfate gels, this has a size similar that reported by Thoenes&Stein (178), but in manyof its characteristics it appearsto be very different fromthe Fcreceptorsisolated by others(183). It is the first receptor for whichan intrinsic activity has been claimed (phospholipaseA2activity), an activity stimulatedby interaction with IgG in solution, particularly with aggregatesof IgG(172, 175). Themechanism by whichIgGstimulates this activity remainsto be clarified. The same grouphas reported recently that similar results can be obtained for the receptor specific for IgG2bon the P388D~ line of mousemacrophages (174). Nonclonal

Systems:

Complement

CLASSICAL PATHWAY The activation of the classical pathway of the complementcascade provides a unique opportunity to study an effector systemactivated by antigen-antibodycomplexes.Thereceptor of this system, C1, can be readily isolated in amountsadequateto performrigorous chemicalanalyses(140), electron micrographs are yielding detailed pictures of its complexstructure (168), andsensitive molecularassaysare available to assess its activation rapidly in solution (29). Nevertheless,it has been extraordinarily difficult to define precisely what is going onmasobering spectacle to those interested in eventhe simplestcellular system! Sites on lg with whichC1interacts Definingthe site of interaction of the globular heads of the Clq subunit of C1with Ig is complicatedby the observationthat there maybe several peptides derived fromor related to immunoglobulins, whichin their isolated form can interact with Clq (127, 128). Therefore, studies with fragmentsgeneratedby proceduresthat involve eventransient exposureto denaturants(e.g. those used to generatean

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Facbfragment)mustbe interpreted cautiously. Nevertheless,the weightof evidencefavors interaction of native IgGvia the Cn2domains,although only one such domainin intact IgGappears to be boundby Clq at a time (127). Oneof the mostconvincingpieces of evidencefor the importance this region is that mutantIgO’s, such as the humanproteins DobandLec, fail to bind andactivate C1(95). Theseproteins are missingthe hingeregion but seemotherwiseintact (see above).It appearslikely that changesin the disposition of the Fab regions with respect to the Cy2domainsis responsible (95)---an explanationvirtually the sameas that usedto rationalize the failure of certain mouseIgGmutantsto bind to Fc receptors(above),as well as for a variety of observations on the effect of reduction of the hinge disulfides in intact and fragmentedIgG(reviewedin 52). Burtonet al investigated the site on rabbit IgOthat interacts with Clq by using specific inhibitors, chemicalmodification,andan analysis of sequenceconservationandof accessibility (24). Basedonthese results, as well as the workof others, they proposedthat the site principally involves chargedresidues in the two carboxyterminal~-strands of the C~2domains (residues 318-322;332-337).Agroupin Madrid(16) correlated controlled conjugation of carboxylate groups on humanIgG with inactivation and implicatedprimarilyglutamicacid residues 258 and293. Thelatter is in a region proposedby Brunhouseand Cebra from analysis of sequencedata (20). Workwith synthetic peptides led to the proposal that His 285, Lys 288, and Lys 290 were important in humanIgG(138). Onthe other hand, deglycosylationof rabbit IgGalso has beenreported to decreaseClq binding (191). Results with concanvalinA also suggestedthat exposureof the carbohydratemightbe importantin one instance (99) but not in another (184). Noneof the approachesused distinguishes betweendirect and indirect effects. Theresults are depictedin figure 4, whichshowsthe a carbons of the implicatedresidues. Notably,all these residuesare well removed from the binding site for protein A of Staphylococcusaureus. Althoughthe residues proposedby the three groupsdiffer, they could all be correct. Firstly, the terminal atomsof the side chainsmaybe topologicallyconsiderably closer than the positions of the ¢ carbonsof the sameresidues. Secondly, the site of interaction with Clqcould be extensive, involvingmany aminoacid side chains (cf Figure3). Onlyproceduresgivingdirect data will yield moreprecise information:either crystallographicvisualization, or the use of cross-linkingreagentsandisolation of peptidesthat containresidues from IgOand Clq cross-linked to each other. Becauseof someunusualaspects of the activation of C1by IgM(below), the site(s) on the latter that interact with Clq are of interest. TheC#4 domain is stated to fix C1efficiently (21, 71), whichsupportsearlier results with fragments of this domain(82). Nevertheless, as noted above with

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respect to IgG, these results could be quite misleading. Althoughthe findings of Siegel &Cathou(159) do not rule out the possibility that the C/x4domainsare active, these workersinterpreted their results on the effect of heating IgMas implicating the C/~2domains. Mechanism of activation C1 by lg Whereassomedata in support of the needfor aggregationof IgG(36) havebeenreinterpreted (166), sufficient other results indicatethat Fcclustering is critically importantfor efficient activation (109, 110, 190). Newobservationson the lack of effect of antigen alone on the binding IgGto Clq (101, 181) and C1activation (180) provide further evidenceagainst allosteric effects beingcritical. Thatunivalentrabbit Fab/c fragments(Fab-(C72, C’Z3)2)efficiently inducedcomplementmediatedlysis of the lymphocytes to whichthey werebound(77) is a strong argumentagainst a distortive mechanism. " Tschopp has observedthat the relative et~cacyof artificial oligomersto activate C1parallels closely their rdative affinity for Clq(179). Apreparation of monomersof IgG fit the same pattern, but Tschoppcould not determine whether or not this was due to contaminationwith traces of aggregates. It remains possible then that monomeric IgG could induce a signal either solely by its interaction with the globular portion of Clqor perhaps by reacting with the nearby (168) Clr. The role of aggregation could then simplybe that of enhancingthe binding by multipoint attachment (such as has been proposed for endocytosis by macrophages;see above). It wouldbe useful to knowwhether or not with monomerieIgG morerigorously freed of aggregatesactivation wasnevertheless observed. Moststudies with IgMsuggest that only antigens that engagemorethan onesubunit are capableof stimulatingits ability to activate C1(93, 137, 158), but the workof Koshland’sgroup suggests otherwise(19, 30, 31). Althoughhaptens wereineffective, they foundthat larger univalent antigens initiated IgM-mediated activation of C1. Themechanism is unclear by whichthese antigens, whichare substantially bigger than what can be accommodated in a combiningsite, producethis effect. Theauthors propose severalalternatives(31). Anewrole for IgGin its interaction with the classical pathwayhas been described (25). It appears that C4 forms a covalent bond with the C~I domainduring the activation of the cascade. Whetheror not the IgGplays morethan a passive role in this effect is not known. ALTERNATIVE PATHWAY Activation of the alternative pathway of complement fixation remainsas the single exceptionto the rule that antibodies stimulate effector mechanismsvia their Fc regions. It has been repeatedly demonstratedthat F(ab~)2 fragmentsare active--as active as

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intact IgG--but since this pathway can be initiated in the total absence of immunoglobulins (117), interpretations of the role of the antibody become more uncertain. Several studies suggest that the monovalent Fab’ or Fab are inactive or much less active (59, 72, 133, 163), but a recent study reports otherwise (1). Different assays were used and the experts in this field will have to sort this matter out. One aspect about which there does seem to be agreement is that activation requires multiple molecules of antibody attached to the antigen. ACKNOWLEDGMENT We wish to thank Enid Silverton,

who prepared Figures 1, 2, and 4.

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GENETICS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX: THE FINAL ACT J. Klein, F. Figueroa, and Z A. Nagy AbteilungImmungenetik, Max-Planek-Institut t’fir Biologic,7400Tiibingen, FederalRepublicof Germany THE MASTER TRICKSTER Lookingbackat the 45-year history of the majorhistocompatibility complex (MHC),one cannot help but be amazedat the tricks the MHC has pulled on its students. Thefour majortricks whichconfusedimmunologists werethese: Trick numberone: the MHC is discovered to control the presence of antigens on the surface of red bloodcells andis regardedas a bloodgroup locus (26). A wholenewdimensionis addedto serological studies through analysis of the erythrocyteMHC antigensbeforeit is realized that red blood cell expressionis the least significant feature of the MHC andhas nothing to do withits function(42, 47). It turns out, in fact, that severalvertebrate species do not expressMHC moleculeson erythrocytesat all, and those that do, do so only becauseof leftover informationremainingin the cytoplasm after expulsionof the nucleus(28, 89). Trick numbertwo: the MHC moleculesare recognizedas a major obstacle to a free exchange of grafts betweenindividualsof the samespecies(27), and this recognition becomesa strong impetus for the developmentof transplantationbiologyas a separatefield of study(42, 89). So firm becomes the tie betweenMHC and transplantation biology that for years immunologists are unable to dd themselvesof the habit of referring to MHC molecules as transplantationantigens. Yet, again, it turns out that its influence on the outcomeof grafting is only on the fringe of MHC physiological function. 119 0732-0582/83/0410-0119502.00

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Trick numberthree: the MHC affords an impressionof being the site of mysterious, immuneresponse (Ir)-governing genes distinct from the MHC itself but somehow magicallybondedto it (67). Themomentum this impression generates is the greatest immunology has known,but it too turns out to be generatedby false premises.Eventually,one realizes that Ir genesare nothing but another MHC disguise: The Ir genes turn out to be MHC genes themselves(53, 54). However,the imprint of this trick on immunologists is so profoundthat as its consequence,immunology maynever rid itself of terms such as Ir genes, I region, and Ia antigens. Trick numberfour: in a numberof vertebrate species, the MHC is found to be intimately associated with genes coding for complementcomponents (7, 58, 69, 87), and this finding aloneis enoughto convinceimmunologists that the complement genes are part of the MHC. Yet, a search provides no evidencethat ties the two groupsof loci together functionally andevolutionarily. This fourth trick maybe the last one of the mastertrickster, unless it turns out that in our present impressionof the true MHC function wehave beentricked again. Bethat as it may,there is no needto feel foolish because of the tricks. Each of them has had an enormouslypositive impact on immunology and has led to a series of importantdiscoveries. Andin the end they all have brought us closer to an understandingof what the MHC is really about.

TROUBLE WITH DEFINITIONS In this reviewwedefine the MHC as a cluster of loci codingfor proteins that provide context for antigen recognition by T lymphoeytes.By"context" wemeanthat a T cell does not recognizean antigen alone; it recognizes the antigen in the context of the MHC molecules of the antigen-presentingcell. Weare awarethis definition doesnot meetthe approval of other immunologistswhotraditionally viewthe MHC as a group of loci that occupycertain arbitrarily delineated chromosomal segments.Thelatter must be a viewbased on Montaigne:"Whatevervariety of herbs there maybe the wholething is includedunder the nameof salad." Ourobjection to this traditional viewof the MHC is preciselythis salad principle, namely, that one lumps together herbs (loci) that mayhave nothing in common except their occupation of the same bowl (chromosomalsegmen0.The mainconsequenceof our definition is that it excludesthe complement loci from the MHC.Wejustify this exclusion thus: ~st, the complementcomponents controlled by loci linked to the MHC clearly have a different function than do MHC proteins. The former are involved primarily in humoralimmunity;they are plasma proteins that becomemembrane-bound

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only secondarily, and they function as typical enzymes. The latter are involved in cellular immunity; they are membraneproteins that mayappear in the plasma secondarily, and they have no demonstrable enzymatic activity. Second, there is a considerable degree of homologyamongthe various MHC genes (and proteins), indicating they are all directly related through their origin from commonancestor genes. No such homology could be demonstrated, as yet, between the MHCand the complementgenes. Wedo not wish to go so far as to maintain that no homologyexists between the two groups, for it may well be that as more DNAand protein sequences becomeknown, particularly for the complementgenes and proteins, some evolutionary relationship between the MHCand complement genes may becomeapparent. But this relationship, if found, almost certainly wouldbe of a different magnitudethan that knownto exist amongthe different MHC genes. Current studies suggest that the MHC genes are related to immunoglobulin (Ig) genes (76); yet, nobodyseems willing to consider Ig genes part &the MHCor vice versa. If homology were found between MHCand complement genes, it would probably not be greater than that between MHCand Ig genes. Hence, if we do not consider the Ig genes as part of the MHC,why should we bestow this privilege on the complement genes? Third, although the C4 genes are closely linked to the MHCin many species, the C3 gene is only loosely MHC-linkedin some species and apparently is not linked at all in others (1). Since C3 and C4 are homologous genes that are almost certainly evolutionarily related, if we consider one (C4) to be part of the MHC, we should consider the other (C3) as well. However, where do we then place the borders of the MHC? If we include the C3 locus in the MHC,we must abandon the salad principle anyway because, otherwise, soon half of the genome would becomethe MHC;and if we abandon the principle, there is no longer any compelling reason to include the complementgenes in the MHC.Fourth, recent work indicates that the mouseMHC contains a locus coding for the enzymeneuraminidase (Neu-1, see below). According to the salad principle, this locus, too, must be included in the MHC.The absurdity of this inclusion is obvious. One possible justification for considering the complementloci as part of the MHCis based on the notion of the supergene. Snell (88) and Bodmer (6) have argued that the MHC and loci linked to it mayconstitute a unit for natural selection and so, from a population genetics point of view, may form a single supergene. However,this notion remains pure speculation, particularly in view of the fact that no effects of natural selection on the MHC have been unequivocally demonstrated--and will be extremely diiticult to demonstrate. Moreover, even if the MHC and the linked loci were to form a unit of selection, what would be the advantage of using this

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observation to defineagcncclustcr in immunology? A MHCsupcrgcnc may bc a uscful concept whcndiscussing oncparticular aspcct of thccomplcx, butto useit asa basisfordefining theMHCwould onlyleadtoconfusion. In summary, wcbelieve it isbesttoignore tradition andtodcfinc thcMHC thcwaywc define virtually allothergencclusters--on thebasisof functional similarity andevolutionary homology.

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MHC CLASSES Usingthecvolutionary definition, onecandividetheMHClociintotwo classes, which~ wcdesiguatc class I andclass II(44,46).Locibelonging thcdiffcrent classes differ intheproperties andfunction ofthcir respective products. The characteristic features of class I glycopeptides are a molecularweight of 44,000, a chain length of some 340 to 350 aminoacids, the presence of three extramembranedomains(Figure 1), association with f12 microglobulin, and functional preoccupation with cytolytic T (Tc) cells (25, 42, 106). The characteristics of class II glycopeptides are a molecular weight of 28,000 (fl chains) and 34,000 (a chains), a chain length of about (a chain) and 220 (#8 chain) amino acids, the presence of two extra membranedomai~ns(Figure 1), dimerization of a and fl chains, and functional preoccupation with helper T (Th) and suppressor T (Ts) cells (53,

96). Molecular Weight The figures given are for glycosylated polypeptide chains. The class I polypeptide may have one or two carbohydrate side chains attached to it, CLASSI

CLASSII

Figure1 Diagram showing the domainorganizationof class I andclass II MHC molecules. flxm, $2 microglobulin; othercombinations of Greekletters andnumbers indicateindividual MHC domains; M, membrane.

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depending on the species and probably also on the particular allele. Each side chain consists of some12 to 15 invariant sugar units, arrangedin a fixed order. Each class II polypeptide chain has one side chain attached to it, apparently organized in a mannersimilar to that of the class I side chains. The difference in the molecularweight of the ~ and # chains probably arises partially from a difference in the length of the polypeptide chain and partially from a difference in the carbohydrate side chains.

Chain Length Thefigures givenrepresentan averagelength; the actual lengthmayvary by several residues, depending on the particular locus and allele. In some class I or class II genes, several codonsor even wholeexons can be deleted and the polypeptide chains thus can be shortened correspondingly. Most of the deletions occur in the chain’s intracellular region.

Domains The basic topological unit of the MHC molecule is the domain, a polypeptide some90 amino-acid residues long and containing cysteines at its opposite ends. The polypeptide probably folds characteristically around a disulfide bridge formed by the eysteines. The compartmentalization of the MHC molecule into domains has its foundation in the segmentation of the MHCgene into exons and introns (Figure 2). Similar domains have also been found in the f12 microglobulin (one domain), immunoglobulins(from two to six domains per chain), Thy-1 (one domain), and other proteins (102). The presence of this basic structural element in different proteins usually interpreted as evidence for the commonorigin of these proteins. CLASSI Chain

¯

m LS

mmm mmm

~1 ce2 (x3

¯ m

m m m m m m

TM CP CP CP

CLASS][ mm m mm. m m Chain m m ¯ ¯ LS o11 0(2 TMICPI3’UT 3’UT

/32M

¯ ¯

LS

m m

¯ ¯ m ¯

/3’2M3’UT3’UT

~’gure2 Organization of class I andclass lI MHC genes,o 1 -t~3, exonsencoding extrarnembrahedomainsof MHC chains; B2m,/~2 microglobulin; LS, leader sequence;TM,transmembrahesequence; CP,cytoplasmic sequence; 3’UT,3’untranslatexi sequence; blackboxes,exons; interconnecting lines, introns.

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However,since similar domainsoccur also in proteins such as the superoxide dismutase (84), for which no other evidence is knownsuggestive of relationship to MHCor Ig molecules, presumably the presence of these domains does not always signify commonancestry of proteins. The domain mayrepresent an optimal solution to some basic topological problems of protein structure, a solution independently arrived at by different protein families through convergent evolution. Chain ~4ssociation To be stable, apparently, the MHC molecule must contain four cxtramcmbrane domains. To achieve this configuration, the three-domain class I chain combines with the one-domain f12 microglobulin and the two-domain class II a chain associates with the two-domainfl chain. The associations are always noncovalent and always between a polymorphic and an oligomorphic chain. (The polymorphismof the/~2 microglobulin and the class II a chains is far lower than that of the class I chain and the class II fl chain.) The function of the oligomorphicchains appears to be the stabilization of the MHCmolecule. To what degree the chains also contribute to the immunologic functions of the MHCmolecule is unclear. The class II molecules are also associated noncovalcntly with a third chain that is evenless variable than the a chains and, therefore, is referred to as invariant (35). Its function is unknown;however, since it is not controlled by the MHC (57) and is associated with a and fl chains only the cytoplasm but not on the cell surface (57a), its role may be nonimmunologic,perhaps concerned with the integration of the class II chains into the membrane. Functional Specialization Although both class I and class II molecules provide context for antigen recognition, a certain division of labor exists betweenthemin that, generally, the former provide the context for antigens recognized by Tc cells, whereas the latter provide context for antigens recognized by Th and Ts cells (54). WhenMHCmolecules themselves serve as antigens (in an allogcncic situation), the samepredilection for the stimulation of ditferent T c¢11 sets can often be observed. However,in the latter situation the specialization is not absolute because class I alloantigens can stimulate not only cytolytic T cells, but under certain circumstances also proliferating (presumablyTh) cells; similarly, class II alloantigens stimulate not only proliferating T cells (ina mixedlymphocyte culture), but,againundercertain circumstances, alsocytolytic cells. A corollary to thisdivision oflaboristhatclassI molecules provide context fortherecognition ofantigens thatareanintegralcomponent of theplasmamembrane (e.g.minorhistocompatibility antigens orviralantigens), whereas classIImolecules provide recognition

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context for soluble antigens pickedup by macrophage-like antigen-presenting cells. Still another corollary mightbe that the cells presenting the antigen in the context of class I moleculesand those presentingit in the contextof class II moleculesmaynot be the same.Thesethree observations, pertaining to the type of the stimulated T cell, the formof the presented antigen, and the type of the antigen-presentingcell, are probablyinterrelated, but the causeandthe effect in this relationshipis not clear. UNITY Wepredicted some10 years ago that the present-day class I and class II loci wouldturn out to be functionally homologous (43, 52). At that time no biochemicaldata available could be used to comparethe products of these loci, not to mentionthe loci themselves,and the prediction seemed to go against the then-current conceptof the MHC. Thegeneral consensus then wasthat the class II werea special groupof loci, in somemystedons wayrelated--through the Ir genes--to the T cell receptor, and therefore weremoreworthyof study than the prosaic transplantation antigen-encoding class I loci. Although someof this/-region mystiquepersists to this day, the prosaic viewbeginsto prevail. There can no longer be any doubtthat the class I andclass II loci forma single familyin termsof their organization and function. Theyprobably are no farther apart than are the Ig light-chain fromthe heavy-chaingenes (61). A major step in MHC evolution must have been the acquisition by the class I genesof an extra exoncodingfor the third extramembrane domain. Thereorganization of the MHC moleculethat followedthis step must have beena consequence of this acquisition. Thereorganizationmusthaverepresented an adaptation to a newfunctionnavariant of the function the MHC geneshad beencarrying out until then. Whetherthis adaptationrelates to T cell function (regulation versus killing), antigen processing(processed versus indigenousantigens), or type of antigen-presenting cell (macrophage-likecell or a lymphocyte) shouldbecomeapparentonceit is resolved whycertain antigens are presentedin the context of class I and others are presentedin the contextof class II molecules.Alternatively,the evolution might have proceededfrom a three- to two-domainchain, or--and this possibility is the mostappealing--thetwochain types evolvedconcurrently from a common ancestor. EXPRESSIONuTHE TOLLING ALIEN SPECIFICITIES

OF THE BELLS

FOR

Anotherimportant step in the evolution of the MHC must have been the acquisition of separate controlling elementsfor the different groupsof

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genes,resultingin a differentialexpression of thesegenesin differenttissues. Three principal modesof expressionhave been recognizedthus far (47). First, someclass I loci (e.g. the K, D, andL loci in the mouse)manifest themselvesin most, if not all, somatictissues, but moststrongly in the lymphoidtissues, in particular in the lymphocytes(both T and B). The difference in expression betweenlymphoidand nonlymphoid tissues is at least 10-fold(2). Second,certain other class I loci (e.g. the QaandTla loci in the mouse)preferentially expressthemselvesin the T cell lineage andin ceils representingcertain stagesof this lineage.However, as wediscusslater, the restriction to the lineageand stage is not absolute andthe differences in tissue distribution maybe largely quantitative rather than qualitative. Third,the known class II loci expressthemselvespreferentiallyin the B cell andmonocyte lineages. In these lineages, again, only somecells mayexpress the genes,whereasother cells expressthemfar less or not at all. Theremay also exist a fourth modein whichcertain class II genesexpressthemselves in certain stagesof T cell differentiation. This modeof expressionlias been debatedextensively over the last decadeand evidenceboth supportingand disclaimingit has beenput forward(29). A newtwist to these discussions froma recent report states that there maybe a special groupof class II loci, perhapsdistinct fromthose alreadyidentified, preferentially expressedin T cell subsets(32). Curiously,moleculargeneticists thus far havenot noticed anysign of these extra loci. MHC moleculesnot expressedon all tissues are sometimesreferred to as differentiation antigens, but wefind this designationmisleading.Theterm differentiation antigen is otherwiseused to meana moleculeinvolved in differentiation, i.e. drivingit. Weare not awareof anyevidenceimplicating any MHC molecules, including the mouse Qa and Tla molecules, in differentiation. Someof the moleculesmayserve as markersof differentiation, but this fact does not qualify themas differentiation antigens. Still another modeof MHC expression has been claimedby the proponents of the alien-specificities hypothesis.Accordingto this hypothesis, each individual possesses most of the MHC genes characterizing a given species (5). Of these, normallyonly a few becomeexpressedat any given time, but duringan aberrantdifferentiation, suchas that characterizingthe generationof tumorcells, unexpectedlysomeof the previouslysilent genes becomeactivated, whichleads to the expression of MHC molecules norreally foundin other individuals(alien specificities). Thehypothesishas beensupportedby findings fromseveral laboratories (78), all claimingthe demonstrationof alien specificities in a variety of tumorsby a variety of methods.Wehavecriticized these findings by pointingout that the experimental methodsused had not been properly controlled, that the purported origin of the tumorlines usedhad not beensatisfactorily demonstrated,and

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that other explanationsof the findingsweremorelikely than those proposed (45). However, the alien-spe~ificities research gainedconsiderablemomentumas attested by the numberof papers publishedon this topic and the numberof meetingsorganized. Moleculargenetics put a period behindthe wholeaffair: Clearly not enoughgenesexist in the MHC to codefor all the allomorphs(allelic forms of MHC proteins) possessedby a species.

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ORGANIZATION Figure 3 depicts the organization of the MHC in the H-2d mice, as determinedby molecular genetics methods(94; L. Hood,personal communication). Thereare 37 class I and five class II loci on this map--theclass II loci all groupedin onecluster, the class I loci split in at least twoand perhapsthree clusters. Preliminarystudies with other 1-1-2 haplotypessuggest that the numberof MHC loci mayvary from one inbred strain to another, andthat the 1-1-2a strains may,perhaps, carry moreloci than do other strains. The//-2a mapitself maynot be complete,particularly in the class II region, but it is unlikelythat large numbers of genesstill remain hidden. Howdoes this mapcompareto that obtained by classical genetics methods? Thefirst thing one notices is the conspicuousabsenceof B, C, and J loci, andtherefore it is these loci that wediscussnow.

The B Locus Thepostulate of a separate B locus (region, subregion)wasoriginally made by Liebermanand co-workers(62) to explain the genetic control of the antibody response to a myelomaprotein MOPC173 (presumablya response against an allotypic determinanton the IgG2amolecule).Thecritical 1-1-2 haplotypes involved in the mappingof this locus were 1-1-2a, ~, //-2 H-2h~, andH-2i~, of whichthe first wastyped as a low responderand the remaining three were typed as high responders to MOPC173. Takinginto account the origin of//-2 h4 and H-2i5 from//-2a///-2 b heterozygotes, Liebermanet al proposed that in H-2~4 the recombination took place proximallyand in//-2 i5 it took place distally to a hypothetical B locus controlling the immuneresponse to MOPC173. The involvementof the B locus waslater postulated for immuneresPonses to at least five other antigens: lactate dehydrogenaseB (LDHB) (68), staphylococcal nuelease KA~ApE~"E/;’E~,

D L

d haplotype. Figure3 MHC genespresentin the H-2

Oa-Tta

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(Nase)(64), oxazolone(12), the male-specificantigen (H-Y,as detected skin grafting) (33), and trinitrophenylated mouseserumalbumin(TNPMSA)(97). Themappingof the genescontrolling these responsesrevolved in all these cases aroundthe four critical//-2 haplotypesusedby Lieberman and her co-workers(Table 1). Wehavereanalyzedin detail two of the six responsespurportedly controlled by the B locus---the responsesto LDH protein B and to the myeloma MOPC173 (3). Theanalysis revealed a peculiar interplay of Thand Ts cells: TheTh cells of the//-2a strain recognizethe antigen in the context of the Ak molecules, but they cannot respondbecausethey are suppressed by Ts cells that recognizethe sameantigen in the context of the Ek molecule.As a result of this interplay the//-2a miceare lowresponders,not becausethey carry a low-responder allele at somehypotheticalB locus, but becausethey arc inhibited in their responseby a simultaneousactivation of Ts cells. Hence,the genetic control of the response to LDHB and to the myeloma protein MOPC173 can be explained without the postulate of a separate B locus, distinct fromthe previouslyidentified loci codingfor the A and E molecules. Onthe basis of these data, wesuggested that the B locus be removedfrom the genetic mapof the 11-2 complex. Onemight ask, however,what of the other responses? Canthey too be explained by similar mechanisms?The response to H-Yand to TNP-MSA probablycan be, since the responsivenesspattern is identical to that of LDHB and MOPC173. The responsivenessto oxazolonedisplays a different pattern, but the responsesare highlyvariable. Not only are there discrepancies amongthe reports fromdifferent laboratories (12, 79), but evenin the samelaboratory there is a considerable variation fromone experimentto another, particularly in micecarrying the tt-2 k haplotype(N. Ishii, Z. A. Nagy,unpublisheddata). Until this variation is broughtundercontrol, the mappingof the genes involved in the response to oxazoloneremains in Table 1 Immune responses complex

Strain B10.A C57BL/10 B10.A(2R) B10.A(4R) B10.A(5R) B10.BR

originally

believed

to be controlled

by a B locus

H-2 aResponse to antigen Alleles at H-2loci haplotype K Ac~ A# E# Ec~ D MOPC173 LDHB Nase H-Y Oxazolone _ + _ .9 a k k k k k d b

b

b

b

b

b

b

+

+

-

h2

k

k

k

k

k

b

h4 i5

k b

k b

k b

k b

b k

b d

-

-

+ +

+ +

k

k

k

k

k

k

k

-

a+, responder; -, nonresponder. For references see text.

in the H-2

TNP-MSA _

+

-

+

+

-

+

-

-

+ +

-

+ +

+

-

+?

-

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doubt. As for the responseto Nase,Berzovskyand his colleagues (4) have established recently that part of the responseis controlled by the A loci (~1~ andA~), but theyinsist that the other part is controlledbythe B locus. Wesuggest as an alternative explanation that Naseis recognizedin the context of A molecules in someH-2 haplotypes and in the context of E moleculesin other haplotypesandthat this switchingof recognitioncontext creates the illusion of a B locus. At anyrate, the importantfact is that the molecular geneticists are unable to find any DNA correspondingto the purportive B locus (L. Hood,personal communication),so there must someother explanationfor all the responsesfor whichthe existenceof the B locus has beenpostulated. Thereis certainly no justification for keeping the locus in the tt-2 map. The C Locus Evidenceof the existenceof the C locus is threefold. First, an H-2h2antiH-2h4 antiserum,originally usedto define the C locus (9), has recently been reproduced by Sandrin & McKenzie(86). Second, according to Rich and her co-workers(82, 83), antisera purportedlycontainingC-specificantibodies react with a suppressor factor producedin an allogeneic mixedlymphocyte reaction (MLR).Third, Okuda& David(74) have reported a that occursin congenicstrain combinationspresumablydiffering at the C locus andan inhibition of this reaction by anti-C sera. Mapping by classical methodshas suggesteda position of the C locus betweenE~and the genes coding for the CAcomponent.Althoughthis chromosomal segmenthas not yet been fully exploredby moleculargenetics methods,the data available thus far do not lend any support for the existence of a C locus near the E,, locus. Ourownexperienceagrees with these negative results: Wehave never been able to obtain any activity in the C-definingH-2h2 ~ anti-H-2 combination,althoughwehave tested this combinationrepeatedly by serological methods, MLR,graft-versus-host reaction, cell-mediated lymphocytotoxicity (CML),and grafting of tissues (36, 63; J. Klein, unpublisheddata). If the entire E~---C4interval indeeddoes turn out to be devoidof class II loci, then someother explanationwill haveto be found for the positive results obtained.Moleculargeneticists havefoundan extra E#-like gene betweenthe E~and the classical E# gene (L. Hood,personal communication), and evidencefor morethan one E moleculehas also been presentedat the protein level (10, 56, 59, 77). Althoughthis newgene on the wrongside with respect to the purportivelocation of the C gene, it mightbe that someas yet not understoodform of interaction betweenthe E genescreates the illusion of the C gene. Anotherpossibility is that the C geneis present only in someH-2haplotypes. A final possibility is, of course, that the C genenever existed in the first place.

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The J Locus In contrast to the C locus, whichhas been championedby only three or four laboratories, the existenceof the J locus has beenaccepted,it seems, by every laboratory exceptours. Supportingthis locus is a massof data: Anti-J sera havebeenproducedin manylaboratories and evenare available commercially(71); J-specific monoclonalantibodies have recently been producedby at least twogroups(37, 99); functionalevidencefor the expression on Ts cells has beenobtainedrepeatedly(71); and, recently, an isolation of RNA bearing the J-encodingmessagehas beenreported (95). Ourreservations havebeenbasedon the inability to obtain anybiochemicalinformation aboutthe purportedJ molecule.If it existed, whywasit so dit~cult to isolate? Therecent inability to find anydifference betweenthe DNAs of the H-2~ and H-2i5 haplotypes (L. Hood,personal communication),the two haplotypesoriginally used to define the J locus (72), of course, puts the scepticismaboutthe J locus on a firm basis. If it can be corroboratedthat there really is no DNA codingfor the J moleculein the class II region, what then is J? Twoprincipal possibilities exist. First, the J genemaybe somewhereelse than in the class II gene interval: It can be outside the H-2 complexor it can be on a different chromosome altogether. However, if the latter werethe ease, it wouldstill haveto be explainedwhythe anti-J sera have been produced by immunizationbetweencongenic lines presumably differing only in a small chromosomal segmentwithin the H-2 complex. Second,the genecould, again, be an illusion arising from an as yet not understoodform of interaction betweenknownclass II genes. But if this were the explanation, howshould one then understand the data on the isolation of J-encodingmRNA? Whateverthe correct explanation (and it mightbe availablebythe time this article is published),it is clear that Jis not what it has been thought to be up to now. Loci Between K and Ae? Recently, Monaco& McDevitt(70) have detected a seres of 16 spots two-dimensionalgels of cell preparations precipitated by anti-MHC sera. The proteins composingthese spots ranged in molecular weights from 15,000 to 30~000.Typingof appropriate H-2 recombinantslocalized the genes coding for the low-molecular-weightproteins in the chromosomal interval betweenthe K and A ~ (A ~) loci. Monaco& MeDevittargue that this interval is occupiedby a newclass of MHC loci that codefor cytoplasmic low-molecular-weight proteins (since the proteins could not be labeled by the lactoperoxidasemethod,the authors assumethey are absent fromthe membrane) (70).. However, evenif these loci couldbe definitely established, what they have to do with the MHC still has to be solved. The fact that

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neither the class I nor the class II DNA probes haveas yet detected them couldsignify they are unrelated structurally and presumably also functionally to the genuineMHC genes. After all, if there are enzyme-encoding loci entrappedwithin the MHC, whynot also other loci, unrelated to the MHC proper?

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The K and D Loci Iv(myi&D~mant (34) recently havedescribeddata suggestingthe existence of at least twoKand at least three Dloci. This suggestionis supportedby DNA cloning, whichestablishes the presence of these loci in the aH-2 haplotype (94). Oneof the Dloci corresponds to D~mant’sL locus and another, perhaps, to the R locus of Hansenand his co-workers(31). Theexistenceof multipleloci in the KandDregions has beeninterpreted occasionallyas contradictingthe two-locusmodelfor the class 1 1-1-2loci. In fact, however,there is no contradictionbecausethe original hypothesis (55) maintainedonly that there are two clusters of loci and that all the then-knownH-2 recombinantsoccurred betweenthese two clusters. This is precisely whatthe methodsof both the classical and moleculargenetics demonstrate.Moreover,in functional tests the H-2complexbehavesas if it consistsof onlytwoclass I loci.

The Qa and Tla Loci The most radical changebrought onto the H-2 mapsby the DNAcloning methodshas occurred in the region occupied by the Tla and Qa loci. Althoughevenclassical methodsliave providedseveral hints of morethan the three or four identified loci, fewof us haveexpectedthe showerof loci triggered by molecularbiologists. Evenif 37 loci in the H-2a haplotype were the maximum numberand other haplotypes had considerably fewer loci, importantquestions concerningthe reasonsfor this multitudeof loci wouldstill arise. In a way,decipheringthe organization, modeof expression, and function of the Qa-Tlaloci is the last major task for immunogeneticists. Becauseof the uncertainties surroundingthem, we now considerthese loci in detail.

THE RIDDLES OF THE QA-TLA LOCI Evidence That Qaand Tla are Class 1 Loci In this review,wehaveclassified the QaandTla as class I loci andwehave doneso for the followingreasons. First, an antiserumproducedin a strain combinationdiffering at the Qa-Tlaloci but identical at the K and Dloci (A.TLanti-A.TH) cross-reacts with at least two K molecules f and

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KP)(13, 14). Wefirst noticedthis cross-reactivitybecausethe antiserum killed 100% of Kf- or KP-beadng cells in the cytotoxictest underconditions in whichonly some30-40% of cells carrying other H-2haplotypeswere killed. Theevidencethat the antiserumwasK-reactivewasprovidedby testing of H-2recombinants andan H-2mutant.Second,one can use strain combinations differing at the Qa-Tlaloci to generateCML, which,unlike that stimulatedby non-MHC antigens,is not restricted by MHC loci (Table 2). Severalpieces of evidenceare nowavailableindicatingthat the target antigensof this CML are controlledby the Qaloci (see below).Third,there is a striking structural homology betweenthe Qa-Tlaandthe K-Dmolecules. This homologyis attested to by the association of Qa and Tla polypeptidechainswiththe B2microglobulin (98), by similar size of the Qa-TlaandK-Dpolypeptidechains (98), by the peptide composition determined by biochemicalfingerprinting(90), by the similarity in gene organization(93), andby nudeotidesequencesimilarity (65, 93). Table 2 Strain combinations in which CML,most likely directed to Qa antigens, has been obtained Possible target aQa-1

Strain combination

References

C3H (AKR or CBA) antioB10.BR B10.HTT anti-B10.S (7R) lor B10.S (9R)] aC57BL/6 anti-B6-T/a

17, 38-40 100

A-Tla b anti-A (BALB/c × C57BL/6) l anti-B10.A (5R) bQa-1

dQa-1 aQa-2

A.TH anti-A.TL B10.BR anti-C3H (AKR, CBA) B10.S (9R) anti-B10.HTT A banti-A-Tla B6-T/a a anti-A.BY/B6 SWRanti-DBA/1 B6-T/a a anti-C57BL/6 B6-T/a a anti-BALB.B B6-Tla a anti-C3H.SW B6-T/aa anti-129 NZBanti-BALB/c C57BL/6 danti-B6-Tla BALB/c By anti-BALB/c J

I. Vu~ak, Z. A. Nagy, J. Klein, unpublisheddata I. Vu~ak, Z. A. Nagy, J. Klein, unpublished data 18 49 15, 40 101 23 23 23 19 19 19 19 16, 17, 82, 83 19 24

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Peculiarities of Qa-Tla Loci Similarities notwithstanding,the Qa-Tlaandthe K-Dloci differ in several respects. Herewepoint out three of these differences, pertaining to the expressionin different tissues, polymorphism, and function. TheTla antigens are expressedonly on thymocytesat a certain stage of differentiation and on certain leukemias(21). TheQaantigens wereoriginally thought to be present only on a subpopulationof T lymphocytes,but this observationno longer holds. Whenoneuses particularly effective complement, one can demonstrate100%killing of lymphnode or spleen cells with anti-Qa sera, an observation suggesting that the Qa antigens are presentnot only on virtually all T cells but also on B cells. Theirpresence on B cells has also beendemonstratedwith the fluorescence-activatedcell sorter (75). However,most of the B cells and someof the T calls must expressonly very little of these antigens becausethey are not killed when one uses weakcomplementthat, however, is strong enoughto kill all lymphoeytesexpressing the correspondingK and D antigens. Thepolymorphism of the QaandTla loci is significantly lowerthan that of the K andDloci. Thebest evidencein supportof this thesis comesfrom the typing of the BI0.Wlines carrying H-2haplotypesof wild mice on the C57BL/10(=B10) background. Although we have found 17 different alleles and 20 different D alldes in these lines, wecould find only three Qa-Ltwo Qa-2, and six Tla alleles (S. Tewarson,F. Figueroa, J. Klein, unpublisheddata). Similar paucity of alldes at the Qaand T/a loci exists also amongthe standard inbred strains and congeniclines (21). Asfor the function of the Qa-Tlaloci, no evidencehas yet beenprovided for their beingable to providecontext for antigen recognition.At the same time, however,it must be emphasizedthat the search for such a function has been hamperedby the lack of suitable recombinantsseparating the Qa-Tlafrom the K-Dloci and the individual Qaloci fromone another. In fact, wecould find only one report in the literature in whichthe function of Qamoleculesas restriction elementshas beentested (39). Organization of the Qa-Tla Loci Five Qaloci (Qa-1 through Qa-5)and one Tla locus have been described with classical immunogenetic methods(20, 22, 30, 91). Of the Qaloci only two (Qa-1and Qa-2)havedefinitdy beenestablished as separate loci (they are separated by recombination and they control distinct polypeptide chains) (21). Theremainingthree loci (Qa-3, Qa-4, and Qa-5)have been postulatedto exist becausethe antigensthey control havea different straindistribution pattern than those controlled by the Qa-1andQa-2loci (22, 30). However, in the past, strain distribution pattern has provedan unrelia-

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ble criterion for separatingloci, andso it seemsbest to i~ostponethe acceptance of Qa-3,Qa-4, and Qa-5as separate loci until they can be matched against the genes identified by recombinantDNA methods. Althoughthe Qa-1andTla moleculeshavea different distribution, this fact aloneis again insufl]cient for consideringthemas controlledby different loci (see below). As already mentioned,strains differing at Qaloci give CML that is not restricted by the MHC loci (24, 49). The Qa-associatedCML has nowbeen obtained in a numberof combinationsin the mouse(Table 2) and in the rat (66). The CML loci were originally thought to b~-distinct from the knownQaloci, in part becauseof an erroneousmappingof the Qa-1locus, but also becauseof certain discrepanciesin the strain distribution of the respective antigens. Nowthat the position of the Qa-1locus has been corrected and. someof the discrepancies havebeen resolved, there is no longer any need to postulate separate CML loci. Furthermore,the identity of the Qaand CML loci is supported by the observation that the CML can be blockedby Qa-specificantibodies (39, 40). Thefew discrepancies that remainno doubtare the consequence of this region’s genetic complexityand of our inability to separate the individual loci by recombination. If weaccept Qa-1and Tla as separate loci, wearrive at an order: D(L) ... Qa-2 ... Tla ... Qa-1. This order is different from that proposed originally and the changehas beenbrought about by a reclassification of certain recombinantsin terms of the Qa-1 locus (92). The chromosome interval containing the Qaand Tla loci also contains loci codingthe C4binding protein (C4-bp)and for the enzymescatalase-2 (Ce-2), phosphoglycerate kinase-2 (Pgk-2) and urinary pepsinogen-1(Upg-1) (51). The order of these loci with respect to oneanother and with respect to the Qa loci is uncertain;the ordergivenin Figure4 is basedonthe typingof B 10.W and someother H-2 congeniclines (41a). Bf C4Sip Neu*lC4-bpQa-2Tta Oa-ICe-2 Pgk-2

Centromere

I

0.3

I

I-2

I

Te|omere

I

Figure 4 Genetic map of the chromosomal segment occupied by the MHC.Thick vertical lines, MHC loci; thin lines, linked loci (only someMHC loci are shown); numbers indicate length of the particular interval in centimorgans;brackets indicate the order of the bracketted loci is uncertain; Bf, complementfactor B; C4, complementcomponent4; C4-bp, C4-binding protein; Ce-2, kidney catalase-2; Glo-I, glyoxalase-l; Neu-l, neuraminidase-1; Pgk-2, phosphoglycerate kinase-2; Sip, sex-limited protein; Thy-2, thymusCell antigen-2;, Upg-l, urinary pepsinogen-1.

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Interpretation Whatcould be the functional significanceof this large cluster of MHC loci? At present, there are twoprincipal waysto viewthe Qa-Tlaloci. Thefirst possibility is that these loci carry out an as yet unidentifiedfunctionthat differs fromthe Kand D loci. Thefunction frequently consideredin this contextis a guidingrole in tissue differentiation. Theonly reasonthis role is consideredis becauseof the differential tissue distribution of the Qa-Tla molecules. However,wefind this proposal unattractive for two reasons. First, becauseit attaches--unjustifiably, in our opinion--exaggerated importanceto a trivial characteristic (tissue distribution), andsecond,because wehavea hard time imaginingthat genesas closely related as are the K-D and Qa-Tlaclusters could performso radically different functions. Thesecondpossibility is that the Qa-Tlageneseither performthe same function as the K-Dgenes but on a morelimited scale, or they represent mostlynonfunctionalclass I genes. Thereasonsthey mightbe only partially functional, or nonfunctionalaltogether, could be, first, becausemanyof themmightbe pseudogeneslacking someimportantpart of the polypeptide chain necessaryfor carrying out the function (93), andsecond, because their reducedexpression. Withmostof the genes, wedo not knowwhether theyare expressedat all; but evenif all of themwereexpressed,it is unlikely that the level of expressionwouldreach that of the K-Dgenes(otherwise, one wouldhavedetected their productslong ago). The conspicuousvariation in one’sability to detect the Qaantigens(21) andthe apparentinfluence of the cell cycle onTla geneexpression(85), suggestthat frommostof these genes only minimalquantities of mRNA molecules are copied and translated into membrane proteins. It appearsthat only cells at a certain favorable stage of differentiation, in certain favorablemetabolicconditions,and certain membranous milieu manageto insert enoughQa or Tla molecules into their membranes to reachthe sensitivity level of our detectionmethods. Mostof the time, the quantity of the expressed moleculesmayalso be insufficient for their servingas restriction elementsin antigenrecognition, but under favorable circumstances someof themmay"leak through" and becomefunctional. This view, of course, leads to anotherimportantquestion. If the Qa-Tla loci are largely nonfunctional, whydo they persist in the genome?To answerthis question,it will be necessaryto find out first howold these genes are. The low polymorphism of the Qa-Tlagenes, combinedwith the suggestion that different H-2 haplotypes mayhave different numbersof Qa-Tla loci, suggeststhat the genesare of a relative recent orgin andthat mostof themarose after the mousesplit fromits nearest ancestors. Thegeneration

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of the cluster thus mayrepresent an evolutionary accident and there may be so manygenes because there has not been enoughtime to dispose of them. Alternatively, one can imaginethat a systemso dependenton polymorphismas the MHC apparently is requires a great deal of experimentation with the availablegeneticmaterialto assure this system’soptimaladaptability. In this respect, one could viewthe Qa-Tlaloci as a "laboratory" in whichgenesare experimentedwith before they are put into production.The low and variable expressionof the Qa-Tlagenes wouldbe compatiblewith this view: Theexpression wouldbe low enoughnot to clutter the plasma membranewith unused proteins, yet at the same time high enough to providean opportunityfor selective mechanisms to find suitable candidates for copingwith newsituations as they arise. Theproblemwith this viewis that it does not explain whythere is such a "laboratory"only for class I andnot for class II genes. Thereis no compellingevidencethat the polymorphism of class II loci is lowerthan that of the class I loci (50), and one wouldexpect that the two classes wouldbe treated the same way. Severalpossibilities maysolve this problem:One"laboratory" could serve both the class I andclass II loci; a class II "laboratory"maybe discovered later in other H-2 haplotypes; or part of the variation of the class II moleculescould be provided by the a chains. ENTRAPPED

LOCI

The mouseneuraminidase-1(Neu-1) locus codes for an enzymethat modifies several other enzymes post-translationallyby removing extra sialie acid residues fromtheir carbohydrateunits. Thusfar, four enzymeshave been identified that are influencedby the Neu-1locus, all presentin liver lysosorues: liver acid phosphatase(60), a-mannosidase(11), acid a-glucosidase (80), andliver arylsulfataseB (8). Certain mutationsin the Neu-1genesresult in the productionof defective neuraminidase that does not manageto removeall the extra sialie acid, and as a consequencethe unmodifiedenzymeshavea different mobility in the electric field in comparisonto enzymesmodifiednormally. It wasthis variation in electrophoretic mobility in the liver acid phosphataseand a-r~annosidasethat wasdiscoveredfirst and wasascribed to the effect of separate loci--Apl (60) andMap-2(11). However,later it wasrealized that mostlikely there are no separate ,4pl andMap-2loci but rather the effects seen werea result of the action of the pleiotropic Neu-1locus (80, 105). Originally, the Apl locus wasmapped at a distance of several eentimorgans from the H-2complexon the telomeric side (104). However,this placement

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turned out to be wrongand newtests have revealed the Neu-I locus to be very closely linked to the H-2complex(103). Ourdiscoveryof a newform of Neu-1variation (41) has allowedus to use several H-2recombinantsfor mappingthe locus, and this testing placedthe Neu-1locus within the H-2 complex,betweenthe Eo and D loci (13). TheNeu-1locus thus resides in the samechromosomal interval as the C4-codinggenes. Althoughit is not certain that Neu-1is a structural rather than a regulatorylocus, there is no evidence it is in any wayrelated to the MHC loci. Wewouldlike to propose, therefore, that Neu-1 and the C4 loci occupya chromosomal segmentthat has been entrapped accidentally betweenthe MHC genes. Passing of long chromosomal segmentsfromancestral forms to presentday species is not an uncommon phenomenon; in fact, it seemsto be a rule in the evolutionary process (48). Homologous linkage groups are being discoveredall over the genomeand in as distant formsas birds and mammals. The preservedchromatinblocks usually contain genes for whichno evolutionaryor functional relationship is apparent:Thepreservedlinkages seemto be the result of accidental conservationof geneassociationsrather than any ontological relationship amongthe genes in the group. The MHC does not seemto be an exceptionto this rule.

THE FINAL ACT Althoughseveral important questions still remain to be answeredwith regard to the genetic organization of the MHC,the astonishingly rapid progressof the moleculargenetics of this systemleaves no doubtthat the answerswill be providedwithin the next few years. It is as if molecular genetics has raised the curtain on the final act of the MHC play, and as in every goodplay this act is turning out to be short and packedwith action. It truly wasa goodplay: dead-earnestmostof the time, hilarious on occasions, andthrilling all the time. Seeingit nearingits end, onecannothelp but feel a sting of nostalgia. For to someof us the play has becomepart of our lives andafter the last curtain call wewill realize, undoubtedly,that somethingmorethan a goodplay is over. ACKNOWLEDGMENTS

Wethank Martha Kimmede and Karina Masurfor help in preparation of this manuscript.Part of the supportfor the experimentalworkcited in this communication has comefrom National Institutes of Health and Deutsche Forsehtmgsgemeinsehaft grants.

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Literature Cited 1. Alper, C. 1981. Complement and the MHC.In The Role of the Major//istocompatibility Complexin Immunobiology, ed. M. Dorf, pp. 173-220. New York: Garland STPM 2. Basch,R. S., Stetson,C. A. 1963.Quantitative studies on histocompatibility antigens of the mouse.Transplantation 1:469-80 3. Baxevanis,C. N., Nagy,Z. A., Klein, I. 1981.A noveltype of T-Tcell interaction removesthe requirementfor I-B region in the H-2 complex.Proc. Natl. Acad. ScL USA 78:3809-13 4. Berzofsky,J. A., Pisetsky, D. S., Killion, D. J., Hicks, G., Sachs, D. H. 1981.Ir genesof different highresponder haplotypesfor staphylococcalnuclease are not allelic, d. Immunol. 127:2453-55 5. Bodmer, W. F. 1973. A new genetic modelfor allelism at histocompatibility and other complexloci: polymorphism for control of geneexpression. Transplant. Proc. 5:1471-75 6. Bodmer, W. F. 1976. HLA:A super supergane.//arveyLectures8er. 72:91138 7. Curman,B., Ostberg,L., Sandberg,L., Malmheden-Eriksson, I., Stalenheim, G., Rask,L., Peterson, P. A. 1975.H-2 linked Ss protein in C4 componentof complement.Nature 258:243-45 8. Darn’el, W. L., Womack,L E., Henthorn, P. S. 1981.Murineliver arylsulfatase B processinginfluencedby region on chromosome17. Biochern. Genet. 19:211-55 9. David, C. S., Shreflter, D. C. 1974. Lymphocyte antigens controlled by the Ir region of the mouseH-2 complex. Transplantation17:462-69 10. Delovitch,T., Barber,B. H. 1979.Evidence for two homologous,but nonidentical Ia moleculesdeterminedby the I-EC subregion. £ Exp. Med. 150:100 11. Dizik, M., Elliot, R. W.1978.Asecond geneaffectingthe sialylation of lysosomalcr-mannosidase in mouseliver. Biochem. aenet. 16:247-60 12. Fachet, J., And6,I. 1977. Geneticcontrol of contactsensitivity to oxazolone in inbred, H-2congenicand intra-H-2 recombinant strains of mice.Eur.d. Immunol. 7:223-26 13. Figueroa,F., Klein, D., Tewarson,S., Klein, J. 1982.Evidencefor placingthe Neu-1 locus within the mouse //-2 complex..L Immunol.129:2089-93

14. Figueroa, F., Zaleska-Rutczynska,Z., Kusnierczyk,P., Klein, L 1983. Crossreactivity between Qa-1 region and H-2Kantigens. Transplantation. In press 15. Fischer-Lindahl,K. 1979. Unrestricted killer cells recognizean antigen controlled by a genelinked toT/a.Immunogenetics8:71-76 16. Fischer-Lindahl, K., Bocchieri, M., Riblet, R. 1980. Maternallytransmitted target antigenfor unrestrictedkilling by NZBT lymphocytes..L Exp. Med. 152:1583-95 17. Fischcr-Lindahl, K., Hausmann,B. 1980. Qed-I--Atarget for unrestricted killing by T cells. Eur. J. Immunol. 10:289-98 18. Fischer-Lindahl, K., Hausmann,B., Flaherty, L. 1982. Polymorphism of a Qa-l-associatedantigen defined by cytotoxic T cells. I. Qed-1a d. and Qcd-1 Eur. Y.. Immunol.12:159-66 Fiseher-Lindahl, K., Langhorne, J. 19. 1981. Medialhistocompatibil/ty antigens. Scand. Y. Immunol.14:643-54 20. Flahcrty, L. 1976.TheTla region of the mouse:Identification of a newserologically defined locus, Qa-2. Immunogentics 3:533-39 21. Flaherty, L. 1981.Tla-regionantigens. In TheRole of the MajorHistocompatibility Complexin Immunobiology,ed. M. Dorf, pp. 33-57. NewYork: Garland STPM 22. Flaherty, L., Zimmermatm, D., Hansen,T. H. 1978.Further serological analysisof the Qaantigens: Analysisof an anti-H-2.28 serum, lmmunogenetics 6:245-51 23. Forman,J. 1979.//-2 unrestricted eytotoxic T cell activity against antigens controlled by genes in the Qa/Tlaregion. ,L Immunol.123:2451-55 24. Forman,J., Flaherty, L. 1978.Identification of a new CML target antigen controlledby a geneassociatedwith the Qa-2 locus. Immunogenetics6:227-33 25. Gachelin, G., Dumas,B., Abastado, J.-P., Carol, B., Papamathealds,J., Kourilsky, P. 1982. Mousegenes coding for the majorclass I transplantation antigens: a mosaicstructure might be related to the antigenic polymorphism. Ann. ImmunoL133:3-20 26. Gorer, P. A. 1936.Thedetection of antigenic differences in mouse erythrocytes by the employmentof immunesera. Br. ,L Exp. Pathol. 17:42-50 27. Gorer,P. A. 1937.Thegeneticandanti-

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MHC GENETICS genie basis of tumortransplantation.Z PathoLBacteriol. 44:691-97 28. GStzc, D.ed. 1977. The Major 11istocompatibility Systemin ManandAnimals. Berlin: Springer 29. Hiimmerling, G. J. 1976.Tissuedistribution of Ia antigensandtheir expression on lymphocytesubpopulations. Transplant. Rev. 30:64-82 30. Hiimmerling,G. J., Hiimmerling,U., Flaherty, L. 1979. Qa-4and Qa-5, new routine T-cell antigensgovernedby the Tla rcgion and identified by mono¢lonal antibodies. Z Exp. Med. 150: 108-16 31. Hausen,J. H., Ozato, K., Melino, M. R., Coligan,L E., Klndt, T. L, Jandinski, L J., Sachs, D. H. 1981. Immunochemicalevidencein two haplotypesfor at least three D region-encodedmolecules, D, L, and R. J. Immunol. 126:1713-16 32. Hiramatsu,K., Ochi,A., Miyatani,S., Segawa,A., Tada, T. 1982. Monoclonal antibodies against unique/-regiongene products expressed only on mature functional T cells. Nature296:666-68 33. Hurme,M., Chandler, P. R., Heterington, (2. M., Simpson, E. 1978.Cytotoxic T-cell responses to H-Y:Correlation with the rejection of syngeneic male skin grafts. Z Exp. Med.147:768-75 34. Ivfinyi, D., D6mant,P. 1981.Serological characterization of previouslyunknownH-2moleculesidentified in the productsof the Ka and I~’ region, lmmunogenetics12:397--408 35. Jones, P. P., Murphy,D. B., Hewgill, D., McDevitt,H. O. 1979. Detectionof a common polypeptide chain in I-A and 1-E immunoprecipitates. Mol.subregion Immuno£16:51-60

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from H-2Kand H-2Dantigens. J. Immunol. 123:1239-44 40. Kastner, D. L., Rich, R. R., Shen, F.-W.1979. Qa-l-associatedantigens. I. Generation of H-2-nottrestdcted cytotoxic T lymphocytesspecific for determinantsof the Qa-1region, d. lmmunol. 123:1232-38 41. Klein, D., Klein, J. 1982. Polymorphismof the Apl (Neu-1) locus in the mouse. Immunogenetics16:181-84 41a. Klein, D., Tewarson,S., Figueroa,F., Klein, J. 1982. The minimallength of the differential segment in 11-2congenic lines. Immunogenetics 16:319-28 42. Klein, J. 1975. Biology of the Mouse Histocompatibility-2 Complex. New York: Springer 43. Klein, J. 1976,Anattemptat an interpretation of the mouse11-2 complex. Contemp.Topics Immunobiol.5:297336 44. Klein, J. 1977. Evolutionand function of the majorhistocompatibilitysystem: facts and speenlations. In The Major I-Iistocompatibility Systemin Manand Animals, ed. D. GStze, pp. 339-78. Berlin: Springer 45. Klein, J. 1978. H-2mutations: Their genetics and effect on immunefunctions. Adv. ImmunoL26:55-146 46. Klein, J. 1979.Themajorhistocompatibllity complexof the mouse.Science 203:516-21 47. Klein, J. 1981.Thehistoeompatibility-2 (H-2) complex. In The Mouse in BiomedicalResearch,ed. H. L. Foster, J. D. Small, J. G. Fox, 1:119-58. New York: Academic 48. Klein, J. 1982. Evolutionand function of the major histocompatibility complex. In 1-1istocompatibilityAntigens: Structure and Function Receptors and 36. Jureti6, A., Nagy,Z. A., Klein,J. 1981. Recognition,Series B, ed. P. Parham,J. Detection of CML determinants assoS. Strominger, 14:221-39 London: ciated with H-2controlled Ea and E. Chapmanand Hall chains. Nature 298:308-10 49. Klein, J., Chiang, C.-L. 1978. A new 37. Kanno,M., Kobayashi,S., Tokuhisa, locus (//-2T) at the D end of the 1t-2 T., Takei,I., Shinohara,N., Taniguehi, complex. Immunogenetics6:235-43 M. 1981. Monoclonalantibodies that 50. Klein, J., Figueroa,F. 1981.Polymorrecognizethe product controlled by a hism of the mouseH-2 loci. ImmunoL genein the 14 subregionof the mouse ev. 60:23-57 H-2 complex,d. Exp. Med. 154:129051. Klein, J., Figueroa,F., Klein,D. 1982. 3O4 11-2 Haplotypes, genes and antigens: 38. Kastner,D. L., Rich, R. R. 1979.11-2second listing. I. Non-H-2genes on nonrestrictedeytotoxie responsesto an chromosome17. lmmunogenetics 16: antigen encoded telomedcto II-2D. d. 285-317 Immunol. 122:196-201 52. Klein, J., Hanptfeld,V., Hauptfeld,M. 39. Kastner, D. L., Rich, R. R., Chu, L. 1974.Involvement of H-2regionsin im1979.Qa-l-associatedantigens.II. Evimunereaetious. In Progress in lmdence for functional differentiation munologylLed. L. Brent, J. Holborow,

~

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3:197-206. Amsterdam Elsevier: genes in the T/a region of mouse North-Holland chromosome17. Nature 295:168-79 53. Klein,L, Jureti6, A., Baxevanis,C. N., 66. Marshal A., Doherty, P. C., Wilson, D. B. 1977.Thecontrol of specificity of Nagy,Z. A. 1981. Thetraditional and eytotoxic T lymphoeytesby the major the newversion of the mouseH-2comhistocompatibility complex(Ag-B) plex. Nature291:455--60 rats andidentificationof a newalloanti54. Klein, J., Nagy,Z. A. 1982. MHC regen system showingno Ag-Brestricstriction andIr genes.Adv. CancerRe~ tion. J. Exp. Med.146:1773-90 37:233-317 55. Klein, J., Shrefller, D.C. 1971.TheH-2 67. MeDevitt,H. O., Chinitz, A. 1969.Genetic control of the antibodyresponse: modelfor majorhistocompatibilitysysRelationship betweenimmuneresponse tems. TransplanLRev. 6:3-29 and histocompatibility (H-2) type. 56. Koch,N., Hiimmerling,G. J. 1981. Ia Science 163:120%8 antigens contain two distinct formsof 68. Melchers,I., Rajewsky,K., Shreflier, //chain. Immunogenetics14:437-44 D. C. 1973. Ir-LDHe:Mapposition and 57. Koch,N., I-Emmerling, G. J., Szymura, functional analysis. Eur. ,£. Immunol. J., Wahl,M. R. 1982. Ia associated I~ 3:754-61 chain is not encodedby chromosome 17 69. Moo,T., Krasteff, T., Shreflter, C. D. mouse. Immunogeneticz of the 1975. Immunochcmical characteriza16:603-6 tion of murineH-2controlled Ss (serum 57a. Koch,N., Koch,S., I-gfimmerling,G. substance)protein throughidentifica1982. Ia invariant chain detected on tion of its humanhomologueas the lymphocytesurfaces by monoclonalanfourth componentof complement.Proe. tibody. Nature299:645 Natl. dead. Set USA72:4536-40 58. Laclunan,P. J., Grennan,D., Martin, 70. Monaco,J. J., McDevitt,H. O. 1982. A., D6mant,P. 1975. Identification of Identification of a fourth class of proSs protein as murine CA.Nature 258: teins linked to the routine majorhis242-43 tocompetibility complex. Proc. NatZ 59. Lafuse,W.P., Corser,P. S., David,C. .4cad. Sc£ USA79:3001-5 S. 1982. Biochemicalevidencefor mul- 71. Murphy,D. B. 1978. I-J subregion of tiple I-E Ia molecules.Immunogenetics the murineH-2gene complex.Springer 15:365-75 Ser, z Immunopathol1:111-31 60. Lalley, P. A., Shows, T. B. 1977. 72. Murphy, D. B., Herzenberg, L. A., Lysosomal acid phosphatasedeficiency: Okumura, K., Herzenberg, L. A., liver specific variant in the mouse.GeMcDevitt,H. O. 1976. A newI subrenetica 87:305-17 gion (I-J) marked bya locus (la-4) con61. Laxhammar,D., Wiman,K., Schentrolling surface determinants onsupning, L., Claesson,L., Gnstafsson,K., pressor T lymphocytes. /,Exp.Med. Peterson, P. A., Rask, L. 1981. Evolu144:699-712 tionary relationship betweenHLA-DR 73.Nathenson, S. G.,Uehara, H.,Ewenantigen//-chains, HLA-A, B, C antigen ¯ stein, B.M.,Kindt, T.J.,Coligan, J.E. subunits and immunoglobulin chains. 1981. Primary structural analysis ofthe Scand. J.Immunol. 14:617-22 transplantation antigens of the murine 62.Lieberman, R.,Paul, W.E.,Humphrey, H-2majorhistocompatibility complex. W.St.,Stimpfling, $.H.1972. H-2Ann. Rev. Biocherr~50:1025-51 linked immune response (It) genes. In- 74. Okuda,K., David,-C. S. 1978. A new dependent lociforIr-lgG andIr-lgA lymphocyte-activatingdeterminantlogenes. Y.Exp.Meal. 136:1231-40 ons expressed on T cells, and mapping 63.Livnat, S.,Klein, J.,Bach, F.H.1973. in I-C subregious. Y. Exp. Med. I47: Graft versus host reaction instrains of 1028-36 mice identical forH-2K andH-2D anti- 75. Okuda,M., Stanton, T. H., Kuppers,R. gem.Nature 243:42-44 C., Henney,C. S. 1981.Thedifferentia64.Lozncr, E.C.,Sachs, D.H.,Shearer, G. tion of cytotoxic T cells in vitro. IV. M.1974. Genetic control oftbe immune Interleukin-2 production in primary response tostaphylococcal nuclease. I. mixedlymphocytecultures involves coIr-Nase: Control oftheantibody reoperation between Qa-1+ and Qa-lsponse to nucleaseby the Ir region of "helper" T cells. J. Immunol.126: the mouseH-2 complex. J. Exp. Med. 1635-39 139:1204-14 76. Orr, H. T., Lancet, D., Robb,R. J., 65. Margulies,D. H., Evans, G. A., FlahLopezde Castro, J. A., Strominger,J. erty, L., Seidman,J. G. 1982.H-2-fike L. 1979. Theheavychain of humanhis-

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MHC GENETICS tooompatibility antigen HLA-B7 contalm an immunoglobulin-likeregion. Nature 282:266-70 77. Ozato,K., Sachs,D. H. 1982.Detection of at least twodistinct mouseI-E antigen molecules by the use of a monoclonal antibody. £ ImmunoL128: 807-10 78. Parmiani,G., Carbone,G., Invernizzi, G., Pierotti, M. A., Semi,M.L., Rogers, M.J., Apella, E. 1979.Alienhistocompatibilityantigensontumorcells. Immunogenetics9:1-24 79. Pelkonen,J. L. T., Kaartinen,M., Karjalainen, K., NFAkele,O. 1979.A hapten-specific Ir gene. d. Immunol.123: 1558-64 80. Peters, J., Swallow,D. M., Andrews, S. J., Evans,L. 1981. A gene (Neu-1)on chromosome17 of the mouse affects acid o-glucosidase and codes for neuraminidase.Gene~Res, 38:47-55 81. Porter, R. R., Reid, K. B. M.1979.Activation of the complementsystemby antibody-antigencomplexes:the classical pathway.,4dv. ProLChem.33:1-71 82. Rich, R. R., Sedberry,D. A., Kastner, D. L., Chu, L. 1979. Primaryin vitro cytotoxic responseof NZBspleen cells to Qa-P-associatedantigenic determinants, d, Exp. Me¢L150:1555-60 83. Rich, S. S., David,C. S., Rich, R. R. 1979. Regulatorymechanisms in cellmediatedimmune responses. VII. Presence of I-C subregiondeterminantson mixedleukocytes reaction suppressor factor. ,L Exp. Med.149:114-26 84. Richardson,J. S., Richardson,D. C., Thomas,K. A., Silverton, E. W., Davies, D. R. 1976. Similarity of threedimensionalstructure betweenthe immunoglobulindomainand the copper, zinc superoxidedismutasesubunit. ,L MoLBioL 102:221-35 85. Rothenberg,E. 1982.A specific biosynthetic marker for immature thymic lymphoblasts. Active synthesis of thymus-leukemia antigen restricted to proliferating ceils, d. Exp. Meg155: 140-54 86. Sandrin, M. S., McKenzie,I. F. C. 1981. Productionof a cytotoxic antiIa.6 antibody. Immunogeneties14: 345-50 87. Shreflter, D. C., Owen,R. D. 1963. A serologically detectedvariant in mouse serum:inheritanceandassociation with the histocompatibility-2locus. Generics 48:9-25 88. Snell, G. D. 1968.TheH-2locus of the mouse:Observationsand speculations concerningits comparativegenetics and

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its polymorphism. Folia BioL 14: 335-58 89. Snell, G.D., Dausset,J., Nathenson, S. 1976. Histocompatibility. NewYork: Academic 90. Solusld,M.J., Uhr,J. W.,Fiaherty,L., Vitetta, E. S. 1981. Qa-2, H-2K,and H-2Dalloantigens evolvedfrom a commonancestral gene..L Exp. Mea~ 153:1080-93 91. Stanton, T. H., Boyse, E. A. 1976. A newserologically definedlocus, Qa-I, in the Tla-region of the mouse.Immunogenetics3:525-31 92. Stanton, T. H., Carbon,S., Maynard, M. 1981. Recognitionof alternate alleles and mapping of the Qa-1locus. ,L ImmunoL127:1640-43 93. Steinmetz, M., Moore,K. W., Frelinger, J. G., Sher, B. T., Shen, F.-W., Boyse,E. A., Hood,L. 1981.A pseudogene homologous to mousetransplantation antigens: transplantation antigens are encodedby eight exonsthat correlate with protein domains. Cell 25: 683-92 94. Steinmetz,M., Winoto,A., Minard,K., Hood,L. 1982.Clusters of genesencoding mousetransplantation antigens. Cell 28:489-98 95. Taniguchi, M., Tokuhisa, T., Kanno, M., Yaoita, Y., Shimizu,A., Honjo,T. 1982.Reconstitutionof antigen-specific suppressor activity with translation products of mRNA.Nature 298: 172-74 96. Uhr,J. W.,Capra,J. D., Vitetta, E. S., Cook,R. G. 1979. Organizationof the immuneresponse genes. Both subunits of routine I-A and I-E/C moleculesare encodedwithin the I region. Science 206:292-97 97. Urba, W.J., Hildemann,W.H. 1978. H-2-Fmked recessive Ir ~eneregulation of high antibodyresponstvenessto TNP haptenconjugatedto autologousalbumin. Immunogenetics6:433-45 98. Vitetta, E. S., Capra,J. D. 1978. The protein products of the murine 17th chromosome:genetics and structure. Adv. ImmunoL26:147-93 99. Waltenbaugh,C. 1981. Regulation of immune responseby l-J gene products. I. Productionand characterization of anti-I-J monoclunal antibodies, d. Exp. Med. 154:1570-83 100. Wernet, D., Klein, I. 1979.-Unrestricted cell-mediated lympholysis to antigens linked tothcTlalocus inthe mouse. Immunogenetics8:361-65 101. Wernet, D., Klein, J. 1981. Cellmediatedlympholysisin H-2K/Diden-

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tical congenicstrain combinations.In er-spccific lysosomalacid phosphatas¢ Controlof CellularDivisionand Develdeficiency (Apl) on mousechromosome opment,Part A, ed. D. Ctmningham, E. 17. MoLGen. Genet. 155:315-17 Goldwasser,J. Watson,C. F. Fox, pp. 105. Womack, J. E., Yan,D. L. S., Potier, 573-77. NewYork: Liss M. 1981.Genefor neuraminidas¢ activ102. Williams, A. F., Gagnon,J. 1982. ity on mousechromosome 17 near H-2: Neuronal cell Thy-1 glycoprotein: Pleiotropic effects on multiple hydrolases. Science 212:63-65 homologywith immunoglobulin.Science 216:696-703 106. Zinkernagel, R. P., Doherty, P. C. 103. Womack,J. E., David, C. S. 1982. 1979.MHC restricted eytotoxieT-cells: Mousegenefor neuraminidaseactivity Studies on the biological role of polymorphicmajortransplantation antlgeus (Neu-1) mapsto the D end of H-2. Immunogenetics16:177-80 determiningT-cell restriction specificity..4dv. Immunol.27:51-177 104. Womack, J. E., Eicher, E. M.1977.Liv-

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Ant~ Rev. Immunol.1983. 1:143-73 Copyright©1983by AnnualReviewslnc. All rights reserved

IMMUNOBIOLOGY OF TISSUE TRANSPLANTATION: A RETURN TO THE PASSENGER LEUKOCYTE CONCEPT Kevin J. Lafferty, Simeonovic

Stephen J. Prowse, and Charmaine J.

Transplantation BiologyUnit, John Curtin Schoolof MedicalResearch, POBox 334, Canberra City, ACT2601, Australia Hilary

S. Warren

CancerResearch Unit, WodenValley Hospital, Canberra, Australia INTRODUCTION OnceMedawar(76) had established the immunologicalbasis of the allograft reaction, tissue antigen was seen to be the barrier to the grafting of tissues and organs. At the root of this notion was the idea that antigen recognition caused the activation of the potentially responsive lymphocyte. Antigen, like an embryonicinducer, was thought to direct the final differentiation of specific immunocyteclones (77). There is no doubt that antigen recognition is involved in the processes of tissue rejection. However,although antigen recognition is responsible for graft rejection, it maynot be correct to deduce that transplantation antigen itself constitutes the majorbarrier to grafting. This is because antigen recognition alone maynot be sut~cient for lymphocyte activation. The idea that tissue antigen was the obstacle to grafting has had a profoundeffect on clinical transplantation. If antigen is the barrier, then we must either matchthe tissues to the recipient or treat the recipient in a way that renders it no longer responsive to the antigenic challenge of the graft. Tissue matching plus recipient immunosuppression have been used with 143 0732-0582/83/0410-0143502.00

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considerablesuccess,moreso the latter, in the clinical development of organ andtissue transplantation(74). In this article, wefirst reviewrecent developmentsconcerningthe mechanism of tissue rejection. Weexplore in some depththe idea that antigenrecognitionaloneis not sufficient for lymphocyte activation. This leads to the conclusion that transplantation antigen, whetherit be class I or class II major histocompatibility complex(MHC) antigen, is not the primarybarrier to grafting, andwereturn to the concept initially proposedby Snell (90), that "passenger"leukocytescarried in the donor tissue provide the major immunogenicstimulus for the host. It therefore makesgoodtheoretical and practical sense to attempt to alter tissue immunogenicity by treating the tissue to be grafted rather than the recipient. Wereview the results of this approachto the transplantation problem. EFFECTOR

MECHANISMS

IN

GRAFT REJECTION

Antibodywasinitially thoughtto play little part in the process of graft rejection (13, 78, 79). It is nowclear that this is not the case. Thefailure of initial attempts to transfer antibodypassively and induce skin graft rejection can be attributed to deficiencies in the complement system of rodents (35); antibody-mediated hyperacuterejection of skin allografts and xenografts has nowbeendemonstratedin mice, but only whenan exogeneous source of complement is transferred along with the specific antibody (21, 75). Different tissues also vary in their susceptibility to antibodymediatedrejection. Allografts prepared by primaryvascular anastomosis are highly sensitive to antibodyand complement andretain this sensitivity for a prolongedperiod (44). In contrast, skin grafts showmarkedvariations with time in their susceptibility to attack by antibodyand complement (36, 50, 51). This effect related to the replacementof donor endotheliumin the skin by host cells (51). Jooste et al (50, 51) showed that soonafter grafting micewith rat skin, rat cell surfaceantigens could be demonstrated on the graft vasculature.As the graft becameless sensitive to anti-rat antibody-mediated damage,mouse cell surface antigens weredetected on the endothelium.Eventually,all the graft vasculature was of recipient origin, and the graft wasresistant to passivelytransferred antibody(51). Administrationof immune serumcausesrapid rejection of islet allografts in tolerant rats (83). However,whenendotheliumis destroyedby a period of organculture prior to transplantation (88), grafts are resistant to the action of antibodyand complement given either at the time of transplantation (M. Agostino, unpublishedobservations) or approximately100 days after grafting (1). Thesefindings suggest that antibody-mediated damage

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a vascular phenomenonand that tissue parenchymal cells may be less sensitive to this form of damage. Endothelial cells are readily lysed by alloantiserum or xenoantiserum and complement(26), and the target antigens for this antibody-mediated damageare likely to be any expressed on the surface of vascular endothelium. In man, both class I and class II MHC antigens as well as blood group antigens are expressed on endothelial cells and could provide a target for antibody-mediated damage(46). Mouseskin can be acutely rejected by antibody directed against class I alloantigens (27, 75), but not by antisera against class II antigens (75). This probably reflects the lack of Ia antigens expression on mouseendothelium (89). A mechanismfor antibody-mediated graft rejection has been described by Bogmanet al (15). Small amountsof antibody binding to the vessel wall fix complement,resulting in endothelial damageand the release of chemotactic factors. This results in the attraction and binding, through complement component receptors, of polymorphonuclear cells, which cause further endothelial damage. Following the exposure of subendothelial tissue, thrombosis occurs. In the presence of large amountsof antibody, the endotheliumis likely to be rapidly destroyed exposing thrombogenictissue and thus causing thrombosis without polymorphonuclearcell involvement. Whereanimals are deficient in complementcomponents, hyperacute rejection will not naturally occur.

Cellular Rejection Mechanisms The idea that allograft rejection was a cell-mediated phenomenoncame from early studies of Mitchison (79) and Billinghamet al (13). Accelerated rejection of an allogeneic tumorgraft could be transferred to a naive animal by cells from the draining lymph nodes of tumor-bearing animals (79). Billingham et al (14) showedthat immunityto a skin allograft could also be adoptively transferred in a similar manner. It was later shown that neonatally tolerant mice carrying skin allografts of the tolerated genotype would reject these grafts wheninjected with normal lymphoidcells, syngeneic with the recipient; if the lymphoid cells came from a sensitized animal, then a "second set" rejection occurred (14). There is nowgood evidence that graft rejection can be triggered by the interaction of sensitized T lymphocytesand graft antigens. Transfer of cell populations to sublethally irradiated rats carrying heart allografts demonstrated that long-lived, recirculating, Ig-negative cells were responsible for initiating rejection (39); the transfer of cells fromsensitized animalsshowed that memorywas carried by long-lived, non-recirculating, Ig-negative cells (40). Loveland and co-workers studied the rejection of skin grafts from adult thymectomized, irradiated, bone-marrow reconstituted (ATXBM)

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mice by transfer of cells from sensitized donors and showedthat the Lyt 1÷2- T cell could trigger rejection (70, 71). These authors went on conclude that Lyt 1+2+ T cells were not involved in the rejection process (70). Talmage’sgroup (112), working with cultured thyroid allografts, served a long delay in rejection following adoptive immunizationand a lack of correlation with cytotoxic activity in the transferred cells. They also suggested that cytotoxic T cells maynot be required for tissue rejection. Although Lyt 1+2- cells may mediate skin graft and possibly thyroid rejection, it wouldseem unwiseon the basis of the aboveevidence to exclude completely a role for Lyt 1+2÷ cells. Pancreatic islets can be transplanted to normalallogeneic miceif the islet tissue is first cultured in an oxygen-rich atmosphere(16-18). This tissue is functional and will reverse chemically induced diabetes (17, 18). In this system, the transfer of sensitized Lyt 1÷2+ T cells is required to initiate acute graft rejection (Figure 1). The subclass of T lymphocyteresponsible for graft rejection maydepend on the type of tissue transplanted, whichin turn reflects the particular class ~ 30

A

I~ ~ 20 ~uO 10

10

~30! ~

20 30 40

5O

6b

70

3 2

10

20

30

40 50 60 70 0 10 20 30 TIMEAFTER TRANSPLANTATION (DAYS)

40

50

60

~

Figure 1 Streptozotocin-induced diabetic CBAmice were grafted with cultured islet clusters. Blood glucose levels ([]) and body weight (1) were monitored. Mice became normoglycemic within 12 days. 5 X 107 CBAanti-PSI5 immunespleen ceils or remaining cells after treatment with antibody and complementwere injected i.v. (indicated by arrow). The immunecells were untreated (.4), treated with anti-Thy- 1.2 and complement(B), or treated with anti-Lyt 2.1 complement(C). The untreated cells had a total cytotoxic activity of 5.2 log~o cytotoxic units. The treated cells had a cytotoxic activity which was less than 3.9 log10 cytotoxic units. The results presented are for one mousein each group and are representative of the four mice in each treatment group. The normal range for blood glucose for CBAmale mice (mean +2 SD) is indicated by the darkly hatched area (see 87).

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TISSUE TRANSPLANTATION 147 of alloantigenexpressedon the graft. Culturedislet allografts expressonly class I antigens(89), whereasceils presentin skin grafts expressbothclass I andclass II antigens(41). ClassI andclass ii antigenscausethe activation of Lyt 1+2+ and Lyt 1+2- cells, respectivdy(24). Lyt 1+2+ could mediate graft rejection by a cytotoxic mechanism (24, 68). Also, both Lyt 1+2+ and Lyt 1+2 T cells, and their humanequivalents, havebeenshownto produce lymphokinewhenappropriately triggered (3, 72). Thus, cells of either subclass could mediategraft rejection via lymphokine release and an associated inflammatory response.Theprotracted rejection of thyroidallografts (112) by adoptively immunizedanimals wouldbe consistent with such mechanism. Theacute rejection of islet allografts, mediatedby Lyt 1+2+ T cells, wouldbe moreconsistent with a direct cytotoxiceffect of the immune calls. Weshould stress, however,that at this stage wedo not knowthe actual mechanismby which Lyt 1+2+ T cells mediate pancreatic islet rejection. Thedifferential sensitivity of skin, thyroid, andislet tissue to rejection by transferred immune T cells requires further investigation. AlthoughT cells mayinitiate graft rejection, other inflammatory cells enter the rejection site and greatly outnumberT cells as the process of rejection develops(5, 48, 106). Cytotoxicactivity in rejecting allografts has been shownto be mediatedby both T cells and other non-thymus-derived leukocytes(4, 5). Cells involvedin antibody-dependent cell-mediatedcytotoxicity havealso beenfoundin rejecting grafts. Thegeneral picture that emergesis oneof antigen-specific,T cell-mediatedtriggering of a rejection processthat is probablymediatedby multiplespecific andnonspecificeffector mechanisms. THE TRANSPLANTATION

BARRIER

WhenSnell in 1957(99) suggested that leukocytes associated with the transplanted tissue were a major source of tissue immunogenicity, he was formally stating the common observation that immunizationof a recipient animal with donor spleen or lymphnodecells wouldsensitize to a tumor allograft, whereasantigen extracts of the tumorwereonly weaklyimmunogenic and, undercertain conditions (52), wouldpromotethe growthof the tumorgraft. It wasthought, at the time, that antigen density or the form of antigen presentationon the surface of viable leukocytesrenderedthese transplantation antigens more immunogenic. Studies of the graft-versus-hostreaction (GVHR), carried out in the late 1960s,rekindledinterest in the role leukocytesin the tissues play in alloreactivity (29-31, 63). Elkins’investigationsin rats implieda majorrole for leukocytesin the inflammatoryreaction producedwhenparental strain lymphocyteswere introduced under the kidney capsule of F1 recipients

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(29-31). Steinmuller showedthat allogeneic leukocytes carried within skin graft were highly immunogenic.Skin isografts taken from strain A micerenderedneonatallytolerant of strain B, and transplantedto naive A mice, immunized the latter against strain B skin allografts (102). Sincethe tolerant donors were hemopoeticchimeras, the phenomenon wasexplained by the passivetransfer of allogeneicleukocytesvia the primaryskin isograft. In support of this proposal, the immunizing capacity of such skin isografts wasshownto be a function of the numberof allogeneic leukocytespresent and the degreeof leukocytechimedsm of the donors(103). Ourownstudies in the chicken embryolead us to concludethat the interaction of lymphocyteswith lymphoreticularcomponents in the graft wasrequired for the activation of the allograft response(63). Thus,the transfer of adult lymphoid cells to syngeneic embryosgrafted with allogeneic tissues would confer on the embryothe capacity to reject the allograft. Strongreactions wereseen with grafts of bone,with its associatedmarrow,spleen, andfetal liver. However, heart muscleallografts, cleared of bloodcells by perfusion prior to transplantation, werenot destroyed.In contrast, perfusion of the embryonic heart with leukocytesprior to grafting resulted in the activation of a violent allogeneicreaction that resulted in total allograft destruction. Theintensity of the allograft reaction wascorrelated with the lymphoreticular cell contentof the tissue transplanted. Theidea that somethingover and abovesimple antigen recognition was involvedin the triggering of the allograft responsecamefroma study of the species specificity of the GVHR in the chicken embryo.Simonsen(98) had clearly shownthis reaction to be an immunephenomenon initiated when lymphoidcells fromadult birds wereintroducedinto genetically different embryos that wereunable, becauseof their immaturity,to reject the grafted cells. However, these reactions provedto be species specific. For example, pigeonspleen cells failed to inducea GVHR wheninjected into the developing chickenembryo(63). This failure to react wasnot due to a failure the xenogeneiccells to survive in the embryonichost; pigeonspleen fragmentssurvivedquite well whengrafted to the chorioallantoic membrane of the chicken embryo,yet causedno damageto the undoubtedlyforeign host (63). Moreover,pigeoncells werecapable of expressingimmunologic function in a xenogeneic(chicken) environment.Pigeon spleen cells rejected allogeneic pigeonbonegrafts transplanted to the samechickenembryohost (63). Weconcludedfrom this evidencethat the failure of the xenogeneic pigeon cells to cause damagewhenintroduced into the chicken embryo meantthat contact with foreignantigen wasnot a sufficient requirementfor the initiation of transplantationreactions. Similar conclusionscan be derivedfromthe study of T cell activation in vitro. WhennormalT cells are cultured with allogeneic leukocytes, some

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TISSUE TRANSPLANTATION 149 are activatedto proliferate anda proportionof the activatedcells differentiate into eytotoxic cells reactive to MHC antigens of the stimulating call. Early attempts to explain this phenomenon assumedthat antigen binding by receptors on the T cells causedtheir activation (77). Accordingto such a model,one signal (antigen binding)wassufficient for lymphocyte activation, andthe presentationof alloantigenaloneshouldinducethe differentiation of lymphocyte clones. This prediction of the one-signalmodelwasnot supportedby experimentalobservation.Thecapacity of allogeneic cells to stimulateT cell activationis a functionof metabolically active cells (42, 45, 64, 69, 92). Cells killed by anyof a variety of proceduresdo not stimulate, althoughthey can be shownto express antigen (Table 1). Furthermore,not all viable calls havethe capacity to stimulate allogeneicT cells in vitro. Antigen-bearingnon-lymphoid cells such as fibroblasts, erythrocytes, or platelets do not stimulatenormalallogeneiclymphocytes in vitro (43). Also, the capacityto stimulateis phylogeneticallyrestricted andthe responseto nonconeordantxenogeneie leucocytes is generally poor or nonexistent (113). Clearly, weare faced, onceagain, with evidencethat somethingother than simple antigen recognition is involved in T cell responses to MHC antigen. The Two-Signal Model For T-Cell Activation In an attempt to explain these phenomena,Lafferty & Cunningham (62) took up the Bretseher-Cohnsuggestionthat two signals were required for lymphocyte activation (19) and developeda two-signalmodelfor the initial step in T cell activation (Figure2). This modelpostulatedthat a stimulator cell (S+) wasrequired for the presentation of antigen to the potentially responsiveT cell. Antigenbinding by the T call providessignal one for T cell activation. Thesecondsignal waspostulated to be an inductive moleTable 1 Twosignals, antigen and a source of CoS activity, activate the in vitro T-cell response to alloantigen Cells used for in vitro stimulation of C57B1 b) lymphocytes (H-2 +) -r-Irradiated P815b (S UV-i~radiated P815 (S-) b (S-) -t-Irradiated CaD2 None

Cytotoxicactivity aNo CoS activity added 5.4 < 2.5 < 3.7 < 3.7

are required to

(loglo CU/culture) CoS activity added 6.0 6.1 6.0 < 3.7

aCoSactivity provided by the supernatant of Con A-activated spleen cells (87, 111). Reproduced with permission (64a). bp815 is a lymphoreticular tumor and CaD2is an epithelial tumor of the DBA/2mouse strain.

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cule, said to express co-stimulator (COS)activity. Oncethe responsive cell was activated, factors maybe released that greatly amplify the overall level of proliferation in the population; this modelwas only concerned with the events leading to the activation of the responsive cell (25, 62). The species specificity of mixedleukocyte reactions was seen to be expressed at the level of the second signal. Provision of signal one alone does not cause T-cell activation and mayhave a negative, i.e. suppressive, effect (Figure 2) (see Appendix).

TwoSignals Required for Cytotoxic T-Cell Activation There is nowclear evidence in support of the two-signal modelfor T-cell activation by alloantigen (see Table1) and it has been shownthat the species specificity of alloreactivity is expressedat the level of the secondsignal, CoS activity (113). Using cloned tumorlines as the source of allogeneic stimulator cells, Talmageet al (111) demonstrated the existence of stimulating (S+) and nonstimulating (S-) tumor lines. Tumorsof lymphoreticular origin expressed the S+ phenotype, whereasthe S- tumor was an epithelial cell line. The stimulator cells must be metabolically active to express their S+ function (111); metabolic inactivation of the + population with U-V irradiation destroys its capacity to activate a cytotoxic T-cell response. Table 1 shows the experimental evidence for the two-signal model of cytotoxic T cell activation. The lymphokineused as a source of the second signal is the

Figure2 Diagrammatic representationof the requirement for T-cellactivation.Bindingof antigen,x, bythe T-cellreceptordoesnotcauseT-ceilactivation.T-cellactivationoccurs whentwosignalsare provided for the responsive T cell. Antigen bindingbythe T-cellreceptor providessignal1 (1), anda stimulatorcell +) provides an inductive mol ecule, CoS activity, which is the second signal(2) for T-ceilactivation.Interaction withthe controlstructure(dark blocks)onthe surfaceof the stimulatorceil triggersCoSrelease.

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supernatant of ConA-activated spleen cells (87, 111), whichprovides sourceof CoSactivity. Neither antigen providedon the S- tumorcells nor CoSactivity alone provideda sut~eient requirementfor eytotoxie T cell activation. However, a strong cytotoxic T cell responsewasactivated when both antigen andCoSactivity wereprovidedfor the potentially responsive Tcell. Clearly,the S- cells provideall the antigenicrequirements for T cell activation, but alloantigenalonedoesnot activate the eytotoxiecell. Acareful kinetic analysis of cytotoxicT cell activation has demonstrated two phasesin the activation process(59). 1 T

)T’ 2 T’------> nT’ 2

(a). (b).

RestingT cells (T) require twosignals for their conversionto the differentiated state (T’); T’ cells canexpresscytotoxicactivity. T’ cells proliferate in the presenceof lymphokiiae(elonal expansion).Interleukin 1 (ILl) beenshownto providethe secondsignal for reaction (a) and interleukin (IL2 or T cell growthfactor) will induceclonal expansionof the activated T cell, reaction (b) (2a); see appendix. Mitogen-induced activation of T cells has also been shownto be a twosignal processin whichmitogen(ConA) providessignal oneand + spleen cells are requiredfor the generationof the secondsignal (2, 66), whichalso appears to be providedby ILl (66).

Role of Class I and Class H MHCAntigens Bach et al (7), arguing from an immunogeneticviewpoint, developed alternative two-signalmodalfor allogeneicT cell activation in whichrecognition of the Ia epitope on the allogeneic cell activated the helper T cell subset, whichthen providedthe secondsignal for cytotoxic T cell activation. Thereis nowevidence,however, that recognitionof the class II antigen is not requiredfor eytotoxieT cell activation, the clearest beingthe demonstration of cytotoxicT cell activation in the situation wherethe stimulator and responderstrains differ only in the K region of MHC (6-8, 82). Also, Batchelor’s group (10) has examinedthe response of lymphocytesto the class II MHC antigen and shownthat recognition of the Ia epitope alone is not a sufficient requirementfor activation of the helper lymphocyte subset. + T cells responsive to class I MHC antigens belong to the Lyt 1+2+3 cytotoxic subset whereasthose responsiveto class II MHC antigens are part of the Lyt 1+2-3- helper subset (24). Similar signalling requirementsare requiredfor the activation of cells of either subset (59). Thatis, reactions

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(a) and (b) describe the activation of both cytotoxicandhelper T cells. nowalso knowthat cells of both the cytotoxie and the helper subsets produceequivalent amountsof lymphokine whentriggered by antigen alone (3, 72). Theclearly establishedsynergybetweenclass I andclass II reactive T cells for the in vitro generation of cytotoxic T cell response can be attributed to the augmentationof IL2 production in the culture system. However,such synergistic effects havenot been clearly demonstratedin vivo and allografts are promptlyrejected whenclass I antigenic differences only are recognized(100, 105). This does not meanthat Ia + ceils are unimportantin the process of allogeneicT cell activation. Inactivation of these cells with anti-I-region antibody and complementremovesthe stimulating capacity of the cell population(93, 114). However, not all + cells st imulate (37, 67, 115), an someIa- cells, suchas P815,do stimulate(Table1). Thatis, althoughthere + is a strong correlation betweenIa positivity and the expressionof the S phenotype,exceptionsto the correlation showthat recognitionof Ia antigen itself cannotprovidethe sourceof the secondsignal required for cytotoxic Tcell activation. Nevertheless,the Ia+ call is probablythe cell responsible for the stimulationof both class I andclass II MHC-reaetive T cells in vivo. Themostlikely candidatefor the physiologicallyrelevant stimulatorcell is the Ia-rich dendriticcell (67). Sucha modelfor immunocyte induction has a profoundinfluence on the wayweviewallogeneicinteractions. In the case of allogeneicT-cell activation, the antigenis built into the surfaceof the allogeneiccell. Cells of the S+ phenotylaehave the capacity to produceCoSactivity; other cells are S-. Thus,only antigen-bearing S+ cells will stimulatethe allogeneicT cells, since it is only in this situation that the two signals required for T cell activation can be provided.Thus, alloantigen on the surface of metabolically active S+ cells will be highlyimmunogenic for T cells in vitro, whereas the sameantigen presentedon the surface of cells of the S- phenotypewill not be immunogenic (see Appendixfor detailed treatment). Since the + phenotypeis only expressedby cells of lymphoreticularorigin (42, 43, 63, 111), wecan see whyleukocytesare strong stimulatorsof allogeneicT cells in vitro. Passenger Leukocyte and Graft Rejection It follows fromthe abovediscussion that leukocytes carried within the grafted tissue play a major role in regulation of tissue immunogenicity. Stimulatorcells (dendriticcells?) will activate recipient T ceils to generate a graft-specific response(see Appendix).Antigencardedon graft parenchymal cells will not activate T cells directly becausethe parenchymal cells cannotprovidea sourceof CoSactivity. Free antigen, shed fromthe graft,

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could be carried to the immunesystem of the recipient and there presented on recipient stimulator cells to recipient T cells. Such indirect antigen presentation wouldnot lead to the generation of graft-specific T cell response; T cells activated by indirect antigen presentation wouldbe specific for graft antigen in association with the MHC of the recipient (see Appendix). Thus, it is stimulator cells in the graft that constitute the major immunogenicstimulus of the graft and therefore represent the major barrier to tissue grafting. Since stimulator cells of the graft, such as dendritic cells, are Ia +, it follows that Ia + cells carried within the grafted tissue will constitute a major immunogeniccomponentof the graft. This is not because Ia antigen itself is highly immunogenic (9, 10), but rather that the class antigen is a marker for the cell that expresses the S+ phenotype(93, 114), i.e. the cell that can provide a source of CoSactivity. Againstthis theoretical background it makes practical sense to attempt a reduction of tissue immunogenicityby removal of leukocytes from the graft prior to transplantation (see Appendix).

MODULATION OF TISSUE

IMMUNOGENICITY

OrganCulture Prior to Transplantation In the early 1970s, several groups investigated the effect of organ culture on the immunogenicityof endocrine organs and certain tumor tissue (65, 79). The idea that organ culture might reduce tissue immunogenicity was not new. There were several reports in the 1930s suggesting clinical benefit when parathyroid tissue was held in organ culture for a period prior to transplantation to patients with hypoparathyroidism (104). However,these studies were not genetically controlled, and without the support of an adequate theoretical base, enthusiasm for the experiments quickly waned. Our interest in organ culture was stimulated by a report from Summerlin et al subsequently not confirmed--that organ culture prior to transplantation could facilitate the grafting of skin to normal allogeneic recipients (108). Jacobs (49) suggested that antigen expression was modified during the period in organ culture. Such an explanation did not seem very likely, and against the above theoretical background, the effect of organ culture could be readily explainedif bloodcells in the tissue died or wereinactivated during the culture period. The organ culture technique has proved spectacularly successful in the case of endocrine organ transplantation. Mousethyroid can be transplanted under the kidney capsule of isogeneic thyroidectomized recipients, where its ability to concentrate radioactive iodine can be used as a measureof graft function (60). Organ culture of thyroid tissue in an atmosphere of 95%

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O2and 5%CO2for 14 days extends allograft survival (61), and after culture period of 3-4 weeks,allografts showno evidenceof rejection over an observation period of 100 days (60). Pretreatmentof the tissue donor with cyclophosphamide (300 mg/Kg)4 and 2 days prior to the harvest tissue reducedthe period of organculture required to facilitate allograft survival; after this treatment, tissue cultured for approximately2 weeks could be transplanted to normalallogeneie recipients whereit functioned indefinitely (65). Underthese conditionsboth thyroid andparathyroidshow indefinite survival in allogeneic recipient mice(65). Similar results were reported with other species (84) and the procedurehas beenshownto work for xenotransplantationof thyroid andislet tissue fromrat to mouse(58, 101). Whatis the explanation for this phenomenon? Doesantigen modulation occurin organculture as suggestedby Jacobs(49), or doesthe effect result from the postulated loss of passenger leukocytes? Duringorgan culture there is a rapid degenerationof the vascular bed and bloodelementswithin the cultured tissue (88). Thetissue retains antigen, recognizableby immunoferritinlabeling (88), and the tissue is rejected whenas few as 103 viable peritonealcells of donororigin are injected into the recipient animal at the time the cultured tissue is transplanted(110). Clearly, the cultured allograft carries functionallyrecognizableantigen. Establishedthyroidallografts are also rejected whena secondunculturedthyroid of donororigin is transplantedto the recipient (65). Thiseffect is antigen-specificsince unculturedthird party graft can be rejected, but its rejection doesnot affect the integrity of the establishedculturedallograft (65). Hirschberg & Thorsby (46) suggested that vascular endotheliummay provide a major stimulus of allograft immunity. Vascular endothelium degeneratesduringorganculture (88) and its destruction could accountfor the reduction in tissue immunogenicity achievedby organ culture. Endothelial cells express MHC antigens and would,therefore, providea primary target for the attack of activatedgraft-specificT cells or graft-specificantibody(see above). The question to be addressed is whether or not endotheliumprovidesa majorsourceof stimulatingactivity for recipient T cells. Cyclophosphamide treatment of animals for 2 days prior to the harvest of tissues causesa profounddropin the capacityof spleencells to stimulate allogeneie T cells in culture (60). This treatment of the donorwith eyelophosphamide has no obviouseffect on thyroid endothelium.However,after this treatment alone, approximately30%of thyroid allografts function normallyover a prolongedobservation period (65). If donor endothelium were a major source of tissue immunogenicitywe wouldnot expect to observesuch an effect. Batehelor’sstudies using kidneytransplantation(9, 11) led to similar conclusions(see below).

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Pancreatic lslet

Transplantation

Initial attemptsto applythe organculture pretreatmentto pancreaticislet transplantationwerefrustrated by technicaldifficulties associatedwith this procedure(16, 56). Theloss of tissue immunogenicity during organculture is an oxygen-dependent phenomenon, thought to result fromthe sensitivity of leukocytesto oxygentoxicity (109). Pancreatic islet tissue cannot treated in the sameway.Singleislets, isolated fromthe adult pancreas,are extremelysensitive to oxygentoxicity andrapidly degeneratewhencultured in 95%oxygen.This toxicity problemcan be overcomeby allowing groups of approximately 50 islets to aggregateand fuse together (16, 18, 58). Theislet clusters are moreresistant to oxygentoxicity andcan be successfully allotransplantedin miceafter 1 weekof culture underthese conditions (16, 18, 58). Followingorganculture in 95%oxygen,islet allografts of to 7 islet clusters havebeenshownto reverse streptozotocin-induceddiabetes in mice. Bloodsugar levels of transplantedanimalsrapidly return to normal, and the animals becomeaglycosuric and respond normally to a glucosechallenge(17, 18). Unculturedislets, on the other hand, temporarily reverse diabetes, but recipient animalsreturn to the diabetic state within4 weeksof transplantation(17). Lacy’sgroup(56) achievedsimilar results the rat. However, in their initial studies, islets werenot culturedin 95%O2 and allograft acceptancewas only achieved whenrecipient animals were treated with a single dose of ALSat the time of transplantation. Similar results havenowbeenreported in the case of rat islet xenotransplantation to mice(57, 58). Faustmanet al (32) demonstrateda dependenceof islet immunogenicity on the presenceof Ia + cells in the transplanted tissue, by achievingallograft survival followinganti-Ia serumandcomplement treatmentof the donor tissue prior to transplantation. However,Ia antigen recognitionis not required;Morrow et al (80) haveshown that islet allograft rejection occurs whenthere is I region compatibility betweendonor and recipient. Thefact that rejection is dependenton the presenceof an la+ cell in the tissues is consistentwiththe notionthat the S+ stimulatorcell carries Ia antigenon its surface (93, 114). Fetal Pancreas Transplantation Fetal pancreascan be used as a sourceof islet tissue for transplantation. Followingorgan culture or transplantation, the exocrinepancreasdegenerates whereasthe endocrinetissue continues to differentiate and develop endocrinefunction. In rodents, transplantationof onefetal pancreasto the kidneycapsulehas beenshownto reversediabetes (20, 47, 73, 95). However, early attemptsto conditionfetal pancreasfor allotransplantationby organ

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culture prior to transplantationmet with little or no success(73). When comparedthe immunogenicity of fetal pancreasand islets isolated fromthe adult pancreas,in the samestrain combinations,wefoundthe fetal pancreas muchmoredifficult to preparefor allotransplantation(94). This difference in behaviorof different tissues wasrelated to the large amountof lymphoid tissue associatedwith the fetal pancreas(94). It is possibleto reversediabetes in CBAmice with tissue from two BALB/c fetal pancreases cultured for 20 days in 95%02 (Figure 3). However,the success rate of allografts is low; approximately 40%of recipients reverse their diabetes. Tissuecultured in this wayis slowto reversediabetes evenin an isogeneicsituation (95). However, the fact that culturedtissue can reversediabetesin isogeneic animals, sometimesas late as 6 monthspost-transplantation, providesevidencethat the cultured fetal pancreashas a capacity for continuedgrowth anddifferentiation after transplantation(95). Immature precursorsof islet tissue, pro-islets, can be isolated fromthe pancreasby collagenasedigestionand culture in air for 4 days (96). Following transplantation, this tissue has the capacity to growanddifferentiate into functionalislet tissue that will reversestreptozotocin-induced diabetes (97). In the digestion procedurethe fetal pro-islets appearto be partially separated fromlymphoidelementsassociated with the fetal pancreas. Pro-

¯ ~ 30

o o 10 GRAF~REMOVEO

~o

~6o

1~o

260

TIMEPOST-TRANSPLANT (Days) Figure 3 Noafasting blood glucose respoa~of diabetic CBAmice following allotransplantation of two 20-day cultured BALB/cfetal pancreases (six 20-day cultured fetal pancreas segments) under the kidney capsule. The shaded region defines the nonfasting blood glucose range of normal CBAmice (mean _+95%confidence interval). Grafts were removed excision of the graft-bearing kidney. Approximately 40%of 20-day cultured fetal pancreas allog~s are successful in reversing streptozotocin-induccd diabetes.

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islets are less immunogenic than the wholefetal pancreasand mayprovide a moresuitable sourceof tissue for clinical transplantation(97). Reversal of Juvenile-Onset Diabetes Thereare suggestionsthat spontaneousjuvenile-onsetdiabetes mayhavean autoimmune etiology (23, 86) and, if this is the case, a transplant could succumb to the samefate as the animal’sownislet tissue. Naji et al (85), workingwith the spontaneousdiabetic BBrat, found that MHC-compatible transplants failed whenanimalsweregrafted soonafter the onset of overt diabetes. Transplants appearedto fare better whenanimals were grafted somemonthsafter the onset of disease. These findings suggest that the transplant maybe morevulnerableto attack whengrafted duringthe acute phaseof the disease. Inflammatorydamageof islet tissue maybe virally mediated(86). Thus, if a virus growsspecifically in the B cells of the pancreas, the immune responseto the virus-infected calls could irreversibly damagethe infected B cells. If such a process wasinvolved in the induction of spontaneous diabetes, one wouldexpect an allogeneic graft to be less susceptible to damagethan the MHC-compatible graft. MHC restriction of the T cell responseto virally infected host ceils wouldprotect the allograft froma cross-reactiveattack mediatedby activated T cells of the diabetic animals. In our laboratory wehavestudied a case of spontaneousjuvenile-onset diabetes that occurred in a youngCBA mouse(Figure 4). This animal was transplanted with a cultured BALB/callograft soon after the onset of diabetes. Withina weekof transplantation, the blood sugar levd returned to normal and remainednormal for a further 180-odddays (Figure 4). Duringthis period, the animalunderwenttwo normalpregnancieswith no evidenceof abnormalbloodsugar responses. This animalwaskilled at the completionof the study and both its ownpancreas and the transplanted tissue were examinedhistologically. Theanimal’s ownpancreas contained fewislets of Langerhans andthose islets that wereseen weregrossly abnormal with few granulated/3cells (Figure 5). Thetransplantedtissue, on the other hand, showedno evidenceof damage(Figure 5). Thesefindings are very promisingfromthe clinical viewpoint;althoughthere maybe technical problemsassociated with the transfer of this technologyto the treatment of juvenile-onset diabetes, there are no conceptualproblems. Organ Transplantation Initial attemptsto removepassengerleukocytesfromtissues prior to transplantation werenot successful. Theseattempts (56, 78, 107) involved the induction of leukopeniain the tissue donor by proceduressuch as whole

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32

16

0

~

-20

~

~

0

20

~

I

40

60

!

I

I

80 100 120

I

I~0 160 180

TIME (days) Figure 4

Nonfasting blood glucose concentrations (open squares) and body weight (closed

circles) of a spontaneously diabeticCBA/H femalemouse transplantedon day0 witheight clustersof culturedBALB/c islets. Pregnancy is indicatedbythe sharpbodyweightincrease. Theshadedarea showsthe rangeof normalbloodglucoseconcentrations(mean4-2 SD). body irradiation, cydophosphamidetreatment, or treatment with anti-lymphocyte serum. At best, only marginal effects were observed for tissues transplanted across a major histocompatibility barrier (38, 55, 107), and rat heart allografts transplanted across multiple minor differences (34). Stuart et al (107) attempted to removepassenger leukocytes by first allografting rat kidneys to passively enhanced intermediate hosts. At 60 to 300 days post-transplantation, the kidney grafts were retransplanted to naive recipient rats isologous to the intermediate host. Only a delay in the onset of rejection was achieved. These marginal or weak effects led to someconfu-

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sion over the extent to whichpassengerleukocytes contributed to tissue immunogenieity (12). Nevertheless,the clinical successreported with renal allografts taken from cadaver donors pretreated with cyclophosphamide and methylprednisolonehelped maintain someinterest in the passenger leukocyteconcept(37a). Studies fromBatchelor’slaboratoryhavenowclarified the role passenger leukocytesplay in renal allografting (9-11, 67). This group demonstrated that long-surviving, immunologicallyenhancedMHC-incompatible rat kidney grafts, whentransplanted froma primaryto a secondaryrecipient of the samegenotype, do not elicit T-cell alloimmunityin the secondary recipient. In contrast, normalprimarykidney allografts in the relevant donor/recipientcombinationare regularly rejected in 12 days. Thefailure of long-survivingkidneygrafts to activate a T-cell responsecould not be attributed to a lack of either class I or II MHC antigens (67), and Batchclot’s group suggestedthat the effect resulted from a lack of .passenger leukocytes. Theythen went on to show, quite convincingly, that donor strain dendritic cells in very lownumberswouldtrigger rejection of kidneys taken froman intermediateenhancedrecipient (67). Theearlier failure see such a dramaticeffect probablyresulted fromthe strain combinations usedin these studies (107). When discussingthis strain-dependentvariation in survival time, Lechler &Batchelor(67) emphasize,and wewouldagree, that acute graft destruction is not a precise measureof a recipient’s response. In those strain combinationswhereacute graft destruction occurs, no quantitative comparison has been madeto determine the relative strength of responsesinducedby normalallografts and those depleted of passenger leukocytes. Theygo on to predict that minimalimmunosuppression at levels ineffective for normalgrafts wouldmaintainpassengercelldepleted allografts in goodfunction. This proposal deserves careful investigation. Development of Tolerance Culturedallografts of both thyroid andpancreaticislet tissue carry recognizable antigen and are promptlyrejected if recipient animals are challenged with donor leukocytes at the time of transplantation. However, animals carrying allografts for a prolongedperiod (~>100days) become progressivdymoreresistant to challengewith donorcells (18, 28, 116). the case of the thyroid wehavedemonstrated that the grafted tissue retains antigen, andthat the adaptation of the graft to its host results fromthe development of tolerance in the adult recipient (28). Wearguedthat this phenomenon mightdevdopas the result of the slow leakage of free antigen into the immunesystem of the recipient, possibly leading to tolerance induction in a mannerakin to active enhancement.Somesupport for this

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TISSUE TRANSPLANTATION 161 notion has beenobtainedin the pancreatic islet system.Whentransplanted animalsare injected with UV-killeddonorspleen cells, a sourceof alloantigen on nonstimulatingcells, around 30 days post-transplantation, graft rejection is not stimulated;administrationof viable donorcells at this time promptlyactivates a rejection reaction. Thatis, the UV-irradiated cells are not immunogenic.Whenanimals that received UV-irradiated cells were subsequentlychallengedwith viable donorspleen cells, the allografts were not rejected. TheUV-irradiatedcells, whichprovide a source of antigen alone, have stimulated the developmentof tolerance in the allografted animals.Thespecificity of this tolerance wasdemonstrated by the fact that these animalswouldaccept an unculturedthyroid allograft of donororigin but wouldreject a third-party thyroid grafted to the samerecipient. We haveshown,in the case of both thyroid- and islet-transplantedanimals,that this phenomenon is not a deletion form of tolerance. Lymphocytes from "tolerant" animals respondnormallyto donor alloantigen in vitro. More recently, Faustmanet al (33) havesuggestedsuch unresponsivenessmay dueto the actionof suppressorcells; the role of suppressorcells in allograft tolerance has beenreviewedelsewhere(91). A similar phenomenon has been observedfollowing heart allografting under cover of immunesuppression with cyclosporin A (81). Whenthe cyclosporin Ais withdrawn,the heart continuesto function but can be rejected if the recipient is appropriatdy challenged withdonortissue; at this stage, the heart allograft is in a metastable condition. Withthe further passage of time, a stable form of donor tolerance gradually devdops(81). In summary, wecan nowsay it is possible to either eliminate or reduce tissue immunogenieity by removalof leukoeytes(dendritic cells) from the transplant prior to grafting. This phenomenon has beendemonstratedto be effective with both endocrinetissue andlarger organs, such as the kidney. Thereis somevariation in the effectivenessof the techniquewith different tissues, whichcan be related to the degree of leukocyte contamination. Long-standing allografts of pretreatedtissue inducea state of specific unresponsivenessin the adult recipient. Althoughthe mechanism of this "tolerance"is not fully understood,the existenceof the phenomenon is oneof the most encouragingrecent developmentsin transplantation biology. THEORETICAL

APPENDIX

Immunobiology requires a theoretical frameworkon whichto mangethe experimental findings of cellular immunology.Such a frameworkmust comfortablyexplain whyit is the MHC complexantigens play the dual role of immune regulation andcontrol of allograft rejection. In the past, this relationship has been explained by the "immunesurveillance" concept,

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whichpostulated that the immunesystem developedto recognize components closely related to "self," including foreign MHC antigens, so that tumors wouldbe readily detected and eliminated from the system. The experimental observation that tissues expressing foreign MHC antigens survive in non-immunosuppressed recipients and showno sign of lymphocyticinfiltration (65) points out the inadequacy of the surveillanceidea. Thefollowinganalysis attempts a solution to this problem. Theory Ourtheoretical development accepts the postulates of "clonal selection" (22), namely,that lymphocyte receptor diversity is generatedby a random process,and that self-reactivity is forbiddenby a mechanism learned during developmentand enforced throughout the life of the animal. Wedevelop a theoretical system based on a further two postulates that govern the processof clonal selection andexpansion.Wethen use a symbolicterminology, whichis abstract yet precise, to derive the characteristics of the immunesystem that follow from these postulates.

Terminology In the followingdevelopmentthe accessory cell required for immunocyte ¯ activationis called the stimulatorcell, S. S.~) is a stimulatorcell that carries on its surfacea control structure, c, anda receptorfor antigen, x; the term (x) is read as: receptor for x. This receptor maybe a product of the stimulator cell or could be a cytophylic productof someother cell. When S(Cx)interacts with x, it can behaveas antigen-presentingcell Sx~, whichpresents x to the potentially responsive immunocyte.Wecan write this reaction as S ~ e. + x------>S (x)

x

Thecontrol structure is a genetically determinedproductandcan be used to define the genotypeof the animalproducingthe cell; weuse c to define a genotypein the samewaythat blood group antigens are used for this purpose. T~x)is a resting T cell that carries a receptorfor x. T’~x)is an activated cell that carries a receptorfor x. Thegenotypeof the resting or activated ¢ ! ¢ T cell can be represented as ¯Tf and T ,. (x) respectively; these are resting x~ and activated cells of x spec~fictty, derived from animalsof c genotype. ActivatedT calls expresseffector functionssuchas the expressionof cellular cytotoxicity or lymphokine production. ActivatedT cells release a number of lymphokine activities (3, 53), and lymphokinerelease is triggered

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TISSUE TRANSPLANTATION 163 signal 1 alone(59). Thatis, 1 > T~ + l.

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T’(x) +

Herel is usedas a generictermto covera number of lymphokine activities. Cell surface antigens other than those that formthe control structure, c, are representedby y. Thatis, (c + y) representsthe sumof all cell surface antigens. Theterms c, x, and y can be used in a generic sense. Particuhr examples of eachare representedas c, c1, c2, c3 ... etc.; x, Xl, x2, x3 ... etc.; y, Yl, Y2,Y3... etc.

Postulatesof the theory Thefirst postulate maintainsthat T cells do not respondto contact with antigen alone, but respondto antigen in conjunctionwith a secondsignal providedby an inductivemolecule,said to expressCoSactivity (Figure 2). FIRSTPOSTULATE Twosignals-antigen activity and CoSactivity-are requiredfor initiation of T cell activation. T

2

1 v. )T

T, the resting T cell, is convertedto the activatedstate Tv followingantigen bindingby the T-cell receptor (deliveryof signal 1) in the presenceof CoS activity, whichprovidessignal 2. Since CoSactivity is a cellular product, a corollaryof the first postulateis the idea that a stimulatorcell is required for T cell activation (Figure2). Thesecondpostulate concernsthe regulation of stimulator cell function. SECOND POSTULATE: A+ control structure on the surface of the S (stimulator) cell regulatesthe release of CoSactivity; CoSactivity is only releasedwhenthis structure is engagedby the potentially responsiveT cell.

Initiation of the lmmuneResponse In the resting immune system, spontaneousT cell activation does not occur becauseT cells do not expressa functional receptor for self-antigens, and c, the controlstructure, is a self-antigen.Thus,the resting situation in the immunesystem can be written c +T(C ) S(x )

> -Ve,

whereS(~) is a stimulator cell that carries a control structure c and

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receptor for x. T.( ~ is a T cell of the samegenotypethat has a receptor specificity definedaf this stage only as beingunresponsive to self-antigens such as c. Thatis, T c is unableto interact with the control structure of S(~. andso trigger ~o~release. ~onsider, now,the entry of foreign antigen, x, into the system. Antigen x), can interact with S<xc the antigen-presentingcell:

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S(Cx) x

> S~,

if Sxc c. ~S Thatis, ff x interacts with c in sucha waythat c.x (the interaction between c and x on the antigen-presentingcell) is no longer equivalent to c, then the followingsituation will hold: c s;+r(.x)

1

2

~c

Thatis, T cells of c’x specificity will interact with c.x on the surface of the antigen-presenting call. This will providesignal 1 for the T ceil. Signal 2, CoSactivity, is released from the stimulator cell becausethe control structure is nowengaged in the interactionwith the T cell of c" x specificity. FIRSTINFERENCE The first inference we draw from our postulates is that the specificity of the T cell responseto exogenous antigen, x, is defined by both x and the control structure, c. That is, T cell responsesto x are restricted by c.

Alloreactivity Ananalysis of alloreactivity definesthe nature of c, the control structure. Considerin vitro allogeneicinteractions in whichT cells of one genotype interact with allogeneicstimulator cells (Figure6). Thereare basically two different classes of suchallogeneieinteractions: Thosethat involvedifferences in the control structure (differences in c), and those that involve differencesin other cell surfaceantigens (differencesin y) (Figure Allogeneicinteractions betweenceils of different c-type havethe form YsC + "T(~) 1 21

..,YT*Cl

: - (¢)’

andT cells specific for the control structure on the stimulatorcell, c, are generated. Onthe other hand, allogen¢icinteractions betweencells of the samec-type, but differing in y-type antigens, do not proceedbecausethe

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responsive T cell is unable to interact with the control structure on the stimulator cell (Figure 6). That is,

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ysc + yl T c -~)

1

~ -Ve.

The potentially responsive T cell maycarry a functional receptor for y, the alloantigen on the stimulator ceil, but an interaction with the stimulator cell via y will not involve the control structure c. As a result, CoSactivity will not be released from the S+ cell, thus depriving the system of a source of signal 2 (see Figure 6). There is another important characteristic of c-type antigens. Allogeneic T cells respond to these antigens in an unrestricted manner: st+ T(~)I

1 T, tl 2 ) (c) Responses to other antigens, whether they be minor histocompatibility antigens (y-type) or other foreign antigens, x, will alwaysbe restricted the control structure of the antigen-presenting cell: c S~v) c+ y ---->S

If c, SCy ~ S

Figure 6 Alloreactivity.

T cells are activated when they interact with c-type antigens (closed blocks) on the surface of viable + cells b ecause t his i nteraction p rovides s ignals 1 and 2 required for lymphocyte activation, y-Type antigens (closed circles) on the surface of metabolically active S+ ceils are not immunogenic under these conditions because CoS activity is not released from the S+ cell. The symbofic representation of each interaction is set out below the diagrammatic sequence.

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1 ~ T’ c (c.x)" 2 ! ¢ Similarly, T cell responses to x antigens are of the type T (c.x)"

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then Sc~ + ) T(c~

SECOND INFERENCEThe second inference we derive from the postulates is that there are twoclasses of alloantigen, c-type and y-type antigens. c-Type antigens are highly immunogenicfor allogeneic T cells in vitro, whenthey are carried on the surface of metabolically active stimulator cells. y-Type antigens can be seen by T cells in an unrestricted manner, whereas y-type antigens are not immunogenicunder these conditions, c-Type antigens are seen in association with the control structure of the antigen presenting cell. Thus, we can use alloreactivity to define experimentally the nature of c, the control structure, c-Type structures will be that group of alloantigens that are highly immunogenicfor T cells in vitro. Experimentally we know that the MHC antigens behave in this way. That is, MHC antigens constitute the control structures of the immunesystem. Complexity of the Control System The MHC of the species is complexand consists of at least two classes of cell surface antigens that showstrong homologieswithin the class and also display a characteristic tissue distribution. Klein (54) has called these antigens class I and class II MHC antigens. In this analysis we refer to class I antigens as K-type antigens and class II antigens as I-type antigens. Weknowfrom the analysis of alloreactivity amongcell populations that differ only in K-type or I-type antigens that K-type and I-type antigens control the activation of different T cell subsets (24): 1 T, ~ ~ 2 > (K)

Sx + T(~I

+ T cell of the cytotoxic subset (24). In the mouse, T’ ~ ~ is an Lyt 1+2+3 Similarly: 1 2 ~T,(~I, wherein the mouseT’(~)~ is an Lyt 1+2-3- T cell of the helper subset (24). Both T’(~1 and T’¢~1 release lymphokine (l) when they are triggered antigen ~il6ne (3, 53~72). That is: SI + Tc(1) ~

1

T’f~ l + K

’el d- l, >T -- K

and T’eI+ (1)

I

1

> T’el -I--I

1.

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Response to MHC-IncompatibleTissue Grafts The above analysis showsthat the control structures of the immunesystem behave as MHCantigens in the mixed leukocyte reaction. Let us now consider howthese antigens behave in the case of tissue transplantation. The in vitro response to a tissue graft is more complexthan the in vitro unidirectional mixed leukocyte interactions. The transplanted tissue will contain antigen-bearing parenchymalcells (Figure 7). The tissue will also carry lymphoreticular cells, either as fixed tissue elements or as passenger leukocytes. Thesecells provide a source of antigen-bearing S+ cells (Figure

7). The simplest way to analyze the allograft situation is to consider separately the types of interaction that mayoccur in the recipient’s immune systemand in the transplanted tissue itself.

Interactions in the Recipient’s ImmuneSystem Antigen-bearing S+ cells from the grafted tissue will be carried to lymph nodes draining the transplantation site by way of the lymphatic system. In the node, the control structure (i.e. MHC antigens) of the donor (c genotype) will be presented directly to the recipient’s T cells (ct genotype): 1 T, C l > (c) 2 If the tissue donor differs from the recipient in both K- and I-type control structures, the activated T cells will be specified as T’ .c_l and T’.C..l This ( direct stimulation of the recipient, s T cells will therefor~tead to the generation of T cells reactive to both the K- and I-type antigens of the donor. S c + T<~) 1

pK

pK

p~

K O

Figure7 Diagrammatic representation of cellularcomponents of allograftbefore(above)and after (below)removal of passenger leucocytes.Parenchymal cells pt expressK-typeantigens. Passenger leukocytes are made up of stimulatorcells SK~that expressbothK-typeandI-type antigensandhavethe capacityto produce CoSactivity. Bloodcells that respond to lymphokine (R~u)also expressbothK-typeandI-typeantigens.

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Asecond type of recipient response will occur whentissue antigens, designated collectively as x, reach the lymphnode either as membrane fragmentsor as free antigen. In the lymphnode, these antigens will be presentedto recipient (c genotype)antigen-presentingcells. This process indirect antigen presentationhas the form:

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Sf~)~ + x-~---~ SxCl .if sCl ;~ Sc 1, then

c~ SCx1 "~ T(c cx)

>T’c(cox)" ~

Theactivated T cells generatedare of specificity (Cl"X)and are not graft specific; graft antigens are x alone. Thatis, indirect antigenpresentation doesnot lead to a graft-specific T cell response. Reactions in the Transplanted Tissue In the case of a solid tissue transplant, the graft will consist of tissue parenchymal calls, including vascular endothelium,that carry K-typeantigens, andlymphoreticularcells that carry both K-typeand I-type antigens. Reactions in the transplanted tissue will be regulated primarily by the interactionof recipient T cells withgraft antigens.ActivatedTcells specific for graft K-typeantigenshavethe potential to expresscytotoxicactivity and in this waydamagegraft endotheliumand parenchymalcells. In addition, bindingof these cells to l~-typeantigenson target cells will trigger lymphokine release andso activate a nonspecificinflammatory processin the transplant. Tcells specific for 1-typeantigensof the graft are unableto interact directly with graft parenchymal cells whichdo not expressI-type antigens. However,these cells could play an importantrole in activating donor-type lymphoreticularcells in the transplant by wayof lymphokine release (Figure 7): ,r T, (1) C I +R

2

>R,~r.

Thus,graft damage couldresult froma cytotoxiceffect directedagainst cells of the transplant and/or a severe nonspecific irdtammatoryresponse. For this reason c-type antigens carried on stimulator cells and other lymphoreticular cells of the graft (passengerleukocytes)constitute the major barrier to tissue transplantation(Figure 6), andtheir removalfroma graft prior to transplantation will greatly reducethe immunogenic strength of the graft (Figure7).

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ACKNOWLEDGMENTS We are most grateful to Jane Dixon for her assistance. This work was supported by Grant No. AM28126from the National Institutes of Health, United States Public Health Service, and grants from the Kro¢ Foundation and the Kellion Foundation.

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Literature Cited 1. Agostino,M., Prowse,S. J., Latferty, K. long surviving,passivelyenhancedallo. J. 1981.Resistanceof establishedislet grafts to provokeT-dependentalloimallografts to re~cctionby antibodyand munity. I. Retransplantation of (ASx complement. ,,lust. J. Exp. Bio£ Med. AUG)F t kidneys into secondary ASreSc~ 60:219-22 cipients. J.. F~xp.Med.150:455-64 2. Andersson,J., Gronvik, K., Larsson, 12. Billingham,R. E. 1971. The passenger cell concept in transplantation unE., Coutinho, A. 1979. Studies on T mphocyte activation. I.Requirements munology.Cell, Immunol.2:1-12 r themitogcn-dependcnt production13. Billingham,R. E., Brent, L., Medawar, of T cell9:581-87 growthfactors. Eur. J. ImP. B. 1954.Quantitativestudies ontismunol. sue transplantation immunity.II. The 2a. Andrns,L., Lalferty, K. J. 1982.Inhiorigin, strengthanddurationof actively bition of T cell activity by cyclosporin and acquired immunity. Proc.adoptively R. SCc, Lond. BioL 143:58-80 A. Scand. d.. Immunol.15:449-58 3. Andrus,L., Prowse,S. J., Lafferty, K. 14. Billingham,R. E., Silvers, W.K., WilJ. 1981. Interleuldn 2 production by son, D. B. 1963. Further studies on both Ly2+ and Ly2T cell subsets. adoptivetransfer of sensitivity to skin Scand. Z ImmunoL13:297 homografts,d.. Exp. Mea~118:397-419 4. Ascher,N. L., Ferguson,R. M., Hoff15. Bogman,M. J., Berden, J. H., Hageman,R., Simmons,R. L. 1979. Partial mare1,J. F., Marass,C. N., Koene,R. characterisationof cytotoxiccells infilA. 1980.Patterns of vascular damage in trating spongematrixallografts. Transthe antibody-mediated rejection of skin plantation 27:254-59 xenograftin the mouse..4r~d. Pathol. 5. Ascher,N. L., Hoffman,R., Chen,S., 100:727-37 Simmons, R. L. 1980. Specific andnon16. Bowen,K. M., Andrns,L., Lafferty, K. specificinfiltration of spongematrixalJ, 1980.Successfulallotransplantation lografts byspecificallysensitizedcytoof mousepancreatic islets to nonimtoxic lymphocytes. Cell ImmunoL52: munosuppressedrecipients. Diabetes 38--47 29:98-104 6. Bach,F. H., Alter, B. J. 1978.Alterna17. Bowen,K. M., Lafferty, K. J. 1980.Retive pathways of T-lymphocyte activaversal of diabetes by allogeneic islet tion. ~.. Exp. Med.148:829-34 transplantation without immunosup7. Bach,F. H., Bach,M.L., Sondel,P. M. pression. ~lust .L Exp. BioL MecL 1976.Differentialfunctionof majorhis58:441-47 tocompatibilitycomplexantigens in T18. Bowen,K. M., Prowsc,S. J., Lafferty, lymphocyteactivatton. Nature 259: K. J. I981.Reversalof diabetesby islet 273-81 transplantation: vulnerability of the 8. Bach, F., Widmer,M. B., Segall, M., established allografl. Science 213: Bach, M. L, Klein, J. 1972. Genetic 1261-62 and immunological complexityof major histocompatibilityregions.Science176: 19. Bretscher, P., Cohn,M.1970. A theory of self-nonsclf discrimination.Science 1024-37 169:1042 9. Batehelor,J. R. 1978.Theriddle of kidney graft enhancement. Trans)71antation 20. Brown,J., Heininger, D., Kuret, J., Mullen,Y. 1981.Islet cells growafter 26:139-41 transplantation of fetal pancreas and 10. Batchelor,J. R., Wel~h,K. L, Burgos, control of diabetes. Diabetes30:9--13 H. 1978. Transplantation antigens per 21. Burdick,J. F., Russell, P. S., Wim~, se are poor immunogens within a speH. cies. Nature273:54-56 J. 1979. Sensitivity of long standing ll. Batchelor, J. R., Welsh,K. I., Mayxenograflsof rat hearts to humoral antinard, A., Burgers, H. 1979. Failure of bodies. J. Immunol,123:1732.

~oY

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pressive agents. Transplan~Proc. 3: 580-82 35. French,M.E. 1972.Theearly effects of alloantibody and complementon rat kidneyallografts. Transplantation13: 447-51 36. Gerlag,P. G., Opel,P. J., Hageman, J. F., Koene,R. A. 1980.Adaptionof skin grabs in the mouse to antibodymediated rejection. Z lmmunol.125: 583-86 37. Glimcher,L. H., Kim,K. J., Green,I., Paul, W.E. 1982. Ia antigen-bearing Bcell tumourlines can present protein antigen and allo-antigen in a major histocompatibility complex-restricted fashion to antigen-reactiveT cells. Z Exp. Med. 155:445-49 37a. Guttmun, R. D., Beadoin, J. G., Moorehouse, P. D., Klassen, J., Knaack,J., Jeffery, J., Chassot,P. G., Abbou,C. C. 1975. Donor pretreatmerit as an adjunct to cadaverrenal allotrausplantation. TransplantProc.7: 117-21 38. Guttman, R. D., Carpenter, C. B., Lindquist,R. R., Merrill, J. P. 1967.An immunosuppressivesite of action of heterologous antilymphocyte serum. Lancet 1:248-49 39. Hall, B. M., Dorsch,S., Roser,B. 1978. Thecellular basis of allograft rejection in vivo I. Thecellular requirementsfor lirst-set rejectionof heartgrafts.J, Exp. Med 148:878-89 40. Hall, B. M., Dorseh,S., Roser,B. 1978. Thecellular basis of allograft rejection in vivo II. Thenature of memory cells mediatingsecondset heart graft rejection. J. Exp. Mec~148:890-902 41. Hanunerling,G. J. 1976.Tissuedistribution of ia antigens andtheir expression on lymphocyte subpopulations. Transplan~Rev. 30:64 42. Hardy,D. A., Knight, S., Ling, N. R. 1970. The interaction of normal lym.taihnocytes andcells19:329-42 fromlymphoid cell es. Immunology 43. Hardy,D. A., Ling, N. R. 1969.Effects of some cellular antigens on lymphocytes and the nature of the mixed lymphocyte reaction. Nature 221: 545-48 44. Hart, D. N.J., Winnearis, C. G., Fabre, J. W.1980.Graft adaption: studies on possible mechanisms in long-term survivingrat renal allografts. Transplantation 30:73-80 45. Hayrey, P., Andersson, L. C. 1976. Generationof T memory cells in onewaymixedlymphocyteculture. IV. Primaryand secondaryresponses to solu-

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C. J., Latferty, K.J. 1982. of renal allografts. Transplan~Proc. 96. Simeonovic, The isolation and transplantation of 3:461-64 foetal mouseproislets. Aus~J. Exp. 108. Summerlin,W. T., Broutbar, C., FoBioL Med. ScL 60:383-90 anes, R. B., Payne, R., Stutman,O., 97. Simeonovic, C. J., Lafferty,K. J. 1982. Hayflick, L., Good,R. A. 1973. AccepImmunogenicityof isolated foetal tance of phenotypicallydiffering culmouseproislets. ~lus~J. Exp. BioLMed~ tured sign in manand mice. Transp. Sc£ 60:391-95 Proc. 5:707-10 98. Simonscn,M. 1962. Graft versus host 109. Talmage, D. W., Dart, G. A. 1978. reactions. Theirnatural history andapEffect of oxygenpressureduringculture plicabilityas tools of research.Prog.~41on survival of mousethyroidallografts. ler~y. 6:349-467 Science 200:1066-67 99. Snell, G. D. 1957.Thehomograftreac- 110. Taknage,D. W.,Dart, G., Radovich,J., tion. An~Rev. MicrobioL11:439 Lafferty, K. J. 1976. Activation of 100. Sollinger, H. W.,Bach,F. H. 1976.ColTransplant Immunity:Effect of donor laboration betweenin vivo responsesto leukocyte, on thyroid allograft rejecLDand SDantigens of majorhistocomtion. Science 191:385 patibility complex.Nature259:487-88 111. Talmage, D. W., Woolnough,J. A., 101. Sollinger, H. W., Burkholder, P. M., Hemmingsen, H., Lopez, L., Lafferty, Rasmus,W.R., Bach,F. H. 1976. ProK. J. 1977. Activation of cytotoxic T longedsurvival of xenograftsafter orceils by nonsthnulatingtumorcells and san culture. Surgery81:74 spleencell factor(s). Proc. NatL,4cad. 102. Steinmuller, D. 1967. Immunisation Sc/. US2174:4610-14 with skin isografts taken from tolerant m~ce.Science 158:127-29 112. Vesole,D. H., Dart, G. A., Talmage,D. W.1982. Rejection of stable cultured 103. Steinmuller, D. 1969. Allografl immuallografts byactive or passive(adoptive) nity producedwith skin isografls from immunization. Proc. Nail Acad. Sc£ immunologically tolerant mice. Transp. USA79:1626-28 Proc. 1:593-96 J. A., Misko, I. S., Laf104. Stone,H.B., Owin~s, J. C., Grey,G.O. 113. Woolnough, ferry, K. J. 1979.Cytotoxicandprolif1934.TransplantaUon of living grafts of erative lymphocyteresponsesto allothyroid and parathyroid glands. ~4n~ geneicandxenogeneic antigensin vitro. Surg. 100:613 .4ust. £ Exp. Biol. Med.Sc~57:467-77 105. Streilein, J. W., Toews,G. B., Bergstresser, P. R. 1979.Cornealallografts 114. Yamashita,U., Shevach,E. 1977. The expression of Ia antigem on imfail to express Ia antigens. Nature munocompetent cells in the guineapig. 282:326-27 II Ia antigenson macrophages. ,L. 106. Strom,T. B., Tilney,N.L., Paradysz,J. munoL119:1584-88 M., Bancewicz,J., Carpenter, C. B. 115. Zitron, I. M., Ono,J., Lacy,P. E., Da1977.Cellular components of allograft rejection:identity, specificity andtyrovie, J. M.1981.Thecellular stimulifor the rejection of establishedislet allotoxic function of cells infiltrating grafts. Diabetes30:242-46 acutelyrejecting allografts. J. Immuno£ 118:2020-26 116. Zitro~n,I. M., Ono,J., Lacy,P. E., Da107. Stuart, F. P., Bastien,E., Holtcr,A., vie, J. M. 1981. Activesuppressionin Fitch, F. W,Elkins, W.L. 1971. Role the maintenance of pancreaticislet alloof passengerleukocytesin the rejection grafts. Transplantation32:156-58

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AUTOIMMUNITY- A PERSPECTIVE Howard R. Smith

and Alfred

D. Steinberg

Section on Cellular Immunology, Arthritis Branch, National Institute of Arthritis, Diabetes, and Digestiveand KidneyDiseases, NationalInstitutes of Health, Bethesda, Maryland20205 AUTOIMMUNITY The conceptual framework for understanding the basis for autoimmune diseases has changeddrastically in the past few years (Table 1). This change has allowed what were previously thought to be mysterious disorders to take their place alongside those with less complexity. Paul Erlich viewed autoimmunityas the "horror autotoxicus," which the organism was to avoid at all costs. Dysfunction of Burner’s thymic censor, which was supposed to prevent self-reactive cells from emerging, provided the primitive conceptualbasis for disorders characterized by self-reactivity. In addition, immunologywas deeply immersed in departments of microbiology, which held Koch’spostulates as sacred long after their usefulness had passed. As a result, autoimmunedisorders were thought to be caused by single agents, which one day wouldbe uncovered. The discovery of slow viruses addedfuel to this line of reasoning. Nevertheless, this conceptual framework left autoimmunedisorders shrouded in a cloak of mystery, In the past 10-15 years there has emerged a new conceptual framework for viewing autoimmunediseases. Although never explicitly stated, the frameworkcan be readily outlined (Table 1). The theoretical considerations have not yet produced a complete picture. Moreover,the precise details of the pathogenesis and etiology of most autoimmunedisorders have not been elucidated. Nevertheless, there is sufficient information, coupled with the new conceptual framework, to movesuch diseases from the realm of the mysterious to the realm of the comprehensible. The shift from mystery to understanding has been complex. At least one important factor is the advance of information in immunology.Another is the recognition that such diseases may not have a single etiology. Since immunologyemerged from 175 0732-0582/83/0410-0175 $02.00

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Table1 Autoimmune diseases: conceptualframework Era

Beliefs

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I. DARKAGES

Horrorautotoxicusof Ehrlich Thymic censorof Burnetpreventsemergence of forbidden clones Qualitativeabnormalities lead to disease Singlecause(especiallyviral) II. ENLIGHTENMENT Self-reactivityis normal Self-self reactionsformthe bases for bothnormalimmune reactivity and normalimmune regulation Diseaseresults fromquantitativeabnormalities (e.g. amount of stemcell proliferation, amountof autoantibody,quantity of immune complexes) Multifactorialetiology Genetic Individualgenespredisposeto particularabnormalities Diseaseis polygenic Environmental factors Stimulate immune system Interfere with normalimmune regulation Hormonal factors (modifydiseasemanifestations) Abnormal immune regulation(mayresult, in part, from the above) microbiology, there was a strong prejudice in favor of a single causative agent, or at least a unifying cause, for a disease. That view maybe fine for manyinfectious diseases; however, Koch’spostulates are useless for a disease in which host susceptibility is the most important factor. They are further compromisedby the need for two or three additional factors for disease to becomemanifest. The role of individual infectious agents in manyautoimmunediseases is still uncertain. The experiences with syphilis and Whipple’s disease still render us sutficiently humbleto acknowledgethat an organism mayyet be found for each autoimmunedisease. In fact, the observation that Lyme arthritis is causedby an infectious agent has recently underlined this possibility. Nevertheless, the best evidence points strongly to a multifactorial etiology for manyautoimmunediseases. If an organismis one of the factors, it is only one. In this paper, we try to define and discuss autoimmune disease in the light of modernimmunologicconcepts and on the basis of the most recent studies. The immunesystem displays a complexity, which includes a requirement for self-recognition. Internal regulation of the system occurs through that self-recognition and constitutes the most basic form of autoimmunity. Regulation mayinvolve cells, antibodies, amplification systems (e.g. comple-

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ment), and combinations of these dements. Thus, certain cell-cdl interactions do not occur efficiently unless there is a recognition by onecell of specific sdf-determinants on the other. This self recognition is a form of autoimmunity. In addition, production of antibodies (Abl) to an antigen maylead to regulation of that response through (auto)antibodies (ABE) specific for Abl. Such anti-idiotype antibody (Ab2) production represents another form of autoimmunity. In health, the regulatory circuits flow smoothly. They regulate the production of antibodies to foreign organisms and play an important role in normal homeostasis. Thus, the horror autotoxicus of Paul Erlich does not necessarily follow from anti-self immunity. However,alterations in the normal functioning of the immuneregulatory circuits maycause dramatic and sometimesdeleterious results. Whereasthe regulated production of autoantibodies appears to be consistent with health, uncontrolled production of autoantibodies to certain antigens maylead to disease. The sameis true of cells, whichrecognize self determinants on other cells. Diseases characterized by abnormal and/or excessive anti-self immune responses have been termed autoimmunediseases. In this paper we discuss mechanismsby which autoimmunediseases may occur. A numberof diseases serve to illustrate particular principles. Thereafter, we discuss two in greater depth: autoimmunethyroiditis (AIT) and systemic lupus erythematosus (SLE). Although we concentrate on human disease, we rely upon animal models where necessary. The Immunological Basis for Disease Before embarking, we wish to classify autoimmunediseases. These are summarizedin Table 2. A defect in the afferent limb of the immunesystem Table 2 Classification

of autoimmunediseases Description

Class A

A defect in the afferent limb of the immunesystem initiated ment for a specific external agent (SLE) With important genetic requirements Without important genetic requirements

without a require-

A defect in the afferent limb of the immunesystem initiated ternal agent (acute rheumatic fever) With important genetic requirements Without important genetic requirements

by a specific ex-

A defect in effector mechanisms of immunity initiated without a requirement for a specific external agent (hereditary angioedema) A defect in effector mechanismsof immunity initiated by a specific external agent (certain central nervous system viral infections) Combinations of the above

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initiated withouta requirementfor a specific external agent(class A) characterizes the classic autoimmune diseases such as gLE,AIT,and rheumatoid arthritis. In them,the primaryevents are presumed to be initiated by immune processesdirected against self antigens (Table3). Theabnormality maylie in the self antigen, the immune process, or both. Thus,onemayhave (a) an abnormalself antigen with a normalimmune response to it, (b) abnormalresponse to a normalself antigen, or (c) an abnormalresponse to an abnormalantigen. Antibodiesreactive with self determinantscan be causedby an exogenous agent. Acuterheumaticfever is an exampleof a class B disease. Theinitiating agentis a groupAbeta-hemolyticstreptococcus.In susceptibleindividuals, a severe infection with the organismleads to an immune response, whichincludesresponsivenessto certain self antigens. In this case the self antigensinclude heart antigens, whichcross-react with those of the infecting organism;thus, the disease process ensues(50). For a variety of formal andhistorical reasons, diseases of class C tend not to be included amongautoimmune diseases. In hereditary angioedema, a defect in the functional capacity of the inhibitor of the first component of complement (becauseof either a structural aberration or a quantitative reduction)leads to edema(initiated by trauma, endotoxinrelease, or other stimuli), whichcan be life threatening (55). Thus,the disease is immunemediated (by componentsof the immunesystem); moreover, it can lethal. However,mosttextbooks and teachers do not include it amongthe autoimmune diseases. Webelieve there is a bias in favor of including among autoimmune diseases those in whichthere is apparentspecificity. This bias comesfromthe historical bias amongimmunologists that specificity is the most important aspect of the immunesystem and that disorders of the immune systemshould be manifestedin terms of alteration in specificity, if not qualitatively, at least quantitatively. In other words,there shouldbe specific antibodyor cell-mediatedimmunitythat is heightenedto account for the diseaseprocess.Unfortunately,not all diseasesfit into these preconceivednotions. It has beenpointedout that the mostnonspecificaspects of immunitymayaccountfor disease (112). Thus,in a strictly formal sense, class C diseases could be regarded as perfectly acceptable autoimmune disorders. The class D disorders are clearly immunemediated; however,they are initiated by known exogenousagents. It is sometimes ditficult to determine the extent to whichthe intlammatoryresponse to exogenousagents is beneficial andthe extent to whichit is detrimental.If moredetrimentalthan beneficial, it could be termedpathologic. Sucha designationwouldbe very broad, probablytoo broad to be practical. Nevertheless, it is useful to recognizethat manytoxins (e.g. froman insect) induce inflammatory reac-

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Table 3 Self-antigens in autoimmunediseases of class A Self-antigen

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Intracellular Nucleolus DNA RNA Sm-RNA Histones Ribosomes Cytoplasm Mitochondria Lysosomes Microsomes Melanosomes Filaments/tubules Cell membranereceptors Thyroid receptor Acetylcholine receptor Insulin receptor Cell membranes Red blood cells Lymphocytes Neutrophils Platelets Muscle Neurons Myelin components Epidermal ceils Spermatazoa Ovum Extracellular Basement membranes Intercellular substance Plasma proteins Immunoglobulins Complement components Clotting factors Hormones Insulin Thyroid hormones Glucagon Intrinsic factor

Disease SLE, Sjogren’s syndrome, scleroderma, polymyositis SLE, chronic active hepatitis, Sjogren’s syndrome SLE, Sjogren’s syndrome SLE, mixed connective tissue disease, scleroderma, polymyositis SLE SLE Insulin-dependent diabetes, autoimmunethyroid disease, idiopathic Addison’s disease, pernicious anemia, SLE SLE, primary biliary cirrhosis, Sjogren’s syndrome, chronic active hepatitis SLE Autoim~nunethyroiditis, idiopathic Addison’s disease, pernicious anemia Vitiligo Chronic active hepatitis, gluten-sensitive enteropathy, SLE, mixed connective tissue disease Graves’ disease (stimulates), Myasthenia gravis Insulin-dependent diabetes

AIT (blocks)

Autoimmune hemolytic anemia, SLE SLE Autoimmune neutropenia, SLE Idiopathic thrombocytopenic purpura, SLE Rheumatoidarthritis, SLE, cirrhosis, polymyositis SLE Multiple sclerosis Pemphigus vulgaris Autoimmune(male and female) infertility Femaleinfertility Goodpasture’s disease, bullous pcmphigoid, discoid lupus Pemphigus vulgaris Rheumatoidarthritis, SLE, Sjogren’s syndrome, chronic active hepatitis SLE, rheumatoid arthritis, dense deposit disease SLE Insulin-dependent diabetes AIT Insulin-dependent diabetes Pernicious anemia

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tions; it couldbe arguedthat in the absenceof the body’sreaction, morbidity would be minimal. An extreme exampleof an apparently misplaced responseto an exogenousagent is the inflammatoryresponseof the central nervoussystemto certain viral infections. Theviruses maynot be limited in any importantwayby the responseand they maynot be very pathologic by themselves;however,the responseto the infection maybe disabling or fatal. Is this an autoimmune response?In the absenceof knowledgeabout the virus and with the presence of hypergammaglobulinemia, antiglobulins, anti-nuclear or antilymphocyteantibodies, and/or circulating immune complexes,such diseases woulddefinitely be labeled autoimmune.Our knowledgeof the role of the virus allows us to philosophize about the relative merits of including such immunediseases amongthe autoimmune ones.

Unfortunately,the classification shownin Table2 is an oversimplification. Manydiseases that are immune-mediated (all diseases?) are influenced to a greater or lesser extent by the geneticbackground of the individualand the state of the immune system. Theseand other factors maybe critical to the expressionof illness or the degreeof abnormality.In addition,there marc be morethan one class of defects or mechanisms that mayoperate in a given personwith a particular disease. Therefore, the multifactorial nature of diseases, especially of autoimmune diseases, mustbe appreciated. Although this adds a degreeof complexity,it helps to explain the great variability among patients with the samesyndrome.It also helps to explain variability in the expressionof diseases amongclosely related individuals, identical twins, or evenhighly inbred rodents. Disease Mechanisms Manyautoimmunediseases have commonfeatures such as arthritis or glomerulonephritis.In part, this maybe due to the limited outcomespermissiblein the affected tissues. It mayalso be dueto the relatively limited mechanismsthat mediate autoimmunedisorders. The immunemechanisms involved with the pathogenesisof autoimmunity are those as described by Coombs &Gell (18): type I, anaphylactic; type II, cytotoxic; type III, antigen-antibody complexes;and type IV, cell mediated. These specific mechanisms can be augmentedby nonspecific measures:amplification systemsinvolving either cellular (via lymphokines)or humoral(i.e. complement,coagulation,kinin, and fibrinolytic) systems.Theoverall interaction of these complexprocessesleads to the specific outcomesof each disease. Thecytotoxie mechanism type II encompasses the reaction of circulating autoantibodywith antigenfixed either to the surface of cells or to tissue. Theantigen maybe the cell membrane, a receptor of that membrane, or an

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AUTOIMMUNITY 181 antigen suchas a virus or drug that has becomeattached to the cell/tissue surface. Thepathologyof manyautoimmune diseases is initiated by this mechanism. The final outcomeof the cytotoxic process maybe mediatedby complementas in the reaction of C’-fixing antibodywith cell-boundantigen, or maybe mediatedby cells as in antibody-dependent cell-mediated cytotoxicity (ADCC).ADCC may be the most important mechanismin most autoimmune diseases. Thefinal effect, irrespective of mediation,is usually cell lysis, elimination, or inactivation. This is the mechanism of many autoimmune hematologicdisorders. However,antibodies to receptors can lead to stimulation. In Graves’disease, long-actingthyroid stimulator, an autoantibody [immunoglobulin G (IgG)] to the thyroid stimulating hormonereceptor, stimulates the receptor and thus mimicsthe function of the hormoneintendedfor that receptor (3, 23). Anopposite effect is seen myastheniagravis. Anautoantibodyto the acetyleholine receptor at the neuromuscularjunction combineswith that receptor and thus blocks the neurotransmitter,acetylcholine.Besidesblockingthe receptors, these antireceptor antibodies accelerate the degradationof the receptor (22). The autoimmuneendocrine diseases commonly have antibodies to receptors. Insulin-dependentdiabetesmayhaveautoantibodiesto the islet cell surface or cytoplasm,insulin receptors, or insulin (48). Insulin maythus be bound by anti-insulin antibodyleading to decreasedavailability of insulin. (This boundinsulin mayform immune complexesthat lead to additional disease manifestations.)Antibodiesto the insulin call surface receptor as seen in insulin-dependentdiabetes and acanthosis nigricans can block responsiveness or occasionally can lead to hypoglycemiaby mimickingthe insulin effect. Immunecomplexescomprise the type III mechanism.The formation of the complexof antigen plus antibody mayfix complementand thereby initiate inflammatoryprocesses. Anyorgan in whichsuch complexesare depositedmaybe subjected to inflammationand ultimately destruction. The original antigen can be either exogenous,as in microbialdiseases, or endogenous,as in manyautoimmune diseases. Nucleicacids often serve as the antigen in SLE.Thevasculitis of polyartedtis nodosaresults, at least in somepatients, fromimmunecomplexdeposition of antibody and hepatitis B surface antigen (39). Often, deposition occurs in the glomerulusand accounts for the immunecomplexglomerulonephritis of manyautoimmune disorders. Localization of the immunecomplexis not limited to the vessels and glomerulus.Theuveal tract can be involvedwith deposition in the ocular basementmembrane,thus leading to uveitis. Thechoroid plexus has been

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demonstratedto be a site for a type III mechanism in SLE.Manyautoimmuneskin diseases result fromthis process,includingcutaneousvasculitis, erythemanodosum,and discoid lupus. Type IV reactions comprise immunemechanismsthat are mediated by interactions of cells or their soluble produceswith antigen rather than by antibodyand complement. Theclassical example,delayedhypersensitivity, is characterizedby a reaction that is time-dependent,has a specific histologic sequencein termsof cellular infiltration and inflammation,andcan only be transferred by cells and not by serum. Theeffector mechanism of cytotoxicity can include direct cell interaction with antigenor elaboration of lymphokines.Thelatter include a numberof mediatorsthat havediverse functions. Theyprimarily amplifythe initial reaction by nonspecifically recruiting inflammatorycells such as neutrophils and macrophages to the reaction area. At that inflammatorysite, cells becomeactivated, undergo transformationand proliferation, and further secrete lymphokines,thus generating a cascade effect. Accordingly,in vitro assessmentof T cell ¯ responsesinclude both quantitative and qualitative measurements of lymphokineproductionas well as proliferation of effector cells. In a similar manner,the humoralsystemhas amplification systemsthat intensify the type I, II, andIII reactions. This systeminvolvesthe cascades of coagulation,complement, kinin, andfibrinolysis, all of whichprimarily interact intravascularly to maximizethe effect of antibody. Althoughthe cellular, humoral,and amplification mechanisms can be dissected, in vivo these reactions tend to act in concert.

SPONTANEOUSTHYROIDITIS Spontaneous thyroiditis waschosento represent organ-specificdiseases. It has beenstudied extensively in chickens, rats, and humans.

OS Model The obese chicken (OS) represents a modelfor the spontaneousdevdopmentof thyroiditis. The disease resembles the humandisease AIT(Hashimoto’s)with regard to histopathology, serological abnormalities, and phenotypiccorollaries. Boththe distinct anatomicalseparationof the B and T cell generative organs and well-characterized immunogenetics of Gallus gallus has allowedotherwiseinaccessible explorationinto the pathogenesis of this disorder. Cellular manipulationsof T or B cells in the OSmodelare madepossible by thymectomyor bursectomy, respectively. It has been repeatedly demonstratedthat neonatal thymectomy accelerates thyroiditis whereasneonatal bursectomyprevents disease (128). Transfer experiments havesubsequentlybeenperformed to elucidate further the site of the cellu-

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lar defect. Bursalcells fromOSchickensor their normalcounterparts (CS chickens) were transferred to neonatally, chemically(cyclophosphamide) bursectomizedOSand CSchicks (85). Thereconstituted normalCSchicks did not developdisease or autoantibodies, whereasthe reconstituted OS chicks still developedthyroiditis and autoantibodies.In contrast, T cell transfers fromOSchickensto normalT cell-depleted(irradiated) CSchickens causedthyroiditis, whereasthe reverse (CST cells into irradiated OS chickens)did not cause disease (95). Thesecellular studies indicate that although B cells are required for disease expression, they are probably normal;however,the T cells appearto be defective. Further investigations have characterized the T cell defect to reside in the T suppressor cell population(95). It has beenpostulatedthat T suppressorcells fail to migrate adequately to the peripheral lymphoidtissue secondaryto delayed thymicmaturation, and/or that relative unresponsivenessto T suppressive actions exists at the target tissue (126). The OSchicken modelhas a genetic predisposition to the development of disease. This appearsto be underthe polygeniccontrol of at least three loci (127): the major histocompatibilitycomplex(B locus), a non-B(minor histocompatibility)locus, anda thyroid locus. Other modelsof spontaneousautoimmune thyroiditis (SAT)exist (beagles, marmosets),but mostnotable is that of Buffalorats (105). Results that modelare quite similar to those foundin OSchickens;again, neonatal (but not adult) thymectomy promotesthyroiditis, whichsuggestssignificant T suppressorrestraint of disease. In contrast, experimentallyinducedautoimmune thyroiditis (EAT)of rabbits, guinea pigs, andmiceappears to have a different cellular basis for disease. Theinductionof diseasewith homologous thyroglobulin has been abrogated by neonatal thymectomy,thus indicating that the induceddisease is thymic(helper) dependent.Further differences exist betweenEATand SAT:thyroid destruction is largely T-cell mediatedin EATas opposedto importantantibody-mediatedmechanismsin SATand humanAIT. Althoughit can be helpful for elucidation of immunogenetic details, the usefulness of EATas a modelfor humanAIT maybe limited. HUMAN AUTOIMMUNE THYROIDITIS

(AIT)

Antibodies Thefinding of antibodiesto thyroglobulinin the serumof patients with AIT by Roitt et al (93) in 1956markedthe beginningof the investigations into the autoimmune nature of this disease. Rose& Witebsky(96) wereconcurrently able to demonstratethat similar lesions wereproducedin animalsby immunizationswith homologousthyroid antigens mixed with Freund’s

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adjuvant. Subsequently,a large numberof antibodies to normalthyroid tissue components werediscoveredin patients’ sera. Themajorantibodyis to thyroglobulin.Antibodiesagainst other thyroid antigens includethose to the secondcomponentof colloid, microsomalantigen, nuclear component, thyroxine, triiodothyronine, and thyroid-stimulating hormonereceptor (26). Theantibodyto thyroglobulinis usually foundin high titers in patients with AIT,but it mayalso be foundin Graves’disease(70), in lowtiters other autoimmune disorders, and evenin normalpersons. As with the other autoantibodiesagainst thyroid antigens, thyroglobulin-antibodyis usually of the IgGclass. The exact role of these various antibodies in the pathogenesisof the disease is unclear. By themselves, they appear unable to cause thyroid lesions. Studies haveshownthat transfer of serumfromindividuals with AITto monkeys does not cause disease and that newbornswith high titers of antibodies obtained by transplacental transfer from motherswith AIT also do not developdisease. Eventhough complement-dependent cytotoxicity by thyroid microsomal antibodyagainst thyroid cells occursin tissue culture, the role in vivo mayfollowinitial cell damageby anothermechanism(52). However,there is a relationship betweenthe levels of serum anti-thyroid antibodies and lymphocyticinfiltration of the thyroid gland (131). Theimportanceof these autoantibodiesmaylie in their involvement in immunecomplexformation (49) or ADCC (17), mechanismsthat be involved in thyroid tissue damage.In AIT, both soluble immunecomplexes in serumand electron-densedeposits in thyroid follicular basement membrane occur and presumablyrepresent thyroglobulin-anti-thyroglobulin antibody (49). Recently, ADCC activity and K cell numbershave been shownto be increasedin AIT(7); their increase corresponded to the appearanceof the thyroid lesion (56). Althoughseveral mechanisms for the induction of inflammationappear to be responsible for AIT, ADCC appears to be a critical component.

Cells Becausea pathogenetic role could not readily be demonstratedfor serum autoantibodiesin AIT,investigators turned their attention to the cellular basis for the disease. Byuse of monoclonal antibodies against cell (sub)populations,defects in T cell numbersand in suppressorT cells havebeen foundin the peripheral bloodof patients with AIT(81). Nonspecificsuppressor T cell function appears to be normal,whereasantigen-specific T suppressor dysfunction recently has been demonstrated(52). The latter points to the possibility that altered immunoregulation via loss of and/or defective T suppressorcells maycontribute to AITby allowing unhindered B cell autoantibodyproduction.

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AUTOIMMUNITY 185 Further defects in tests of cell-mediated immunityin AITraise the possibility that other mechanisms participate in the pathogeneticevents. T lymphocytes and monocyte-macrophagesmay be involved. Lymphokines are generatedby sensitized T lymphocytes in AIT,indicating specific recognition of thyroid antigens. T lymphocytes fromAITpatients can be directly cytotoxic for thyroid cells in tissue culture, for mousemastocytoma cells coatedwith thyroid microsomes,or for heterologousred bloodcells coated with thyroglobulin. Monocyte-macrophage function in AITis not well characterized: however, it maybe important for tissue inflammation.Monocytespresented with cytophilic anti-thyroglobulin-antibody becomearmed. If presented with thyroglobulin-coatedred bloodcells they are able to lyse the target cells by an extracellular mechanism (68). Anti-microsomal antibodies can also be cytophilicfor monocytes (68). In addition, the interaction of macrophages with lymphocytesdiffers in autoimmune thyroid disorders as compared to normals:Thereis an increase in macrophage-lymphocyte rosettes. Thus, the defective interaction of macrophageswith both monocytesand lymphocytesmaycontribute to disease. Summary of AIT Spontaneously occurring autoimmunethyroiditis in humansmayhave more than one etiology and morethan one mechanism of inflammationand glandulardestruction. Theanimalstudies indicate that at least three genes are necessaryfor disease production. Thehumandisease is significantly associated with HLA-D3,howeverwith only a moderaterelative risk, whichsuggests that additional genes maybe involved. In somesusceptible individuals, variousinitiating events maytrigger the diseaseprocess.These havenot yet beenelucidated. In other individuals, an inherited thyroid defect maybe sutScientto trigger the diseasein the presenceof the appropriate genes for the immuneabnormalities. SYSTEMIC

LUPUS ERYTHEMATOSUS (SLE)

Introduction In contrast to AIT,SLEis an exampleof a widespreadorgan nonspecific disorder. This autoimmune disease is characterizedby B-cell hyperactivity, whichresults in hypergammaglobulinemia and in the production of a variety of antibodies reactive with organ-nonspecificantigens such as DNA, RNA,and cell membranestructures. Disease signs and symptomsmay occur in virtually any organ in the body. It is widely held that immune complexesof antigen and antibodyand complement components initiate the inflammatoryprocess in manyorgans; however,alternative or additional mechanisms are likely. A major problemin the study of SLEisdetermining

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whichimmuneabnormalities result from the illness and whichlead to disease. This aspect has beenespecially difficult to dissect in patients who usually appearwith the syndromealready present. However,the availability of miceknown to developthe illness allowsfor studyprior to the onset of overt symptoms or signs of disease. In the past few years, workwith the experimentalanimalsthat develop an illness resembling humanSLEhas contributed substantially to our knowledge.In addition, important advanceshave been madein the study of the humandisease. Mostof these advanceshave emphasizedpathogeneric factors in the induction of the syndrome.Theysuggest that multiple factors play a role in the development andexpressionof illness, including genetic factors, immune factors, environmentalfactors, and hormones.Although the exact mechanismshave not been completelyelucidated, broad outlines are nowpossible.

Murine Lupus Anumberof different strains of mice develop spontaneouslyan autoimmunedisorder with features of humanSLE. However,the clinical syndromesvary substantially amongthe various murinemodelsof SLE.Some of the features of the mostwidelystudiedstrains are shownin Table4. NZB miceproduceanti-T cell antibodiesfromearly in life. Thereafter, the pace of their illness is very slow. Thesemicedo not die until 15-20monthsof age. In contrast, MRL-lpr/lprmicehavea rapidly progressiveillness characterized by massive lymphadenopathyand death by 7 months of age. BXSB malesdie between6 and10 monthsof life, whereastheir sisters live well into year 2. The opposite occurs in the (NZBX NZW) FI: Thefemales die of immune complexrenal disease at about 10 monthswhereasthe males live until 15-20 months. Therefore, although any one mousemaybe a modelof humanSLE, they, like the patients, vary tremendouslyamong themselves. B CELLHYPERACTIVITY All of the strains mentionedhave B cell hyperactivity: hypergammaglobulinemia and increased numbersof antibodyformingcells. This hyperactivityappearsto be polyclonal;it is not limited ~ to the production of autoantibodies. As a result, murineSLEcould b~ viewedas merelya manifestationof polyclonalB cell activation, with some of the B cells producingautoantibodiesthat lead to injury. Thecauseof the B cell activation is uncertain;moreover,it mayvary fromstrain to strain. NZBmice produce enormousamounts of IgM; MRL-lpr/lpr mice produce IgG1 and IgG2a in great excess; BXSBmice produce large amounts of IgG1and IgG2b.Nevertheless, the informationfor the polyclonal B cell activation resides in the bonemarrowstemcells. In all of the strains, marrowstem cells can transfer the characteristic disease to irradiated

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histocompatiblerecipients (5, 25, 74, 121). Therefore,the problemis, least in part, proximalto the matureB cell. T cells normallyserve to regulate the magnitudeof B cell responses. In the autoimmune mousestrains, it is possible that B cell hyperactivitymight result, in part, from T cell regulatory dysfunctions. It appears that in different strains different T cell functionsare prominent.Therefore,it is necessaryto examineT cell function before conclusionsregardingthe B cell hyperactivityare possible. T CELLFUNCTIONS IN MURINE LUPUSThe most glaring examples of T cell abnormalities are found in the MRL-lpr/lpr mouse. This mouse develops massive lymphadenopathy(77). The predominant cellular increase consists of Ly l+,2-,Thy 1+ (helper phenotype)Tcells (63). ever,the intensityof stainingof these Tcells withanti-Ly1 is low;therefore, the cells maynot be representative of the normaldistribution of Ly1+ T cells. Thepresenceof such enormousnumbersof Ly 1+ T cells suggests the possibility that such T ceils mightdrive B cells to produceautoantibodies. In the absenceof the neonatal thymus, lymphadenopathy does not occur; moreover,anti-DNA,renal disease, and death are markedlyretarded (116). If a thymusgraft is provided, the autoimmune syndromecan occur (121). Thus, the stem cells of the MRL-lpr/lprmousegive rise to abnormalT cells. Suchbonemarrowstemcells give rise to pre-T cells that mustmature in a thymus. Thus, the defect that leads to autoimmunityin MRL-lpr/lpr mice is not in the thymus,it is merely thymic dependent. The abnormal stem cell needs a proper environmentin whichto mature. NZBmice also have both B cell and T cell abnormalities. Although nu/nu NZBmice develop disease (32) and stem cells can transfer it non-autoimmune-prone recipients (74), the addition of NZBT cells to NZB marrowcells in neonatally thymectomized radiation chimerasincreases the autoimmune disease (120). Recent studies have demonstratedthat a marrowpre-T cell contributes to the abnormalityin experimentaltolerance (61). This cell can mature in a non-NZB thymus. Therefore, the T cell abnormalityof NZBmice, like that of MRL-lpr/lprmice, is based uponan abnormalstem cell that requires a thymusfor maturation. ThemaleBXSB mousehas not only polyclonal B cell activation, but also B cell hyperplasia(121). Unlikethe T cell adenopathyof the MRL-lpr/lpr mouse,the lymphadenopathy of the BXSB mouseconsists largely of B cells. Recently, the B cell hyperplasia of the male BXSB mousehas been found to be underT cell regulation (106a). Neonatalthymectomy of male, but not female, BXSB mice leads to a markedincrease in lymphadenopathy;the enormouslyenlargednodes contain large numbersof B cells. Of interest, these neonatally thymeetomized BXSB mice contain very few Lyt2+ calls,

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AUTOIMMUNITY 189 suggestingthat T cells of the suppressorcircuits normallyserve to regulate the B cell hyperactivity. (NZBX NZW) F1 T cells also have been found to regulate the pace illness. Their effect is morelike that seen in BXSB mice than in MRLlpr/lpr mice~neonatalthymectomy tends to accelerate disease (I 13, 114, 116). However,thymectomy at 6 or 10 weeksof life does not (114). Therefore, whateverserves to regulate the pace of illness mustworkwithin the first few weeksof life for it is no longer present in older (NZB× NZW) FI mice. Consistentwith these cellular studies is suppressorfactor production deficiencyin these mice(86), whichmaybe replacedwith somebenefit. STUDIESOF MARROW STEMCELLSIn view of the ability of marrow fromall of the autoimmune strains to transfer disease, it appearsthat the information necessary to produce abnormal lymphocytesis contained within the marrowstem cells. Stem cells from NZBmice were found to produce large numbersof endogenousgranulocyte and monocytespleen colonies (75, 122, 125). However, lethally irradiated NZB micegivena fixed numberof syngeneic marrowcells did not have an increase in colonies (125). Twoexplanationsare possible: Spleenstemcells manifestexcessive endogenous colonyformation; marrowstem cells, whichgive rise to colonies, are increased in absolute numberbut not percentagein NZB mice. The latter possibility wouldexplain whystudies of a fixed numberof marrow cells leads to fewerabnormalitiesthan study of the wholemarrow potential. Nevertheless, NZBmarrowcells were able to generate increased numbers of B cell coloniesin vitro (53), whichwereresistant to suppressivefactors such as prostaglandins or IgM(54). The increase in B cell colonies observedvery early in life but becomessubnormalby 15 weeksof age (47). These studies suggest that the NZBmarrowstem cells are capable of excessiveproductionof B cell offspring;however, if that processplaysa role in disease, there mustbe a longlag between the peripheralizationof the cells and their ultimate contribution to disease. Perhapsthe increased marrow production of B cell colonies gives rise to an abnormaldistribution of peripheral B cells, whichultimately explains the unusualIgMproduction, + cells, hyperdiploidcells, and the autoantibody the increase in Ly 1÷, IgM production characteristic of NZBmice. It mayalso explain the splenic localization of the abnormal cells after the first fewmonthsof life. GENETIC STUDIES A numberof genetic factors have been found to influence the expressionof autoimmunity in various strains of mice.Thelpr/lpr geneis a single genelocus that causes lymphoproliferation.In homozygous form, this gene is responsible for the lymphoproliferationseen in MRLlpr/lpr mice. It has been suggestedthat this is a single gene modelfor

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autoimmunity(77) and that a specific defect mightbe the cause (130). However,the MRL+/+ mice are not normal. They spontaneously produce antinuclear antibodies. In addition, MRL mice have phenomenalhypermaturity. Wehave observedthat they are born morethan twice as big as miceof other inbred strains and are able to reproduceas early as 5-6 weeks of age (whichcontrasts with the 8-12 weeksin other inbred strains). Thus, there appear to be backgroundgenes unrelated to lpr that maypredispose to hypermaturity, accelerated aging, and autoimmunity.This is perhaps best demonstratedby examinationof other strains of micewith the lpr/lpr gene. In the absence of the MRL backgroundgenes, C57BL/6-lpr/lprmice manifestmuchless vigorousautoimmune disease (77, 130). However,in the presenceof appropriate background genes fromanother source (e.g. NZ.B), the pace of illness inducedby lpr is markedlyaccelerated. In other words, lpr/lpr has its majoreffect in accelerating disease whenbackgroundgenes for autoimmunity are present. The backgroundgenes for autoimmunityhave been studied best in the NZB strain. Here,at least six genesare responsiblefor the full-blownillness (87). Someof these genesappearto codefor individual abnormalitiesin the mice.Thus,a single geneis responsiblefor the predispositionto anti-DNA productionand another unlinked geneallows anti-T cell antibodies to be producedin large amounts(87, 90). Thereappears to be no linkage to H-2 or allotype in the RIlines (13). In the NZB micethere is no single genethat predisposesto, or causes, autoimmunity; rather, a numberof genescontribute (113). The BXSB mouseinitially appearedto have a single gene for autoimmunity(78). This wasa Y chromosome-linked factor. Recent studies have clearly demonstratedthat the Y-linked factor cannot, by itself, induce autoimmunity. For example, crossing a BXSB male with a non-autoimmunefemale leads to male offspring that do not manifest autoimmunity (113); however, crossing a BXSB male with an autoimmune-prone female leads to maleoffspring with disease that is accelerated relative to their sisters (121). Thus, the Y chromosome-linked factor operates in concert with backgroundgenes to induce accderated autoimmunity.Thus, like the MRLand NZBmice, BXSBmice have background genes for autoimmunity.In all of these mice, the lpr/lpr or BXSB Ychromosome can cause markedacceleration of disease. Theexact nature of the backgroundgenes and the precise modesof action of lpr/lpr or the BXSB Y remain to be determined. Characterization of the gene products wouldbe a major advance in our understandingof the basis for autoimmunity. B CELL SUBSETS RESPONSIBLE FOR AUTOANTIBODIES More extensive studies of B cell subsets responsiblefor autoantibodyproductionhave

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AUTOIMMUNITY 191 been performed with NZBmice and their hybrids. Congenic NZB.x/d miceare micethat are largely NZBbut bear the xid gene, a genethat originated from the CBA/NX chromosome.Mice that bear xid in homozygousor hemizygousform are deficient in a subset of mature B cells (Lyb+, Lyb 5+), which is r esponsible for immuneesponses r ot polysacchafide antigenswith repeatingstructures (103). NZB.xid micefail to manifest the autoimmunesyndromecharacteristic of NZBmice; they have a much reducedquantity of autoantibodiesandlive almosta normallife span(119). The same is true for (NZBX NZW)Fl’xid (117), BXSB.xM and (NZB X BXSB) F~’xidmice (H. R. Smith, T. M. Chused,A.D. Steinberg, submitted for publication).Therefore,the xid geneappearsto inhibit markedly the developmentof autoantibodies in NZBand BXSB mice and their hybrids. Thesimplest explanationis that the Lyb3+,5+ B cells are necessaryfor autoantibodyproduction(they makethe autoantibodies)and that in their absence fewer antibodies are made. Onecaveat remains, however.It is possible that xid +, has additional effects besides the deletion of Lyb3 5+ cells. In fact, it appearsthat it also alters macrophage markers(97). Therefore,it is possible the xid genemoreindirectly reducesautoantibody production by eliminating an important macrophage-B cell interaction. Nevertheless,it is clear that the Lyb3+,5+ B cell subset limbof the immune response is very important for autoantibody production and that in its absence autoantibodyproduction is reducedmarkedly.The reduction is, however,not absolute. Lowlevels of autoantibodiesoccur spontaneouslyin xid-bearing NewZealand mice (80, 94), and prolongedadministration polyclonal immuneactivators can stimulate such mice to produceautoantibodies (106). Thus, although the Lyb3+,5+ B cell subset appears to be largely responsiblefor autoantibodyproduction,other B cells can produce autoantibodiesif stimulatedsufficiently. The B cell subset responsible for autoantibody production in MRLlpr/lpr and BXSB mice has not been completelyelucidated. Wehave now developedfully congenicmicebearingxid and suchstudies are in progress. Studies with morelimited inbreeding suggest that the hyperactivity of MRL-lpr/lprmiceis not as completelyinhibited by xid as is the case with NZB mice. Thus,the Lyb3-,5- cells mayplay a larger role in autoantibody productionin the MRL mice(51). However, it is possible that the abnormal T cells of the MRL-lpr/lprmousedrive the B cells to produceantibodyvery muchas the exogenouslyadministeredpolyclonal immuneactivators drive the B cells in the NZB.xidmice (106). Asubset of B cells has been described with an unusualphenotype.This Ly1+ B cell is quantitatively increasedin NZBmice(66). In addition, is increased in neonatally thymectomizedBXSBmice with accelerated autoimmunity (106a). It is possiblethat this B cell subset is responsiblefor

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autoantibodyproduction. Therelationship betweenthis B cell subset and the Lyb3+,5+ subset remainsto be determined. DEFECTS IN TOLERANCE The development of widespread autoimmune disease in NZB, (NZBX NZW)F1, MRL-lpr/lpr, BXSB,and (NZB BXSB) F1 micemaybe viewedas a loss of self tolerance. This loss has been studied not only by assessing the development of autoantibodies, but also by assessing experimentallyinducedtolerance. Toleranceto heterologous gamma globulins has been studied most extensively. Thesemice have been foundto be defective in the development of experimentaltolerance to both bovine gammaglobulin (BGG)and humangammaglobulin (41, 62, 113). Thedefect can be transferred to lethally irradiated recipients in both the NZBand BXSB by marrowstem cells (25, 41, 61, 62, 120). In the NZB mouse,the T cells and non-Tcells are both abnormal;however,the marrow pre-T cell wasfound to be sut~cient to producethe defedt (61, 62). contrast, studies in the BXSB mice suggest that a non-TB cell might be most abnormal (41). Earlier studies in (NZBX NZW) F~ mice indicated that marrowstem cells contained the informationfor intolerance to BGG (111). Of interest, the young(NZBX NZW) F~ thymuscontains cells promotetolerance or preventthe escape fromtolerance to BGG;these cells also retard spontaneousanti-DNAproduction (114). Therefore, defects experimentaltolerance, like the disease, can be transferred by stemcells. Theexact contributions of different maturelymphoidpopulations to the defect mayvary fromstrain to strain or maybe dependentuponthe details of experimentaldesign. Other forms of tolerance have been less completelystudied. Recently NZBmice were found to have a defect in the developmentof systemic tolerance followinggastrointestinal exposureto antigen (18a). Antibodymediatedsuppression of immunityhas been found to be defective in NZB and MRL-lpr/lpr mice, but not in BXSBor (NZBX NZW)F1 (18b). Therefore,these functions maybe irrelevant to the development of autoimmunity; alternatively, the differences amongthe strains with regard to certain immune functions mayunderlie the differences in their expression of the autoimmunesyndrome. SEXMostpatients with SLEare females. In fact, during the childbearing years of females (approximatelyages 12-40 years) morethan 90%of the patients developingSLEare females.Asa result, there has beenan attempt to understand the female predominancein humanSLEby studying sex hormonesin mice. Initial studies of (NZB× NZW) F~ mice demonstrated only partial effects of sex hormone reversal. Suchstudies involvedmicethat were5 weeksof age. Subsequentstudies demonstratedthat the age at which

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sexual alteration was carried out was critical to the outcome.Castration of females and males at 2-3 weeks of age and treatment with the opposite sex hormoneled to accelerated disease in males and retarded disease in females (98, 115). In these studies it appeared that androgensretarded disease a greater extent than estrogens accelerated disease; however,both processes were operative. To the extent that patients were like (NZB× NZW) mice, a hormonal explanation for the female predominance of SLEwas possible. The cellular basis for the result is not completely understood. It has been found that androgens tend to favor the development of thymic suppressor cells and that estrogens favor helper cell differentiation (E. A. Novotny, E. S. Raveche,M. Ottinger, A. D. Steinberg, submitted for publication). Studies of experimental tolerance have further shownthat in the presenceof estrogensa single cellular defect is sufficient to transmit a defect in tolerance; in the presence of androgens,two cellular defects are necessary for a tolerance defect (62). Therefore, females, by virtue of their estrogens, might be expected to have a predisposition to SLEwith more modest cellular abnormalities and males would be protected by their androgens unless they had profound cellular abnormalities. There are muchless markedsex differences in the severity of disease in MRL-lpr/lpr and NZBmice than in NZBhybrids. Nevertheless, androgens tend to retard and estrogens accelerate disease in these strains, even if it is quantitatively less impressive than in the NZBhybrids. A notable exception is a gene for androgeninsensitivity in NZBmice that regulates anti-T cell production (113). BXSB mice, superficially, represent an exception to the above. The male BXSB mice have markedlyaccelerated disease relative to their female littermates. This effect is unrelated to sex hormones (24) and appears to caused by the stem cell defect that requires the BXSBY chromosome(25). However, in crosses of CBA/Jmice with BXSB,the female offspring have more severe disease than do the males whether the BXSBis the mother or the father (113). Thus, only when the BXSBY chromosomecan act upon other suitable background genes does the male predominancebecomemanifest. VIRUSESIN MURINE SLE There has been considerable interest in the search for environmental agents, such as viruses, to trigger the immune system and cause SLE. Both high titers of xenotropic type C RNAvirus and circulating levels of the murineretroviral envelopeglycoprotein, gp 70, are found in murine SLEstrains (121). However,features of autoimmunity have been dissociated from viral expression in F2 and RI mice (19). Some viruses, such as lymphocyticchoriomeningitis, can accelerate disease (123) whereasothers, such as lactate dehydrogenasevirus, can ameliorate disease

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and prolongsurvival (83). Althoughavailable evidencedoesnot support etiologic role for viruses, it appearsthat antibodyformationto virus and/or viral antigens causes immunecomplexeswhichcan deposit in the kidney and contribute importantly to the developmentof glomerulonephritis. PRIMARY VERSUS SECONDARY DEFECTS Despite an intensive effort over the course of a quarter century~ it remains unclear as to whether individual cellular abnormalitiesare primaryor secondaryto other defects. Many abnormalitiesappearto result fromthe illness rather than inducethe illness. Theseinclude reducedprimaryantibodyresponsesin vivo, reduced responses of T cells to mitogensin vitro, reducedIL-2 production, and impaired-cell mediatedimmunity(130). Manyof these are also observed patients with active SLE(see below).Thesedefects mayprovideinsight into the effects of ongoingautoimmune disease. Theymayalso provide a basis for therapy; however,they are unlikely to provide insights into the early stages that precede the developmentof the autoimmunesyndrome. SUMMARY OF MURINE SLE There are a number of different mouse strains with spontaneousautoimmunity. Theyall havedefects in self-tolerance (they producepathogenicautoantibodies) and in experimentaltolerance. However,the details of their clinical syndromesand the basis for illness in the mature lymphocytesvary amongthem. The major common feature is the ability of lymphoidstem cells fromthe miceto carry the informationnecessary to transmit the disease. Nevertheless, a variety of environmental factors can modifythe expressionof illness, as can a variety of background genes. Finally, oncethe illness is fullblown,multipleimmune defects maybe measuredthat bear little relationship to the primary(preclinical) underlying process. The relationship to humanSLEis discussed below.

Human SLE Human SLEvaries from a mild disorder that mayrequire little treatment to a rapidly progressive and possibly fatal disorder. If one examinesthe spectrumof diseases that characterize the murinemoddsof SLEand adds to them the offspring of matings amongthemselvesand with another 20 inbred strains, one begins to developa picture of the spectrumof human SLE.Neverthdess, certain differences maycharacterize humans.Disease manifestationsoccurin manyhumans early in life, especiallyin the first half of the reproductivelife span of females. In contrast, manymicewith SLE do not developillness (as opposedto serologicalabnormalities)until a later time. Onlylpr/lpr femalesdie during their reproductive period and share this feature with humans. Rashes are more commonin humans, as are

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serositis andalopecia. Theseare not characteristicof the mice.Joint disease is a feature only of MRL-lpr/lprmice, but it occurs in the majority of patients. Onevery importantdifference betweenmiceandpeopleis that the studies in the miceusually are performedin the absenceof therapy, whereas the peopleoften havebeentreated previouslyor are currentlybeingtreated. Aseconddifference is the ability to study the miceprior to the onset of disease. Finally, a variety of manipulations are possiblein mice, but not in humans. B CELLSANDB CELLFUNCTIONS IN SLE Patients with active SLE have reducedabsolute numbersof circulating B cells (37) but increased percentagesof cells staining with anti-B1(72). PolyclonalB cell hyperactivity characterizesthe great majorityof SLEpatients with active disease. Theyhavemarkedlyincreased numbersof antibody-formingceils as determinedby reverse plaqueassay, especially those of the IgGand IgAclasses (10, 35). This increase in antibody-forming cells is highly correlated with the degreeof diseaseactivity (10). TheseSLEB ceils producelarge quantities of autoantibodies, such as to DNA;however,they also produceincreased numbersof antibody-forming cells that produce antibodies to irrdevant chemicalhaptens (16, 71). Althoughpatients with SLEmayhave generalized antibody production, it maybe especially directed towarda limited numberof antigens. A stemcell defect could accountfor the increase in B call function as SLEpatients haveincreased numbersof B cell colonies generatedby peripheral blood non-Tcells (57). Paradoxically, whenSLEperipheral blood B cells are stimulated with B cell mitogens, they do not necessarily producemoreimmunoglobulin than normal,and they actually mayhavea markedlydepressedin vitro response to pokeweedmitogen(27). However,in short term unstimulated cultures of fresh peripheral blood lymphocytes,immunoglobulin synthesis by SLE cells has beenfoundto be muchgreater than that by normalcells (35, 45, 79) and parallels SLEdisease activity (35). It has recently beenobserved that SLEbloodgives rise to greater numbers of B cell colonies,a defect that mapsto B but not T cells (57). T CELLSANDT CELLFUNCTIONS IN SLE A considerable number of studies havebeen performedon this subject (88). Lymphopenia, including reductionsin circulating T cells, characterize mostpatients with active SLE.The abnormalityis muchless markedwhenthe disease is quiescent. Varioussubsets of T cells have been reported to be reducedin patients with SLE. These include active or early rosette-forming cells, To calls, and morerecently T calls that rosette with humanerythrocytes (Tar). Thesecells mayall overlapto a substantialdegree.TheTar subpopula-

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tion of T cells has beenfoundto havethe surfaceandfunctionalcharacteristics of post-thymicprecursorT ceils, can generatesuppressionfunction and natural killer cell activity, andcanact as the respondingcell in the autologous mixedlymphocytereaction (AMLR).The decrease in Tar ceils active disease maybe due, in part, to antilymphocyte antibodies,as antibodies specific for suchcells havebeenfound;of interest, partial correctionof the defect occurredwhensuch cells wereincubatedwith fresh humanserum (or serumthymicfactor) (84). This result suggests that the Tar precursor cell was present or that the erythrocyte receptor wasblockedor not expressed. Therelationship betweencirculating T cells and the humantotal body T cells is unknown.Althougha reduction in peripheral blood T cells is foundin SLE,it is not knownwhetherthis represents a migrationof cells fromthe circulationto the tissue or a decreasein absolutetotal bodyT cells. Manypatients with SLEhave impaireddelayed hypersensitivity responses to a variety of antigens (11). In addition, they haveimpairedability to sensitized(28). At least part of this defect mayresult fromthe lymphopenia. Anadditional componentderives from suppression, perhaps by macrophages and their products. Wehavefound that treatment of patients with azathioprine or cyclophosphamide in moderatedosage often leads to normalizationof skin test responses. Variableresults havebeenreported regardingthe ability of SLET cells to respondto mitogens. Someof the variability stems from use of whole peripheral bloodmononuclear cells as opposedto morepurified cell populations. However, it is often foundthat responsivenessto the T cell mitogen concanavalinA (ConA)is reduced in patients with active SLEand that there is a return towardnormalin less active disease. It is thoughtthat there is a defect in cell-cell communication in patients with active SLE.This is perhaps best exemplified by the impairmentin the AMLR (100). However, that reaction also returns towardnormalin disease quiescence(101). At time whenB cell hyperactivity is mostabnormal,helper T cell function is mostdeficient in SLE(21). Therefore,the maintenanceof B cell hyperactivity does not appear to be dependentuponhelper T cells. In the absenceof excessivehelper function, a defect in suppressorfunction has been sought. A numberof papers have provided evidence for a defect in suppressorfunction in patients with SLE(1, 14, 99). However, evenwhensuch a defect has beendescribed, somepatients did not havea defect andothers had a defect with regard to somesuppressorfunctions but not others (99). Finally, a defect in suppressor function has also been denied. Animportantserial study demonstratedthat both the defect in the AMLR and the defect in suppressor function becamenormalized whenthe diseasebecameless active (101). Asa result, mostof the studies of T cell

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AUTOIMMUNITY 197 functionsappearedto be analysesof defects that resulted fromthe disease rather than those that lead to the disease. The value of ConA-induced suppressor cells has been questioned. However, a defect in suppressor function has beendemonstratedeven in the absence of ConA(58a). Moreover,antibodies reactive with T calls are producedspontaneously by manypatients with SLE(and by NZBmice). Suchantibodies are capable of interfering with T cell functions. BothIgM (129) and Ig~ (58, 82) antibodies maybe capable of eliminating T cells. Suppressorfunction has been reducedor abolished by such antibodies in vitro (102). Therefore,it is possiblethat defectsin T cell functionsmaynot lead to disease, but, rather, result fromthe diseaseprocess. Nevertheless, such antibodies mayplay an importantrole in perpetuatinga process that otherwisemightbe self-limited. GENETICSTUDIES A genetic

predisposition to the developmentof SLE has been established both by familial studies and by analysis of major histocompatibilitycomplexassociations. Althoughrelatives of patients with SLEhave an increased frequencyof developingthe disease (approximately 5%)(12), concordanceof SLEin identical twin pairs is about 70%(12). Thereis someevidenceto suggestthat environmentaleffects contribute to the familial occurrenceof SLE(8, 15); however,the concordanceof SLE in dizygotictwins is no morethan that of first’ degreerelatives (12). Although a high incidence of serologic and immunoregulatory abnormalities occurs in clinically unaffected relatives of patients with SLE,including diminishedsuppressorcell function (67), reducednumbersof erythrocyte C3breceptors (43, 69, 128a), antinuclear antibodies (65), lymphocytotoxic antibodies, andanti-RNAantibodies (20), the possible role of environmental factors in someof these defects has not beenfully elucidated. Recent studies suggestthat a geneticdeficiencyof C3breceptors maybe responsible for impaired clearance of immunecomplexes.Previously it wasthought that circulating immunecomplexeswere responsible for decreased C3b receptors. Populationstudies indicate that particular groupssuchas American blacks (104) and AmericanSiouxIndians (76) have an increased incidence of SLE. Analysisof the relationship betweenthe majorhistocompatibility complex and SLEdiscloses an association betweenthe disease and genes that are groupedin the HLA(humanleukocyte antigen) region of the sixth chromosome. In animalspecies, genesof this region play a fundamentalrole in the control of normalimmune responses(9). Initial investigations into the associations betweenSLEand certain HLA Aor B locus antigens failed to highlight any strong ones. However,the study of the D region antigens showeda high incidence of two alloantigens, DRw2 and DRw3 (34, 92).

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Aneven greater association was found with an Ia-like antibody: 715. In family studies, inheritance of two of the markersDR2,DR3,or 715 was highly associated with features of SLE(91). Aninteresting association exists betweenSLE(and similar disorders) and a variety of hereditary deficiencies of complement components,primarily C2. A substantial percentage of womenwith homozygous C2deficiency, perhaps as manyas 60%,develop a lupus-like illness (4). It has been speculatedthat the C2-deficientstate mayplay a role in the development of the rheumaticdisease, possibly by allowinga predisposition to certain infectious agents that stimulate the immunesystem and thus favor the developmentof SLE. However,a strong linkage is found between the hereditary C2deficiency and the DR2antigen, whichitself is associated with SLE.Thus,the C2defect maybe a markerfor a disease susceptibility gene. Arelated preliminaryfinding is a strong association betweencertain C4alleles andsusceptibility to SLE(124). SEX Hormonalfactors

play a role in certain murine SLEmodels (see above)and, similarly, it is becomingclear that sex hormones are important in both normal and abnormal humanimmunoregulation. HumanSLEis predominantlya femaledisease, especially betweenthe ages of 12 and 40. Exacerbations of humanSLEare often seen with hormonalchanges, such as during or after pregnancy(I09), at menarche,or with oral estrogencontaining contraceptive use (46). It has been shownthat both male and femalepatients with SLE(60), SLE-Klinefelter’spatients (118), and first degreerelatives of SLEpatients (59) all havean increase in hydroxylation of estrogen to the potent estrogenicurinary 16 alpha metabolites. In addition, maleSLEpatients haveelevated ratios of estradiol/testosterone (44). Thus, somepatients with SLEmayhavea metabolicabnormalitythat leads to excessive production of certain estrogenic components.Themechanisms wherebyestrogens exacerbate disease have not been entirely elucidated. Nevertheless, sex hormonesand their metabolismprovide yet another factor that modifiesdisease expressionin SLE. HETEROGENEITY IN SLE Amongthe various murine models of SLE, there is heterogeneitywith regardto the genetics,cellular basis, andexpression of disease. Theseanimalmodelshave revealed that similar outcomes maybe the sequelae of different perturbations of immunoregulatory pathways(113). Therefore it wouldnot be surprising to find that humanSLE wassimilarly heterogeneous.Althoughthe widespectrumof varied clinical presentations and serologic abnormalities of patients with SLEhas long beenappreciated, mostphysiciansgenerally consider SLEa single illness. However,like the mousemodels, humanSLEdemonstrates heterogeneity

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AUTOIMMUNITY 199 for (a) genetics (HLAtypes, C receptors, immune responses), (b) cellular basis (variations in helper functions, suppressor function, AMLR, antibody-forming cells), and(c) expressionof disease(types of autoantibodies, organsysteminvolvement).Recently,patients havebeendividedinto clinical subgroupsbasedupondifferencesin their peripheral bloodT cell subset composition(108). Certain clinical characteristics of patients with SLE wereassociated with the ratio of these regulatory T cells. Thesestudies providea clue to relationshipsbetweenclinical subsetsof diseaseanddifferencesin cellular abnormalities.Thus,SLEmaynot be oneillness, but rather a symptomcomplexwith commonmanifestations. MECHANISMS OF DISEASEIN SLE The various immuneabnormalitiesB cell hyperactivity with autoantibodyformation,concomitantT cell aberrations, anddefectiveamplificationsystems(i.e. complement)-all interact culminatein tissue pathologyand disease manifestations.In both routine and humanSLE, antibody appears to be the primary initiator of tissue damage.Immune complexes(IC), whichare found in various sites including kidney, blood vessels, and skin, are both locally deposited fromthe circulation and formedin situ. The sites are determinedby local tissue factors, such as vascular permeability, whereasmultiple properties of IC accountfor their pathogenicity(includingsize, avidity for antibody,ability to fix complement,and clearance of IC by the mononuelearphagocytic system). The mononuclearphagocytic system in SLEhas been demonstrated to be defective in the immunospecific clearance of IgG-coatedautologous erythrocytes, whichwouldallow increased levels of circulating immunecomplexes(30, 84a). Thecompositionof IC includes antibody DNA/anti-DNA. Anysuperimposedinflammatoryprocess or viral or bacterial infection wouldfurther stress the system. Recently, low-molecular-weightDNA fragments similar to those found in DNA/anti-DNA antibody IC were found to accumulate in peripheral blood lymphocytesin SLEpatients, suggesting that they mayserve as a primarysource of autoantigen for anti-DNAproduction and/or pathogenic IC (73). Not all anti-DNAmaydeposit in the form of IC. DNA mayfirst bind to basementmembranes and circulating antibody secondarily bind to the DNA to initiate the inflammatoryprocess. Other autoantibodies, such as to platdets, red blood cells, or lymphocytes,can accountfor disease manifestations such as thromboeytopenia,hemolytic anemia, and lymphopenia, respectively. In addition to specific anti-lymphocyte antibody,antiribonucleoprotein antibody from SLEsera can penetrate into suppressor TGceils via their Fcreceptors,bindto their nuclei, causetheir deletion, and abrogatetheir suppressorfunction (6). Furthermore,disruption of normal\ amplification systems(e.g. prostaglandins)by autoantibodiesoccurs. SLE

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patients produce antibodies to an inhibitor of phospholipase A2, lipomodulin, whichinterferes with lipomodulin’sinhibition of cell triggering. Thus, such an autoantibody could lead to abnormalcellular activation characteristic of patients with SLE(42). Cellular mechanismsmay be involved in the formation of SLEdisease. Damageto humansuppressor T cells by anti-T cell antibodies can be effeeted in the absence of complement by ADCC(58), whereas anti-DNA antibodies in SLEserum have been shownto act as specific antibodies with a model target cell (i.e. cell-mediated lysis of DNA-coatedcells) (33). Althoughnot as wall characterized, NKactivity is defective in SLE(40), as is macrophagefunction. Titers of alpha interferon, which can be produced by NKcells, are increased in active SLE(31) and other interferons maycause increases in macrophageactivation and Ia expression as well as suppressor T cell defects. SUMMARY OF THE PATHOGENETICFACTORS IN SLE Humans with SLEhave B cell hyperactivity. In most individuals, such hyperactivity is measurable as hypergammaglobulinemia,autoantibody production, and increased numbers of antibody-forming cells in the blood. Similarly, mice with SLE have hypergammaglobulinemia, autoantibody production, and increased numbers of antibody-forming cells (usually measured in the spleen). The basis for the B cell hyperactivity is difficult to study in vitro. In particular, the cells of the mice and the patients do not excessively respond to B or T cell signals in culture; rather, they are more likely to hyporespond.Therefore, an in vivo stimulus to B cell hyperactivity is lost in culture, but is present in the body. This stimulus maybe a T cell in the case of MRL-lpr/lpr mice, as in graft-versus-host disease in mice. It may be a T cell in a small numberof patients with SLE. However,in most mice and patients, it is difficult to demonstrateexcessive helper T cell function. In addition, defective suppressor T cell function is not a uniform finding. That is not to say that either or both are not important in perpetuating a process. However,it suggests that another cause of B cell hyperactivity is operative. Perhaps the antigen-presenting cell is defective, providing excessive stimulation to B cells. There is no doubt that macrophagedefects are present in SLE. All of the murine models have defects in macrophage function. It is possible that factors are producedby somecell that drives the B cells in SLE. Alternatively, the B cells maybe endogenouslyhyperactive. This could be a result of a primaryB cell defect. Alternatively, it could be a result of excessive production of B cells (individual B cells wouldbe normal). SLEpatients, even whenthe SLEis inactive, have excessive proliferation in their B cell-enriched fraction of leukocytes (37). Mostof the autoimmune mice also have excessive spontaneous lymphoid proliferation (89). There-

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AUTOIMMUNITY 201 fore, SLEcould be viewedas a disorder of proliferation. That wouldput it in the class of diseases in whichmalignanciesare placed; however,the proliferation is polyclonal rather than monodonal.In the mice, the increasedproliferation is foundat the precursorstemcell level. At least one study of humanshas found evidencefor abnormalpre-B stem cells in SLE (57). Moreover,in that study T cells werenot necessaryfor expression the defect. Therefore,excessivestemcell turnovermaybe sufficient to lead to excessive proliferation. A secondfactor maybe necessaryto result in differentiation. This factor is whatturns a patient with inactive SLEinto onewith manifesteddisease activity. Anyof a variety of triggers maylead to the signals that allowthe excessivelyproliferatingB cells to differentiate into antibody-producing cells. This formulationmayallow for a synthesis of diverse SLE-likediseases. Graft-versus-hostdisease mayprovidesuchan extremestimulusto proliferation and differentiation that autoimmunity mayoccur in the absenceof a genetic predispositionin either mice(36, 64) or people (scleroderma-like illness). In addition, severe chronic stimulation of the immune systemby polyclonal immune activators such as nucleic acids or endotoxin(29, 107) or by certain viruses (Epstein-Barr, aleutian mink)maybe sufficient producedisease. Finally, a variety of geneticdiseasesmaylead to an individual with a predispositionto the development of diseasebyvirtue of excessive stemcell activity. Additionalsignals wouldlead to differentiation and the disease. Genesfor high responsivenessto certain autoantigenswouldallow specific autoantibodiesto be produced.However, a sufficient disruption of normalimmune regulatory processescould lead to a variety of autoantibodies despite no strong geneticsusceptibility. Viewedin this way, SLErepresents a syndromethat maybe brought about by a great numberof immuneperturbations. Somepatients may inherit a very strong predispositionsuch that trivial environmental factors (or evenendogenous factors) maybe sufficient to inducethe syndrome. the other end of the spectrum,a personwhois not particularly susceptible mayreceive a sufficiently strong stimulus to induce disease. There are probablyseveral genesthat particularly predisposeto, or protect against, the developmentof the syndrome.However,their predisposing or protective ability maybe influenced by other genes or the environment.Thexid gene in NZBmice is a good example. NZBmice are protected against autoimmunity by virtue of the impositionof the xid gene (119). However, removalof normalT cell regulatory function by neonatal thymectomy will allow disease to occur. Alternatively, stimulation of the immune system with polyclonal immune activators will allow the syndrometo occur. Thus it is possible that anyof several perturbations of the immune systemwill allow protective functions to be overwhelmed. Oncethe syndromeof polyclonal B cell activation and hypergamma-

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globulinemiais induced, a great numberof secondaryfactors operate to perpetuate the disease process and to producedisease symptoms. Activated T cells andother cells produceinterferon. Interferonmaylead to increased Ia or DRexpression on macrophagesand moreefficient macrophagefunction. Initially, this wouldbe expectedto heightenimmune responses.Ordinarily, interferon might be expected to suppress antibody production; however, autoimmuneNZBmice, and perhaps patients with SLE, appear to be relatively resistant to those suppressiveactions of interferon. As a result, only the stimulatoryactivities are observed.Ultimately,excessive macrophagefunction suppresses new immuneresponses whereas the autoimmuneprocess continues unabated. Someof the autoantibodies producedperpetuate disease. Antibodiesreactive with T lymphocytestend to. impair regulatory function; antibodies reactive with B lymphocytesmay stimulatefurther B cell proliferation. Whyis the autoantibodyresponse of SLEpatients not regulated as arc normal immuneresponses? In periods of remission it maybe. Normal suppressorfunctions mayserve to limit the extent of autoantibodyproduction. Reductionin suppressor function in BXSB miceby neonatal thymectomyor in normalmicegiven polyclonal B cell activators tends to allow accelerated disease. In addition, anti-idiotype antibodies maybe produced duringremissionbut maybe ineffective duringdisease activity (2). It appears that the degreeof immune stimulationthat triggers the active disease process maybe sufficiently strong to overcomeany normalregulatory processes. In individualswith impairedregulatoryprocesses,it is eveneasier to trigger disease. Moreover,as indicated above, the process tends to be self-perpetuating. Onceunderway, the autoimmune process tends to favor continued stimulation of autoantibody production and thereby continued inflammation,whichtends to favor stimulation of the process. Furthermore,if B cells already are excessivelyproliferating and require only a signal for differentiationto initiate disease, manyregulatoryprocessesthat operate on proliferation havealready been circumvented.Therefore, once the disease is full-blown, a very strong immunosuppressive intervention maybe necessary to bring the entire system back toward normal. AUTOIMMUNITY

AND AUTOIMMUNE

DISEASES

Whatcan the analysis of AITand SLEtell us about autoimmune diseases? They suggest that whereas autoimmunity maybe normal, autoimmune diseases havea variety of features that lead to pathology.Agenetic basis for disease maybe a uniquegene; this could be true of certain HLA-B27associated diseases. Even there, additional genes and an environmental trigger maybe required. In AITand SLE,a greater numberof genesappear

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to be necessary to predispose to disease. Somegenes may code for cell surface determinants (e.g. a thyroid structural determinant in AIT or reduced or abnormal surface C receptors in SLE). Others maycode for high responsiveness to certain antigens (thyroglobulin or the thyroid stimulating hormonereceptor in AIT, nucleic acids or circulating cells in SLE). Still other genes maypredispose to immunedefects such as B-cell hyperactivity or impaired immuneregulatory processes. In view of the great complexity of the immuneregulatory circuits (110), a numberof different regulatory defects could all predispose to disease. A variety of different genes maybe very important in modulating the extent of inflammatory processes through the regulation of a variety of molecules that are so important in those processes. In addition to genetic factors, environmental factors mayserve to promote or retard disease. Environmental immuneactivators may be critical to the triggering of disease in certain patients with SLE. Femalesex hormones may predispose to, and male sex hormones may retard, disease. Nutritional factors maydetermine the degree of inflammation or autoantibody production (110). Different factors maybe moreimportant in different individuals. In AITand SLE,and especially in other diseases, such as some of the arthritides, chronic immunestimulation mayinitiate and/or perpetuate illness. Suchstimulation maybe viral (e.g. Epstein-Barr) or bacterial. Bacterial stimulation can occur through chronic release of stimulatory factors (e.g. endotoxin) or the persistence of nondegradablecell wall components. Does any of this help us to plan for the treatment or prevention of disease? It is possible that a complete understandingof the pathogenetic and etiologic mechanismsof disease might not immediately lead to prevention or specific therapy. Onthe other hand, it is possible that a direct approach to specific therapy or prevention might be apparent or at least theoretically possible. Appreciation that a subset of B cells is responsible for autoantibody production, and that elimination of that subset prevents disease in NZB, BXSB, (NZB X NZW)1, a nd ( NZB XBXSB) F1 mic e wit h SLE-like illness, suggests that a related approachmight be possible for patients with SLE. If a subset of humanB cells analogous to the Lyb3+,5+ cells of mice is found to be responsible for disease, that subset could be specifically eliminated by a monoclonal antibody specific for that subset. Such an appproach might be entirely nontoxic and might not interfere with most other normal immuneresponses. Recognition of the importance of antireceptor antibodies in myasthenia gravis could lead to specific prevention of the synthesis of those autoantibo-

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An appreciation that certain viruses or bacteria act to stimulate the immune systems of many people could lead to a vaccination program. We believe that the Epstein-Barr virus may be a very important underlying stimulus to excessive immuneactivity in a variety of situations. Vaccination against that virus alone might substantially reduce the number of people aflticted with a variety of autoimmune diseases. A similar attack on other stimulatory viruses, or bacteria with nondegradable but stimulatory cell wall components, could be carried out. In addition to preventing a variety of autoimmune diseases in many people, such an approach might reduce the severity of disease in others. Despite the possibility of specific intervention, study of autoimmune diseases should provide a deep respect for the variety of mechanisms responsible for normal immune regulation and therefore the multiple mechanisms by which those may be rendered abnormal. Nevertheless, the autoimmune diseases appear to be losing their mystery. Some are relatively simple; others are less so. They are complicated because of the multifactorial nature of the disorders, the complexity of the immune system, the multigenetic bases for disease, and our imperfect understanding of these and their interrelationships, Nevertheless, substantial insights are being gained into all of these. Knowledgeof the bases for individual autoimmunediseases is increasing rapidly. As more is learned, a greater number of details will fit into an even more understandable whole. Literature Cited 1. Abdou,N. I., Sagawa,A., Pascual, E., Hebert,J., Sadeghee,S. 1976.Suppressor T call abnormalityin idiopathicsystemic lupus erythematosus. Clin. lmmunol, lmmunopathol.6:192-99 2. Abdou, N. I., Wall,H., Lindsley,H.B., Halsey,J. F., Suzuki,T. 1981.Network theory in autoimmunity.In vitro suppression of serum anti-DNAantibody binding to DNA by anti-idiotypic antibodyin systemiclupus erythematosus. Z Clin. lnvest. 67:1297-304 3. Adams,D. D., Kennedy,T. H., Stewart, R. 1974.Correlationbetweenlongacting thyroid stimulator protector level andthyroid t31I uptakein thyrotoxicosis. Brit. Med.Z 2:199-201 4. Agnello, V. 1978. Associationof systemic lupus erythematosus and systemic lupus erythematosus-like syndromeswith hereditary and acquired complement deficient states. Arthritis Rheum. 21:S146-$60 5. Aldzuki,M., Reeves,J. P., Steinberg, A. D. 1978. Expression of autoim-

munity by NZB/NZW marrow. Clin. Immunol. Immunopathol.10:247-50 6. Alarcon-Segovia, D. 1982. Antibody penetrationinto living cells. III. Effect of antiribonucleoprotein IgGon the cell cycle of human peripheral blood mononuclearcells. Clin, lmmunol,lmmunopathol.23:22-33 7. Amino,N., Hidemitsu,M., Yoshinori, I., Seishi, A., Izumiguchi,Y., et al. 1982. Peripheral K lymphoeytesin autoimmune thyroid disease: Decreasein Graves’ disease and increase in Hashimoto’sdisease, or. Clin. Endo.Met. 54:587-91 8. Arnett, G. C., Shulman,L. E. 1976. Studies in familial systemiclupus erythematosus. Medicine55:313-22 gene 9. Benacerraf, B. 1981. Role ofMHC ¯ productsin immune regulation. Science 212:1229-38 10. Blaese, R. M., Grayson,J., Steinberg, A. D. 1980. Elevated immunoglobulin secreting cells in the bloodof patients with active systemlupus erythematosis: correlationof laboratoryandclinical as-

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AUTOIMMUNITY sessment of disease activity. A~ J. Med. 69:345-50 11. Block,S. R., Gibbs,C. B., Stevens,M. B., Shulman, L. E. 1968. Delayed hypersensitivity in sytemiclupus erythematosus. Ann. Rheum. Dig 27: 311-18 12. Block,S. R., Loekshin,M.D., Winfield, J. B., Wecksler,M.E., Iamura,M., et al. 1976. Immunologic observations on 9 sets of twinseither concordant or discordant for systemic lupus erythematosus. Arthritis Rheum. 19:545-54 13. Bocchieri, M.H., Cooke,A., Smith,J. B., Weigert,M., Riblet, R. J. 1982.Independentsegregation of NZBautoimmune abnormalities in NZB× C58 recombinant inbred mice. Eur. J. Immunol. 12:349-54 14. Bresnihan,B., Jasin, H. E. 1977. Suppressor function of peripheral blood mononuclear cells in normalindividuals andpatientswithsystemiclupuserythematosus.Z Clin. lnvest. 59:106-16 15. Buckman,K. J., Moore,S. K., Ebbin, A. J., Mavis,B. C., Dubois,E. L. 1978. Familial systemiclupus erythematosus. Arch. lnter~ Med. 138:1674-80 16. Budman, D. R., Merchant,E. B., Steinberg, A. D., Dolt, B., Gershwin,M.E., et al. 1977. Increasedspontaneousactivity of antibodyformingcells in the peripheralbloodof patients withactive systemiclupus erythematosus.Arthritis Rheum.20:829-33 17. Calder, E. A., Penhale, W.J., McLennan, D. 1973. Lymphocyte-dependent antibody-mediatedcytotoxicity in Hashimoto’s thyroiditis. Clin. Exp. Immunol. 14:153-58 18. Coombs, R, R. A., Gall, P. G. H. 1975. Classificationof allergic reactionsresponsiblefor clinical hypersensitivity anddisease. In Clinical Aspectsof Immunology,ed. P. G. H. Gell, R. R. A. Coombs,P. J. Lachmann,p. 761. Oxford: Blackwell 18a. Cowdery,J. S., Curtin, M. F. Jr., Steinberg, A. D. 1982. Theeffect of prior intragastrie antigen administration on primary and secondary antiovalbtunin responses of C57BL/6and NZBmice. J. Exp. Med. 156:1256-61 18b. Cowdery, J. S., Steinberg,A. D. 1982. Regulation of primary, thymusindependent,anti-hapten responsesof normal and autoimmunemice by syn~2eneic antibody. J. lmmunol. 129: 50-55 19.Datta, S.D.,Owen, F.L.,Womack, J. E.,Riblet, R.J.1982.Analysis of

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recombinantinbred lines derived from "autoimmune"(NZB)and "high leukemia"(C58)strains: independentmultigenic systemscontrol B cell hyperactivity, retrovirus expression, andautoimmunity. J. lmmunol.129:1539-44 20. DeHoratius,R. J., Messner,R. P. 1975. Lymphoeytotoxic antibodies in family membersof patients with systemic lupus erythematosus,d. Clin. lnvest. 55:1254-58 21. Delfraissy,J. F., Segund,P., Galanaud, P., Wallon,C., Massias,P., et al. 1980. Depressedprimaryin vitro antibodyresponsein untreatedsystemiclupus erythematosus: T helper cell defect and lack of defective suppressorcall function. J. Clin. lnvest. 66:141-48 22. Draehman,D. B., Adams,R. N., Josifek, L. F., Self, S. G. 1982.Functional activities of autoantibodiesto acetylcholinereceptorsandthe clinical severity of myastheniagravis. N. Engl. J. Meal. 307:769-75 23. Drexhage,H. A., Bottazo, G. F., Bitensky, L., Chayen,J., Doniaeh, D. 1981. Thyroidgrowth-blockingantibodies in primary myxoedema.Nature 289:594-98 24. Eisenberg, R. A., Dixon, F. J. 1980. Effect of castration on male-determined acceleration of autoimmune disease in BXSB mice. J. Immunol.125:1959-61 25. Eisenberg,R. A., Izui, S., McConahey, P. J., Hang, L., Peters,C. J., et al. 1980. Male determined accelerated autoimmunedisease in BXSB mice: transfer by bone marrow and spleen cells. J. lmmunol. 125:1032-36 26. Endo, K., Kasagi, K., Konishi, J., Ikekubo, K., Okuno,K., et al. 1978. Detectionand properties of TSH-binding inhibitor immunogiobulins in patients with Graves’ disease and Hashimoto’s thyroiditis. J. Clin. Endocrinol. Metab. 46:734-39 27. Fauci, A. S., Steinberg,A. D., Haynes, B. F., Whalen,G. 1978. Immunoregulatory aberrationsin systemiclupus erythematosus. J. Immunol.121:1473-79 28. Foad,B. S., Khullar,S., Freimer,E. H., Kirsner,A. B., Sheon,R. P.. 1975.Cellmediated immunityin systemic lupus erythematosus:alterations with advancing age. J. Lab. Clin. Med.85:133-39 29. Fournie, G. J., Lambert, P. H., Miescher, P. A. 1974. Release of DNA in circulating blood and induction of anti-DNAantibodies after injection of bacterial lipopolysaccharides. Z Exp. Med. 140:1189-206

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30. Frank, M.M., Hamburger,M. I., Lawley, T. J., Kimberly, R. P., Plotz, P. H. 1979.Defectivereticuloendothelialsystem Fc-receptor function in systemic lupus erythematosus.N. Engl. J. Med. 300:518-23 31. Friedman,R. M., Preble, O., Black,R., Harrell, S. 1982.Interferon production in patients with systemic lupus erythematosus.Arthritis Rheum.25:802-3 32. Gershwin,M.E., Castles, J. J., Erickson, K., Ahmed,A. 1979. Studies of congenitally immunolo#cmutant New Zealandmice.II. Absenceoft cell progenitorpopulationsandBcell defects of congenitally athymie(nude) NewZealand Black (NZB)mice. J. ImmunoL 122:2020-25 33. Gershwin,M.D., Glinski, W., Chused, T. M., Steinberg, A. D. 1977. Lymphocyte dependent antibody mediated cytolysis of DNA-anti-DNA coated target cells using humanand routine effector populations. Clin. lmmunoZ lmmunopathol.8:280-91 34. Gibofsky, A., Winchester, R. J., Hansen,J., Patarroyo, M., Dupont,D., et al. 1978. Contrasting patterns of newerhistocompatibility determinants in patients with rheumatoidarthritis and systemic lupus erythematosus.Arthritis Rheum.2h134s-38s 35. Ginsburg, W.W., Finkelman, F. D., Lipsky, P. E. 1979. Circulating and pokeweed mitogen-induced immtmoglobulin-secretingcells in systemlupus erythematosus. Clin. Exp. lmmunol 35:76-88 E., Issa, P., VanElven,E. 36. Gleichmann, H., Lamers, M. C. 1978. The chronic graft-versus-hostreaction: A lupuserythematosus-like syndromecaused by abnormalT-Bcell interactions. Clin. Rheum.Diz 4:587-602 37. Glinski, W., Gershwin,M.E., Budman, D. R., Steinberg, A. D. 1976. Studyof lymphocytesubpopulations in normal humansand patients with systemic lupus erythematosus by fractionation of peripheral bloodlymphocyteson a discontinuousficoll gradient. Clin. Exp. Immunol. 26:228-38 38. Glinski, W., Gershwin,M.E., Steinberg, A. D, 1976.Fractionationof cells on a discontinuous ficoll gradient.Study of subpopulationsof humanT-cells using anti-T-cell antibodiesfrompatients with systemic lupus erythematosus.£ Clin. lnvesL 57:604-14 39. Gocke, D, J., Hsu, K., Morgan,C., Bombardiere,S., Lockshin,M., et al. 1970.Associationbetweenpolyarteritis

and Australia antigen. Lancet 2: 1149-53 40. Goto, M., Tanimoto,K., Horiuehi, Y. 1980. Natural cell mediatedeytotoxicity in systemic lupus eqahematosus. Suppression by antilymphocyteantibody. Arthritis Rheum.23:1274-81 41. Hang,L., Izui, S., Slack, J. H., Dixon, F. J. 1982.Thecellular basis for resistance to inductionof tolerance in BXSB systemic lupus erythematosus male mice. J. lmmunoL129:787-89 42. I-lirata, F., del Carmine,R., Nelson,C. A., Axelrod,J., Sehithnan,E., et al. 1981. Presence of autoantibody for phospholipase inhibitory protein, lipomodulin,in patients with rheumatic diseases. Pro¢. NatL Aca~S¢£ US.4 78:3190-94 43. Iida, K., Mornaghi,R., Nussenzweig, V. 1982. Complement receptor deftciency in erythroeytes from patients with systemic lupus erythemtosus..L Exp. MetL155:1427-38 44. Inman, R. D., Jovanovie, L., Dawood, M. Y., Longcope, C. 1979. Sy~tmmie lupus erythematosusin the male: A genetic and endocrine study. Arthritis Rheum. 22:624 45. Jasin, H. E., Ziff, M. 1975. Immunoglobulin synthesis by peripheral blood cells in systemic lupus erythematosus. Arthritis Rheum.18:219-28 46. Jungers,P. J., Dougados, M., Pelissier, C., Kuttem,F., Tron,F., et al. 1982. Influenceof oral contraceptivetherapy on the activity of systemiclupus erythematosus. Arthritis Rheur~ 25: 618-23 47. Jyonouehi, H., Kincade, P. W., Landreth,K. S., Lee,G., Good,R. A., et al. 1982. Age-dependentdeficiency of B lymphocytelineage precursors in NZB ttfice. £ Exp. Med.155:1665--78 48. Kahn,C. R. 1980. Roleofinsulinrecq~tors in insulin-resistant states. Metabolism 29:455-66 49. Kalderon,A. E. 1980. Emergingrole of immune complexes in autoimmune thyroiditis. PathoLAnn.15:23--35 50. Kaplan, M. H., Svec, K. H. 1964. Immunologic relation of streptococcal and tissue antisens. III. Presencein human sera of streptococcal antibody crossreactive with heart tissue, Association with streptococcalinfection, rheumatic fever, and glomerulonephritis.J. Exp. MecL119:651-65 51. Kemp,J. D., Cowdery, J. S.,.Steinberg, A. D., Gershon, R. K. 1982. Genetic controls of autoimmunediseases: In-

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AUTOIMMUNITY teractions betweenMdand lpr. Z ImmunoL128:388-92 52. Kidd,A., Okita, N., Row,V. V., Volpe, R. 1980. Immunologic aspects of Graves’ and Hashimoto’sdiseases. Metabolisra 29:80-99 53. Kincade,P. W., Lee, G., Fermmdes, G., Moore,M.A. S., Williams,N., et al. 1979.Abnormalitiesin clonable B lymphocytesand myeloidprogenitorsin autoimmuneNZBmice. Proc. Natl. Acad. ScL USd76:3464-68 54. Kincade, P. W., Paige, C. J., Parkhouse, IL M.E., Lee, G. 1978. Characterization of routine colony-formingB cells. I. Distribution,resistanceto antiimmunoglobulinantibodies, and ressionof Ia antigens. J. IraraunoL 20:1289-96 55. Kohler, P. F., Percy, J., Campion,W. M., Smyth,C. J. 1974.Hereditaryangicedema and "familial" lupus crythematosus in identical twin boys. J. Meal56:406-11 56. Kotani, T., Komuro,K., Yoshiki, T., Itoh, T., Aizawa,M.1981. Spontaneous autoimmune thyroiditis in the rat accel~rated by thymectomy and low dos~ of irradiation: mechanisms implicated in the pathogenesis. Clin. Exp. ImraunoZ 45:329-37 57. Kumagal,S., Sredni, B., House, S., Steinberg,A.D,, G-r~°n,I. 1982.Defective re~ulation of B lymphocytecolony formation in patients with systemic lupus erythematosus..LIramunoZ128: 258-62 S., Steinberg,A. D., Green,I. 58. Kumagai, 1981.Antibodiesto T cells in patients with systemiclupus erythematosusmediated~ADCC against humanT cell. d.. Clir~ Im~st 67:605-14 58a. Kumagai, S., Steinberg, A. D., Green, L 1981. [mmtm¢ responses to kaptenmodifiedself and their regulation in normal individuals and patients with SLE. ,L Imraunol 127:1643-52 59. Lahita, R. O., Bradlow,L., Fishman,J., Kunkea,H. G. 1982. Estrogen metabolism in systemiclupus eryth~matosus: patients andfamily members. ,4rthr/t/s Rheum.25:843-46 60. Lahita, R. G., Bradlow,H. L, Kunkel, H. G., Fishman,J. 1981. Increased 16 e-hydroxylation of estradiol in systemic lupus erythematosus. J. Cli~ EndocrinoL Metab. 53:174-78 61. Laskin,C. A., Snmthers,P. A., Reeves, J. P., Steinberg,A.D. 1982.Studies of defective tolerance induction in NZB mice: Evidencefor a marrowpre-T cell defect. Z Exp. Mea~155:1025-36

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62. Laskin,C. A., Taurog,J. D., Smathers, P. A., Steinberg,A. D. 1981.Studiesof defective in murinelupus. Z Iraraunol.tolerance 127:1743-47 63. Lewis,D. E., Giorgi,J. V., Warner,N. L. 1981. Flowcytometryanalysis of T cells and continuousT-cell lines from autoimmune MRL/1 mice. Nature 289:298-300 64. Lewis, R. M., Armstrong, M. Y. K., Andre-Schwartz,J., Muftooglu, A., B~ldotti, L,, et al. 1968.Chronicallogeneie disease. I. Developmentof #omerulonephritis. Z Exp. Mad.128: 653-67 65. Loweustein, M. B., Rothfield, N. F. 1977. Familystudy of systemic lupus erythematosus:analysis of the clinical history, skin immunofluorescence, and serologic parameters.Arthritis Rheum. 20:1293-303 66. Manohar,V., Brown,E., Leiserson, W. M., Chused,T. M.1982. Expressionof Ly-I by a subset of B lymphocytes.J. Iramunol. 129:532-38 67. Miller, K. B., Schwartz,R. S. 1979. Familial abnormalities of suppressor cell function in systemic lupus erythematosus.N. Engl. J. Med.301:803-9 68. Mitsunaga,M., Suzuki, S., Natagawo, O., Hirakawa, S., Miura,H., et ai. 1980. Cytophilic antibodies and monocytemediatedcytotoxicity in Hashimoto’s thyroiditis. In ThyroidResearchVIIL ¢d. J. R. Stockigt,T. Nagataki,pp. 78992. NewYork: Pergamon 69. Miyakawa,Y., Yamada,A., Kosaka, K., Tsuda,F., Kosugi,E., et al. 1981. Defective immune-adherence (C3b) receptor on erythocytes frompatients with systemic lupus erythematosus. Lancet 2:493-97 70. Moil, T., Kriss, ~. P. 1971. Measurementsby competitivebinding radioassay of serumanti-microsomaland antithyroglobulinantibodiesin Graves’disease and other thyroid disorders, J. Clin. Endocrinol.Metab.33:688-98 71. Morimoto, C., Abe, T., Hara, M., Homma, M. 1977. In vitro TNPspecific antibodyformationby peripheral lymphocytesfrom patients with systemiclupus erythematosus.Scand.d. IramunoL6:575-79 72. Morimoto,C., Reinherz,E. L., Nadler, L. M., Distaso, J. A., Steinberg,A. D., et al. 1982.Comparison in T- and B-cell markersin patients with Sjogren’ssyndrome and systemic lupus erythematosus. Cli~ ImmunoL Immunopatho£22:270-78

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73. Morimoto, C., Sano, H., Abe, T., Homma,M., Steinberg, A. D. 1982. Correlationbetweenclinical activity of systemic lupus erythematosusand the mounts of DNAin DNA/anti-DNA antibody immunecomplexes. J. ImmunoL129:1960-65 74. Morton,J. I., Siegel,B. V.1974.Transplantation of autoimmune potential. I. Development of antinuclear antibodies in H-2histocompatible recipients of bone marrowfrom NewZealand black mice. Proc. Natl. Acad. Sci. USA71: 2162-65 75. Morton,J. I., Siegal,B. V.1978.Transplantation of autoiinmunepotential. III. Immunologicalhyper-tesponsiveheSSand elevated, endogenousspleen colonyformationin lethally irradiated recipients of NZBbone marrowcells. Immunology34:863-68 76. Morton, R. O., Gershwin, M. E., Brady,C., Steinberg,A. D. 1976.Incidenceof systemic lupus erythematosus (SLE) in North AmericanIndians. Rheum. 3:186-90 77. Murphy,E. D. 1981. Lymphoproliferafion (LPR)and other single-locus models for routine lupus. In Immunologic Defects in LaboratoryAnimals,ed. M. I~. Gershwin,B. Merchant,2:143-73. NewYork: Plenum 78. Murphy,E. D., Roths, J. B. 1979. A Y-chromosome associated factor in strain BXSB producingaccelerated autoimmunityand lymphoproliferation. Arthritis Rheum.22:1188-94 79. Nies, K. M., Louie,J. S. 1978.Impaded immunoglobulinsynthesis by peripheral blood lymphocytes in systemic lupus erythematosus.Arthritis Rheum. 21:51-57 80. Ohsugi, Y., Gershwin,M. E., Ahmed, A., Skelly,R., Milich,D.R. 1982.Studies of congenitally immunologic mutant NewZealand mice IV. Spontaneous and inducedautoantibodiesto red cells and DNAoccur in NewZealand Xlinked immunodeficientmice without phenotypie alterations of the Xia geneor generalized polyclonal B cell activation. J. ImmunoL 128:2220-27 81. Okita, N., Kidd,A., Row,V. V., Volpe, R. 1981.SuppressorT lymphocytedeficiency in Graves’ disease and Hashimoto’s thyroiditis. Z Clit~ EndocrinoL Metab. 52:528-33 82. Okudalra,K., Seades,R. P., Tanimoto, K., Hoduchi,Y., Williams, R. C. Jr. 1982. T lymphocyteinteraction with immunoglobulinG antibody in sys-

temiclupus erythematosus.J. Clin. Invest. 69:1026-38 83. Oldstone,M.B. A., Dixon,F. J. 1972. Inhibitionof antibodiesto nuclearantigen and to DNAin NewZealand mice infected with lactate dehydrogenase virus. Science175:784--88 84. Palacios, R., Alarcon-Segovia, D., Llorente, L., Ruiz-Arguelles, A., DiazJouanen, E. 1981. Humanpostthymic precursorcells in healthanddisease.II. Their loss and dysfunctionin systemic lupus erythematosusand their partial correction with serumthymicfactor. J. Clin. Lab. Immunol.5:71-80 84a. Parris, T. M., Kimberly,R. P., Inman, R. D., McDougal,J. S., Gibofsky,M. D., Christian, C. L. 1982.DefectiveFc receptor-mediated function of the mononuclearphagocytesystemin lupus nephritis. Ann.Int. Med.97:526-32 85. Polley, C. R., Bacon,L. D., Rose,N. R. 1977.Failure of functional bursa cells transplanted betweensusceptible OS and resistant CSstrains of chickensto influence spontaneous autoimmune thyroiditis. Fed.Proc.36:1207 86. Ranney,D. F., Steinberg, A. D. 1976. Differencesin the age-dependent release of a low molecular weight suppressor (LMWS)and stimulators by normal and NZB/W lymphoid organs. J. Immunol. 117:1219-25 87. Raveche, E. S., Novotny, E. A., Hansen,C. T., Tjio, J. H., Steinberg,A. D. 1981. Geneticstudies in NZBmice. V. Recombinantinbred lines demonstrate that separate genes control autoimmune phenotype. J. Exp. Med. 153:1187-97 88. Raveche,E. S., Steinberg, A. D. 1979. Lymphocytes and lymphocyte functions in systemic lupus erythematosus. Clinics Haematol.16:344-70 89. Raveche,E. S., Steinberg, A. D., DeFranco, A. L., Tjio, J. H. 1982. Cell cycle analysis of lymphocyte activation in normal129:1219-26 and autoimmunestrains. J. Immunol. 90. Raveche, E. S., Steinberg, A. D., Klassen, L. W., Tjio, J. H. 1978. Genetic studies in NZB mice. I. Spontaneous production. J. Exp. Med.autoantibody 147:1487-502 91. Reinertsen,J. L., Klippel,J. H., Johnson, A. H., Steinberg,A. D., Decker,J. L. 1982. Family studies of B lymphocytealloantigens in systemiclupus erythematosus.J. Rheumatol.9:253-62 92. Reinertsen,J. L., Klippel, J. H., Johnson, A. H., Steinberg,A. D., Decker,J. L. et al. 1978. B-lymphocyte all0anti-

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gens associatedwithsystemiclupus ery- 103. Sober,I., Steinberg,A. D., Berning,A. D., Paul, W.E. 1975. X-linkedB-lymthematosus. N. Engl. d. Med. 299: phocyte immune defect in CBA/N 515-58 mice. II. Studies of mechanisms under93. Roitt, I. M,, Doniach,D., Campbell,R. lying the immune defect. J. Exp. Med. N., Hudson,R. V. 1956. Autoantibo142:637-50 dies in Hashimoto’sdisease. Lancet 104. Siegel, M., Holley, H. L., Lee, S. L. 2:820-21 1970. Epidemiologiestudies on sys94. Romain,P. L., Cohen,P. L., Fish, F., temic lupus erythematosus. Arthritis Ziff, M.,Vitetta,E. S. 1980.Thespecific Rheura. 13:802-11 B cell subset lacking in the CBA/N mouseis not required for the produc- 105. Silverman, D. A., Rose, N. R. 1974. Neonatalthymectomy increases the intion of autoantibody in (CBA/N cidence of spontaneous and methylNBZ)Ftmale mice. d.. lmmunoL125: cholanthrene-enhaneedthyroiditis in 246--51 rats. Science 184;162-63 95. Rose, N. R., Kang,¥. M., Okayasu,I., Giraldo,A. A., Beisel, K., et al. 1981. 106. Smathers, P. A., Steinberg, B. J., Reeves, J. P., Steinberg, A. D. 1982. T-cell regulation in autoimmune Effects of polyclonal immune stimulathyroiditis, lmraunol.Rev. 55:299-314 tors upon NZB’xideongenie mice. J. 96. Rose,N. R., Witebsky,E. 1956.Studies Immunol. 128:1414-19 on organ specificity V. changesin the thyroidglandof rabbits followingactive 106a. Smith, H. R., Chused,T. M., Smathors, P., Steinberg,A. D. 1983.Evidence immunizationwith rabbit thyroid exfor thymicregulation of autoimmunity tracts. J. lraraunol.76:417-27 in BXSB mice: Accelerationof disease 97. Roscnwasser,L. J., Huber,B. T. 1981. by neonatal thymectomy.J. lmmunol. The xid gene controls ia. W39130:Inpress associated immune responsegene func107. Smith,H. R., Green,D. R., Raveche,E. tion. J. Exp. Med.153:1113-23 S., Smathers,P. A., Gershon,R. K., et 98. Roubinian, J. R., Papoian,R., Talal, N. al. 1982.Studieson the inductionofan1977. Androgenichormonesmoderate ti-DNAin normalmice. ,L. Iraraunol. autoantibody responses and improve 129:2332-34 survival in mudnelupus. Z Clit~ Invest. 108. Smolen, J. S., Chused,T. M., Leiserson, 59:1066-70 W.M., Reeves, J. P., Ailing, D. W. 99. Sakane,T., Steinberg,A. D., Green,I. 1982. Heterogeneityof immunoregula1978. Studies of immunefunctions of tory T cell subsetsin systemiclupuscrypatients with systemic lupus erythematosus.Correlation with clinical thematosus.I. Failure of suppressorT Am, £ Med.72:783-90 cell activity related to impairedgenera- 109. features. Smolen,J. S., Steinberg, A. D. 1981. tion of, rather than responseto, supSystemic lupus erythematosus and pressor cells. Arthritis Rheura.21: pregnancy. Clinical, immunological 657-64 and theoretical aspects. In Reproductive 100. Sakane,T., Steinberg,A. D., Green,I. Iramunology, ed. N. Oleicher, pp. 2831978.Failure of autologousmixedlym302. NewYork: Liss phocytereactions betweenT and non-T 110. Smolen,J. S., Steinberg, A. D. 1982. cells in patientswithsystemiclupuseryDisordersof immune regulation. In The thematosus.Proc. Natl. Acad.ScL USA Pathophysiology of Huraan Ira. 75:3464-68 raunologic Disorders, ed. J. Twomey, 101. Sakane,T., Steinberg,A. J., Green,I. 10:173-98. Baltimore: Urban & 1980. Studies of immune functions of Schwarzenberg patients with systemic lupus ery111. Staples,P. J., Steinberg,A.D., Talal,N. thematosus.V. T-cell suppressorfunc1970. Induction of immunological toltion and autologousMLR during active erance in older NewZealand mice andinactive phasesof disease. Arthritis repopulated with young spleen, bone Rheurr~23:225-31 marrow or thymus, d. Exp. Med. 102. Sakane,T., Steinberg,A. D., Reeves,J. 131:1223-38 P., Green,I. 1979. Studies of immune 112. Steinberg, A. D. 1980. Autoimmunity. functions of patients with systemic In Strategiesof lrarauneRegulation,ed. lupus crythcmatosus. Complement deE. E. Sercarz, A. J. Cunningham,p. pendentimmunoglobulin M anti503. NewYork: Academic thymus-derived cell antibodies prefer-113. Steinberg, A. D., Huston,D. P., Tanentially inactivate suppressor cells..L rog, J. D., Cowdery,J. S., Raveehe,E. Clin. lnvest. 63:954-65 S. 1981.Thecellular and genetic basis

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of routine lupus. Immunol. Rev. 55:121-54 114. Steinberg, A. D., Law,L. W., Talal, N. 1970. The role of the NZB/NZWFt thymus in experimental tolerance and autoimmunity. Arthritis Rheum‘ 13: 369-77 A. D., Melez, K. A., 115. Steinberg, Raveche,E. S., Reeves,J. P., Boegel, W. A., et al. 1979. Approachto the study of the role of sex hormones in autoimmunity. Arthritis Rheum-22:1170-76 116. Steinberg, A. D., Roths, J. B., Murphy, E. D., Steinberg, R. T., Raveche, E. S. 1980. Effects ofthymectomy or androgen administration upon the autoimre.tree disease of MRL/MF-lpr/lpr trace. £ Immunol. 125:871-73 117. Steinberg, B, l., Smathers,P. A., Frederiksen, K., Steinberg, A. D. 1982. Ability of the xMgene to prevent autoimmunity in (NZB X NZV OFt mice durhag the course of their natural history after polyclonal stimulation or following immunization with DNA.J. Clin. Invest. 70:587-97 118. Stem, R., Fishman, J., Brusman, H,, Kunkel, H. 13. 1977. Systemic lupus erythematosus associated with Klinefelter’s syndrome. :trthritis Rheun~20: 18-22 119. Tautog, J. D., Raveche,E. S., Smathers, P. A., Glimcher, L. H., Huston, D. P., et al. 1981. T cell abnormalities ha NZB mice occur independently of autoantibody production. £ Exp. Med. 153: 221-34 120. Tanrog, J. D., Smathers, P. A., Steinberg, A. D. 1980. Evidence for abnormalities in separate lymphocytepopulations in NZBmice. J. lmmunoL 125: 485-90 121. Theofilopolous, A. N., Dixon, F. L 1981. Etiopathogenesis of murine systemic lupus erythematosus, lmmunoL Re~. 55:179-216 122. Till, J. E., McCulloch,E. A. 1964. Reo air processes in irradiated mouse ematopoletic tissue. Ann. NY Acad. Sci. 114:115-25 123. Tonietti, G., Oldstone, M. B., Dixon, F. J. 1970. The effect of induced chronic viral infections on the immunologicdis-

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eases of NewZealand mice..L Mee£ 132:89-109 124. Walport, M. J., Fielder, A. H. L., Batchdor, J. R., Black, C. M., Rynes, K. I., et al. 1982. I-ILA linked complement allotypes and genetic susceptibility to systemic lupus erythematosus. Arthritis Rheum.24:s41 125. Warner, N. W., Moore, M. A. $. 1971. Defects in hematopoietie differentiation in NZB and NZC mice. £ Exp. MecL 134:313-34 126. Wick, G., Boyd, R., Hala, K., Thunold, S., Kotler, H. 1982. Pathogenesis of spontaneous autoimmunethyroiditis in obese strain (OS) chickens. Clin. Exp. ImmunoL 47:1-18 127. Wick, G., Gtmdolf, R., Hala, K. 1979. Genetic factors in spontaneous autoimmunethyroiditis in OS chickem. ,L Immuno. Genet. 6:177-83 128. Wick, G., Kite, J. H. Jr., Witebsky, 1970. Spontaneous thyrolditis in the obese strain of chicketm. IV. The effect of thymectomy and thymo-bursectomy on the development of the disease. lmmunoZ 104:54-62 128a. Wilson, J. G., Wong, W. W., Schur, P. H., Fearon, D. T. 1982. Modeof inheritance of decreased C3b receptors on erythrocytes of patients with systemic lupus erythematosus. A~. EngL £ Med. 307:981-86 129. Winfield, J. B., Winchester, R. J., Wernet, P., Fu, S. M., Kunkel, H. G. 1975. Lymphocytotordc antibodies to lymphocyte surface determinants in systemic lupus erythematosus. Arthritis Rheum. 18:1-8 130. Wofsky, D., Murphy, E. D., Roths, B., Dauphinee, M. J., Kipper, S. B., et al. 1981. Deficient interleukin 2 activity in MRL/Mpand C57BI/6J mice bear. ing the lpr gene. J. Exp. Mee£ 154: 1671-80 131. Yoshida, H., Amino, N., Yagawa, K., Uemura,K., Satoh, M., et al. 1978. Association of serum antithyroid antibodies with lymphocyticinfiltration of the thyroid gland. Study of 70 autopsied eases. J. Clin. EndocrinoL Metab. 46: 859-62

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MECHANISMS OF T CELL-B CELL INTERACTION Alfred Singer and Richard J. Hodes Immunology Branch, National CancerInstitute, National Institutes of Health, Bethesda, Maryland20205

INTRODUCTION The demonstration that interactions amongdistinct immunocompetentcell types were required for the generation of most immuneresponses signalled the beginning of a new era in cellular immunology.The subsequent demonstrations that these cellular interactions were regulated by cell surface gene products encoded by the major histocompatibility complex (MHC)provided investigators with a precise and potent tool for the dissection of immunecell interactions. In addition, these observations provided the basis for truly dramatic insights into the reasons for the existence of an MHC. Thus, modemday cellular immunologyis the offspring of the union between cell biology and immunogenetics. In this review, we focus on the genetic regulation of the cellular interactions required for the T helper cell-dependent activation of B cells to secrete immunoglobulin.However, it is becomingincreasingly clear that the general principles that apply for the cellular interactions involvedin B cell activation also apply for the cell interactions involved in a broad variety of immuneresponses.

ACTIVATION OF ANTIGEN-SPECIFIC B CELLS REQUIRES INTERACTIONS WITH T HELPER CELLS The first demonstrationthat cell interactions were required for the generation of immuneresponses came from the seminal experiments of Claman et al (21), whichwere soon followed by the experimentsof Davieset al (22) and of Mitchell &Miller (64, 66, 67). Clamanand co-workers demonstrated that the antibody response to sheep erythrocyte (SRBC)antigen by lethally irradiated mice could be fully reconstituted by the adoptive transfer of spleen cells, but could not be reconstituted by the adoptive transfer of either 211 0732-0582/83/0410-0211 $02.00

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bone marrow or thymus cells. However, the antibody response to SRBC antigen in lethally irradiated mice could be fully reconstituted by the simultaneous transfer of both bone marrowcells and thymus cells. Thus, the transferred bone marrowand thymus cells cooperated synergistically, resulting in the generation of an anti-SRBCresponse. These investigators suggested that the bone marrowwas the source of antibody-secreting cells and that the thymus was the source of an "auxilliary" cell. Definitive evidence that the precursors of antibody-producing cells were contained within the bone marrow population and not within the thymus population was provided by the experiments of Miller & Mitchdl (64, 66, 67). Using an adoptive transfer system in which the bone marrowcells and the thymuscells were derived from H-2 different strains, these investigators observedthat all antibody-secreting cells expressed the H-2 type of the bone marrow. Three conclusions could be drawn from these experiments. First, antibody-secreting cells were derived from the bone marrow(referred to as B cells). Second, thymus cells (referred to as T cells) were required "helper" cells for the activation of B cells. Andthird, histoincompatible T and B cells could cooperate with each other.

EFFECTIVETH-B CELL INTERACTIONREQUIRES TH CELL RECOGNITIONOF CARRIER DETERMINANTSAND B CELL RECOGNITION OF HAPTENIC DETERMINANTS The experiments that provided the first understanding of the mechanismby which T helper (Tu) cells and B cells interacted were performed by Mitchison (68), by Rajewskyet al (82), and by Raft (81). Assessingthe anti-hapten antibody response stimulated by carrier-hapten conjugates, these investigators observed that optimal IgG anti-hapten secondary responses required that the T cells be previously primedto carder and the B cells be previously primed to the hapten. They concluded that the T cells recognized carrier determinants whereas the B cells recognized haptenic determinants. Furthermore, they noted that optimal anti-hapten responses were only elicited when the carder and hapten were physically linked on the same molecule. Thus, these investigators proposed the key concept that effective TH-Bcell collaboration involved the physical joining of a THcell with its partner B cell via an antigen bridge. It was clear from this modelthat the antigenic determinants recognized by the Tt-i cell could be (must be?) quite distinct from the determinants recognized by its partner B cell (i,e. the haptenic determinants).

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INVOLVEMENT OF MHC ENCODED GENE PRODUCTS IN TrI-B CELL COLLABORATION Next,the possibility that the interaction betweenTit cells andtheir partner B cells mightbe genetically regulated wassystematically addressed. In contrast to the repeated observation from a numberof laboratories that histoincompatibleTn cells and B cells could cooperatewith each other, experimentsreported by Kindred& Shreflter suggestedthat thymus-derived cells could not effectively cooperatewith histoincompatiblenudeB cells (54). Katzet al then providedcritical insights into this issue by demonstrating that the mechanismby whichhistoincompatible Tn and B cells collaborated was distinct from the mechanism by whichhistocompatible Tn and B cells collaborated(30, 50). Usinga carrier-hapten response, they observedthat anti-hapten B cell responsesresulting fromthe interaction of histoincompatible TI~andB cells did not require carrier primingand did not require carrier-haptenlinkage. In contrast, they observedthat anti-hapten responsesresulting from the interaction of histocompatibleTit cells and B cells did require carrierprimedTI~cells anddid require carrier-hapten linkage. Theseexperiments wereinterpreted as demonstrating that the activation of B cells requiredtwo distinct signals, oneresulting fromthe bindingof antigenby the B cell and onederivedfromthe Tit cells. Thus,the activationof antigen-specificB cells by either histocompatibleor histoincompatibleTit cells involvedB cell bindingof the haptenas well as B cell triggeringby a Tit cell-derivedBcell activation signal. It wasin the mechanism by whichthe Tn cell-derived activation signal was dicited from the Tn cells that the two responses fundamentally differed. In the case of histoincompatible Tncells, the activation signal waselicited fromthe Tit cells by their recognitionof the allogeneic MHC determinants expressed by the B cells. This mechanismwas termedthe allogeneiceffect anddid not require THcell recognitionof any carrier determinants.In contrast, in the case of histocompatibleTri cells, the activation signal waselicited fromthe Tit cells by their recognitionof carrier determinantssimultaneouslywith their recognition of syngeneic MHC determinants expressed by the B cells. This latter mechanismwas termed phyfiologic cooperation because it was presumablythe mechanism by whichTrrB cell interactions occuredin a normalanimal. It shouldbe emphasized that the experimentsby Katzet al did not refute the ability of Tit cells to activate histoincompatibleB cells, but rather demonstratedthat such an interaction resulted froman entirely different mechanism than that involving histocompatibleTit and B cells. Katz et al went on to showthat physiologicinteractions betweenTn and B cells did

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not require completeMHC compatibility, but only required I-region compatibility (49). Since it wasuncertainthat the I-region-encoded structures involvedin TI~-Bcell interactions wereidentical with the serologieally defined Ia antigens, they referred to these structures as cell interaction molecules. Nearlysimultaneouslywith the reports of Katzet al, Rosenthal& Shevachreported that T-accessorycell interactions weresimilarly regulatedby MHC encoded genes in that only MHC compatible accessory cells could presentantigento antigen-primed T cells (84). In add, ition, these investigators demonstratedthat anti-MHC antisera effectively blockedT-accessory cell interactions, formallydemonstrating that this cellular interaction was mediated by MHC encodedgene products that were expressed on the cell surface, andstronglysuggestingthat the cellular interaction moleculeswere likely to be the serologically defined MHC antigens themselves. MHC REGULATION OF Tn-B CELL INTERACTION IS MEDIATED BY A RECEPTOR-LIGAND INTERACTION Therequirementfor Ia compatibility for the physiologiccooperationbetweenTHcells andB cells still neededto be explained.Onepossibility was that a like-like interaction betweenIa encodedcell surface moleculesexpressedon Tri cells and B cells wasrequired to activate B cells. Asecond possibility wasthat carrier-specific THcells neededto recognizevia their cell interaction structures the Ia-encodeddeterminantsexpressedby B cells. Thus,the requirementfor MHC compatibility betweenTri and B cells could haverelteeted either a like-like interaction or a receptor-ligandinteraction in whichcartier-specific THcells could only recognizesyngeneic,but not allogeneic, Ia encodedB cell interaction structures. Theanswerwasprovidedquite clearly by the experimentsof von Boehmeret al (111). Theseinvestigators constructeddouble-parentalradiation bone marrowchimeras of the type PI+P2 ~ (PIXP2)FI. The lethally irradiated Fi mice were completely repopulated by lymphoid elements derived from bone marrowstemcells of both parents. Thesechimeric mice were then primedin vivo to SRBC,and the SRBC-primed chimeric T ceils were assayedfor their ability to cooperatewith SRBC-specific B cells of P1 or P2 type. Sincesomeof the chimericT cells wereof PI origin andsome of P2 origin, these investigatorsused strain-specific alloantisera plus complement(C) to eliminateT cells of either P1 or P2 origin selectively. Upon isolating the P1 T cells fromthe chimeras,it wasfoundthat, as a result of the chimerization,P~ T cells weretolerant to both parental haplotypesso that these T cells could not activate either parental B cell populationvia

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T CELL-B CELL INTERACTION 215 an allogeneic effect. WhenSRBC-primed P1 T cells were assayedfor their ability to cooperatewith B cells of either P1 or P2 origin, it wasobserved that they couldeffectively, andphysiologically,cooperatewith either parental B cell populationfor the generationof anti-SRBC responses.Thus,since chimeric P1 T cells could cooperate with P2 B cells, these experiments demonstratedthat physiologiccooperationbetweenTHcells and B cells did not require MHC identity and, therefore, that TH-Bcell interactions could not be mediatedby a like-like mechanism. Althoughthe experimentsof yon Boehmeret al effectively ruled out a like-like interaction mechanism, their results werealso apparentlydiscrepant with the experimentsof Katzet al in that they failed to showany role for MHC at all. Thus, these experiments provokedrenewedskepticism about the central role of the MHC in the regulation of physiologiccooperation betweenTHcells and B cells. After all, Katzet al haddemonstrated that, in the absenceof allogeneiceffects, histoincompatible THandB cells could not cooperate. Thefield wasat an impasse. The wayout of the impasse was provided by Katz, whoproposed that the conflict betweenhis and von Boehmer’sexperimentalresults derived fromthe fact that the chimericP~ T ceils that cooperatedwith allogeneic P2 B cells were not simply tolerant to the allogeneic MHC determinants expressedby the P2 B cells. Rather,becausethese T cells haddifferentiated from stem cell precursors in a P2 MHC environment,they had "learned" to cooperatewith P2 B cells, a processhe termedadaptivedifferentiation (47). This hypothesisacceptedthe conclusionthat like-like interactions werenot the mechanism of TrI-B cell interaction. Moreimportantly, however, it proposedthat the physiologicinteractive capacity of Tri cells was "plastic" and could be changedby the environmentin whichthe Tri cells haddifferentiated. Renewed support for a central role of the MHC in regulating Tn-Bcell interaction wasthen providedby the elegant experimentsof Sprent (97). Sprentutilized as a Tncell sourcenormalstrainAadult T cells that hadbeen in vivo primedwith SRBC and then depleted of T cells that werealloreactive to strain B MHC determinantsby filtration throughan (A X B)F~.Such SRBC-specific Tri cell populationswerenon-alloreactiveto either strainA or strainB B cells andcould not mediateallogeneiceffects in responseto either haplotype.However, the strainA Tr~ cells (whichhad beenpalmedin a strainA environment)only cooperatedwith strainA B cells. Thus, these results confirmedthe earlier studies of Katzet al andreaffirmedthat MHC playeda central role in regulating physiologicTn-Bcell interaction. Thecentral role of the MHC in regulating Tta-Bcell interactions wasnow firmly established. Furthermore,the role of the MHC in these interactions couldnot be explainedas a like-like interactionbetween Tncells andB cells,

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but wasinsteadmostlikely a reflection of a receptor-ligandinteraction with Trt cells expressingreceptorsspecific for the recognitionof self-MHC determinantsexpressedby B cells. In contrast to allorecognition, this sort of "physiologic"recognition of MHC maintainedthe requirementfor recognition by THcells of carrier determinants,and has beentermedstir-recognition. Compellingevidencefor the reeeptor-ligand-mediatedself-recognition mechanismof TH-Bcell interaction was provided by several different groups and was based upon Burnet’s clonal selection theory (19). The fundamentaltenet of Burnet’s clonal selection theory wasthat receptor expression by immunocompetent and antigen-specific lymphocytesis clonally excluded.That is, somelymphocyte clones expressone receptor specificity whereasother clonesexpressother receptorspecificities. In contrast, non-receptor cell surface componentssuch as MHC determinants are not clonally excludedand are expressedby all members of a givencell type. For example,essentially all B cells froma given individual express the same genetically encodedIa determinants. Thus, if the interactive capacity of THcells wereclonally excluded,it wouldbe direct evidencethat the interactive capacity of the Trt cells was due to a receptor mechanism.Paul and co-workersdemonstratedthat antigen-specific (A X B)F1T cells consisted of at least two distinct subpopulations,one able to respondto antigens presented by parentAaccessorycells and one able to respondto the same antigens presentedby parentBaccessorycells (77). Thus,their results demonstrated that the ability of antigen-specificF1 T cells to cooperatewith parental accessorycells wasclonally excludedand, hence, wasmediatedby receptors specific for parental (i.e. sel 0 MHC determinants. Sprent (98, 99) and Swierkioszet al (102) madeuse of’this observation to select positively for (A × B)F1Hcells s pecific for the self-recognition of the Ia determinantsof one or the other parent. In the case of Swierkiosz et al, (A X B)F~THceils were precultured on antigen-pulsed parentA accessorycell monolayers.TheT cells that adheredto the monolayerswere then assayedfor their ability to cooperatewith B cells of either parental haplotype. It wasfound that the adherentT cells weremarkedlyenriched in their ability to cooperatewith parental B cells that wereMHC identical to the accessorycall monolayer.Thus, these experimentsprovidedstrong direct evidencethat the ability of antigen-specificTHcells to cooperatewith B cells was clonally excludedand, therefore, was mediated by an MHCspecific receptor mechanism. Fromthis perspective, the original experimentsof Katzand Sprent in whichin vivo primedstrainA THcells had failed to cooperatephysiologically with allogeneicstrainBB cells did not, in retrospect,necessarilyrettect an intrinsic inability of strainA Tr~cells to cooperatephysiologicallywith allogeneic strainB B cells. Rather, it is possible that their results with

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strainA Tn cells, like those with (A X B)Fl Trt cells, specifically reflected the failure of TH cells that had been primed on strainA accessory cells to cooperate with strainB B cells. Thus, if it were possible to prime strainA Tr~ cells on allogeneic straina accessory cells in the absence of allogeneic effects, such Tri cells might be able to cooperate physiologically with allogeneic strain~ B cells. Such an experimental outcome would be consistent with the concept that MHC-restdcted Tn-B cell interaction never reflects a requirement for MHC identity betweenTH cells and B cells, but instead reflects the existence of a receptor-ligand interaction in which TrI cells are required to recognize the MHC determinants expressed by their partner B cells. Additional evidence that the antigen-dependent Tn-Bcell interaction was mediated by Tn cell receptors that recognized B cell Ia determinants came from chimera experiments that had been based on the exciting findings of Zinkernagel et al (114, 115) and Bevanet al (15, 25). These investigators had shownthat for cytotoxic T lymphocyte (CTL) responses in (A X F1 -~ A radiation bone marrowchimeras, the only F1 T cells that differentiated into functional competencewere those capable of recognizing antigens in conjunction with host-type straing MHC determinants. In addition, these investigators had shownthat the specific host elementsresponsiblefor determining the self-MHCspecificity of the T cells in (A X B)F~ -~ chimeras was the thymus in which the T cells had differentiated. These studies, together with later studies in A -~ B fully allogeneic radiation bone marrowchimeras(27, 56, 63, 93, 94), confirmedthe plasticity of the T cell self-MHCrepertoire proposed by the adaptive differentiation concept of Katz. Sprent was the first to assess the ability of Tn cells from such (A B)F~ -~ A chimeras to cooperate with B cells from each parental strain (100). He found that even though the chimeric T cells were genotypically (A X B)F~and tolerant to both parental haplotypes, they were only able cooperate with parentA B cells for the generation of anti-SRBCresponses. These results were soon confirmed by two other groups (45, 91). Thus, it was quite clear that TI~ cell self-recognition of MHC determinants was a critical event in the immunecell interactions required for the generation of antigen-specific B cell responses. The precise identification of those cell interactions that required Tn cell recognition of self-MHCwas to be the next area of controversy.

TI-I CELL RECOGNITION OF MHC DETERMINANTS ON ACCESSORY CELLS VERSUS B CELLS The experiments reviewed above demonstrated unequivocally that Tn cells were required to recognize the self-MHCdeterminants expressed by cells contained within the B call population. However, these previous experi-

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ments had not attempted to distinguish whether this self-recognition requirement actually existed betweenthe THcells and the responding B cells, between the TH cells and the accessory cells contained within the B cell population, or both. Indeed, the existence of MHC restrictions for the activation of T cells by accessory cells had been described virtually simultaneously with the existence of MHC restrictions for the activation of B cells. As a result, it was possible that the only cellular interaction in which TH cell recognition of self-MHCdeterminants was required was that between Tr~ cells and antigen-presenting accessory cells. Katz et al reasoned that in their experiments, TH cells were indeed required to directly recognize B cell MHC determinants since their experiments had been performed in lethally irradiated F1 recipient mice, which should have provided F1 accessory cells that would have fulfilled any MHC requirements for the activation of the THcell populations (48). Furthermore, the finding of a requirement for carrier-hapten linkage in their responses was interpreted as providing additional supportive evidence for a direct interaction betweenTHcells and B cells. Similarly, Sprent also reported (98, 99) that secondary adoptive transfer responses to SRBCinvolved MHC-restricted TH-Bcell interactions since he found that this restriction could not be fulfilled by the intentional addition of accessorycells that were appropriate for the activation of the TH cells. Similar observations were reported by Swierkosz et al (102). Thus, these results suggested rather elegant modelfor.B cell activation in which THcells were initially activated by the recognition of antigen in the context of accessory cell MHC determinants and were then triggered to provide their helper signals by the subsequent recognition of the identical antigen and the identical MHC determinants expressed by B cells. However, conflicting data were simultaneously reported from a number of other laboratories. Erb & Feldmann(24) observed that H cells w ere MHC restricted in their interaction with accessory cells, but, once activated, were not MHC restricted in their interaction with B cells. Similar results were reported by McDougal& tort (61) and Singer et al (91-93) for antigen-primed THcells and unprimedTHcells, respectively. Finally, Shih et al (90) extended these previous reports by demonstrating that MHC unrestricted TH-Bcell interactions for IgG secondary antibody responses could still require carrier-hapten linkage. Thus, these investigators suggested that the activation of THcells required the recognition of antigen and accessory cell MHC determinants, but that, as a result of this interaction, the Tit cells were already triggered to provide their helper signals and did not need to recognize again the identical MHCdeterminants on B cells. Both sets of investigators agreed that the initial activation of Tr~ cells required T~I cell recognition of antigen and accessory cell MHC determi-

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nants. However,they were clearly divided over whether a similar requirement existed for Tri cell recognition of antigen and B cell MHC determinants. This conflict did not appear to correlate easily with experimental variables such as the use of primed versus unprimed Tn cells or B ceils, the use of in vitro or in vivo responseconditions, or the use of soluble versus particulate antigens. The field was again at an impasse. In an attempt to understand why Tn-B call interactions were clearly MHCrestricted in some experimental systems but not others, Shreier, Andersson, Melchersand colleagues (3, 62, 85) attempted to study the early events involved in the T cell-dependent activation of B cells. Theseinvestigators found that the transformation of small resting B cells into B cell blasts required an antigen-specific and MHC-restricted interaction with THcells. In contrast, B cell blasts could then be further driven into antibody secretion by Tn cells by an antigen-nonspecific and MHCunrestricted mechanism.Thus, these results suggested that the conflict in the field resuited fromthe fact that small resting B cells had been inadvertantly utilized in those studies demonstrating MHCrestricted Tn-B cell interactions, whereaspartially activated B cell blasts had been inadvertantly utilized in those studies that failed to observe MHC restricted TrI-B cell interactions. Although these studies provided important insights into some of the mechanisms of Tr~ cell activation of B cells, they did not appear to be able to resolve the discrepant results from the various laboratories. For example, if the discrepant results were due to the use of small, resting B cells versus partially activated B cell blasts, it wouldbe expectedthat those studies that utilized B cells recently immunizedwith TNP-LPSwould have failed to observe MHC restricted Tn-B cell interactions and those laboratories that had utilized unimmunizedand unmanipulated B cells would have successfully observed MHCrestricted TH-Bcell interactions. Contrary to these expectations, experiments reported by the Kappler-Marracklaboratory frequently utilized B calls that had been recently primed with TNP-LPS (102), yet the investigators consistently observed MHC restricted TH-Bcell interactions, whereas Singer and Hodes had consistently failed to observe MHC restricted TH-Bcell interactions even though they had utilized unprimed, unmanipulated B calls (91, 92). Anotherpossible resolution to this conflict was suggested by experiments demonstratingthat optimal Tit cell function required two distinct interacting T cell subsets, whichcould be distinguished by their sensitivity to adult thymectomyor to anti-lymphocyte serum (5, 43). The results of experiments by Keller et al (46) and by Tada et al (103) further suggested one Tn call subset was MHCrestricted in its interaction with B calls whereas the other one was not, a concept extended by the work of Bottomly &Mosier(18). However,in these experimentsthere existed a strict require-

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ment for the function of the B cell-restricted TH cell subset; the second THcell subset appeared to augment,rather than initiate, B cell responses. Thus, these experiments could not readily explain the failure of a number of laboratories to observe MHC restricted TH cell activation of B cells. Furthermore, it has more recently been shown that monoclonal TH cell populations, which only consist of the progeny of a single T cell, are sul~cient to activate B cells and can trigger B cells without recognizing the MHCdeterminants expressed by the responding B cells even though they are required to recognize the MHCdeterminants expressed by antigenpresenting accessory cells (34). It should be emphasizedthat even though heterogeneity of TH cell subsets appears insut~cient to fully resolve the conflict over the role of MHC in TH-Bcell interactions, it is likely that such heterogeneity does exist.

DISTINCT B CELL SUBPOPULATIONS DIFFER IN THEIR GENETIC REQUIREMENTS FOR ACTIVATION BY TH CELLS One facet of the problem of THcell-dependent B cell activation that had not been examinedwas the fact that B cells consisted of distinct B cell subpopulations that fundamentallydiffered in their requirements for activation. It had been shown for T-independent B cell responses that such responses were mediated by two distinct B cell subsets that differed in their ontogenyas well as in their expression of a numberof B cell differentiation antigens, such as Lyb3,5, and 7 (1, 2, 39, I01). Thus, LybS-B cells were present from birth in normal mice and responded to T-independent antigens such as TNP-LPS and TNP-Brucella abortus (TNP-BA), which were grouped together as TI-1 antigens (70, 72). In contrast, Lyb5+ B cells appeared at 2-3 wks of age and responded to T-independent antigens such as TNPoFicoll and TNP-Levan,which were grouped together as TI-2 antigens (70, 72). In addition, it had been shownfor these TI responses that LybS- and Lyb5+ B cell subpopulations differed in their cellular interaction requirements for activation such that Lyb5+ B cell responses to TI-2 antigens required Ia + antigen-presenting accessory cells (16, 17, 20, 71), whereas LybS- B cell responses to TI-1 antigens proceeded without accessory cells (16, 17). An early suggestion that alternate pathwaysof B cell activation might also exist for T-dependent responses came from the work of Pierce & Klinman(78). In their work, it was observed that I-region syngeny between Tri cells and B cells was necessary for IgGt responses by unprimedB cells, but was not necessary for IgMresponses by unprimed B cells. In addition it was observed ihat I-regionsyngeny between THcells and B cells was also

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T CELL-B CELL INTERACTION 221 not required for the IgGl responsesof primedB cells in spite of a strict requirementfor carrier-hapten linkage in these responses. Thepossibility that MHC-restrictedand MHC-unrestrictedT-dependentactivation of B ceils mightbe mediatedby different B cell subpopulationswasfirst suggested by the workof Lewiset al (57). Theseauthors separated haptenprimedB cells into complementreceptor positive (CR+) and complement receptor negative (CR-) fractions and assessed the presence or absence MHC restrictions in the activation of each subpopulationby carder-primed + B ceils wereactivated by T ceils across Ta ceils. It wasobservedthat CR allogeneic barriers, whereasCR-B cells wereactivated only by I-regioncompatibleT cells, findings interpreted by the authors as suggestingthe existence of alternative cooperativepathwaysbetweenT cells and distinct B cell subpopulations. Compellingevidencethat distinct B cell subpopulationsexisted that fundamentallydiffered in their requirementfor MHC-restricted interaction with Tit cells camefrom experimentsthat assessed the MHC restriction + and Lyb5-B cells, the two B cell subsets that had requirementsof Lyb5 beendefined by differences in their responsivenessto T-independent antigens. A primaryTit cell-dependentin vivo adoptive transfer response to SRBC was utilized (96) in whichthe unprimedTit cell population came from(A × B)F1-~ A radiation bone marrowchimerasso that these T cells weretolerant to both parental haplotypesbut were only able to recognize the self-MHC determinantsof parentA;also utilized wereunprimedB cells fromthe "inappropriate" parent (parentB), whichwere selected such that they containedeither Lyb5-B cells exclusivelyor containedboth Lyb5-and Lyb5+ B cells. It was found that uponthe addition of parentAaccessory cells, the (A × B)F~~ Achimeric Tit cells could collaborate with the parentB B cell populations that contained Lyb5+ B ceils but could not collaborate with parentB B cells that were devoid of Lyb5+ B cells. In contrast, normal(A × B)F1Tit cells, whichcould recognizethe self-MHC determinantsexpressedby parentBB cells, could collaborate with either parenta B cell subpopulation.Thus, in a single systemof immune response to SRBC, these experimentsdemonstratedthat the interaction of Tit calls with Lyb5+ B cells did not require Tit cell recognition of B cell MHC determinants, whereasthe interaction of Tn cells with Lyb5-B cells did require Tn cell recognition of B cell MHC determinants. Similar results have also recently beenobservedin vitro for the generation of anti-SRBC responses(Ono,Yaffe,Singer, manuscriptin preparation)as well as for the generation of anti-TNP-KLH responses(10). It has also beenobserved(9) that a single antigen-specific monoclonalTri cell populationthat is MHC restricted in its interactionwith accessoryceils could,underdifferent condi+ B cells, but that its activation of tions, activate both Lyb5-and Lyb5

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LybS- B cells requires recognition of the MHC determinants expressed by LybS- B cells, whereas its activation of Lyb5+ B cells does not require recognition of the MHCdeterminants expressed by Lyb5+ B cells. These experiments provided a novel resolution for the long-standing dispute over the requirements for THcell recognition of B cell MHC determinants. Essentially, they demonstrated that the same Tri cells could interact with B cells in either an MHC restricted or MHC unrestricted way, but that the B cells whichwere triggered in each case were different (Figure

1).

However, since MHCunrestricted Lyb5+ B cells were present in all unfractionated normal B cell populations, it remained to be explained why manyof the experiments had failed to observe MHC unrestricted activation of this B cell subset. The answerapparently involves the existence of still greater complexities in the cell interactions involved in the activation of B cells by THcells. In most experimental systems, it appears that the conditions utilized predominantly,if not exclusively, favor the activation of one or the other B cell subset, but rarely result in the simultaneousactivation of both B cell subsets. Thus, in vitro primary responses to T-dependent antigens that utilize,unprimed TH and unprimed B cell populations are almost exclusively mediated by Lyb5+ B cells and do not require an MHC restricted TH-Bcell interaction (16, 96); secondaryin vitro responses to low concentrations of T-dependent antigens that utilize primed THand primed B cells appear to be almost exclusively mediated by LybS- B cells and require an MHC-restdctedTrI-B cell interaction (10); in contrast, augmented primary in vitro responses to T-dependent antigens that utilize palmed TH but unprimed B cells are mediated by both Lyb5+ and LybSB cells, at least to SRBC(Ono, Yaffe, Singer, manuscript in preparation). Thesedifferences are likely to result from several different factors, at least one of which involves the participation of regulatory T-suppressor (Ts) MHC

MHC Restricted

~

~

~ ~

Figure I Existence of distinct B cell subpopulations that differ in their genetic requirements for activation by Tn cells. The interaction of Tn cells with accessory cells is MHC restricted, as indicated by a solid arrow. Onceactivated, the interaction of Ta cells with LybS-B cells is also MHC restricted. In contrast, the interaction of activated Tn cells with Lyb5+ B cells need not be MHC restrieted and can be mediated by soluble Tn cell.derived B cell activation factors such as TRF(indicated by broken arrow).

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cells, since it has been shown(8) that heterogeneousT~I cell populations contain Ts cells able to differentially suppress the activation of LybS-but + B cell responses. Thus, the B cell subset that is actually triggered not Lyb5 in a given experimental situation must be directly assessed, since it cannot be assumedthat a given response will be mediated by both B cell subsets. For the purposes of this review, the complexities involved in Tn-Bcell interactions can be reduced with reasonable accuracy to the fact that distinct B cell subpopulations exist that differ in their requirements for an MHC restricted interaction with THcells. This difference is precisely corre+ but not Lyb5- B cells to respond to antigenlated with the ability of Lyb5 nonspecific Tn cell factors, a feature of B cell activation considered in greater detail below.

ROLE OF MHC-LINKED GENES IN TH-B CELL

IMMUNE RESPONSE INTERACTIONS

In parallel with studies examiningthe general role of the MHC in Tn-Bcell interactions were a large number of studies examining the role of MHClinked immuneresponse (It) genes in Tn-Bcell interactions. The earliest studies investigating mechanismsof Ir gene function attempted to identify those cell types whose immunefunctions were regulated by Ir genes. In T-dependentantibody responses, the first studies of this question variously concludedthat Ir genes controlled the function of Tr~cells, B cells, or both (12, 13, 44, 65, 73-75, 80, 87, 88). Appreciationof the role of accessorycells in T-dependent responses further complicated this issue by requiring the potentially difficult distinction of Ir gene control of accessorycell function versus B cell function. Morerecently, a large bodyof experimental data has suggested that regulation of immuneresponses by MHC-linkedIr genes is an antigen-specific facet of the antigen-nonspecific regulation of immune cell interactions by MHC genes. Thus, the fact that MHC restrictions result from a receptor-ligand interaction in which Tn cells, regardless of their antigen specificity, are required to recognize self-Ia determinants expressed by the cells with which the Tn cells directly interact implied that Ir gene function might better be understood as regulating the interaction with B and accessory cells of those Tt-i cells involved in the generation of Ir gene regulated immuneresponses. A numberof studies concluded that Tn-Bcell interactions are regulated by MHC-linkedIr genes. For example, it was observed that (Responder X Nonresponder)F~/(R X NR) F~] Tri cells cooperated with R but not parental B cells for the generation of antibody responses (51, 59, 113). Furthermore, NRTri cells that had matured in a responder environment, i.e. NR--> (R X NR)F~chimera, could in most (45) but not all (60)

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also cooperatewith R but not NRB cells. Sinceit wasfoundin these studies that R strain accessorycells did not reverse the unresponsiveness of NRB cells, it wasconcluded that TH-B cell interactionfor these Ir gene-controlled responseswasregulated by Ir genes. In contrast, analogousstudies wereperformedby other investigators in whichprimaryIr gene-controlledresponseswere analyzedin vitro and in whichTHcells, B cells, andaccessorycells wereseparatedinto independent cell pools and mixedtogether in three-cell mixingexperiments(31). It was found that Tr~ cells of R, (RxNR)F~,or NR-~(RxNR)Florigin activated by accessorycells that expressedR Ia determinantsbut werenot activated by accessorycells that only expressedNRIa determinants. However, onceactivated by accessorycells of R phenotype,the THcells could collaborate equally well with either R or NRB cells. Thus, these studies concludedthat T~i-accessory cell interactions were regulated by MHClinked Ir genesbut that, in contrast, TH-B cell interactions werenot regulated by MHC-liiikedIr genes. Thereare obviousparallels betweenthis conflict over Ir generegulation of TH-Bcell interactions and the conflict over MHC regulation of TH-Bcell interactionsin general.Essentially,all investigatorsagreedthat Ir genesdid regulate TH-accessorycell interactions but strongly disagreed whetheror not Ir genesalso regulatedTH-B cell interactions. In fact, thoselaboratories that foundIr generegulation of TH-Bcell interactions wereprecisely the samelaboratories that had also foundMHC regulation of TH-Bcell interaction; whereasthose laboratories that did not observeIr generegulation of TH-B cell interaction wereprecisely the samelaboratories that hadalso not found MHC regulation of TH-Bcell interaction. As wasthe case for MHC regulationof TH-B cell interactions, the wayout of this impasseis probably also providedby an appreciationof B cell heterogeneity. Experimentswere performedin whichcarrier-primed THcells and hapten-primedB cells werechallengedin vitro with either low or high concentrations of Ir gene-controlled antigens. As for nonolr gene-controlled antigen responses, low in vitro antigen concentrationspredominantlyresuited in IgOresponses.mediatedby LybS-B cells, whereashigh antigen concentrations predominantly resulted in IgMresponses mediated by + B cells Lyb5+ B cells (11). Responsesmediatedby either LybS-or Lyb5 required the presence of R accessorycells, demonstratingagain that Tnaccessorycell interactions wereregulated by Ir genes. However,following their activation by R accessorycells, the THcells could only collaborate with R Lyb5-B cells and could not collaborate with NRLyb5-B cells. Thus, Ir genesregulate the interaction of THcells with Lyb5-B cells. In contrast, oncethey wereactivated by R accessorycells, the THcells could

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T CELL-B CELL INTERACTION 225 + B cells, demonstrating collaborate equally well with either R or NRLyb5 + B cells. that Ir genesdo not regulate the interaction of Tncells with Lyb5 Theselatest findings dramatizethe parallel betweenantigen-specific Ir gene regulation of cell interactions and antigen-nonspecificMHC regulation of cell interactions. Specifically, both regulate Tn-accessorycell interactions, both regulate Tn-LybSB interactions, and neither regulates + B cell interactions. TrrLyb5 Althoughit is nowgenerallyacceptedthat Ir generegulation represents an antigen-specific regulation of those cell interactions regulated by MHC genes,it remainscontroversialhowthe antigen-specificityof Ir generegulation is derived.Threeschoolsof thoughtcurrentlyexist that can be characterized in the followingway.Oneviewpointis that THcells only recognize a molecularassociationof Ia andantigen and that Ir gene-controlledantigens are unable to form an immunogenic complexwith NRIa determinants even though there exist NRT cells that wouldbe capable of recognizing such a complex,were it formed. This viewpointhas been referred to as determinantselection (14, 83). Asecondviewpointis that the problem the NRdoesnot lie in the failure to forman Ia-antigencomplex,but rather that there do not exist in the repertoire of the speciesTHcells capableof recognizingthe complexcreated betweenNRIa and antigen (89). Thethird viewpointrepresents a middlegroundand suggests that antigen can form a complexwith NR-Iamolecules but that the particular complexformed cannot be recognizedby THcells that had maturedin an environmentthat wouldhave tolerized themto NRIa determinants (42, 79, 86, 93). This latter viewpointhas beenreferred to as clonal deletion (86). Whichif any of these perspectivesis correct remainsunclear, but each is fundamentallycompatiblewith the data that presently exist. However, whicheveris correct, it is nowclear that antigen-specific Ir genesonly regulate those cell interactions that are regulated for all antigens by MHC genes. Indeed,MHC regulation of cell interactions can be viewedas identical with,Ir generegulation for all antigen responses,eventhose for which a nonresponder strain does not exist. ROLE OF ANTIGEN-SPECIFIC CELL ACTIVATION

TH FACTORS IN B

Analternative and potentially powerfulapproachto the study of Tn-Bcell interactions is the study of soluble TH cell productsthat possess someor all of the functionalpropertiesof THcells. Indeed,bothantigen-specificand antigen-nonspecific Tri cell factors havebeendescribedthat are either partially or completelyTncell-replacing.

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Thestudy of Tn cell factors in general and of antigen-specific Tn cell factors in particular has proceededthrougha relatively stereotypedgeneral experimentalapproach. It has been necessary to generate soluble T cell products, to establish a T-dependentB cell response system, and then to assess the functional activity of soluble T cell productsin that response system.Among the first studies of antigen-specificTn factors werethose of Taussig and co-workersand Mozeset al (74, 75, 108), whocharacterized factors specific for synthetic polypeptides such as TGAL.Thesehelper factors were generated by the education of thymocytesthrough transfer with antigenin irradiated recipients, andthe subsequentin vitro stimulation of these educatedT cells with specific antigens. Thesoluble supernatant fromsuchcultures wasthen assayedfor its ability to trigger, in the absence of Tr~ cells but in the presence of antigen, antibody production by bone marrowcells adoptively transferred into irradiated hosts. Theresults of such studies demonstratedthat soluble products derived from educated THcells could help in an antigen-specificmannerthe productionof antibody by bone marrow-derived cells. It wasalso shownthat MHC-linked Ir genes influenced these responsesboth by determiningthe ability of T cells to respondto antigen for the generationof active factor, and by determining the ability of antigen-specificB cells to be triggeredby the active factor. No MHC restriction was observed, however,betweenthe strain of origin of active T cell factor and the strain of high responderB cell. Theconclusion that suchantigen-specificT cell factors acted directly on B cells wasbased uponthe additional observation that B cell-containing populations were uniquelyable to absorbout the functional activity fromthese factor preparations. Theseearly studies openedthe wayfor the pursuit of a numberof critical issues. First, there wasa needfor a morerigorousidentification of the cells producingactive Tn cell factors, andof the activation requirementsfor its production.Second,the nature of the target cells directly triggered by TH factors required moreprecise identification. Finally, the THfactor itself requiredcharacterizationat the biochemicalandserologic, as well as functional, level. In particular, the demonstration of soluble, antigen-specificT cell factors offered a promisingsystemin whichto evaluate the character of the T cell receptor, of MHC restricted recognition in general, and of TH-B cell interactions in particular. Amorepreciseidentificationof the T cell origin of helperfactors wasfirst facilitated by the workof Feldmann and colleagues, whowereable both to primemudnespleencells in vitro andstimulate the release of helper factor in vitro (36). Themostdefinitive approachto this questioncame,however, fromthe workof Mozes,Lonai, Feldmannand their colleagues, whoestablished cloned T cell hybddomas that producedactive helper factors (4, 26,

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T CELL-BCELL INTERACTION 227 58). Althoughthese findings establishedthe ability of hybridoma T cells to synthesizeactive factors, they did not resolve the question of whatcell interactions mightnormallyresult in the triggering of factor productionby THcells. Otherinvestigators havesuggestedthat accessorycells andeven B cells maybe involvedin the productionof helper factors by T ceils (55). It wasalso unclearfromthe initial studies in this area whetherT helper factors couldfunctionto activate B cells directly, or whetheradditionalcall interactions wererequired for B cell activation. Howie& Feldmann demonstrated that antigen-specific helper factors producedby heterogeneousT cell populationscouldhelp T cell-depleted spleen cells, suggestingthat T cells did not play an essential intermediaryrole in the activation of antigenspecific B cells by antigen-specific THfactor (36). However,it has been suggested by Howie& Feldmann(37) that adherent accessory cells are required for B cell activation in the presenceof antigen-specificT helper factors, andindeedthat genetic restrictions betweenB cells and accessory calls can be observedunder these conditions. Thus, although THfactors mayact in the absenceof additional T cells, the activation of B cells may require additional signals providedby accessory cells. The question of whetheror not geneticrestrictions exist in the ability of T cell factors to help B cells wasapproachedin part in the early studies by Taussig and colleaguesdescribedabove.Morerecently, this issue wasanalyzedby Lonai et al employingthe soluble productsof monoclonal T cell hybridomas (58). It wasfoundin these studies that there existed a strict H-2restriction (mappingto the Kor I-A region) betweena factor of H-2b origin and the B cells with whichthe factor cooperated.Asimilar restriction wasobserved in the ability of B cells to absorbfactor. Thesestudies did not formally distinguish whetherthe observedrestrictions werebetweenT cell factor and B cells, or betweenfactor andaccessorycells containedin the B cell population. Biochemicaland immunochemical analyses of T helper factors havebeen carried out in recent years both for the productsof heterogeneousT cell populations and for the products of monoclonalhybridomas.Howieet al reported that factors derived fromconventionalTi~ cell populationsbound antigen specifically, expressedI-A- or I-J-encoded(Ia) determinants,and reactedwith a chickenanti-mouseIg reagent, raising the possibility that T cell factors express Ig-like determinants(38). Apte et al found that TGAL-specific factor boundantigen, althoughwith a fine specificity distinct fromthat of antibody(4). ThisT cell factor expressedidiotypic determinants cross-reactive with those expressed by anti-TGALantibodies, reacted with an anti-Vnreagent, and consistedof two chains, one of which wasVrt positive andthe other of whichwasIa positive. Lonaiet al similarly

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reported that a CGG-specific hybridoma factor expressed both Vrt and I region products (58). Thus, antigen-specific T cell factors, including those obtained from monoclonalT cell populations, appear to interact with B cells in a manner analogous to that demonstrated for Tn-Bcell interactions. The behavior of antigen-specific TrI cell factors for activating distinct B cell subpopulations such as those represented by LybS- and Lyb5+ B cells remains to be examined. The "physiologic" role of such factors in the cooperation of Trt cells and B cells remains a reasonable but unconfirmed hypothesis.

ROLEOF ANTIGEN-NONSPECIFICTH FACTORS IN B CELL ACTIVATION A g(eat deal of experimental data has recently been accumulated assessing the role of antigen-nonspecific factors in B cell triggering. These studies have attempted to identify the pertinent lymphokineselaborated as a result of Tn-accessory and TrrB cell interactions involved in activating responding B cells. A variety of such factors have been identified and include interleukin 1 (IL-1), intedeukin 2 (IL-2), B cell growth factor, and T replacing factor (TRF) (69). It remains unclear at this time whether or all of these factors are involved in B cell triggering, and it remains controversial in what sequence these factors act on responding B cells. An interesting model has been proposed by Hoffman(35), whosuggested that the accessory-cell-derived lymphokineIL-1 first binds to B cells, resulting in the B cells becomingreceptive to T cell-derived lymphokines. Althoughthe identification of the precise lymphokinesinvolved in B cell activation remains uncertain, studies with soluble supematants that probably contain multiple lymphokineshave provided a number of insights into the mechanismof Tn-B cell interactions. One of the earliest antigen-nonspecific factors studied was derived from the supernatants of mixed lymphocyte reactions directed against allogeneic stimulator cells (51). These supematants were designated as allogeneic effect factor (AEF), and were able to trigger B cells in the absence of Tri calls but still required the presence of antigen (6, 7). As originally described, AEFwas capable activating B cells of any MHChaplotype as well as B cells of any antigen specificity. Thus, activation of B cells by AEFwas remarkably similar to the activation of B cells by histoincompatible Tn cells. Indeed, it seemed likely that secretion of AEFby alloreactive Tn cells was the molecular mechanismby which B cells were triggered by histoincompatible TH cells.

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T CELL-B CELL INTERACTION 229 Morerecent studies with AEFby Delovitchet al have shownthat it is possibleto obtain"restricted" AEFcapableof activatingB ceils of a specific MHC haplotype (23). If the MLR from which the AEFsupernatants were obtainedconsists of T cells depletedof Ia+ cells andstimulatorcells devoid of T cells, the resulting AEF specificallyactivatedB cells of stimulatortype. It was suggested that the restricted AEFconsists in part of molecules secreted by the respondingT cells, whichrecognizethe Ia determinantsof the stimulator cells. Thus,the ability of restricted AEFto trigger B cells depended in part on its anti-Ia specificity. Thesestudies provideda molecular modelfor TH-Bcell activation and confirmedthe importanceof Ia recognitionin the cell interactions requiredfor B cell triggering. In light of the controversyover Tn cell recognitionof Ia determinants expressedby accessorycells versus B calls, it wasdearly of interest to determinewhetherthe specificity of restricted AEFwasfor accessorycell Ia determinants, B cell Ia determinants, or both. It has recently been observed(Delovitchet al, submittedfor publication) that restricted AEF is in fact specific for the Ia determinantsexpressedby accessoryceils but is not specific for the Ia determinantsexpressedby the respondingB ceils. Asa result, eventhoughrestricted AEFis Ia specific andis able to trigger B cells, these studies stronglysupportthe existenceof TIt-Bcell interaction pathwaysthat do not require THcell recognitionof B ceil Ia determinants. Giventhe recent awarenessof B cell heterogeneityin Tri ceil-dependent antigen responses, it wouldbe informativeto knowwhichB cell subset is activated by restricted AEF.Indeed,Delovitchet al havesuggested(submitted for publication) that restricted AEFactivates Lyb5+ but not Lyb5-B cells. Thus, restricted AEFbehavesin a mannerconsistent with the observationsthat Tn cell recognitionof B cell Ia determinantsis not + B cells but is requiredfor T cell required for Tri cell activation of Lyb5 n activation of Lyb5-B cells. Asecondapproachto the studyof B cell activation is illustrated by the studies of Huberet al, whoutilized Lyb3-specific antisera in Tnceil-dependent antibodyresponses(39). TheLyb3determinantis non-ailelie andis only expressedon a late-appearingB cell populationthat is essentially identical with the B cells that express Lyb5.Theseinvestigators demonstratedthat Lyb3antisera significantly augmented the TI-I cell-dependentB cell response to suboptimaldoses of SRBC.As wouldbe expected, the augmentationof anti-SRBCresponsesby Lyb3antisera wasonly observedin B cell popula+ B cells. Thus,these investigators suggested tions that containedLyb3+5 the possibility that the augmentation of anti-SRBC B cell responsesby Lyb3 antisera was due to the fact that the Lyb3determinantwas part of the B cell membrane receptor for Tn cell-derived B cell activation signals.

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A third and exceedingly informative approach has been the study of antigen-nonspecificsupernatant factors that replace THcell function in "physiologic,"rather than allogeneic, TH-Bcell interactions. Suchsupernatant factors havebeentermedT cell replacing factors (TRF),andare both antigen-nonspecificand haplotype-nonspecific(84a). TRFhas been shown to activate B cells via a mechanism that is MHC unrestricted and that does not require carrier-haptenlinkage (40, 41). Among the mostelegant studies with TRFwere those performed by Takatsu, Hamaoka,and co-workers, whoutilized a monoclonalTRFproducedby a T cell hybridoma(104-106). In addition, these investigators haveproducedantisera specific for the TRF acceptormoleculeexpressedby B cells (104). Thus,these investigators have beenable to identify the distinct B cell subpopulations that are able to bind TRFas well as to functionally respondto TRF.Their studies demonstrated that the expression of the TRF-acceptormoleculewas genetically controlled and that there exists a mousestrain, DBA/2Ha, whichis both specifically deficient in the expressionof this receptor moleculeand also functionally unresponsiveto TRF(109). Moreover,these investigators further demonstratedthat there exist in normalmousestrains two distinct B cell subpopulations, one of whichis TRF-acceptor.positive and one of whichis TRF-aeeeptornegative. The TRF-acceptorpositive B cells are responsive to TRFand can be triggered by an unlinked carrier-hapten mechanism. In contrast, TRF-acceptor negative B cells are unresponsiveto TRFand require a linked carrier-hapten mechanism for their activation. Interestingly, the TRF-acceptor positive B cells appearto be predominantly +, whereasthe TRF-acceptornegative B cells appear to be predomiLyb3+5 nantly Lyb3-5-.The relationship betweenthe TRF-acceptormoleculeand the Lyb3determinant is unknown,but anti-TRF acceptor antibody, like anti-Lyb3 antibody, augmentsthe response to suboptimal doses of SRBC (107). Thus, the functional parallels betweenanti-TRF-acceptorand antiLyb3antisera are quite striking, and it mightbe suspectedthat the Lyb3 determinant is a componentof the TRFacceptor molecule. The effect of TRF-containing supernatants on MHC-restricted and MHC-unrestricted TH-Bcell interactions has also been directly examined. Studies have been performedutilizing supernatants from either ConAstimulatedspleencells (29, 96) or fromantigen-specific,Ia-restricted Tncell clones(33, 34). In both cases it wasobservedthat suchsupernatantsin the + Bcells but did not trigger Lyb5-B cells. presenceof antigentriggeredLyb5 Thestudies with TRFderivedfromantigen-specificandIa restricted THcell clones wereespecially informativein sorting out someof the complexities of Tn-Bcell interactions and the potential role performedby soluble TRF in these interactions. As mentionedpreviously, the THcell clones, in re+ B cells via sponseto lowin vitro concentrationsof antigen, activated Lyb5

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T CELL-BCELL INTERACTION 231 an MHC restricted mechanism and the sameTn cell clones, in response to + B cells via an MHC high in vitro concentrationsof antigen, activated Lyb5 unrestricted mechanism.Underhigh antigen dose conditions, the Tn cell clone was able to secrete soluble TRF,which functioned in a manner analogousto the clone itself underhigh antigendose conditionsin that the + B cells, and activated these B cells in an MHC TRFonly activated Lyb5 unrestricted manner(90a). Thus,these studies provideddirect evidencethat the secretion of TRFby Tn calls was the probable mechanismby which + B cells in an MHC such Tri cells activated Lyb5 unrestricted manner.In addition, these studies suggestedone further complexitythat had not been previouslyappreciated.Specifically, they demonstratedthat in responseto low concentrationsof antigen, the Tri edl clones wereactivated to function as helper cells but werenot activated to secrete detectable quantities of soluble TRF,suggestingthat a monoclonal Tn cell populationcould potentially exist in twodistinct activationstates, onein whichit is able to activate B cells via an MHC restricted Ta-Bcell interaction, and one in whichit secretes TRFand is able to activate B cells via an MHC unrestricted TrrBcell interaction. Takentogether, the studies with antigen-nonspecificfactors have provided importantinsights into the possible molecularmechanisms underlying MHC-unrestrictedTH-Bcell interactions. Specifically, they have + provided strong evidence that the MHC-unrestricted activation of Lyb5 B cells by TI~ cells is mediatedby soluble TRFsecreted by THcells which + B cells. In contrast, these is then boundby receptorson the surfaceof Lyb5 studies have not provided any insights into the mechanismsof MHCrestricted activation of Lyb5-B cells by Tncells. A prior, it wouldseem a reasonable presumptionthat the MHC-restrictedactivation of Lyb5-B cells by Tn cells wouldalso be mediatedby the local release of small quantities of TRF.However,such B cells appear not to expressdetectable quantities of TRF-acceptormoleculesand fail to be triggered by soluble TRF.Consequently,the possibility needsto be consideredthat contrary to current presumptions,the Tnsignals involvedin the MHC-restrieted activation of Lyb5-B ceils maynot only be quantitatively distinct but mayalso be qualitatively distinct from the Trt signals involved in the MHC-unre+ B cells. stricted activation of Lyb5 Arecent study has examinedthe consequences,but not the mechanism, of MHC restricted Tn-Lyb5-B cell interactions (Ono,Yaffe, Singer, manuscript in preparation). This study assessed the TRFresponsivenessand MHC restriction requirementsof SRBC-specificLybS-B cells after they had beeninitially activated by an MHC-restricted TrrB cell interaction. Unprimedmutant CBA/N (i.e. Lyb5-) B cells specific for SRBCwere unresponsiveto TRFand were triggered in vitro by Tn cells only via an

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MHC restricted TH-Bcell interaction. In contrast, CBA/NB cells that had been previously primed in vivo with SRBCand so had potentially undergone an MHC-restdctedTH-Bcell interaction in vivo were responsive to TRFand were triggered in vitro by TH cells via an MHC-unrestricted TH-Bcell interaction. These results are concordant with the observation that SRBC-primed CBA/NB cells express the TRF-acceptor molecule (104). These results suggest that TRF-aeceptornegative LybS-B cells can be induced by an MHC-restricted interaction with THcells to express the TRF-acceptor molecule and becomefunctionally responsive to TRF. However, the molecular mechanisminvolved in the initial activation of Lyb5B cells via an MHC-restdctedinteraction with Tn cells remains obscure. It nowseems unlikely that insights into the molecular mechanismsinvolved in MHC-restdctedTH-Bcell interactions will comefrom studies of antigen-nonspecific Tn factors. Rather, it seems more likely that such insights will comefrom the isolation and characterization of THcell factors that are both specific for antigen and specific for B cell Ia determinants.

DO ANTIGEN-SPECIFICB CELLS RECOGNIZE SELF-MHC DETERMINANTS? Thusfar, the role of the MHC in regulating TH-Bcell interactions has been discussed from the perspective of a receptor-ligand interaction in which antigen-specific THcells express receptors specific for the recognition of self-MHC determinants expressed by the cells with which the THcells directly interact, i.e. accessorycells and someB cells. However,the possibility can also be considered that antigen-specific B cells also express receptors specific for self-MHCdeterminants expressed by cells with whichthe B cells might directly interact, i.e. accessory cells and THcells. It has generally been presumedthat an MHC-restricted self-repertoire is not expressed by B cells because B cells express on their surface, as well as secrete, Ig, which is capable of binding free antigen directly. However, several groups of investigators have reported that it is possible to devise experimentalsystems in which MHC-restricted self-recognition by B cells can be observed. The first suggestion that the interactive phenotype of B cells can be MHC-restdctedcame from experiments by Katz et al (53). These investigators assessed the ability of B cells from F~ -> parent radiation bone marrow chimeras to accept help from THcells. It was observed that such B cells preferentially interacted with Tr~ cells syngeneicto the chimeric host rather than with THcells syngeneic to the chimeric donor. Thus, the chimeric Fr B cells appeared to distinguish THcells according to the MHC determinants the TH expressed. Subsequently, Gorezynsld et al utilized an anti-SRBCresponse system, which required accessory cells but in which the usual requirement for T H

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cells could apparently be replaced by low concentrations of LPS(28). In this response system these investigators observed that the responding B cells that had been previously primed in vivo to SRBCcould distinguish among different populations of accessorycells such that the B calls only interacted with accessory cells that were syngeneic to the haplotype of the environment in which the B cells had originally been primed. In a more conventional anti-SRBCresponse utilizing a short-term adoptive transfer system, Nisbet-Brownet al observed th~at optimal Tn cell-dependent responses were obtained when the irradiated host, which presumably provided accessory cells, wassyngeneicto the respondingB cells (76). Again, these results were consistent with the concept that antigen-specific B cells could distinguish the MHCdeterminants expressed by different accessory cells. In an effort to more simply analyze MHC restrictions between a specific subpopulation of ar~t~gen-speclfic B cells and accessory cells, Singer and Hodes analyzed the response stimulated by the polysaeeharide antigen TNP-Ficoll (32, 95). Antibody responses stimulated by TNP-Ficoll are mediated exclusively by Lyb5+ B cells, require Ia + accessory cells, and exhibit both a T-independent and a T-dependent component. For the Tindependent component of the TNP-Ficoll response (95), it was observed that spleen cells from (A X B)F1 ~ parentA and from full allogeneic A B radiation bone marrowchimeras were MHC-restricted in their interactions with accessory cells such that these chimeric B cells only cooperated with host-type, but not donor-type, accessory calls. Suchrestrictions could not fully be accountedfor by restricted Tta cell recognition of accessorycell Ia determinants, because the intentional addition of appropriate Tn cells consistently failed to alter the observed MHCrestrictions. Moredirect evidence that such MHC restrictions actually resulted from the restricted recognition by B cells of accessory cell MHCdeterminants came from experiments in which "unrestricted" strain A spleen cells from normal mice were cultured together with "restricted" strain A spleen cells from A ~ B chimeras and stimulated with TNP-FicoI1.Thus, in addition to TNP-FicolI, these co-cultures contained strain A T~t cells, strain A accessory cells, and strain A B ceils derived from both normal strainA mice and A -~ B chimeric mice. If the MHCrestrictions observed in TNP-Ficoll responses only refleeted the MHC requirements for triggering Tia cells, despite the fact such responses appeared to be T-independent, then the normal strain~, Tit ceils in these cultures wouldbe triggered by the strain A accessory cells and in turn should activate both the normalas well as the chimeric strainA B cells. Alternatively, if the MHC restrictions had in fact resulted from the MHC recognition requirements of the TNP-Ficoll responsive B cells, then the strainA accessory cells present in these co-cultures wouldonly be recognized by the normal strainA B cells but would not be recognized by the chimeric strainA B cells; hence, only the normal strainA B cells would be activated

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in these co-cultures. It was observed that in these co-cultures only the normal strainA B cells, but not the chimeric A -~ B strainA B cells, responded to TNP-FicoI1. These experiments provided strong evidence that TNP-Ficoll-responsive Lyb5+ B ceils, at least Lyb5+ B cells from fully allogeneic chimeras, recognize accessory cell MHCdeterminants. For the T-dependent component of the TNP-Ficoll response, it was observedthat both the Tr~ cells as well as the TNP-Ficoll-responsiveB cells were required to recognize self-MHCdeterminants expressed on accessory cells, but were not required to recognize each other (32). Thus, for dependent responses to TNP-Ficoll, the Trt-accessory cell interaction was MHC restricted, the B-accessory cell interaction was MHC-restricted, but the TI-I-B cell interaction was not MHC-restricted.Indeed, it seemedlikely that the TH-Bcell interaction actually occurred via an accessory cell intermediary. It was also observed in these experiments that the addition of soluble TRFto the responding cultures obviated any requirement for accessory cells and obscured any requirement for B cell recognition of accessory cell MHCdeterminants. As a result, it was concluded that T-dependent responses to TNP-Ficoll differed from classical T-dependent responses such as those to TNP-KLH in that the participating THcells did not secrete detectable quantities of TRF.Thus, the existence of a cell interaction pathway involving self-recognition of accessory cell MHC determinants by both + B cells (32a) wouldbe obscured in classical T-dependent TH cells and Lyb5 responses which result in the elaboration of TRF(Figure 2). Takentogether, all of these different experimentssuggested that antigenspecific B cells, like antigen-specific THcells, might express a requirement

]

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SOLUBLE

Figure 2 T-dependent responses to TNP-Ficoll reveal the existence of a cell interaction pathwayrequiring MHC-restrictedrecognition of accessory cells by both Tn cells and B calls. (A) T-dependent responses to TNP-Ficoll require the self-recognition of accessory cell MHC determinants by both Tn cells and TNP-Ficoll responsive Lyb5+ B n cells. Since soluble T cell-derived factors can overcomeany requirement for B-accessory cell interactions, the existence of this cell interaction pathwayin TNP-Ficollresponses suggests that TNP-Ficoll, unlike classical T-dependent antigens, does not induce the release of TRFfrom Tn cells. (B) For classical T-dependentantigen responses that stimulate Tu cells to secrete TRF,it is likely that any genetic restrictions between Lyb5+ B cells and accessory cells wouldbe obscured.

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T CELL-B CELL INTERACTION 235 for recognition of MHC determinants expressed by the cells with which they directly interact. It wouldbe simplest to imaginethat a requirement for MHC recognition by B cells wouldbe mediated, not by a separate receptor, but by its sIg. Indeed,it mightbe conceivedthat, in the absence + B cells such as those of THcell-derived B cell activation signals, Lyb5 respondingto TNP-Ficollelicit activation signals fromthe accessorycells with whichthey interact by expressingslg that recognizesTNPin conjunction with accessorycell Ia determinants. Evidenceconsistent with this possibility has recently beenreported (110, 112). For example,Wylieet observedin the splenic focusassay that the antibodieselicited in response to vitally infected fibroblasts wereto a large extent not specific for free virus, but were specific for virus in conjunction with the MHC determinantsexpressed by the infected fibroblasts. Althoughit is uncertain that such a result would have been obtained in response to immunization with free virus, this studydemonstratesthat it is at least possible to manipulateB cell responsesto reveal the existence of MHC-restrieted antibodies. Recognitionof self-MHC determinants by antigen-specific B cells increases enormously the complexitiesof Tn-Bcell interactions. Indeed, it raises the provocativepossibility that eachMHC-restricted cell interaction involved in the activation of B cells mightsimultaneously involve two discrete sets of receptor-ligandinteractions in whicheachpartner cell expresses MHC determinants as well as anti-MHCreceptors.

CONCLUSIONS The enormouscomplexities involved in TrrB cell interactions are still emerging.Indeed, the history of this research area consistently demonstrates that areas of controversyand disagreement reflected the existenceof greater complexitiesthan wereappreciatedat the time. Thus,B cell activation required cell interactions amongdistinct immunocompetent cell populations. These cell interactions were regulated by MHC-encoded gene products,reflecting a requirementfor a receptor-ligandinteraction in which the Tn cells recognizedMHC determinantsexpressedon the surface of cells with whichthey directly interacted. Theheated controversyover Tn cell recognition of B cell MHC determinantsnowappears to have been due to the existenceof distinct B cell subpopulations that differed in their ability to respondto antigen-nonspecific and MHC-unrestricted soluble factors secreted by activatedTHcells. Themajorarea of controversythat is developingat this timeinvolvesthe ability of antigen-specificB cells to recognize self-MHC determinantson the cells with whichthe B cells directly interact. Whatever the final resolutionto this issue, it is likely to revealstill greater levels of complexitythan are nowappreciated.

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Literature Cited 1. Ahmed,A., Scher, I. 1979. MurineB tion of B cell subpopulations.MHC recell heterogeneitydefinedby anti-LybS, stricted and unrestricted B call re~ an alloantiserumspecific for a latesponsesare mediatedbydistinct B cell appearingB lymphocytesubpopulatinn. subpopulations. Z Exp. Med. 154: In B Lymphocyte and Immune Re1100-15 sponses, ed. M.Cooper,D. E. Mosier, 11. Asano, Y., Singer, A., Hodes, R. J. E. S. Vitetta, I. Seher,pp. 117-24.New 1983. Role of the majorhistocompatiYork: Elsevier-North Holland bility complexin T cell activation of B 2. Ahmed, A., Sober, I., Sbarrow,S. O., cell subpopulations.Ir generegulation Smith,A.H., Paul, W.E., Sachs,D.H., of the T cell dependent activationof disSell, K. W. 1977. B lymphocyte tinct B cell subpopulations.Z lmmunol. heterogeneity: developmento fan al130:67-72 loantiserumwhichdistinguishes B lym- 12. Bechtol, K. B., Freed, J. H., Herzenphocytedifferentiation alloantigens. £ berg, L. A., McDevitt,H. O. 1974. GeExp. Med. 145:101 netic control of the antibodyresponseto 3. Andersson,J., Schreier, M.H., Melchpoly-L(TYR,GLU)-poly-D,L-ALA--pers, F. 1980. T-call-dependentB-cell oly-L-LYSin C3H<-> CWB tetraparstimulationis H-2restricted and antiental mice. J. Exp. Med.140:1660-75 en dependentonlyat the resting B-cell 13. Beehtol, K. B., MeDevitt,H. O. 1976. vel. Proc. Natl. Acad. Sc~ USA77: Antibody response of C3Ho (CKB 1612-16 CWB)F~ tetraparental mice to poly4. Apte, R. N., Eshhar, Z., Lowy,I., L(TYR,GIn)-poly-D,L-ALA--poly-LZinger, H., Mozes,E. 1981. CharacterLYS immunization. Z Exp. Med. istics of a poly(Ltyr,LGlu)-poly(D144:123-44 LAla)~poly(LLys)-specific helper factor derived from a T cell hybridoma. 14. Benacerraf, B. 1978. A hypothesis to relate the specificity of T lymphocytes Eur. Z lmmunol. 11:931-36 andthe activity of I region-specificIr 5. Araneo,B. A., Marrack(Hunter), genes in macrophages and B lymC., Kappler, J. W. 1975. Functional phocytes. J. lmmunoL120:1809-12 heterogeneity amongthe T-derived 15. Bevan,M. J. 1977.In a radiation chimlymphocytesof the mouse. II. Senera, host H-2antigens.determine the imsitiwty of subpopulations to antimuneresponsivenessof donorcytotoxic thymoeyte serum. Z Immunol. 114: cells. Nature269:417 747-51 16. Boswell,H. S., Ahmed, A., Scher, I., 6. Armerding,D., Katz, D. H. 1974.ActiSinger,A. 1980.Roleof accessorycells vation of T andB lymphocytes in vitro. in B cell activation.II. Theinteragtion II. Biologicaland biochemicalproperof B cells withaccessorycells results in ties of an allogeneiceffect factor (AEF) the exclusiveactivation of an LybSq-B active in triggering specific B lymcell subpopulation. Z lmmunoL phocytes. Z Exp. Med. 140:19-37 125:1340-48 7. Armerding,D., Sachs, D. H., Katz, D. 17. Boswell,H. S., Nerenberg,M.I., Seher, H. 1974. Activation of T and B lymI., Singer, A. 1980. Roleof accessory phoeytesin vitro. III. Presenceof Ia cells in Bcell activation. III. Cellular determinants on allogeneiceffect factor. analysis of primary immuneresponse Z Exp. Med. 140:1717-22 deficits in CBA/N mice: presenceof an 8. Asano,Y., Hodes,R. J. 1982. T-cell accessoryeell-Bcell interactiondefect. regulationof Bcell activation. T cells J. Exp. Med. 152:1194-209 independentlyregulate the responses mediatedby distinct B cell subpopula- 18. Bottomly,K., Mosier,D. E, 1981. Antigen-specifichelper T cells requiredfor tions. Z Exp. Med. 155:1267-76 dominantidiotype expression are not 9. Asano, Y., Shigeta, M., Fathman,C. H-2 restricted. J. Exp. Med. 154: G., Singer, A., Hodes,R. J. 1982. Role 411-21 of the majorhistocompatibilitycomplex in T cell activationof Bcell subpopula- 19. Buruet, F. M. 1959. The Clonal Selection Theory of Acquired Immunity. tions. Asingle monoclonal T helper cell Cambridge:CambridgeUniv. population activatesdifferent Bcell sub20. Chused,T. M., Kassan,S. S., Mosier, populations by distinct pathways.J. D. E. 1976. Macrophagerequirement Exp. Meal. 156:350-60 for the in vitro responseto TNP-Ficoll: 10. Asano, Y., Singer, A., Hodes, R. J. 1981. Role of the majorhistocompatia thymic independentantigen. J. Imbility complex(MHC) in T cell activamunol. 116:1579

~e

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T CELL-B CELL INTERACTION both mappingto the K-endof the H-2 complex.J. Exp. Med.147:1159-74 100. Sprcnt,J. 1978.Restrictedhelper function of F~ ~ parent bonemarrowchimeras controlled by K-endof H-2complex. Z Exp. Meal.147:1838-42 101. Subbarao,B., Ahmed, A., Paul, W.E., Scher, I., Licberman,R., Mosier,D. E. 1979. Lyb-7, a newB cell alloantigcn controlled by geneslinked to the Ig Ca locus. J. lmmunol.122:2279 102. Swierkosz,J. E., Rock,K., Marrack,P., Kapplcr, L W.1978. The role of H-2linked genesin helper T-cell function. II. Isolation on antigen-pulsedmacrophagesof two separate pop.ulations of F~helper T ceils eachspecific for antigen and one set of parental H-2products. J. Exp. Med.147:554--70 103. Tada, T., Takemori,T., Okumura,K., Nonaka, M., Tokuhisa, T. 1978. Two distinct types of helperT cells involved in the secondaryantibodyresponse:independentandsynergisticeffects of Iaand Ia+ helper T cells. J. Exp. Med. 147:446-58 104. Takatsu, K., Hamaoka, T. 1982. DBA/2Hamice as a model of an Xlinked immunodeficiency whichis defective in the expressionof TRF-acceptor site(s) on Blymphocytes,lmmunol. Rev. 64:25 105. Takatsu, K., Tominaga,A., Hamaoka, T. 1980.Antigen-induced T cell-replacing factor (TRF).I. Functionalcharacterization of a TRF-producing helper T cell subset andgenetic studies on TRF production. J. Imraunol.124:2414-22 106. Takatsu, K., Tanaka, K., Tominaga, A., Kumahaxa,Y., Hamaoka,T. 1980. Antigen-induced T cell-replacing factor (TRF).III. Establishment of T cell hybrid clone continuouslyproducingTRF and analysis of released TRF.functional J. lmmunol. 125:264-65 107. Takatsu,K., Sano, Y., Tomita,S., Hashimoto, H., Hamaoka, T. 1981. Antibodyagainst T cell-replacingfactor aeceptor site(s) augmentsin vlvo primary IgMresponses to suboptimaldoses of

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heterologouserythrocytes. Nature292: 360-62 108. Taussig,M.J., Mozes,E., Isac, R. 1974. Antigen-specific thymuscell factors in the genetic control of the immune response to poly-(TYROSYL,GLUTAMYL ) - POLY- D, L- ALANYL -POLY-LYSYL.J. Exp. Med. 140: 301-12 109. Tominaga,A., Takatsu, K., Hamaoka, T. 1980.Antigen-induced T cell-replacing factor (TRF).II. X-linkedgenecontrol for the expressionof TRF-acceptor sites(s) on B lymphoeytesand preparation of specific antiserumto that acceptor. Z Immunol.124:2423-29 110. van Leeuwen, A., Goulmy,E., van Rood,J. J. 1979. Majorhistocompatibility complex-restricted antibodyreactivity mainly, but not exclusivelydirected against cells frommaledonors. J.. Exp. Med.150:1075-83 111. yon Boehmer,H., Hudson,L., Sprent, J. 1975. Collaborationof histocompatible T and B lymphocytesusing cells from tetraparental bone marrowchimeras. J. Exp. Med.142:989 112. Wylie,D. E., Sherman,L. A., Klinman, N. R. 1982.Participation of the major histocompatibilitycomplexin antibody recognitionof viral antigens expressed on infected cells. J, Exp. Med.155:403 113. Yamashita,U., Shevaeh, E. M. 1978. Thehistocompatibilityrestrictions on macrophageT-hdper cell interaction determinethe histocompatibility restrictions on T-helpercell B-cell interaction. J. Exp. Med.148:1171-85 114. Zinkernagel,R. M., Callahan, t3. N., Althage,A., Cooper,S., Klein, P. A., Klein, J. 1978. Onthe thymusin the differentiation of"H-2serf-recognition" by T cells: evidencefor dual recognition? Z Exp. Med.147:882-96 115. Zinkernagel, R. M., Callahan, G. N., Althage,A., Cooper,S., Streilein, J. W., Klein, J. 1978. The lymphoreticular systemin triggering virus plus selfspecificcytotoxicT cells: evidencefor T help. Z Exp. Med.147:897-911

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Ann.Rev.Immunol. 1983.1:243-71 Copyright ©1983by AnnualReviews Inc. All rights reserved

COMPLEMENT LIGAND-RECEPTOR INTERACTIONS THAT MEDIATE BIOLOGICAL RESPONSES Douglas T. Fearon and Winnie W. Wong Department of Medicine,HarvardMedicalSchool,and the Department of Rheumatology and Immunology, Brighamand Women’s Hospital, Boston, Massachusetts 02115 INTRODUCTION Complement is the principal humoraleffector systemof inflammation.It is comprised of 18 proteins that are presentin plasmaand, at lesser concentrations, in extravascular fluids. The systemcan be activated through the classical pathwayby immunecomplexescontaining immunoglobulinIgG or IgMand throughthe alternative pathwayby particulate material having certain chemicalcharacteristics, suchas the absenceof sialic acid (23, 78, 93). Duringactivation of these pathways,proteases that hydrolyzecomplement proteins are assembledand produce cleavage fragments that then serve as ligandsfor specific receptors on certain cells, includingpolymorphonuclear leukocytes, eosinophils, monocytesand macrophages,mast cells, andlymphocytes.Theseligand-receptorinteractions elicit cellular responses that mayculminate in an inflammatoryreaction. The importantrole of the complement systemin host resistance to microbial infection relates to its capacityto induceinflammation at a tissue site. Twotypes of ligands are generatedduring complement activation to serve this function: those boundto the microorganism,and those freely diffusible in the fluid phase.Examples of the formerare Clq, C4b,andC3b, whichmaybe considered bifunctional since they can attach both to the target of complement activation andto effector cells bearingthe appropriate receptors. Ligand-receptorinteractions of this type directly promotethe 243 0732-0582/83/0410-0243502.00

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removalof microorganisms throughphagocytosisby leukocytes. Thediffusible ligands, whichare low-molecular-weight cleavagepeptides such as C3a and CSa,act as local hormonesthat cause receptor-mediatedcellular responsesof secretion, altered metabolism,and migrationin the microenvironment. These ligand-receptor interactions indirectly promote the clearance of infectious agents by augmentingthe accumulationof large numbersof phagocyticceils, thereby synergistically enhancingthe effects of the complementligands boundto microorganisms. This reviewconsiders the molecularreactions that lead to formationof these twoclasses of ligands, their cellular receptors,andthe cellular reactions inducedby occupancyof the receptors. MOLECULAR REACTIONS LEADING TO THE GENERATION OF COMPLEMENT LIGANDS FOR CELLULAR RECEPTORS Ligands Attached to the Target of Complement Activation C3 C3is

comprisedof two polypeptide chains, the molecular weights of whichare 120,000(a-chain) and 75,000(B-chain) (Figure 1). Cleavage the a-chain at several sites yields six fragmentsthat can interact with cellular receptors. All of these cleavageproductsexcept C3aand C3eare covalentlyboundto the target of complement activation and are therefore bifunctionalligands. Activation of the classical or alternative complement pathwaysleads to the assemblyof their respective C3-cleavingenzymes,C4b,2aand C3b,Bb, whichhydrolyze the Arg 77-Ser 78 bond in the o~-polypeptide of C3 to generate the Mr 9000 C3a peptide and the Me 186,000 C3b molecule (Figure 1). This proteolytic reaction causes a rapid conformationalchange in C3bthat is associatedwith an increasein its surfacehydrophobicity (55) andexposureof the internal thioester (113) betweencysteinyl and glutamyl residuesin the a-polypeptide.This structure’ mediatesthe covalentattachmentof C3bby transesterifying to nucleophilicsites on the surface bearing the C3convertase, such as hydroxylgroupsof carbohydrates(68). Covalent bindingof C3bis relatively nonspecificas exemplifiedbythe capacityof the moleculeto fix on manydifferent types of cell surfaces and on immune complexes. TheC3bis subject to conversionto iC3bby the serine protease, factor I, whichcleaves a 3000-Mrpeptide fromthe t~-polypeptide, leaving segmentsof 68,000and 43,000Methat are disulfide-bondedto the B-polypeptide (45, 67, 98). This reaction generallyrequires the presenceof factor whichbinds to C3band presumablyinduces the conformationthat is suit-

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C3

OR SH C:O I

C3o+ C3b Annu. Rev. Immunol. 1983.1:243-271. Downloaded from arjournals.annualreviews.org by HINARI on 08/27/07. For personal use only.

$ I

S

® iC3b

C3d,g (e2D)

C3c

C3d+ CSg

S I S I

S I S ~

OR ® I l sHc:o I I

sI

sI o. ® s.~::oI

Figure 1 Proteolytic cleavage of C3 to generate ligands for C3 receptors. The position of the internal thioester capable of formingan ester linkage for covalent deposition is shownon the a-chain. The sequential degradation of native C3 to its biologically active fragments occurs in four steps. Step (~) involves the cleavage of the a-chain at the designated site by the classical or alternative pathway convertases to form C3a and C3b. Step (~) cleavage mediated by factor I in the presence of cofactors Hor C3breceptor and results in formation of iC3b. The source of the enzymes r~_]~onsible for Step @cleavage to form C3d,g and releasable C3cis not well defined. Step (~ involves the cleavage of C3d,g by tryptie enzymes to form C3d and C3g. Not shown in this figure is the production of C3e, which is thought to be derived from C3c by unknownproteolytic reactions.

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able for interaction with factor I (88, 125). Sincethere is impairedbinding of factor Hto C3bthat is covalentlyattachedto activatorsof the alternative pathway(23, 78), C3bat these sites is relatively resistant to factor I. The cofactor function of factor H can also be performedby purified, solubilized C3breceptor (21) and probablyby membrane-associated receptor (73, 74). Since conversionof C3bto iC3bgreatly reducesthe capacity of the protein to bind to the C3breceptor, this receptor, in concert with factor I, has a mechanism for releasing boundligand. TheiC3bis cleaved at another site in the a-polypeptidethat is on the oppositeside of the region containingthe covalentbindingsite, This reaction leads to formationof two newfragments, C3d,g(also termed a2D) 41,000 Mr, that remains boundto the complement-activatingtarget, and C3cof 143,000Mr, that is released (61, 102, 124). Theenzymeresponsible for the physiologiccleavageof iC3bhas not beenconclusivelyidentified. Incubationof iC3bin serumat 37°Cfor up to 24 hr causesslowconversion of the protein to the C3d,gand C3cfragments(61). Althoughplasmincan hydrolyzethe C3d,gfragmentfromiC3b(61, 80), it also splits C3d,ginto the C3dand C3gfragments,whichare not observedafter treatment of iC3b with serum. Moreover,removal of plasmin/plasminogenfrom serum does not diminishthe rate of cleavageof iC3b(61). Themostlikely candidate for the enzymeeffecting the appropriate scission of iC3b is factor I by analogyto its capacity to generate the C4dand C4cfragments from C4b in the presence of C4-binding protein (27, 79). Factor H and the C3b receptormaybe the necessaryfluid phaseandsolid phasecofactors, respectively, since they have beenreported to haveweakat~nity for iC3b (98), whichis muchless than their respectiveaffinities for C3b.In supportof this possibility is the recent demonstrationof cleavageof C3cfromcell-bound iC3bby factor I in the presenceof membrane-associated C3breceptor (73). If this findingis confirmed,the C3breceptorhas the unusualrole of participating in the formationof two ligands, iC3bandC3d,g,for other receptors. Thefinal cleavagereaction involvingC3is that whichgenerates the C3d fragment (Mr 33,000) by release of C3g(Mr 8000) from C3d,g (61). This can be effected by plasmin, trypsin, leukocyteelastase, and cathepsin G. Onlyrecently have investigators distinguished betweenthis fragmentand the a2Dor C3d,gfragment, so that muchof the literature is confusingin this respect. In general, if the "C3d"wasgeneratedby serumenzymes,the fragmentprobablyis C3d,g,whereasin those instances in whichtrypsin was utilized, the 33,000-MrC3dfragmentwasformed.Since C3d,g is the final C3fragmentfound in plasmaof patients with complement activation and on erythrocytes of patients with autoimmune hemolyticanemia,generation of C3dmayoccur only in tissues wherethere is an influx of granulocytes secreting elastase and cathepsin G.

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Thefirst component of the classical pathwayof complement activation is comprisedof three subcomponents, Clq, Clr, and Cls, in a molar ratio of 1:2:2, held together in a calcium-dependentcomplex. The globular heads of the Clq subcomponent bind to the Fc region of IgMand IgG in immunecomplexes,and the collagen-like region binds the Clr and Cls subcomponents.Followingbinding and activation of C 1 by immunecomplexes, viruses or lipopolysaccharides,the Clr and Cls subcomponents are dissociatedfromClqby the C 1 inhibitor that formsthe complex,C lr-C ls(C1 inhibitor)2 (108, 129). TheClq remainsboundto the activator, and collagen-likeregion is nowexposedandcapableof interacting with cellular Clq receptors.

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Clq

FACTORH Factor

H is the regulatory protein of the alternative complementpathwaythat binds to C3band promotesits cleavageby factor I (23, 45, 67, 78, 88, 125). Recently,a receptorfor this protein has beenidentified on leukocytes(63, 66, 87, 104). Since factor H is present in plasma concentrationsof 300 to 500 #g/mland factor H receptors are presumably not continuouslyoccupiedby this ligand, physical alteration in the molecule mustoccur that confers on it receptor-bindingactivity. Hexamers of factor H producedby concentrating the purified protein have been reported to bind to factor Hreceptors on Raji cells (63), suggestingthat conversion the protein into a multivalent ligand capableof multipointattachmentmay be critical for its bindingactivity. Theoccurrenceof solublemultimersof factor H in vivo has not beendemonstrated,but a possible mechanism for polymerization mayinvolve the binding of factor H to immunecomplexes bearingseveral molecules of C3b.In supportof this possibility is the finding of depositionof factor Hat tissue sites corresponding to the location of C3 antigen (10). Since the interaction betweenimmunecomplex-boundC3b moleculesand factor H probablyrepresents the meansfor productionof an effective ligand, factor His consideredas a boundligand rather thana freely diffusibleligand. Diffusible Ligands Releaseof C5afromits parent molecule,C5, occursby a mechanism similar to that already describedfor C3a. C5is comprisedof two polypeptidesof Mr112,000(a-chain) and Mr74,000(B-chain). The classical and alternative pathwayC5convertases,C4b,2a,3bandC3b(n),Bb,respectively, cleave the a-chain at Arg74- x 75 to release the M r 11,000glycopeptide,C5a(23, 78, 93). The C-terminal arginines of C3aand C4aare removedby serum carboxypeptidase Nto producethe desArgderivatives of these peptides that lack biologicactivity. In serum,this reactionoccursso rapidly that the time during whichC3aand C4a can diffuse before losing their capacity to

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interact with their receptors on mast cells is probablybrief. SerumcarboxypeptidaseN also releases the C-terminal arginyl residue of CSato diminishgreatly its capacity to interact with receptors on mastcells, but CSadesArgretains the capacity to bind to receptors on polymorphonuclear leukocytesand monocytes.Therefore, CSadesArgcan diffuse fromthe site of its generationand can establish the concentrationgradient necessaryfor its leukoattractingfunction. A fragmentof C3termed C3ehas been identified by its leukocytosisinducingactivity wheninjected into rabbits (35, 100). It can be generated by prolongedincubation of serumor by treatment of C3with trypsin. This Mr12,000 peptide is thought to be derived from the a-chain of C3cand thus represents a relatively late degradationproduct. MEMBRANE RECEPTORS COMPLEMENT-DERIVED

FOR LIGANDS

Specific cellular receptors for ligands derived fromcomplement proteins (Table 1) have beenidentified in studies with assays that vary in their reliability. For example,the bindingto a cell of antibodymonospecific for a receptor providesstrong evidencefor the presenceof that receptor. However, someotherwisedistinct membrane proteins mayshare antigenic determinants(128). The saturable binding of a purified ligand might also consideredproofof the existenceof a particular receptoron a cell, but some proteins, such as iC3b and C3d,g, apparently can bind to two different receptors on lymphocytes (97). Thebindingto cells of indicator particles, such as erythrocytes, coated with certain complementproteins is a frequently used assay for complement receptors, but it mayyield misleading results: Theligand maybe modifiedduringinteraction with cells (63, 97); morethan onepotential ligand maybe presenton the indicator particle (97); and classical studies of saturability and reversibility of bindingare not possible. Themostindirect assayinvolvesthe inductionby a putative ligand of a biologic response from a cell or mixtureof cells. For example,the capacity of C3ato cause contraction of the guinea pig ileumis considered indicative of C3areceptors on mastcells containedwithin this tissue. Other problemsin experimentsdesignedto demonstratethe presenceof particular receptors on certain cell types are related to impurityof the ligand and inadequate identification of cells whenmixedpopulations, such as lymphocytes,are utilized. For these reasons,this section reviewsin somedetail the methodology used for studies relating to the cellular distribution of receptors.

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Receptors for Ligands Attached to the Target of ComplementActivation ClqRECEPTOR Thecapacityof certain cells to bindsolubleClqwhich waslabeledeitherwith125Ior indirectlywithttuorescein (115,116),and form rosetteswitherythrocytes bearing C1 q (28), hasindicated thepresence of cellular Clqreceptors.Human peripheralbloodgranulocytes, monocytes, mostBlymphocytes, less than8%of Tcells (identifiedbyformation of rosetteswithsheeperythrocytes), anda smallproportion of null cells bound fluoresceinated Clq(116). Inexplicably,in onestudyClq-bearing erythrocytes formed rosettesonlywithB cells butnotwithmonocytes or granulocytes (28). Scatchard analysisof thespecificbinding of 125I-labeled Clqto granulocytes indicatedthe presenceof 150,000-600,000 receptors per cell (116). Bindingof radiolabeled Clqto mononuclear cells and Clq-coated particleswasinhibitedbytypeI collagenandbypepsin-resistant Clqfragments; the intact molecular complex of Clqrswasnottaken Table1 Cell types bearing complementreceptors Receptor Clq

C4a, C3a C3b

CR2 CR3 C3e C5a

Factor H

Cell type Neutrophil Monocyte Nullcells B lymphocytes Mastcells Erythrocyte Neutrophil Monocyte/Macrophage Eosinophil B lymphocyte T lymphocyte Glomerular podocyte B lymphocyte Nullcells? Sameas C3breceptor Neutrophils Mastcells Neutrophil

Cellular response Respiratoryburst Enhanced ADCC Secretion Immunecomplexclearance? production of iC3b and C3d,g? Enhancedphagocytosis; adsorptive endocytosis Sameas neutrophil Enhancedphagocytosis

Enhanced ADCC Sameas C3breceptor? Release from bone marrow Secretion; leukotriene synthesis? Chemotaxis; secretion increasedstickiness; increasedC3breceptor expression Mo’nocyte/Macrophage Chemotaxis;secretion; spreading leukotriene synthesis? B lymphocytes Secretionof factor I; mitogenesis Monocytes Respiratory burst Neutrophils

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up by these cells. Thus, the portion of the Clq moleculeinteracting with receptors was identified as the collagen-like tail region. Thecalciumdependentassociation of Clq with platelets is reported to occur through C ls boundto the platelet andthus is distinct frombindingmediatedby C lq receptors (121). The affinity of the Clq receptor on granulocytes and monocytesfor monomericClq at physiological ionicity was so low that binding was reproduciblydetected only in hypotonicmedia(115, 116). In the presence of a relative salt concentrationof 0.09, the averageequilibriumbinding constant of the receptor for monomericClq on granulocytes was 7.6 X -1 (116). Since C lq wouldusuallybe presentedto cells as an aggregate 106M or array on immunecomplexes,multipoint attachment to Clq receptors wouldgreatly increase the effective avidity and this increase mustaccount for the rosetting of Clq-coatedparticles with leukocytes. Otherthan sensitivity of its function to trypsin, nothingis knownof the physical nature of the receptor that binds Clq. c3 RECEPTORS Four different proteolytic cleavage products of C3--C3b, iC3b, C3d,g, and C3d--boundto the target of complementactivation interact with three types of cellular receptors. Agenerallyacceptedterminology for these receptors is evolving with increasing knowledge of the biochemistryof their ligands. In this review,a receptoris namedfor its preferred ligand, if known.Thus, the immuneadherencereceptor is designated the C3breceptor rather than complement receptor type 1 (CR1).The less descriptive terms, CR2andCR3,are usedfor the other C3receptors because there is someuncertainty in the identification of their biologically relevant ligands. C3breceptor TheC3breceptor (CR1)was discovered almost 30 years ago whenbacteria whichhad been incubated in serumthat contained specific antibody and active complementwere shownto adhere to humanerythrocytes (83). The immuneadherence receptor of the erythrocyte was shownsubsequentlyto be specific for C3bon the immune complex(37) and to reside on other cell types, includingneutrophils, eosinophils,monocytes, macrophages,B and someT lymphocytes,mast cells, and glomerularpodoeytes (7, 30, 43, 59, 69, 95, 118, 126). Apopulation of large granular lymphocyteswith natural killer activity and identified by the monoclonal antibody, anti-HNK-1,lacks C3breceptors (114). Althoughthe presence of C3breceptors on these variouscells is usuallydetectedin rosette assays with sheep erythrocytes that bear C3b, monospecificpolyclonal and monoclonal antibodies to the humanC3breceptor have nowbecomeavailable (19, 22, 53).

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The C3b receptor was first isolated from humanerythrocytes and was shown to be a glycoprotein of Mr 205,000 to 250,000 when assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (21). The higher estimate of Mris based on the more recent observation that the purified C3b receptor migrated slightly more slowly than erythrocyte band 1, which has a Mr of 240,000. Monospecific rabbit antibody to this glycoprotein blocked C3b receptor function on erythrocytes, neutrophils, monocytes, and B and T lymphocytes and immunoprecipitated from each of these cell types a single membraneprotein identical to the glycoprotein initially isolated from erythrocytes (19, 22, 126). This polyclonal anti-C3b receptor also bound exclusively to glomerular podocytes in renal biopsy specimens (59). Thus, in man the C3b receptor of each of these cells represents a commonmolecular entity. This conclusion was supported by other studies with monoclonal antibodies to the C3b receptor (53). Twoforms of the C3b receptor differing in their mobility on polyacrylamidegel electrophoresis in sodiumdodecyl sulfate have recently been identified. This structural polymorphism represents a stable phenotypic characteristic inherited as an autosomal codominanttrait and expressed by receptors on erythrocytes, neutrophils, and mononuclearleukocytes. In the normal population, 65%have only receptors of Mr 250,000, 3%have only receptors of a higher molecular weight, and 32%have both forms of receptors (W. Wongand D. Fearon, unpublished observation). The number of C3b receptors on cells has been estimated by measuring the uptake at saturating concentrations of 125I-labeled dimeric C3b and anti-C3b receptor (3, 127). Erythrocytes have an average of only 500-600 receptors per cell. The numberof receptors expressed by erythrocytes is genetically regulated by two autosomal codominant alleles determining high and low receptor numbers, respectively. Individuals homozygousfor the high allele have erythrocytes with an average of 800 receptors per cell, those homozygousfor the low allele have cells with 250 receptors, and heterozygotes’ erythrocytes have 500 receptors per cell. Of normal individuals, 34 and 12%are homozygousfor the high and low alleles, respectively 027). A markedly different distribution of these phenotypes was found amongpatients with systemic lupus erythematosus, of whomonly 5 and 53%were homozygous for the high and low alleles, respectively. Thus, the low numbersof C3b receptors on erythrocytes of lupus patients was found to be inherited, as had been suggested in another study (76), rather than acquired (53), and mayrepresent a predisposing factor in the development of this disease. An additional abnormality of the C3b receptor in lupus is its absence from glomerular podocytes in proliferative glomerulonephritis (59).

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Twostudies of purified neutrophils have estimated the presence of 20,000-40,000 C3breceptors per cell (3, 22), whereasa third found140,000 receptors per cell (53). Purified monocytes and B lymphocytes haveapproximately the samenumberof receptors as neutrophils, whereasthe 5-25% of peripheral bloodT cells that expressthis glycoproteinhavefewerreceptors (3, 126). Unstimulatedneutrophils and monocytesin peripheral blood express only 10-20%of their total numberof receptors, and the processof purifyingthese cells causestemperature-dependent increases of up to eightfold in the numberof receptors present on the plasma membrane.For example,neutrophils purified at 4°Cexpressedonly 5000C3breceptors per cell; incubationof the purifiedcells at 37°Cfor 15 minled to the expression of 38,000 receptors per cell. Addition to wholeblood of C5adesArgin nanomolarconcentrationsalso causedneutrophils to increase their plasma membrane expression of receptors rapidly in a dose-responsemanner(24). Thus, a minority of C3breceptors of unstimulatedneutrophils and monocytes resides on the plasma membrane while the majority is present in latent, intracellular compartmentsthat can be recruited to the plasma membrane. This observationexplains the capacity of chemotacticfactors to enhancethe formation of rosettes betweenthese cells and sheep erythrocytes (58). In contrast, peripheral blood B lymphoeytes express 30,000-40,000receptors per cell in the absenceof stimulation by purification proceduresor chemotacticfactors (24). Theontogenyof C3breceptors on humanB lymphocytesand neutrophils has beenstudied. Thereceptor is present on only 15%of large pre-B cells and 35-50%of small pre-B cells in the bone marrow.Although60-80% of immatureB cells in bonemarrowand fetal liver and all peripheral blood matureB cells express C3breceptors, maturationto plasmacells is associated with the loss of these receptors(114). Thegradualappearanceof C3b receptorsduringB cell development is consistent with its absencein several lymphoblastoidcell lines and on most B ceils of patients with chronic lymphaticleukemiaarrested early in differentiation. C3breceptors on neutrophils appear later in developmentthan do CR3receptors (96). Equilibriumbinding studies, with dimeric C3beither found in purified preparations of the protein or producedby chemicalcross-linking, demonstrated that the C3breceptor on erythrocytes, neutrophils, monocytes,and B lymphoeyteshad an apparent association constant for these ligands of -1 (3). Theseexperimentsprobablyunderestimatedthe affinity 2-6 X l0T M of the receptor for the bivalent ligand since they did not determinethe percentageof the ligand capableof specific binding. When the active bindable fraction of dimericC3bwaspurified by specific elution fromC3breceptors and was reassessed for uptake by receptors on erythrocytes, an association constant of 109 M-1 was obtained (D. Fearon, unpublished

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observation).Theaffinity of the receptor for monomeric C3bwasestimated -t (3). to be only 2 X 106 M Theform of C3recognizedby the C3breceptor also can be generatedby hydrolysis of the thioester without cleavage of the C3apeptide fromthe a-polypeptidechain, indicating that the conformationalchangesoccurring in C3followingthioester hydrolysisare similar to those inducedby cleavage of the a-polypeptidechain (6). Althoughparticles beating iC3bdo not form C3breceptor-dependent rosettes underconditionsof physiologicalionicity, rosette formationhas beenobservedunderconditionsof low ionic strength, whichsuggeststhe receptorhas weakaffinity for this formof C3(97). This finding mayrelate to the possible eofactor role of the C3breceptor in the proteolytic processingby factor I of iC3bto C3cand C3d,g(73, 74). High concentrationsof C3c, but not C3d,inhibit formationof rosettes between humanerythrocytes and particles bearing C3b, a finding that mapsthe receptor bindingsite in C3bto its C3cregion (99). Althoughthe preferred ligand for C3breceptors is C3b, the samereceptors can also mediateuptakeof particles bearingC4b(16). Receptoraffinity for C4bis consideredto be lowerthan for C3b,since the purified receptor is only one tenth as efficient in dissociating C2afrom C4bas it is in dissociating BbfromC3b(86). Relatively less is knownabout C3breceptors in species other than man. Generally,they reside on the samecall types, with possible exceptionsof erythrocytes and glomerularpodocytes, whichappear to lack receptors in non-primatespecies, andplatelets, whichappearto havethis function, in contrast to man(82). AC3b-binding protein of Mr64,000has beenisolated from rabbit alveolar macrophages, and a membraneprotein of M r 60,000-63,000 on murine leukocytes cross-reacts with anti-human C3b receptor, but these proteins have not beenshownto mediateC3breceptor function (105, 128). C3 receptor type 2 (CR2)Indicator particles bearing C3dform rosettes with essentially all Raji and Daudilymphoblastoid cells, approximately one third of sheeperythrocyte-negativeperipheral bloodlymphocytes,and most of the tonsil B lymphocytes (19). Thus,in peripheral blood, approximately 50%of the B cells carry only C3breceptors, andthe rest expressboth C3b receptors and CR2;tissue B lymphocytes apparentlyexpressboth receptors. T lymphoeytes,monocytes,and neutrophils are generally considerednot to have CR2,basedon absenceof staining by rabbit anti-CR2and absenceof formationof rosettes with particles coated with C3d,althoughone report found C3d-dependent rosette formation with monoeytes(11, 65). In addition, the capacity of target-bound C3dto augmentantibody-dependent cellular cytotoxicity suggeststhat null cells also haveCR2(92).

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TheCR2isolated fromculture supernatants of Raji cells wasa glycoprotein of Mr72,000,and rabbit antibodyto the purified receptor immunoprecipitated fromRaji and BFlymphoblastoid cells a single protein of the samemolecularweight as the immunogen. This antibody blockedall C3ddependentand someiC3b-dependent rosette formation, indicating that the C3dregionis sufficiently exposedin iC3bfor interaction with CR2(19, 64). Further studies on ligand specificity haveindicated that CR2is capableof bindingall three degradationfragmentsof C3b,whichinclude iC3b, C3d,g, and C3d(98). Thus, CR2shares someligands with CR3,but it is the only receptor capableof bindingC3d.Thenumberof receptors on cells and their affinities for these ligandsis not known. C3receptor type 3 (CR3)Theexistence of a third type of receptor for a C3fragmentcovalently boundto the target of complement activation was suggestedby the capacity of sheeperythrocytes bearing iC3b to formrosettes with monocytes, neutrophils, somelymphocytes,and glomerular podocytes(11, 19, 22). Theinability of antibodies specific for the C3b receptor and for CR2to block iC3b-dependent rosette formationof neutrophils, monocytes,and all of the lymphocytesmust be considered strong evidencefor the presenceof this additional receptor (19, 22). Reportseontiiet concerningthe forms of C3capable of binding to CR3. In one study with 125I-labeled C3, sheep erythrocytes bearing only iC3b formedrosettes with monocytesand glomerular podocytes (11). Other investigators have prepared indicator particles by treatment of C3bon sheeperythrocyteswith purified factors Hand I underconditionsgenerally consideredto produceiC3band little, if any, C3d,g. Theseparticles have been shownto adhere to neutrophils and monocytes,implyingagain that the receptor is capable of binding iC3b (5, 22, 107). However,a recent report comparingthe products of treatment of fluid phase and particleboundC3bwith factors H and I found that the former reaction ended at the iC3bstage, whereasthe productsof the latter reaction wereiC3b and an uncharacterized polypeptideof Mr41,000, whichwas slightly smaller than the M r 43,000 fragmentof the a-polypeptidedisulfide bondedto the B-chain in iC3b. Althoughthis Mr 41,000 fragment resembles the C3d,g fragmentby size, this study did not attempt to demonstratetheir common identity (98). WheniC3b and a mixturecontaining iC3band the Mr41,000 fragmentwereeach assessed for binding to CR3,only the mixturehad this function, whichsuggests the fragmentresemblingC3d,gwasthe ligand for this receptor(97, 98). Direct evidencefor the capacityof C3d,g to bind to receptors on neutrophils has beenobtainedwith the purified, solubleformof the protein. C3d,g isolated from agedhumanserumwasreversibly boundin a saturable fash-

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ion by humanneutrophils, and binding was not inhibited by anti-C3b receptor antibody. The numberof receptors for C3d,g ranged from 40,000-60,000 per cell, andthe associationconstantof the receptorfor this ligand was 0.5-1 × 107 M-~ (D. Vik and D. Fearon, unpublishedobservation). Thereceptor responsible for this interaction is probablyCR3,but these studies did not addressthe questionof whetherthis receptoralso binds iC3b. TheCR3could be designatedthe C3d,greceptor if it can only bind C3d,g. The lymphocytesubpopulation expressing CR3includes B ceils, althoughsomeCR3-positivecells were reactive with the monoclonal antibodies anti-Leu-1andOKM-1 (97). Theformerrecognizesa T-cell antigen, and the latter recognizesdeterminantson neutrophils, monocytes, and null cells that havenatural killer activity. Lessthan one third of peripheral bloodB lymphocytesand less than half of tonsil B lymphocytesexpress CR3,indicating CR3resides on only a subpopulationof B ceils. Variableproportions of Raji, Daudi, and BFlymphoblastoidcells express CR3(19). The membraneprotein namedCR3has recently been suggested to be identical to or spatially associatedwith the Mac-1antigen. Rat monoclonal anti-Mac-1inhibited formationof rosettes betweensheeperythrocytesbearing humaniC3b and humanneutrophils and between antibody-sensitized sheep erythrocytes treated with wholemouseserumcomplement and mouse peritoneal macrophages (5). Therewasno inhibition of C3breceptor function. Anti-Mac-1recognizes an antigen present on murine and human monocytes,macrophages,neutrophils, and a subpopulationof lymphocytes havingnatural killer activity. However, Mac-1 doesnot reside on peripheral blood or tissue B lymphocytes(4). This disparity betweenthe different subsets of lymphocyteswith functionally detectable CR3and whichexpress Mac-1antigen mustbe resolvedbefore the latter can be consideredthe CR3. FACTOR H RECEPTOR The presence of receptors for factor H on leukocytes has been demonstratedby the adherenceto these cells of particles coated with factor H and by the binding of radiolabeled factor H. Two groupsof investigators measuringthe binding of labeled factor H to Raji cells reported that 4.2 × 106 (63) and 4 × 104 (104) moleculesof H boundper cell, respectively,at equivalentinputsof the ligand. In the former study, saturation was achievedwith an approximateinput of 3 X 10-7 M of H (63). Subsequently,157,000H receptors wereestimated to be present on Raji cells by measuringthe uptakeof a rabbit anti-idiotypic anti-human factor H antibody that had specificity for factor H receptors (66). The reasonsfor these disparate results are not apparent, althoughexplanations mayinclude the varying degreesof aggregationof the ligand leading to an overestimateof receptors, or the varyingreceptor numberexpressedon Raji

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cells dependingon culture conditions and phase of growth cycle. The anti-idiotypic antibody immunoprecipitated from biosynthetically labeled Raji cells a cross-reactive protein comprisedof polypeptidesof Mr100,000 and 50,000in their unreducedstates and Mr50,000whendisulfide-reduced (66). Asimilar proteinin the spent culture mediaof these cells wasadsorbed by agarose to whichfactor H had been coupled. TheB lymphoblastoidlines Raji and BF, and peripheral blood B cells havebeenshownto express factor H receptors, whereasa T cell line, HSB, did not (63). Anotherstudy found that 94%of tonsil lymphocytesformed rosettes with particles bearingfactor H (104), indicating that T cells may also express these receptors. However,lower percentages of peripheral blood lymphocytes,"nearly all" of whichhad surface immunoglobulin (63, 104), were capable of binding factor H, indicating a possible difference betweencirculating and tissue T cells. Factor H-coatedparticles havealso been found to bind to granuloeytes and monocytes(104), but since the particles used in these experimentsalso carried C3b,rosette formationmay havebeen mediatedby C3breceptors on these phagocyticcells. Moreover, C3bnoncovalentlymodifiedby factor Hhas beensuggestedto bind to novel C3receptors on Raji cells that are distinct from C3breceptors CR2and CR3(87), a finding that further complicatesanalyses of factor Hreceptors by use of particles bearing both C3band H.

Receptors for 9iffusable ComplementLigands C3a ANDC4a RECEPTOR Despite the early recognition that C3a can inducecontractionof the guineapig ileum, increase vascular permeability, andcausedegranulationof mastcells (15, 18), anddespite the relative ease with whichC3acan be purified (51), studies of the bindingof C3ato various cell types are incomplete.Therefore,the conclusionthat specific receptors for C3areside on mastcells is still basedon indirect experiments.Purified C3ainducessecretion of histaminefrompurified mastcells (57). Theguinea pig ileal contraction causedby this peptide is primarilyhistamine-dependent, and this tissue exhibits tachyphylaxis,or desensitization, to C3a(15, 18). Therefore,the contractile responseof the guineapig ileumis considered secondaryto release of histaminecausedby interaction of C3awith specific receptorson mastcells in this tissue. Noequilibrium-binding studies of the interaction of C3awith mastcells of anyspecies havebeenperformed,but an estimate of the affinity of this putative cellular receptor for C3acan be madebased on the capacity of l0 -s MhumanC3ato induce contraction of the guinea pig ileum (15). This concentrationof C3awouldbe achieved cleavage of less than 1%of C3in plasma.Secretion of histamineby basophils in preparations of humanperipheral blood leukocytes required con-

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centrations of humanC3ain the range of 10-7-10-6 M(38); the basis for the 10- to 100-fold higher concentration of C3anecessaryfor the human basophil reaction than for the guineapig ileal responseis not known.The biologic activities of C3aare abolished whenthe C-terminal arginine is removed,and this is presumedto indicate that C3adesArgcannot bind to the C3areceptors. Theprimarystructural features of C3aessential for its biological activity have been examinedby use of synthetic oligopeptides (52). Autoradiographicstudies that involved incubation of 12~I-labeledC3a with humanperipheral blood leukocytes demonstratedbinding of the peptide to eosinophils, neutrophils, and monocytes(38). However,doseresponsecurves describingthe quantitative uptake of ~25I-labeledC3aby preparations of neutrophils and monoeyteswere convexto the abscissa rather than concave,as wouldhavebeenexpectedfor a saturable reaction, and only 2-3%of the ligand was bound. Therefore, additional studies of the binding of C3aby neutrophils, eosinophils, and monoeytes are necessary to establish the presenceof C3areceptors on these cells. Highconcentrationsof C4ainduce contraction of the guinea pig ileum, and the contractile response can be abolished by prior incubation of the tissue with C4aor C3a(41). C4adesArghas no activity. This low affinity interaction of C4awith C3areceptors on tissue mastcells probablydoesnot reflect a physiologicalfunction of C4asince the mieromolarconcentrations of C4arequired for the response could be achievedonly by cleavage of almost the entire content of C4in plasma.However,this finding reveals a functional homology betweenC3aand C4a, whichalso share similar structural features(41). CSa RECEPTOR Cell types that exhibit biological responses to CSaare mast cells, basophils, neutrophils, eosinophils, monoeytes,and macrophages. Evidencethat specific receptors for CSamediatethe responseof mastcells andbasophilsis indirect andis similar to that for C3areceptors on these cells. Treatmentof the guinea pig ileumwith C3aor CSainduces taehyphylaxisto the samebut not the other peptide, suggesting that the receptors on mastcells for these twopeptides are distinct. Concentrations of CSaof 10-1°-10-9 Mcause contraction of the guinea pig ileum, and cleavageof the C-terminalarginine fromCSadecreases its activity by at least 1000-fold(32). Since the serumconcentrationof C5, the parent molecule, is approximately10-6 M,C5adesArgmightnot be present in vivo in concentrationssufficient for interacting with these receptors. Theimpaired bindingof humanCSadesArgis eansedin part by its single oligosaeeharide, becausedeglyeosylation of the peptideaugmented its biologicactivities (31).

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In contrast to the indirect evidencefor C5areceptors on mastcells, the presenceof specific receptors on neutrophils (14) and macrophages (13) beenestablished. Human neutrophils have a saturable binding capacity for 125I-labeled C5a, each cell taking up a maximum of 100,000-300,000 molecules of the glycopeptide with an association constant of 0.3-1 X 910 -1. Similar numbersof receptors on murine peritoneal macrophages M boundhumanC5awith a comparableaffinity. C5adesArgalso binds to C5a receptors on neutrophils and macrophages as indicated by its capacity to inhibit competitivelythe uptake of 125I-labeledC5awhenpresent in 100fold higher concentrations(13, 14, 31). Direct analysis of the binding 125I-labeled C5adesArgto neutrophils demonstratedthe presence of only 40,000-50,000receptors per cell (24). Thedifference in the numbers receptors binding C5aand C5adesArgprobably was caused by different experimental conditions: Althoughbinding of C5adesArgwas performed at 4°C, C5a was incubated with neutrophils at 24°C, which mayhave permittedinternalization of the ligand or expressionof additional receptors cycling between the plasma membraneand intracellular compartments. Saturation of 50%of the neutrophil receptors occurred with 2.5 × 10-8 M C5adesArg,a concentrationthat wouldbe achievedby cleavageof less than 5%of plasmaC5(24). It is not knownwhetherthe C5areceptors on mast cells are similar or identical to those present on neutrophils and macrophages. RECEPTOR Incubation of radioiodinated humanC3e with rabbit bloodfor 1 hr at 37°Cled to the association of 640,000moleculesof the peptide per polymorphonuclear leukocyte and 73,000 molecules per mononuclearleukocyte (35). However,the specificity, saturability, and reversibility of binding wasnot examined.Morerecently, saturable uptake of humanC3e by humanneutrophils was reported and the presence of 60,000receptors per cell wasestimated(33). Theaffinity of these cellular receptors for the peptide wasnot stated. C3e

BIOLOGICAL FUNCTIONS OF COMPLEMENT RECEPTORS Receptors for Complement Ligands that are Attached to the Target of Complement Activation Clq RECEPTOR Interaction of humanneutrophils with latex beads coated with purified Clq stimulated a respiratory burst as measuredby chemiluminescence and NADPH-oxidase-dependenthexose monophosphate shunt activity (117). Nosuperoxideanion wasdetected. Thecellular

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responseto the C lq-coated beadswaspartially inhibited by free monomeric Clq. Thesefindings indicate that either C lq receptorson neutrophils, when cross-linkedby particle-boundClq, mediateda respiratory responseof the cells or, since latex beadshavesomeintrinsic stimulatory capacity, the receptor-ligandinteraction augmentedthe response to latex beadsby promotingtheir attachmentto the neutrophils. Chickenerythrocytes bearing Clq were lysed by Raji cells and Daudi cells in a 20-hrcytolytic assay(34). Forcytolysisto occur,the target cells did not haveto be sensitized with antibody,suggestingthat the reaction was mediatedby Clq receptors rather than Fc receptors on the lymphoblastoid cells. C lq also augmented the cytolysis of antibody-sensitizedchickenerythrocytes by peripheral blood lymphocytespurified by rosette formation with sheeperythrocytes. Althoughthe normaleffector ceils werenot characterized, the presenceof C 1 q receptors on null cells lacking both B and T cell markers(116) supports the possibility that somecells mediating antibody-dependent cellular cytotoxicity mayexpress these receptors. The function of Clq receptors on B ceils is not known. C3 RECEPTORS C3breceptor TheC3breceptor of erythrocytes is a relatively static membrane protein in that it does not internalize boundligand or becomeattached to cytoskeletal elements following cross-linking by multivalent ligands as do receptors on neutrophils and monocytes.However,greater than 85%of the C3breceptors in peripheral blood are found on erythroeytes, so that immune complexesbearing C3bpresent in the intravascular space will mostlikely bind to these cells rather than to leukocytes. Recently, a role for erythrocytes in the clearance of such complexeshas been suggested by the finding that IgG-containingimmunecomplexesadministeredintra-arterially to primatesare taken up by these cells and are then removedas they traverse the liver throughthe portal system(49). The mechanismby which the complexesare released from erythrocytes and transferred to Fc receptor-bearingcells in the liver, whichare presumably Kupfercells, is not known and maybe related to the conversionof complexassociated C3bto iC3bandC3d,gby factor I, with the C3breceptor serving as the cofactor. Additionalbiologic functionsof erythrocyteC3breceptors are related to their factor H-like activity. This receptorwasinitially isolated fromerythrocyte membranes becauseit dissociated Bbfromthe C3b,Bbbimolecular complexand served as a cofactor for conversionof C3bto iC3bby factor I (21). The receptor had analogousfunctions on C4b,2aand C4b(36, 54). Recent studies have also suggested that the C3breceptor mediates the

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cleavageby factor I of cell-boundiC3bto C3d,gand C3c. (73, 74). Thus, erythrocyte C3breceptors mayprotect cells in the circulation fromattack by the classical or alternative pathways,andthey mayfacilitate the conversion of C3bon immune complexesto iC3band C3d,g. Thelatter capability, whichwouldbe sharedby all cells with C3breceptors, creates the ligands for CR2and CR3,thereby recruiting the functional participation of these receptors. C3breceptors on neutrophils, monocytes, and macrophageshave long beenrecognizedas enhancingphagocytosisof IgG-beadngparticles (20). Thesynergyof C3breceptor-mediatedbinding of the target and Fc receptor-mediatedphagocytosissuggests a cooperativeinteraction betweenthe two receptors. Thebidirectional co-cappingof cross-linked C3breceptors and Fc receptors on humanneutrophils (R. Jack and D. Fearon, unpublished observations)indicates that multivalentinteraction of particle-bound C3bwith receptors on a localized area of the plasma membranewould recruit Fc receptors to the sametopographicalsite and enhancethe likelihoodof interaction of Fc receptors with particle-boundIgG. Thecapacity of C3breceptors on monocytesand macrophagesto mediate phagocytosis of sheep erythrocytescoated with C3bis dependenton the state of activation or maturationof these cells. Cultureof humanperipheral blood monocytes for 7 days leads to the acquisition by these cells of a capacity to internalize C3b-coated particles in the absenceof IgG(85). In addition, C3b receptors on both neutrophils and monocytes whencross-linked can rapidly internalize soluble ligands by adsorptive pinocytosis throughcoated pits andcoatedvesicles (1, 25). Thus,the C3breceptor on these phagocyticcells maymediateseveral responses, dependingon the physical state of material bearingthe ligand andon the maturationalstage of the cells. Onestudy reported that serum-treatedSepharosefrom whichall IgGhad beenremovedstimulateda respiratory burst in neutrophils and secretion of granule contents (39). However,these particles probably had iC3b and possibly C3d,gin addition to C3b, so that the cellular response maynot havebeeninitiated by C3breceptors. Indeed,a subsequentstudy utilizing sheep erythrocytes beating only C3bfound that these cells adheredto but did not stimulate neutrophils(84). Direct cross-linkingof C3breceptors neutrophils with F(ab’)2 anti-C3b receptor does not induce secretory respiratory responses (D. Fearon, unpublishedobservation). Although recent study stating that dimedcC3bcaused release of histaminase from humanneutrophils appears to contradict these findings, the concentration of dimericC3brequired to elicit the secretory reaction was60- to 300-fold more than that which caused saturation of 50%of the C3b receptors, raising the possibility that the effect wasmediatedby a minorcontaminant (75).

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The function of C3breceptors on B lymphocytesis unknown.Addition of C3bto guinea pig and humanlymphocytescaused secretion ofa lymphokine-mediating chemotaxis of monocytes, but the cell sourceof this activity wasnot directly shownto be the B cell (60). Anearly report of mitogenesis of mudnespleen cells inducedby C3bis nowattributed to factor H contaminatingthe preparation of C3b(46). C3breceptors haverecently been found to be present in 15-25%of peripheral blood T lymphoeytes.Whether or not these cells are a specializedsubset has not beendetermined,andthey are present in both subpopulations of T cells defined by the OKT4and OKT8 monoclonalantibodies (126). The extensive literature describing depressed antibody responses and developmentof memoryB cells to Tdependentantigens in mice rendered C3-deficient by administration of cobra venom factor cannotbe interpreted at the level of cellular receptors (90). Somerole for C3in the localization of antigen to lymphoidfollicles has beenestablished,but the particular C3receptorinvolvedin this function is not known(89). C3 receptor type 2 (CR2)F(ab’)2 antibody to CR2has been reported to inhibit the proliferative responsesof humanlymphocytesinducedby pokeweed mitogen, antigen, and mixed lymphocyte reactions, but not by phytohemagglutinin or eoncanavalinA, thus confirmingan earlier finding that soluble C3dinhibited antigen-inducedproliferation of humanlymphocytes(62, 103). Sincethese assaysreflect primarilyT rather than B cell responses, and only B cells havebeenconsideredto express CR2,the cell circuits involved in the CR2-mediated suppression maybe complex.However, enhancedkilling by humanperipheral blood lymphocyteswas observed whenantibody-sensitized target cells were also coated with C3d, suggesting that CR2also resides on somenon-Blymphocytes. C3receptor type 3 (CR3)Killing of target cells in lymphocyte-mediated antibody-dependent cellular cytotoxicity is enhancedwhenthe targets carry iC3b(92). This finding does not unambiguously demonstratethat the killer cell carded CR3,since iC3b binds also to CR2.The CR3on humanmonocytes cultured for 7 days can mediatethe phagocytosisby these cells of iC3b-coatedsheeperythrocytes(85). Earlier studies that demonstratedthat lymphokine-stimulated mouseperitoneal macrophagesingest IgM- and wholemouseserum-sensitizedsheeperythrocytes probablywerealso examining CR3-dependentphagocytosis (42). Humanneutrophils incubated with 10-8-10-7 Mof soluble C3d,g, whichprobably binds to CR3,selectively secretedspecific granulecontents, but not azurophilgranuleenzymes, in the presenceor absenceof cytochalasinB. Theseconcentrationsof C3d,g are similar to those causing 50%saturation of cellular receptors (D. Vik

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and D. Fearon, unpublished observation). In addition, sheep erythrocyte ghosts beating iC3b have recently been found to induce a respiratory burst by neutrophils, as assessed by chemiluminescence(107). FACTOR H RECEPTOR Binding of presumably polymeric factor H to B lymphoblastoid cell lines and to peripheral blood lymphocytes and tonsil lymphocytesinduced release of factor I, which was detected antigenically by immunodiffusionagainst goat anti-factor I and functionally by conversion of C3bto iC3b in the presenceof factor H (63). Release of factor I from these cells was calcium- and energy-dependent (63). This finding has been considered by some to account for the capacity of factor H to induce binding of sheep erythrocytes bearing C3b to Raji cells, which have few, if any, C3b receptors but which express receptors for iC3b/C3d,g and C3d (63, 66). As others have noted, however, Raji cells may bind particles bearing factor H linked to C3b directly by their factor H receptors (104). Humanfactor H has recently been shown to induce proliferation of mousespleen cells (44). The possibility that this effect was mediated contaminating lipopolysaccharide was excluded since H had a comparable effect on spleen cells from C3H/Hemice. Removalof spleen cells that formed rosettes with erythrocytes treated with antibody and complement diminished the proliferative response, suggesting that at least someof the responding cells also had receptors for fragments of C3. Approximatelytwo thirds of the responding blast-like spleen cells could be stained with antiIgM. As factor H frequently contaminates preparations of C3 and C3b, these authors considered these findings to be the basis for an earlier report of stimulation of murine B cells by humanC3b (46). Comparableexperiments with humanlymphocytes have not been reported. In support of the finding of factor H receptors on human monocytes (104), incubation of these cells with factor H induced a rapid chemiluminescent response (106). The concentration of factor H eliciting this response was in the appropriate range of 10-7 Msince this receptor-ligand interaction has an association constant of 3 × 106 M-~ (63). The capacity of factor to induce the respiratory burst in neutrophils was not examined, although this group of investigators had reported the presence of factor H receptors on this cell type (104).

Receptors for Diffusable ComplementLigands C3a RECEPTOR The capacity of H1 blocking agents to prevent the contractile response of the guinea pig ileum to C3ahad been taken as evidence that the only receptor-mediated biologic response of C3a was the secretion of histamine-containing granules by mast cells. However,some investiga-

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tors had observed a histamine-independent effect of complementanaphylatoxins on the guinea pig ileum (8), and recently, the contractile response of guinea pig lung strips inducedby 10-8-10-7 MC3awas shown to be relatively unaffected by H1or H2blocking agents (110). It is not knownwhat cell type in this tissue is responding to C3a, nor has the mediatorbeenidentified. However, by analogyto the effects of C5aandof C5adesArgon guinea pig lung tissue, the non-histaminemediatormaybe oneof the leukotrienes(94, 109). C3ahas recently been shownto suppress certain lymphocytefunctions. This first report suggestingthis function for C3afoundthat addition of a low-molecular-weight C3-derivedfragmentto humanperipheral blood lymphocytes in serum-free culture mediasuppressed mitogenesis caused by antigen, pokeweed mitogen, concanavalinA and phytohemagglutinin(81). Therewasno absolute identification of the fragmentas beingC3a, although the activity of the material resembledthat of C3ain beingheat- andacidstable. The presence of humanserumin the culture mediumreduced but did not abolish the inhibitory effect of the low-molecular-weight fragment(s). Thesamegroup also reported that humanor rat C3acould inhibit the secondaryin vitro antibodyresponseof rat lymphocytes in the presence of fetal calf serum(50). Proliferative responseswerenot assessedin this study. A third report from these investigators found that C3ahad no inhibitory effect on mitogen-induced humanlymphocyteproliferation when 5%or higher concentrations of fetal calf serum, rather than autologous serum, were included in the culture medium(122). These findings were extendedby the demonstrationthat highly purified C3a, in concentrations of 0.1-50 #g/ml, suppressedin a dose-related mannerthe plaque-forming cellular response of humanperipheral blood lymphocytesto Fc fragments (77). Noinhibition of proliferation wasobserved,but fetal calf serumwas present in the culture media. Suppressionwas localized to the level of impairedhelper T cell function since normalresponsesoccurredif soluble helper factors wereadded.C3adesArghad no activity in this assay system. This latter finding contrasts with that of the earlier study in whichonly partial loss of inhibitory activity of the low-molecular-weight fragmentof C3, presumedto be C3a, was observed in the presence of humanserum containing carboxypeptidaseN (50). This discrepancymaybe resolved a preliminaryreport suggestingthat C3e,whichis similar to C3ain size and whichis resistant to degradationby enzymespresent in humanserum, may haveaccountedfor the growth-suppressing activity previously ascribed to C3a(123). The mechanismby whichC3amight alter lymphocytefunction is obscuresince these cells are not consideredto possessreceptorsfor this peptide; the possiblerole of basophilsandmastcells that mightbe present in cultures of humanperipheral blood lymphocytesand murine and rat spleen cells has not beenaddressed.

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C5aRECEPTOR As noted previously in this review, binding of CSa to receptorson mastcells andbasophilscausessecretion of their granulesand release of histamine. Recently,two groupsof investigators providedevidence that CSaand CSadesArgalso cause the release of slow reacting substanceof anaphylaxis,whichis comprisedof leukotrienes, fromguinea pig lung andtracheal strips (94, 109, 110). Althoughthe cell type interacting with these peptides is presumablythe pulmonarymast cell, macrophagesalso possess CSareceptors (13) and these cells havebeenshown studies utilizing other stimulantsto be capableof synthesizingleukotrienes (101). Thediverse effects of these productsof arachidonicacid (reviewed in 70) broaden the potential pathobiologic consequencesof complement activation in the lung and mayindicate someof the mechanismsby which CSaand CSadesArgproduce acute pulmonaryinjury (17, 48, 111). Binding of CSaor CSadesArgto receptors on neutrophils induces an array of cellular reactions: chemotaxisalong a gradient of the CS-derived glycopeptides,specific granulesecretion, increasedadhesiveproperties, increased oxygen consumptionand superoxide generation, and enhanced expressionof C3breceptors (24, 26, 47). Thesecellular responsesare not uniqueto CSareceptor-ligandinteractions, as they occur followingbinding of the synthetic formylatedpeptides to different receptors. Since there are several comprehensive reviewsof this extensivelystudied area of neutrophil cell biology(29, 120), only a discussionof the relevanceof these reactions to host defense and inflammationis presented. AlthoughCSadesArginteracts with the C5areceptors on neutrophils with 25- to 50-fold lower affinity than doesCSa,it is probablythe formof the peptidethat is acting in vivo. In the time required for establishing a concentrationgradient and for diffusing fromextravascular sites to the intravascular space in which neutrophils normallyreside, carboxypeptidase-N wouldremovethe C-terminal arginine from CSa. Theserumconcentration of C5is approximately 10-6 M, so that sut~cient C5adesArgcan easily be generatedfor binding to receptorswith an associationconstantfor the peptide in the rangeof 2-4 × 10-7 M-kStudies in vitro have demonstratedthat maximalneutrophil chemotaxisoccurs with approximately10-8 MC5adesArgin the presence of an anionic serumcochemotaxin(91), and that maximalexpression neutrophil C3breceptors is inducedwith nanomolarconcentrationsof the peptide. Theincreased stickiness of neutrophils inducedby C5adesArgis presumablynecessaryfor the cells’ capacityto adhereto endothelialcells preparatory to diapedesis and chemotaxis(56). This cellular response mayalso account for the pulmonaryleukosequestration and transient leukopenia observedin animals to whichcobra venomfactor is administered(71) and in humansundergoinghemodialysisand cardiopulmonarybypass (12, 56),

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situations in whichactivation of C5 occurs via the alternative pathway. The CSa desArg-induced rapid increase in the number of C3b receptors available on the plasma membraneof neutrophils (24, 58) wouldprepare the cell for uptake of C3b-coated complexeswhenit completes its migration to the site of complementactivation. Less is knownabout the biologic responses of monocytes and macrophages elicited by occupancyof their CSa receptors. CSa and CSa desArg induce chemotaxis of these cells and the slow secretion of glycolytic and proteolytic enzymes(72, 119). An uncharacterized factor chemotactic for ncutrophils is also released by rabbit alveolar macrophagesin response to these peptides. It wouldbe of great interest if this factor was shownto be LTB4,an extremely potent chemoattractant, since this would constitute evidence that CSa and CSa desArg induce synthesis of the leukotrienes by a defined cell type. Addition of nanomolar concentrations of CSa or CSa desArg to murine spleen cells augmentedthe primary plaque-forming cell response to antigen (40). This effect was also accomplishedby adding to the cultures peritoneal macrophagespulsed with the peptides, a result that indicates that the macrophages mediated the CSa response. Spreading of human monocytes induced by factor Bb also has been suggested as being secondary to the action of CSa cleaved from cell-associated C5 by Bb (112). C3e RECEPTOR Systemic complement activation in animals and in man is accompaniedby a rapid fall in the numberof circulating neutrophils, a secondary effect of CSaon these cells, followed by a return to levels above those prior to complementactivation. This leukocytosis is thought to be secondary to the C3e fragment of C3, which has been shown to induce release of leukocytes from the perfused, isolated rat femur and to induce leukocytosis in rabbits (35, 100). The absence ofleukocytosis accompanying pyogenic infections in someindividuals genetically deficient in C3 reflects the inability of these patients to generate C3e(2). CONCLUSIONS The complementsystem is a mechanismfor the generation of ligands that elicit a widerange of biologic functions by interacting with specific cellular receptors. These receptors reside in almost all cells that participate in immuneand inflammatory reactions. The proteolytic reactions occurring amongthe complementproteins that lead to the elaboration of these ligands are relatively well understood, but little is knownconcerning complement receptors other than their distribution amongcell types and someof the cellular responses induced by binding of ligands. Only two complement

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receptors havebeenisolated, the C3breceptor and CR2.Essential studies, such as calculation of the numberand at~nities of receptors, have been performedonly for the Clq, CSa, and C3breceptors. Biologic studies of complementreceptors on neutrophils, macrophages,and mast cells have established their roles in eliciting cellular responsesthat contribute to phagocytic,immediatehypersensitivity, and acute inflammatoryreactions. In contrast, the role of lymphocyte complement receptors, such as the C3b, CR2,and CR3receptors, in regulating the immune responsemust be clarified. ACKNOWLEDGMENTS

This workwassupported in part by grants AI-17917,AI-07722,AI-10356, and RR-05669. Literature Cited 1. Abrahamson, D. R., Fearon, D. T. 1983. Endocytosisof the C3breceptor of complement within coatedpits in human polymorphonuclear lcukocytes and monocytes.Lab. Invest. In press 2. Alper, C. A., Colten, H. R., Rosen,F. S., Rabson,A. R., MacNab,G. M., et al. 1972. Homozygous deficiency of C3 in a patient with repeated infections. Lancet 2:1179-81 3. ArnaoUt,M. A., Melamed,J., Tack, B. F., Colten, H. R. 1981. Characterization of the humancomplement(C3b) receptor with127:1348-54 a fluid phase C3bdimer. J. ImmunoL 4. Ault, K. A., Springer, T. A. 1981. Cross-reaction of a rat-antimouse phagocyte-specific monoclonalantibody (anti-MAC-l) with humanmonocytes and natural killer cells. J. Immunol. 126:359-64 5. Beller, D.I., Springer,T. A., Schreiber, R. D. 1982. Anti-MAC-I selectively inhibits rosetting mediatedby the type three complementreceptor (CR3). Exp. Med. 156:1000-9 6. Berger,M., Gaither, T. A., Hammer, C. H., Frank, M.M.1981. Lackof binding of human C3,in its native state, to C3b receptors. J. Immunol.127:1329-34 7. Bianco,C., Patrick, R., Nussenzweig, V. 1970. A population of lymphocytes bearing a membrane receptor for antigen-antibody-complementcomplexes. I. Separationandcharacterization. J. Exp. Med. 132:702-20 8. Bodammer,G., Vogt, W. 1970. Contraction of the guineapig ileuminduced by anaphylatoxinindependentof hista-

minerelease, lnt. Arch. Allergy Appl. Immunol. 39:648-57 9. Bokisch,V. A., Miiller-Eberhard,H. J. 1970.Anaphylatoxin inactivator of humanplasma:its isolation andcharacterization as a carboxypeptidase.J. Clin. lnvest. 49:2427-36 10. Carlo, J. R., Ruddy,S., Sauter, S., Yount,W.J. 1979. Deposition of B1H in renal biopsies frompatients withimmunerenal disease. Arthrit. Rheum. 22:403-8 11. Carlo, J. R., Ruddy,S., Studer, E. J., Conrad, D. H. 1979. Complement receptor binding of C3b-coatedcells treated with C3b inaetivator, B1H globulin and trypsin. Z lmmunol. 123:523-28 12. Chenoweth,D. E., Cooper, S. W., Hugli, T. E., Stewart,R. W., Blaekstone, E. H., Kirklin, J. W.1981. Complement activation during eardiopulmonary bypass. N. Engl. J. Med. 304:497-503 13. Chenoweth, D. E., Goodman,M. Weigle, W.O. 1982. Demonstrationof a specific receptor for humanCSaanaphylatoxin on murinemacrophages.J. Exp. Med. 156:68-78 14. Chenoweth,D. E., Hugli, T. E~ 1978. Demonstration of specific C5areceptor on intact humanpolymorphonuclear leukocytes. Proc. Natl. Acad.ScL USA 75:394347 15. Cochrane,C. G., Miiller-Eberhard, H. J. 1968. Thederivation of twodistinct anaphylatoxinactivities fromthe third and fifth componentsof humancomplement. J. Exp. Med.127:371-86

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28. Gabay,Y., Pedmann,H., Perlmann,P., Sobel, A. T. 1979. A rosette assay for the determinationof Clqreceptor-bearing cells. Eur. J. lmmunol.9:797-801 29. Gallin, J. I., Quie, P. G., ed. 1978. LeukocyteChemotaxis:Methods,Physiology and Clinical Implications. New York: Raven 30. Gelfand, M. C., Frank, M. M., Green, I. 1975.Areceptor for the third component of complement in the humanrenal glomerulus. J. Exp. Med.142:1029-34 31. Gerhard,C., Chenoweth,D. E., Hugli, T. E. 1981. Responseof humanneutrophils to CSa:a role for the oligosacchafide moiety of humanC5a desArg-74 but not of C5ain biologic activity. J. lmrnunol. 127:1978-82 32. Gerhard,C., Hugli,T. E. 1981.Identification of classical anaphylatoxinas the des-Argform of the CSamolecule: evidence of a modulatorrole for the oligosaccharide unit in humandesArgT*-CSa.Proc. Natl. Acad. Sc£ USA 78:1833-37 33. Ghebrehiwet, B. 1982. C3e-induced lysosomal enzymerelease from polymorphonuclearleukocytes. Fed. Proc. 41:966(Abstr.) 34. Ghebrehiwet,B., Miiller-Eberhard,H. J. 1978. Lysis of Clq-coated chicken erythrocytes by humanlymphoblastoid cell lines. J. lrnmunol.120:27-32 35. Ghebrehiwet,B., Miiller-Eberhard,H. J. 1979.C3e:Anacidic fragmentof humanC3 with leukocytosis-inducingactivity. J. lmmunol.123:616-21 36. Gigli, I., Fearon, D. T. 1981. Regulation of the classical pathwayconvertase of human complement by the C3b receptor of humanerythroeytes. Fed. Proc. 40:1172(Abstr.) 37. Gigli, I., Nelson,R. A. Jr. 1968. Complement dependent immunephagoeytosis. Exp. Cell Reg51:45-67 38. Glovsky,M.M., Hugli,T. E., Ishizaka, T., Lichtenstein,L. M., Edckson,B. W. 1979. Anaphylatoxin-inducedhistaminerelease with humanleukoeytes.J. Clir~ Invest. 64:804-11 39. Goldstein,I. R., Kaplan,H. B., Radin, A., Frosch, M. 1976. Independent effects of IgG and complementupon humanpolymorphonuclear leukocyte function. J. Immunol.117:1282-87 40. Goodman,M. G., Chenoweth,D. E,, Weigle,W.O. 1982. Potentiationof the primary humoral immuneresponse in vitro C5a anaphylatoxin. J. lmmunol.by 129:70-75 41. Gorski,J. P., Hugli,T. E., Miiller-Eberhard, H. J. 1979. C4a: Thethird ann-

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phylatoxin of the humancomplement system. Proc. Natl. Acad. ScL USA 76:5299-302 42. Griffin, J. A., Griffin, F. M.Jr. 1979. Augmentationof macrophagecomplementreceptorfunctionin vitro: I. Characterizationof the cellular interactions requiredfor the generationof a T-lymphocyte product that enhancesmacrophagecomplement receptor function. J. Exp. Med. 150:653-75 43. Oupta, S., Ross, G. D., (3ood, R. A., Siegal, F. P. 1976. Surfacemarkersof humaneosinophils. Blood 48:755-63 44. Hammann, K. P., Raile, A., Schmitt, M., Mussd,H. H., Peters, H., et al. 1981. B1Hstimulates mousespleen B lymphocytesas demonstrated by increased thymidine incorporation and formationof B cell blasts, lmmunobiology 160:289-301 45. Harrison, R. A., Laehmann, P. J. 1980. The physiological breakdownof the third component of humancomplement. Mol. lmmunoL17:9-20 46. Hartman,K. U., Bokiseh, V. A. 1975. Stimulation of routine B lymphocytes by isolated C3b. J. Exp. Med. 142: 600-10 47. Henson,P. M., Zanolari, B., Sehwartman,N. A., Hong,S. R. 1978.Intraeellular control of human neutrophilsecretion. I. CSainducedstimulus-specific desensitizationandthe effects or cytochalasin B. J. lmmunol.121:851-55 48. Henson,P. M., McCarthy,K., Larsen, G. L., Webster,R. O., Giclas, P. C., et al. 1979. Complement fragments,alveolar macrophages andalveolitis, drr~ J. Pathol. 97:93-110 49. Hebert, L. A., Cornacoff,J. B., VanAman, M. E., Smead,W. L., Birmingham,D. J. 1982.In vivokinetics of the primate erythroeyte-immunecomplex clearing mechanism.Clir~ Re~ 30: 350A(Abstr.) 50. Hobbs,M. V., Feldbush,T. L., Needleman,B. W., Weiler,J. M.1981. Inhibition of secondaryin vitro antibodyresponsesby the third component of complement. J. lmmunol.128:1470-75 51. Hugli, T. E. 1975. Humananaphylatoxins (C3a) from the third component complement: primarystructure. Z Biol. Chem.250:8293-301 52. Hugli,T. E., Erickson,B. W.1977.Synthetic peptideswiththe biologicalactivities andspecificity of humanC3aanaphylatoxin. Proc. Natl. Acac~Sc£ USA 74:1826-30 53. Iida, K., Mornaghi,R., Nussenzweilb V. 1982. Complement receptor (CRt)

deficiencyin erythrocytesfrompatients with systemic lupus erythematosus. Z Exp. Med. 153:1427-38 54. Iida, K., Nussenzweig,V. 1981. Complementreceptor is an inhibitor of the complement cascade. J. Exp. Med. 153:1138-50 55. Isenman,D. E., Kells, D. I. C., Cooper, N. R., Miiller-Eberhard, H. J., Pangburn, M.K. 1981.Nueleophiliemodification of humancomplementprotein C3: Correlation of conformational chan~eswith acquisition of C3b-like functlunal properties. Biochemistry20: 4458-67 56. Jacob, H. S., Craddock, P. R., Hammersehmidt, D. E., Moldow,C. F. 1980. Complement-indncedgranulo* cyte aggregation. N. Engl. Z Meal. 302:789-94 57. Johnson,A. R., Hugli, T. E., MiillerEberhard,H. J. 1975. Releaseof histaminefromrat mastcells by the complement peptides C3a and CSa. Immunology28:1067-80 58. Kay, A. B., Glass, E. J., Salter, D. MeG.1979. Leukoattractants enhance complement receptors on human phagocyticceils. Clir~ Exp. lmmunol. 38:294-99 59. Kazatchkine,M. D., Fearon,D. T., Appay, M. D., Mandet,C., Bariety, J. 1982. Immunohistoehemicalstudy of the humanglomerular C3breceptor in normalkidneyand in seventy-fivecases of renal diseases. J. Clin. lnvest. 69: 900-12 60. Koopman,W. J., Sandberg, A. L., Wahl,S. M., Mergenhagen, S. E. 1976. Interaction of soluble C3 fragments with guinea pig lymphoeytes.Comparison of effects of C3a,C3b,C3candC3d on lymphokine production and lymphocyte proliferation. J. Immunol. 117:331-36 61. Lachmarm, P. J., Pangburn,M.K., Oldroyd, R. G. 1982. Breakdownof C3 after complementactivation. J. Exp. Med. 156:205-16 62. Lambris,J. D., Cohen,P. L., Dobson, N. J., Wheeler,P. W.,Papamichail,M., et al. 1982.Effect of antibodiesto CR2 (C3dreceptors) on lymphocyteactivation. Clin Res. 30:514A(Abstr.) 63. Lambris,J. D., Dobson,N. J., Ross,13. D. 1980. Releaseof endogenous C3binactivator from lymphoeytes in response to triggering by membrane receptorsfor /31H globulin. J. Exp. Med. 152: 1625--44 64. Lambris,J. D., Dobson,N. J., Ross,G. D. 1981. Isolation of lymphocytemere-

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COMPLEMENT RECEPTORS brahe complementreceptor type two (the C3dreceptor) and preparation receptor-specific antibody.Proc.Natl. Acad. Sci~ USd78:1828-32 65. Lambris,J. D., Ross,G. D. 1982.Assay of membranecomplement receptors (CRt and CR2) with C3b- and C3dcoated fluorescent microspheres.Z Immunol. 128:186-89 66. Lambds, J. D., Ross,(3. D. 1982.Characterization of the lymphocytemembrane receptor for factor H globulin)with an antibodyto anti-factor H idiotype. J. Exp. Med. 155: 1400-11 67. Law,S. K., Fearon,D.T., Levine,R. P. 1979.Actionof the C3binactivator on cell-bound C3b. J. Iramunol. 122: 759--65 68. Law,S. K., Levine,R. P. 1981.Binding reaction betweenthe third humancomplementprotein and small molecules. Biochemistry20:7457-63 69. Lay, W. H., Nussenzweig, V. 1968. Receptors for complementon leukocytes. Z Exp. MecL128:991-1007 70. Lewis, R. A., Austen,K. F. 1981. Mediation of local homeostasisandinflammationby leukotrienes and other mast cell-dependent compounds. Nature 293:103-8 71. McCall, C. E., De Chatelet, L. R., Brown,D., Laehmaun,P. J. 1974. New biologicalactivity followingintravascular activation of the complement cascade. Nature 249:841-43 72. McCarthy,K., Henson,P. M. 1979. Induction of lysosomalenzymesecretion by alveolar macrophages in responseto the purified complement fragmentsC5a and CSa desArg. £ IratnunoL 123: 2511-17 73. Medicus,R. G., Arnaout, M. A. 1982. Surface restricted control of C3blNA dependentC3crelease from boundC3bi molecules.Fea~Proc.41:848(Abstr.) 74. Medof,M. E., Prince, G. M., Mold,C. 1982. A function for immune adherence receptors. FeelProc.41:966(Abstr.) 75. Melamed,J., Arnaout,M. A., Colten, H. IL 1982. Complement (C3b)interaction with the humangranulocytereceptor: correlationof bindingof fluid phase radio-labelled ligand with histaminase release. J. Immunol.128:2313-18 76. Miyakawa,Y., Yamada,A., Kosaka, K., Tsuda, F., Mayumi,M. 1981. Defective immune-adherence (C3b)receptor on erythrocytes frompatients with systemic lupus erythematosus.Lancet 2:493-97

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77. Morgan,E. L., Weigle,W.O., Hugli, T. E. 1982.Anal.phylatoxin-mediated regulation of the ~mmune response. Z Exp. Med. 155:1412-26 78. Miiller-Eberhard,H. J., Schreiber, R. D. 1980. Molecularbiology and chemistry of the alternative pathwayof complement. Adv. lmmunol.29:1-53 79. Nagasawa, S., Iehihara, C., Stroud, R. M. 1980. Cleavageof C4bby C3binactivator: productionof a nickedformof C4b,C4b’, as an intermediatecleavage product of125:578-82 C4bby C3binactivator. Z Immunol. 80. Nagasawa, $., Stroud, R. M. 1977. Mechanism of the C3binactivator: requirementfor a high molecularweight cofactor (C3b-C4blNA eofactor) and production of a new C3bderivative (C3b’). lmmunochemistry14:749-56 81. Needleman, B. W.,Weiler,I. M., 17eldbush, T. L. 1981. Thethird component of complementinhibits humanlymphocyte blastogenesis. J. IraraunoL 126:1586-91 82. Nelson,D. S. 1963. Immune adherence. Adv. lmmunol.3:131-80 83. Nelson, R. A. Jr. 1953. The immune adherence phenomenon.Science 118: 733-37 84. Newman, S. L., Johnston, R. B. Jr. 1979. Roleof binding throughC3band IgG in polymorphonuelearneutrophil function:studies withtrypsin-generated C3b. J. Imraunol. 123:1839-46 85. Newman,S. L., Musson,R. A., Henson, P. M. 1980. Development of functional complement receptors during in vitro maturation of humanmonocytes into macrophages.J. Iramunol. 125: 2236-44 86. Nicholson-Weller, A., Burge, J., Fearon,D. T., Weller,P. F., Austen,K. F. 1982. Isolation of a humanerythrocyte membrane glycoprotein with decay-acceleratingactivity for C3convertases the complementsystem. Z Imrnunol.of129:184-89 87. Okuda,T., Taehibana, T. 1980. Complementreceptors on Raji cells. The presenceof a newtype of C3 receptor. Immunology41:159-66 88. Pangburn, M. K., Schreiber, R. D., Miiller-Eberhard, H. J. 1977. Human complement C3binactivator: isolation, characterization and demonstrationof an absolute requirementfor the serum protein ~IH for cleavage of C3band C4b in solution. £ Exp. Med. 146: 257-70 89. Papamichail, M., Gutierrez, C., Embling, P., Johnson,P., Halborow, E. J.,

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et al. 1975. Complement dependencyof 101. Rouzer,C. A., Scott, W.A., Hamill,A. L, Cohn, Z. A. 1982. Synthesis of localizationof aggregatedIgGin germinal centres. Scan. J. lmmunol. 4: leukotriene C and other arachidonic 343-49 acid metabolites by mousepulmonary maerophages.J. Exp. Med.155:720-33 90. Pepys, M.B. 1974. Role of complement in inductionof antibodyproductionin 102. Ruddy,S., Austen,K. F. 1971. C3binactivator of man.II. Fragmentsprovivo. Effect of cobra venomfactor and duced by C3binactivator cleavage of other C3-reactive agents on thymuscell-bound or fluid phase C3b. Z lmindependent antibodyresponses.J. Exp. Med. 140:126-45 munol. 107:742-50 91. Perez, H.D., Goldstein,I. M., Webster, 103. Schenkein,H. A., Geneo,R. J. 1979. Inhibition of lymphocyteblastogenesis R. O., Henson,P. M. 1981. Enhanceby C3c and C3d. J. lmmunol. 122: mentof the chemotacticactivity of hu1126-33 man C5a desArg by an anionic ("cochemotaxin") in normal serum and 104. Schmitt, M., Mussel, H. H., Hammann, K. P., Scheiner, O., Dierich, M. P. plasma. J. lmmunol.126:800-4 1981. Role of B1Hfor the binding of 92. Perlmann,H., Perlmann,P., Schreiber, C3b-coated particles to humanlymR. D., Miiller-Eberhard,H. J. 1981.Inphoid and phagocyticcells. Eur. Z lmteraction of target cell-boundC3biand munol. 11:739-45 C3dwith humanlymphocytereceptors. 105. Schneider, R. J., Kulczycki, A. Jr., Enhancement of antibody-mediatedcelLaw,S. K., Atkinson,J. P. 1981.Isolalular cytotoxicity. J. Exp. Med.153: tion of a biologicallyactive macrophage 1592-603 receptor for the third componentof 93. Reid, K. B. M., Porter, R. R. 1981.The complement.Nature 290:789-92 proteolytic activation systemsof complement. An~Rev. Biocher~50:433-64 106. Schopf, R. E., Hammann,K. P., Seheiner, O., Lemmel,E.-M., Dierieh, 94. Regal,J. F., Picketing,R. J. 1981.C5aM. P. 1982. Activation of humanmonoinducedtracheal contraction: effect of cytes by both humanfl IH and C3b.Iman SRS-A antagonist and inhibitors of 46:307-12 araehidonate metabolism.J. lmmunol. 107. munology Schreiber, R. D., Pangburn, M. K., 126:313-16 Bjornson,A. B., Brothers,M.A., Miill95. Reynolds,H. Y., Atkinson,J. P., Newer-Eberhard,H. J. 1982. Therole of C3 ball, H. H., Frank, M.M. 1975.Recepfragmentsin endocytosisand extraceltors for immunoglobulinand complelular cytotoxic reactions by polymorment on human alveolar macrophages. J. lmmunol. 114:1813-19 phonuclearleukocytes. Clin. lmmunol. Immunopathol.23:335-57. 96. Ross,G. D., Jarowski,C. I., Rabellino, 108. Sim, R. B., Arland, G. J., Colomb,M. E. M., Winchester,R. J. 1978. These13. 1979. C1 inhibitor-dependentdisquential appearanceof Ia-like antigens sociation of humancomplementcomand two different complement receptors ponent C1 boundto immune complexes. during the maturation of humanneuBiochem.Z 179:449-57 trophils. J. Exp. Med.147:730-44 109. Stimler, N. P., Bach,M.K., Bloor, C. 97. Ross,13. D., Lambris,J. D. 1982.IdenM., Hugli,T. E. 1982.Releaseof leukotification of a C3bi-specific membrane trienes fromguineapig lungstimulated complement receptor that is expressed by C5a desArg anaphylatoxin. J. Immunol. 128:2247-52 on lymphocytes, monocytes, neutrophils and erythroeytes. J. Exp. Med. 110. Stimler, N. P., Brocklehurst, W.E., 155:96-110 Bloor, C. M., Hugli, T. E. 1981. Ana98. Ross,G. D., Lambris,J. D. 1982.Idenphylatoxin-mediated contraction of tification of three formsof iC3b that guineapig lung strips: a non-histamine havedistinct structures and receptor tissue response. J. lmmunol. 126: binding site properties. Mol.Immunol. 2258~il (Abstr.) 19:1399 111. Stimler, N. P., Hugli, T. E., Bloor,C. 99. Ross,G. D., Polley, M.J. 1975.SpeciM. 1980. Pulmonaryinjury induced by ficity of humanlymphocytecompleC3a and 100:327-38 C5a anaphylatoxins. An~J. Pathol. ment receptors. J. Exp. Med. 141: 1163-80 112. Sundsmo, J. S., GiStze, O. 1981. Human 1190.Rother, K. 1972. Leukocytemobilizing monocytespreading induced by factor Bbof the alternative pathwayof comfactor: A newbiological activity from plementactivation. J. Exp. Med.154: the third componentof complement. 763-77 Eur. J. lmmunoL2:550-58

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COMPLEMENT RECEPTORS 113. Tack, B. F., Harrison, R. A., Janatova, J., Thomas,M. L., Prahl, J. W. 1980. Evidencefor the presence of an internal thioester in the third component of complement.Proc. Natl. dead. Sci. USA 77:5764-68 114. Tedder, T. F., Fearon, D. T., Gartland, G. L., Cooper, M. D. 1983. Expression of complement receptor type one (for C3b) on humannatural killer cells, cell and lymphoblastoid cell lines. J. Immunol. In press 115. Tenner, A. J., Cooper, N. R. 1980. Analysis of receptor-mediated Clq binding to human peripheral blood mononuclear cells. J. Immunol. 125: 1658-64 116. Tenner, A. J., Cooper, N. R. 1981. Identification of types of cells in human peripheral blood that bind Clq. J. Immunol. 126:1174-79 117. Tenner, A. J., Cooper, N. R. 1982. Stimulation of a humanpolymorphonuclear leukocyte oxidative response by the Clq subunit of the first complement component. J. Iramunol. 128:2547-52 118. Vrainian, G. Jr., Conrad, D. H., Ruddy, S. 1981. Specificity of C3 receptors that mediate phagocytosis by rat peritoneal mast cell. J. Immunol. 126:2302-6 119. Ward, P. A. 1968. Chemotaxis of mononuclear cells. J. Exp. Med. 128: 1201-21 120. Ward, P. A., Hugli, T. E., Chenoweth, D. E. 1979. Complement and chemotaxis. In Handbookof Inflammation, ed. L. E. 13lynn, J. 13. Houck,G. Weissmann, h153-78. NewYork: Springer 121. Wautier, J. L., Souchon, H., CohenSolal, L., Peltier, A. P., Caen,J. P. 1976.

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C1 and human platelets. Immunology 31:595-99 122. Weiler, J. M., Ballas, Z. K., Feldbush, T. L., Needleman, B. W. 1982. Inhibition of humanlymphocyteblastogenesis by C3: the role of serum in the tissue culture medium. Immunology 45: 247-52 123. Weiler, J. M., Needleman, B. W., Hobbs, M. V., Feldbush, T. L. 1982. Two separate fragments of the third component of complement inhibit immune responses. Fed. Proc. 41:849 (Abstr.) 124. West, C. D., Davis, N. C., Forristal, J., Herbst, J., Spitzer, R. 1966. Antigenic determinants of human BIC and BIG globulins, Z Immunol. 96:650-58 125. Whaley, K., Ruddy, S, 1976. Modulation of the alternative complement pathway by B1Hglobulin. J. Exp, Med. 445:1147-63 126. Wilson, J. G., Tedder, T. F., Fearon, D. T. 1982. Antigenic and functional detection of C3b receptors on humanperipheral blood T lymphocytes. Fed. Proc. 41:965 (Abstr.) 127. Wilson, J. O., Wong,W, W., Schur, P. H., Fearon, D. T. 1982. Modeof inheritance of decreased C3b receptors on erythrocytes of patients with systemic lupus erythematosus. N. Engl. J. Med. 307:981-86 128. Won~,W. W., Fearon, D. T. 1982. Antigemc cross-reactivity between human C3b receptor and a mudnelymphocyte surface protein. Fed. Proc. 41:965 (Abstr.) 129. Ziccardi, R. J., Cooper, N. R. 1979. Active disassemblyof the first complement component, C1, by C1 inactivator. J. Immunol. 123:788-92.

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CYTOLYTIC T LYMPHOCYTES M. Nabholz GeneticsUnit,SwissInstitute for Experimental CancerResearch, CH-1066 Epalinges, Switzerland H. R. MacDonald Ludwig Institute for CancerResearch,Lausanne Branch,CH-1066 Epalinges, Switzerland PREFACE Thepurposeof this reviewis twofold.In the first part wetrace the history of cytolytic T lymphocytes (CTL)in the context of the development immunology as a whole;i.e. weanalyzewhythe knowncharacteristics of CTLwerediscoveredat a particular pointin time. Wethink that suchan analysisis not onlyinterestingbut also useful, becausethe contextof the discoveryof a phenomenon determinessometimesto a large extent its interpretation,andthe distinctionbetween the experimental observations andtheir interpretationbecomes blurredwith time. Are-evaluation of the data on CTLmaybecomeimportantat a time whenthe emphasisof the questionsaskedaboutthese cells changes.We think the discoveryof T-cellgrowthfactor andthe designof methods that haveallowedthe maintenanceof CTLclones in permanentculture have usheredin a periodin whichthe establishedconceptualandexperimental framework of cellular immunology is rapidlycedingto a muchmorecellbiologicallyorientedapproachto CTL.Therefore,in the secondpart, we identify someof the questionsthat arise fromthe workwithCTLclones andthat representthis newoutlook. Ourreferencesare quiteeclectic, andfor someareas-- e.g. lytic mecha. uisms--werefer mainlyto someof the comprehensive reviewsthat have appearedrecently. 273 0732-0582/83/0410-0273502.00

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THE HISTORY

OF CTL

Graft Rejection and Cytotoxic Cells Theobservationthat tissue grafted fromone individual to another is rejected is very old (see 83 for review).Thatit wasmediatedby lymphoid cells wasapparently first suggestedat the beginningof this century, whenthe phenomenon of accelerated rejection of secondarygrafts was also first observed.It seemssomewhat surprising, therefore, that it took almost 50 years before it becamefirmly established, as a consequence of the transfer experimentsof Mitchison(103) and the studies of Algire and his collaborators (149) with diffusion chambers,that in mostsituations cells but not antibodies mediatedgraft rejection. Themainreason for this delay was probablythat after the discoveryof antibodiesand the appreciationof their incredible discriminatorypower,the temptationto ascribe all immune phenomenato these moleculeswas, for mostpeople, irresistible. (Thosewho insist that T-call specificity mustbe controlled by genes of the immunoglobulingenefamilymaybe fighting the last battle of this controversy.) In 1948Medawar looked,unsuccessfully,for evidencefor the destruction of tissue by cells from appropriately immunizedanimals. Several other investigatorsalso failed (see 123for references), but in 1960Govaerts(54) reported that thoracic duct lymphocytesfrom dogs that had received a kidneygraft werecytotoxicfor kidneyepithelial cells fromthe donor, but not for cells from"irrelevant" dogs. This report wasrapidly confirmed(see 155 for review), and it wasshownthat (a) the cytotoxic effects were munologicallyspecific, (b) that they required the contact betweenliving etfector and target cells, (c) that serumfromthe immunized animalwasnot required, and (d) that specific eytotoxic lymphocytescould be raised not only against allografts but also against syngeneictumors(124). Although the first reports on cytotoxicityof leukocytesfor culturedcells dearly suggested that such effects were mediated by lymphocytesthat specifically recognizedthe target cells, the picture subsequentlybecame blurred by reports (see 116, 117 for reviews) that lymphocytesor other leukocytesfromunimmunized individuals could lyse different types of target cells nonspecifically, especially whenthey werestimulatedwith mitogens, and that normal lymphocytes could lyse cells precoated with antitarget antibodies. In addition, immune lymphoeytescould release nonspecifically cytotoxie molecules(lymphotoxins)whenincubated with the relevant antigen. Butthe evidencefor the existenceof specificallycytotoxiccells in animals immunized against foreign tissues wassufficiently strong to survive this period of confusion. From1968on, the situation gradually becamedearer for tworeasons: the introductionof a simple, rapid, quantitative assay for

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CYTOLYTICT LYMPHOCYTES 275 specific cytotoxiccells, andthe identification of cytotoxiccells as T lymphocytes. In the early experiments,the cytotoxic activity of the lymphoidpopulations wasmeasuredby direct microscopicobservationof the target cells, usuallymonolayers of adherentcells (see 155for review). In the search for methodsthat makereliable quantitation possible, the solution that has imposeditself is the measurement of lytic activity as definedby the release of 51Crfromprelabeledtarget cells (20). This assaywasfirst usedto measure complement-dependent, antibody-mediated lysis of nucleated cells (see 117 for references). Whenthe cytotoxic activity of a lymphocytepopulation against tumortarget cells as measuredby this assay is comparedwith the capacity of the samepopulation to reduce the plating efficiency of the targets, a goodcorrelation is obtained(20, 82). The51Crrelease has the advantageof allowingprecise quantitative comparison of the lytic activity of different cell populationsagainst the same target cells, but neither this nor anyother methodcan reliably enumerate the numberof cytolytic cells in a population. In 1968the idea that there weretwoantigen-specificlymphocyte lineages, B cells andT cells, wasfinally acceptedthanksto the experiments of Miller &Mitchell(101) on T-Bcell collaboration. Thus,a clear interpretation all the earlier experimentspointing to a dichotomybetweencellular and humoralimmunityimposeditself, and Brunner& Cerottini (19) rapidly demonstratedthat specific cytotoxicity dependedon T cells, and provided someevidencethat other cell types werenot required for the induction of target lysis. In addition, they showedthat animalsprimedwith allogeneic cells give a faster andstronger cytotoxicresponseto a boost with the same cells. This immunologic memory is long lasting. Severalquestionspresentedthemselvesat this point. Whatwasthe in vivo role of CTL?Werethey indeed the effector cells in graft rejection, and possibly in other immunephenomena such as the graft-versus-host reaction? Howwere CTLgenerated? Did the precursors of active CTL(CTL-P) express the samespecificity as their progeny?Andwhat were the mechanismsby whichthey inducedtarget cell lysis? CTL and the Major I-listocompatibility Complex That tissue graft rejection wasdue to an immune responseto alloantigens on normal or tumor cells was established by the work of Gorer and of Medawar andhis colleaguesin the 1930sandearly 1940s(see 76 for review). Theearly workon the genetics of histocompatibility(//) loci hadalready shownthat there was a very large numberof such genes (the estimates turned around 30). In 1956, Snell concludedfrom his workwith strains congenicfor different Hgenes that there were strong and weakH genes,

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and subsequentlyit becameclear that H-2is the only strong Hlocus in the mouse.Thecytotoxic lymphocytepopulations that could be obtained from animals immunizedwith allogeneic tissue were soon shownto be specific for determinantscontrolled by this locus (18). Theserologicalandhistogenetic analysis of H-2revealedthat the number of alleles at this locuswasvery large (this wasat a time whenit wasstill thoughtthat mostindividualsof a species werehomozygous for the sameallele at mostloci). This finding and the bewilderingserological complexityof H-2 turned its study into a moreandmoreesoteric field, comprehensible, ff at all, only to an exclusive club of specialists. Butin the middleof the 1960sa renaissance(76) of H-2 wasin the offing, stimulated by several developments. Throughimprovementsin surgical techniques, routine kidney transplants becamepossible, and the clinical importanceof graft rejection increased dramatically. Searchfor humanH genes rapidly led to the discoverythat in man,also, a single complexgenetic system, HI, A, wasresponsiblefor strong tissue incompatibility(see 23 for review).This wasthe first evidencepointingto the phylogeneticconvergence of a majorhistocompatibility gene complex (MHC),which, as we nowknow, extends all throughvertebrate evolution. Then,in 1971,Klein and Shreffler re-interpreted H-2 serology into the current bipartite genetic modelwith two alloantigen loci, H-2KandH-2D(77). This model,in general, wascompatible with the findings of HLetserology. Andfinally, in 1972,McDevittand his co-workers(97) reported that a gene controlling the immune response (It) to a specific antigen (It gene) discoveredin 1965waslocated between H-2Kand H-2D,and subsequentreports showedthat the immuneresponse to manyantigens was controlled by genes in this H-2I region. Fromthe studies on H-2it wasalready clear that it wouldbe difficult to developserological methodsthat allowedone to predict the histocompatibility of a particular kidneydonor-recipientcombination,and the developmentof rapid in vitro assays for this purposeseemedurgent. In an outbred species such as man,experimentsin whichthe proliferation of lymphocytes wasstimulatedwith phytohemaggiutinin inevitably led to the discoverythat proliferation occurredalso whenleukocytesfromdifferent individuals were mixed(10, 63). This reaction, called the mixedleukocyte culture (MLC) response,wasimmediately interpreted as an in vitro analogueof an allograft reaction. Experiments in the mouseandin mansoonestablished that strong proliferative MLC responseswere associated with MHC incompatibility (8, 36) and obeyedthe laws of transplantation. [Toavoid two-wayreactions in mixturesof allogeneiccells, one population,the stimulator, is madeunresponsiveby blockingits DNA synthesis (see 110 for references).] But would an MLC responselead to the generationof the cytotoxiccells described,in other systems,as possiblecandidatesfor the effectorcells in graft rejection?

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CYTOLYTICT LYMPHOCYTES 277 In 1968, Ginsburg(48) had shownthat rat lymphocytescultured on mouse embryofibroblast monolayersdevelopedH-2-specific cytolytic capacity, andin 1970,usingthe ~Crrelease assayand culturedcell lines as targets, four groups(57, 61, 64, 139)showed that cytolytic cells weregeneratedalso in MLC. However,the requirementfor fibroblasts or established cell lines as targets effectively precludedan evaluationof the prognosticcapacity of CTLgeneration in MLCfor the outcomeof organ transplants. Normal lymphocyteswererelatively refractory to CTL-induced lysis and showeda high spontaneous~Cr release, but mitogen-activated lymphocytesproved to be adequatetargets (100). Nowthe stage wasset for a genetic analysis of the genesthat controlled the proliferative MLC response, the generation of CTL,and the target determinantsof the latter. After the finding of recombinationevents between the serologically defined HL~genes and those coding for MLC incompatibility in manby Yunis & Amos(157), analysis of MLC and CTL responses betweenmice carrying recombinantH-2 haplotypes led to the conclusion that strong MHC-controlledMLC responses were elicited by H-2I and to a lesser extent by H-2D-regionincompatibility in the mouse (9, 98), and by HLd-DR in man(156). CTL,on the other hand, recognize mainlydeterminantscodedfor by the genescontrolling the classical, serologically defined MHC antigens (H-2K,H-2D,HL/I-.d,B) or by genes very closely linked to these loci (4, 39, 111, 144). The demonstrationof/-region-controlled MLC responses coincided with the discoveryof I-region-associated (Ia) antigens(33, 56, 59, 129), andafter a period of considerablecontroversyit is nowgenerally accepted, but has not beenproved,that the genescodingfor these antigens and MLC determinants largely overlap(see 156for review).Ia antigensare expressedpredominantly on B cells and macrophagesand early experimentscomparingthe stimulatorycapacity of different leukocyteshadindeedsuggestedthat both of these cell types wereefficient stimulators. (When Ia antigenswerefirst detected, the idea that H-2-linkedIr genescodedfor the T cell receptors seemedso convincingthat the first reports on Ia antigens suggestedthat they wereexpressed predominantlyon T calls, and muchless on B cells. The opposite turned out to be true.) However,morerecent studies with more rigorous separation methods show that Ia + macrophagesand/or dendritic cells (141) maybe responsiblefor mostof the stimulatingactivity in spleencell populations(1, 2). Further workmadeit clear, in any case, that the separation betweenthe genes coding for MLC incompatibility and for CTLdeterminants was not complete(see 110 for review). Theclearest evidencefor an overlap comes from the study of mutations in the H-2Kgene, manyof whichlead both to MLC incompatibility and the appearanceof newCTLdeterminants (45, 151).

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In the meantime,sufficient evidencewasaccumulatingto convincealmosteverybodythat the cells respondingspecifically to antigenic stimulation in MLCby proliferation or generation of cytotoxic cells were T lymphocytes(see 110 for discussion). Thegenetic complexityof the responsewasthe first hint at a correspondingcellular complexity,a topic to whichwereturn later. Oneimportantpuzzle raised by these studies coneerned the progenyof cells of a given individual respondingto a given allogeneic MHC haplotype(see 110 for review). Estimatesof this frequency were obtained by determiningthe proportion of cells that wouldrespond to allogeneic stimulators by DNA synthesis or division. There was no way of measuringthe fraction of CTLin this population,but, recently, limiting dilution assays (see below) of CTL-Phave indicated that they represent about 50%of all ceils stimulated in a MLC (86). This problemwasclearly a newaspect of the older question of the biological significance of graft rejection and transplantation antigens. Thefindings that as manydifferent MHC haplotypes could be defined by MLC responses as by serology, and that morethan 1%of all T cells respondedto a single allogeneichaplotype whereasonly MHC locus differences wouldelieit a measurable MLC responseby cells of an unprimedindividual, provokedspeculations that alloreactive T cells werenot monospecific. (In the mousethere is at least one locus, not linked to H-2, that controls the expression of strong MLC determinants,the Mls locus. Thenumber of alleles at this locus is smalland homologous genes have not been found in other species.) But all attempts to demonstratepoly-specificity failed, andthe conclusionthat mostif not all T cells werespecific for MHC alloantigens seemedinescapablebut met understandablywith a great deal of resistance. (Onewayof solving this dilemmawasobviouslythrough the identification and characterization of the structures that were the basis of antigen recognition by T cells. The enormous amountof effort spent on searchingfor the T-cell receptors since the early 1970shas resulted, so far, in negativeor controversialresults. No quantitative antigen-bindingassay for T cells has beendevelopedandtheir specificity can still be determinedonly throughassays of their function.) Onehypothesisthat providedan explanationfor the biological significance of alloreactivity wasJerne’s (67; see also 110)postulatethat the germ-line genesfor lymphocyte antigen receptors codedfor moleculesthat recognized the MHC antigens of the species, and that the repertoire of an adult was generatedby somaticmutationsin the genes codingfor receptors specific for the individual’s ownMHC determinants. Oneof the starting points of Jerne’s speculation wasthe observationthat in xenogeneicMLC an inverse correlation existed betweenthe magnitudeof the response and the phylogenetic distance betweenthe mixedcells (7), the so-called allo-aggression phenomenon,whichclearly indicated an interaction betweenthe genes codingfor MHC antigens and those controlling T-cell specificity.

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CYTOLYTICT LYMPHOCYTES 279 Themostimportantadvancein our understandingof the biological role of MHC antigens has certainly been the discovery of the phenomenon we call MHC restriction. The first experimentalevidencefor a role of MHC antigens in cell-cell interactions in the immune responsecamefromstudies (69, 75) showin.gthat efficient cooperationbetweenT and B lymphocytes in an antibodyresponse, in whichallogeneic effects were prevented, requiredcompatibilityof the cooperatingcells in the H-2Iregion. Katzet al suggested,quite naturally, that efficient cooperationdependedon positive recognitionof self by the interactingcells, self beingdefinedby H-2Iregioncodedcell surface moleculesthat wouldinteract, e.g. in the wayenzyme subunitsself-assembleto formoligomers(69). In the meantime,the realization that in antiviral immunitycell-mediatedphenomena played an important role (see 3 for review) inducedZinkernagel&Doherty(160) to for virus-specific CTLin infected animals. Theycould demonstratevirusspecific cytotoxicity against syngeneic-infectedbut not allogeneic(H-2incompatible)-infectedtarget cells. Sheareret al (133), in the sameyear, reported that the sameheld true for hapten-specificCTLgeneratedin vitro by stimulating lymphocyteswith syngeneichaptenatedcells. Interpretationsof these data in termsof positive recognitionof self MHC determinantsweresoon swept awayby experimentsthat demonstratedthat a virus-specific CTLfroman 1-1-2 heterozygousF1 animalwouldrecognize infected target cells of only oneparent (161)[see also the earlier findings in the guineapig (125, 135)], andthat in irradiated F1 micereconstituted with parental bonemarrowcells these wouldgive rise to virus- or haptenspecific CTL-P able to recognizeinfected or haptenatedtargets bearingthe allogeneicparental MHC haplotype(146, 158): i.e. duringtheir differentiation into CTL-P,stemcells "learn" to restrict their responsesto non-MHC determinantsso that they will recognizesuchantigensonly on cells expressing the MHC haplotypes encounteredduring maturation (16, 147, 159). wasquickly appreciatedthat the rules of MHC restriction applied not only to virus- andhapten-specifieCTLresponses,but also to those against minor H- (15, 53) and tumorvirus-specific antigens (118). Furthermore,the liferative responsesto soluble antigens that could be inducedby exposing T cells from immunizedanimals to antigen-pulsed macrophageswere also MHC restricted (see 145 for review). Whereasthe determinantsrestricting CTLwere coded for, mostly, by the H-2Kand H-2Dgenes (or their homologues in other species), antigen-induced proliferation wasrestricted, in mostcases, by genesmappingin the I region, mostlikely codingfor the samemoleculesas those carrying Ia-determinants. Thesefindings providedan explanationof whythe activity of virus- or hapten-specific CTLcould not be inhibited by non-cell-boundantigen (an obviousa prod requirementfor effeetor lymphoeytes).Theyalso offered, at last, someinsight into the biologicalrole of MHC antigensandstimulated

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the elaboration of newhypotheseson the evolutionary origin of the MHC polymorphism (110, 114). Theattempts to elucidate the structural basis MHC restriction gaverise to two types of models(see 138for discussion): Onepostulatesthat T-cell receptorsare specific for "altered self" determinants formedby interactions betweenMHC and surface-boundvirus, hapten, or cell surface molecules;the other claimsthat T cells carry a "dual recognition"systemconsisting of receptors specific for self MHC determinants and other non-MHC antigen-specific ones. To us, it seems more simple to explain allo-aggression, the MHC polymorphism,and the large fraction of T cells that respondto MHC alloantigens, as well as the crossreactivity of (self) MHC-restricted cells specific for non-MHC determinants with MHC alloantigens (see below),in the context of an altered-self model. However,it seemsclear, by now,that a definitive answerwill comeonly fromthe structural characterization of the moleculesformingthe T cell receptor(s). The Nature of the ~4ntigenic Stimulus for T Cells Oneof the obstaclesto progressin the identificationof the T-cell receptor(s) is the lack of a quantitative specific binding assay with characterizable antigenic material, evenin allogeneicsituations wheregeneticanalysis allowsprecise identification of the genescodingfor the determinantsrecognized. Althoughit is possible to demonstratethat activated alloreactive T lymphocytesspecifically bind membrane fractions from appropriate cells (113), it has not beenshownthat this workswith purified MHC molecules. Onereport indicates CTLcan induce specific ~Crrelease from artificial liposomescontainingcellular glycoproteins(65), but several other groups havehad problemsreproducingthese results. Attemptsto inhibit CTLwith antigen-bearingmembrane fractions haveresulted in failures in mostlaboratories, where,as inhibition withwholecold-targetcells has beenrepeatedly demonstrated. Thesedifficulties in defining the molecularnature of the determinants recognizedby T cells maybe related to the onesencounteredin the investigationsinto the nature of the entities stimulatingthe activation (proliferation or acquisition of cytolytic activity) of T cells. Althoughin oneof the first descriptions of the proliferative MLC response it wasclaimedthat extracts fromallogeneiccells couldstimulatethis reaction (63), this turned out not to be true: In unprimedcells a proliferative or CTLresponsecan only be inducedwith living metabolicallyactive stimulators, althoughthese can be irradiated with lethal doses of Xrays or treated with mitomycin C. But UV-irradiationabolishestheir stimulatorycapacity (see 110 for references). In cell populations from immunized animalsa CTLresponse can be induced by subcellular antigenic membranepreparations (42), but only

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whenadherent accessory spleen cells are present (34). Accessoryspleen cells, probablyIa + macrophages,are also required for the generation of CTLin a primaryMLC against allogeneic or haptenatedsyngeneiccells (2). Theymaybe allogeneic or syngeneicwith the respondercells. Analogywith the proliferative response of primedT cells to soluble protein antigens suggests that one function of the accessorycells maybe antigen presentation, but a definitive picture of the role of these andthe other cells participating in a MLC will almost certainly emergeonly whenwe understand the molecularbases of their interactions. Evidence for Cell-Cell Interaction in the Generation of CTL Theuse of mitogen-induced blast cells as targets madepossible the genetic analysis not only of the target determinantsof CTL,but also of the incompatibilities required for their generationin a MLC. It soonbecame apparent that in someexperimentalsystemsinduction of CTLactivity required, as expected,differencesat loci that codedfor the target determinantsbut, in addition, incompatibilitythat induceda strong proliferative MLC response (4, 38). At the sametime evidencefor the synergistic involvementof two sets of T cells in lethal graft-versus-hostreactionswasreported, as wellas data suggestingthe possibility that the cells that respondmainlybyproliferation to allogeneic stimulators weredifferent fromthe CTL-Pandthat CTL generationwasthe result of a cooperationbetweenthe twodifferent sets (see 22 for references). Experimentsin whichrespondercells were mixedwith two different stimulators madeit clear that the determinantsinducing, a strong proliferative response and those recognizedby the CTLgenerated in the MLC did not haveto be on the samecell (38, 130): i.e. this system did not followthe then-acceptedrequirementsfor hapten-carrierlinkage in T-Bcell collaboration.It became apparentquickly,however,that the influence of incompatibilities inducinga strong proliferative responseon CTL generation wasmorequantitative than qualitative (41, 111) and could not be shownin somesystems. Furthermore,the genescodingfor determinants recognizedby the proliferating cells and those controlling CTLdeterminants werefoundto overlap (112, 151). Convincingevidencefor the existence of twosubsetsof alloreactive T cells wasprovidedby Cantoret al (21, 22). They found that murine CTLand CTL-Pexpressed the cell surface antigens Lyt 2 and Lyt 3 (their results suggestingthat CTLand CTL-P did not express~Lyt 1 turnedout to be a reflection of onlyquantitativevariation in the expressionof this antigen),that the bulkof the proliferatingcells in a MLC did not express Lyt 2 or Lyt 3 and were not cytotoxic, and that the two populations would showsynergism during CTLgeneration--the Lyt 2-3- MLC-responsive cells wouldamplify the CTLresponse. Theyhad the sameLyt phenotypeas helper T cells required for manyantibodyresponses.

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Did they belong to the samepopulation and, if so, did they exert their influence on B cells and CTLby the same mechanism?

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T-Cell GrowthFactor Thesuggestionthat the synergistic effects betweendifferent T-cell subsets during CTLgeneration weremediatedby soluble factors without antigenic specificity wastakenseriouslysurprisinglylate, in spite of the accumulating evidencefor the existence of several biological activities in MLC supernarants (5, 37, 68, 119). Tosomeextent the belated recognitionof the importance of such molecules is almost certainly due to the obsession of immunologists with antigenic specificity. Rather than admit the existence of such uninteresting hormone-likeagents, somepreferred to suggest, for example,that the T-cell growth-stimulatingactivity in supernatants of mitogen-activatedleukocyteswasa mixtureof antigenically specific molecules. Twopapersfinally usheredin whatonemightcall the age of factors (or lymphokinesor interleukins or whatevernameyou prefer). Onewas contributed by a group (105) whosemainconcernhad been the problem growingnormallymphocytesin long-term culture for cell biological and ontological studies. The impact of their discovery that normalT cells required a factor present in supernatants from T-cell mitogen-stimulated leukocytesfor long-termproliferation hit immunologistsreally only when Gillis &Smith(46) reported that with this T-cell growthfactor (TCGF interleukin-2) they could maintaineytolytically active CTLin culture for several months.Virtually at the sametime Ryser et al (126) showedthat in vitro-primedMLC cells (i.e. cells stimulatedseveral weeksearlier in MLC)could be inducedto re-express specific CTLactivity by exposureto supernatants from short-term secondaryMLC cultures. The active principle in this latter systemalmost certainly includes TCGF. Further work on the role of TCGFin the proliferative responses to mitogeniclectins has shownthat resting matureT cells can only respond to TCGF if they are previously activated, e.g. by a short-termexposureto eoncanavalinA (ConA) (81, 136), and it has been assumedthat antigen plays the samerole in a specific response,althoughsomeevidencethat this maybe so has only recently been published (62). ConA mayrender cells responsive to TCGF by inducing them to express TCGFreceptors: several groups showedthat ConA-activated but not resting T cells absorb TCGF (e.g. 81), a result recently confirmedby bindingassayswith purified labded growthfactor (122). TCGF itself is a T cell product (136), and work murineT-cell clones has shownthat mostTCGF-producing cells belong to the non-cytolytic,Lyt 2-3- class (52, 72). [It is possiblethat TCGF-producing cells themselvesrequireTCGF for proliferation (137), i.e. the proliferative responseof all T cells in a MLC maydependentirely on this growth

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CYTOLYTICT LYMPHOCYTES 283 factor, but this hypothesisis not supportedby any direct evidence.] TCGF production, in turn, requires the presence of another lymphokine,lymphocyteactivating factor (or interleuldn-1), a macrophage product (115, 136). This explains, at least in part, the requirementfor macrophage-like accessorycells in the MLC response. It appears, in addition, that lymphocyte-activatingfactor productionmaydependon an interaction between antigen-specific T cells andantigenic or antigen-presentingmacrophages. (Onenotes that use of the terms "stimulation" and "respondingcells" in their original definition becomesmeaningless.) The experimental support for manyaspects of this type of modelfor T-cell activation as proposedby several groupsat the sametime (81, 115, 136) is still shaky, andfurther probingwill undoubtedlyreveal additional interactions. But at least it has become impossibleto ignore the complexity of the T-cell responseto antigenicstimulation,andthe importanceof understandingthe molecularbasesof the cellular interactionsinvolvedis increasingly apparent. T-Cell Clones The discovery of TCGFmadepossible two developments:the estimation of the frequenciesof CTL-P of a givenspecificity in limitingdilution assays; and the establishment, in culture, of cell lines derived from CTLthat continueto express the capacity to recognizeand lyse appropriate target cells. Attemptsto estimate CTL-Pfrequenciesby stimulating themat limiting dilutions had already been madeprior to the discovery of TCGF,but the results of such experimentsonly becameconvincingwhenthe distribution of the amountof 51Crreleased remainedbimodalregardless of the number of respondercells/well, the positive events weredistributed accordingto one-hit Poissonstatistics, andthe frequencyestimates stoppedincreasing from one publication to the next, whenthe cultures, in 0.2-ml wells of microtiter plates, contained,besidesirradiated spleencells as stimulators, TCGF-containing supernatants from secondary MLC-or Con Astimulatedspleen cells (127). Thefirst attemptsto maintainCTLin culture for prolongedperiods were madeby re-exposing the ceils obtained from a primaryMLC periodically to fresh stimulators(87). Eachrestimulationinduceda waveof proliferation that peakedon day 3 or 4 and then declined rapidly. Duringthe first few restimulations the proliferative responsewasaccompanied by a strong rise in cytolytic activity, whichreachedits maximum on day 5 and then abated more slowly than DNA synthesis. Later on, the cytolytic but not the proliferative response wouldrapidly becomesmaller and woulddisappear completelyafter about six to eight restimulations(60). Analysisof the Lyt

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phenotypeof the respondingcells suggests that this decline is due to a gradual dilution of the Lyt 2+3+ CTL-Pduring such culture regimes(H. R. MacDonald, unpublishedresults). Thereport by Gillis & Smith(46) that CTL-containing MLC populations could be grown for several months in TCGF-containingmedia, in the absenceof stimulator cells, withoutloss of specific cytolytic activity was quickly followedby the demonstrationthat CTL-likeclones could be isolated from such long-termcultures and could be established as permanent lines (11, 109, 148). Soonafterward, Glasebrook& Fitch (50) showed CTLclones obtained by the limiting dilution technique described above could also be passagedfor apparently unlimited periods. Wehave comparedthe mouseCTLlines obtained by the two types of protocols elsewhere (106), and we restate here only the most prominent observationsplus our conclusions.Bothtypes of lines dependon TCGF, but the ones obtainedby immediatecloningin limiting dilution cultures (which wehavecalled CTL-A clones) also require the addition of irradiated spleen cells (filler cells) for continuedgrowth.Althoughthe original reports suggested that in the presence of an excess of TCGF-eontaining supernatant s syngeneie filler cells weresufficient (50, 84), recentpapersshowthat at least someCTL-A clonesproliferate only ff the filler cellsare antigenic(6, 121). CTL-Aclones resemble normal CTLmore than the lines derived from populations maintained in TCGF-containing mediaprior to cloning; the latter--we havecalled themCTL-B lines--maybe derived from filler cellindependentvariants that arise andare selected for duringthe first months of culture (55). Thepossibility to clone CTLand CTL-P,andto establish cell lines with the functional characteristics of CTL,has beenexploitedin different ways: (a) it providesa tool for an analysis of the specificity repertoire of CTL. (b) It allows, combined with the use of monoclonal antibodies and fluorescence-activatedcell sorting, a morerigorous characterizationof their cell surface antigen phenotype.(c) Assaysof CTLclones for other functions, in particular productionof soluble mediators,haveled to the realization that CTLare a muchmoreheterogeneouscell population than was previously suspected. (d) Extensionof such analysesto T cell populationsfrom various organs at different stages of developmenthas already becomeimportant in the study of the ontogenyof the immune system.(e) Thepossibility to clone CTL-P and the availability of pure TCGF is giving newimpetus to investigations on the requirementsfor the complexset of events lumped together as T cell activation or CTLgeneration.Finally, (f) CTLlines are being used for attempts to identify the genetic basis of CTLfunction by somaticcell genetic or molecularbiologytechniques.In the followingsections wediscuss someof these topics.

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CYTOLYTICT LYMPHOCYTES 285 THE CELLULAR

BIOLOGY

OF CTL

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The Phenotype of CTL Clones and their

Precursors

SPECIFICITY REPERTOIRE The frequency estimates for anti-allogeneic CTL-Pamonglymphoidcells from unprimedmice obtained whenlimiting numbersof lymphocyteswere cultured in the presenceof H-2-incompatible stimulator cells and TCGF-containing supernatants varied from 1 per 170 to 1 per 1000spleen cells, or 1 per 40 to 1 per 147 lymphnodecells (86). Specific CTL-Prepresent about 50%of all cells that proliferate in this systemin responseto alloantigen(127); thus these estimatesagreevery well with the earlier ones basedon the fraction of cyclingcells in MLCs. Most, if not all, of the CTL-Prespondingto stimulator cells from completely unrelatedstrains are directed against H-2determinants,andthe findingthat the frequencies of CTL-Prespondingto determinants coded for by H-2K or H-2Dalleles, or by H-2Kgenemutations,are all very similar (86, 152) suggests that most CTL-Pare specific for H-2Kor D determinants. Although CTLpopulations and clones specific for H-2I determinants have beendescribed, the frequencyof H-2I-specific CTL-Phas not been determined. Thefrequencies of CTL-Preactive against minorH or tumorvirus antigens could only be accurately determined in cells from immunized animals(86). The possibility of expandingclones obtained in the limiting dilution cultures wasexploitedto examinetheir activity against several targets. The results of these experimentsindicate that the cross-reactivity patterns observed in unelonedpopulations of CTL(see 138 for review) reflect the frequencyof cross-reactiveclones (143). [Theterm cross-reactivity is used entirely in an operational sense: noneof the experimentswediscuss rules out the theorythat the cross-reactivity of an individualcloneis dueto the expressionof several receptorpopulationswith different specificities. Furthermore, the cross-reactivity of a clone maydependon its expressionof molecules,suchas Lyt 2, that do not contribute to the receptor specificity (see below).] Whenthe cross-reactivity patterns of cells respondingto unrelated H-2Kallele against a series of mutations of this allele were compared,a high proportionof all possible different cross-reactivity patterns wasdetected (134). (The conclusionsthat can be drawnfrom these experimentsare limited becausethe estimate of the CTL-Pfrequencywas about 10 times lower than that obtained in other laboratories, and the monoclonality of the positive cultures wasnot ascertained.) Thereactivity of anti-MHCclones does not dependon the non-MHC backgroundof the target cells (80), a finding that arguesstrongly against the hypothesis Matzinger&Bevan(96), accordingto whichalloreactive T cells are specific for non-MHC antigens restricted by the allogeneic MHC determinants.

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AmongMHC-restricted CTL, cross-reactivity between non-MHC (influenza virus) antigens (17), as well as cross-reactivity with regardto restriction element,has beenobserved(55, 150); andthere are several cases of clones specific for non-MHC antigens on syngeneictargets that crossreact with allogeneic targets not carrying the relevant non-MHC antigen (17, 27, 148). Theseexperimentssuggestthat the distinction of allogeneic MHC antigens, non-MHC antigens, and self-MHCrestricting determinants is quite artificial: CTLclones do not seemto distinguish amongthem. PRODUCTION OF SOLUBLE MEDIATORS The role of soluble mediators in cell-mediated immuneresponses had been recognized very early, and soonafter the discoveryof the MLC reaction the presence, in MLC supernatants, of biologicalactivities suchas blastogenicfactor (68) andan activity replacing T calls in B-cell responseswasnoted (37). Butnoneof these activities wasbiochemicallycharacterized, and althoughit wasoften assumedthat they wereproducedby the respondingT cells, no clear evidence to this effect wasprovided.Mostcellular immunologists werenot interested in nonspecificsoluble mediatorsand assumedthat lymphocyte proliferation wasinduceddirectly by their interaction with antigen. (Mitogenicplant lectins werethoughtto be universal antigens.) This viewbecameuntenable with the discovery of TCGF,and it is nowclear that the growthof many if not all matureT cells dependson this lympholdne,whichitself wassoon shownto be a product of T cells (51, 136). In 1979, Ghscbrook& Fitch (50) isolated the first TCGF-producing alloreactive T cell clone, and subsequentlyseveral groupsstarted to test whetherT cell clones producedother soluble mediators[TCGF,interferon (IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage activating factor (MAF),and factors involvedin the proliferation andmaturationof B cells (B cell helperfactors) (see 51for references)]. Initially, the mainpurposeof suchscreeningswasto identify the cellular sourceof variousbiologicalactivities. Theresults showed that T cell clones werevery heterogeneous whenthey wereclassified accordingto the biological activities they produced(51, 71, 78), andthis type of analysis would sometimesallow a functional separation of the moleculesmediatingtwo different biologicalactivities by the fact that certain clonesproducedonly one of them. After the division of T ceils into subclasses(21, 22), it hadbeenwidely assumedthat the biological activities present in MLC superuatants were productsof non-cytolyticproliferating Lyt 2-3- ceils specific for/-region determinants. Comparisonof estimates of the frequency of MHC-reactive T ceils that proliferate but do not give rise to CTL(127) with those of the frequencyof cells that give rise to TCGF-producing clones (85) suggests

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that the two populations largdy overlap. The majority of the TCGFproducers are Lyt 2- (72) and, in a response to H-2-incompatiblestimulators, they respond to/-region determinants, but amongthem are about 10-20% cells specific for K- or D-region antigens (85). AmongK/D-region-reactive Lyt 2+ CTLclones isolated by micromanipulation from a 5-day MLC,13% produced detectable quantities of TCGF(A. Glasebrook, personal communication). They were included in a larger fraction, 25%, which did not secrete detectable amountsof TCGFbut still proliferated in response to stimulator cells without addition of growthfactor. It seemslikely that such clones produce sufficient TCGFto stimulate their own growth, but not enoughto be detectable in the culture fluid. Recent analyses (51, 72) show that most CTLclones produce one or more factors, and that, as far as tested, CTLcan produce the same biological activities as non-cytolytic Lyt 2-3- MLCresponders, although at least for MAFand GM-CSF the frequency of alloreactive (1-1-2 4- Mls) splenic T cells giving rise to producer clones is about three times higher amongLyt 2- negative cells (about 1/100) than amongLyt + ones ( 72). I n a ny case, clones with almost any possible combinationof functional activities can be found. The one exception is IFN and MAF,where a strict correlation has been observed so far (71), but there is no convincing biochemical evidence that these two factors are not the same. The kinetics of appearance of MAF,GM-CSF,and B-cell helper factor in clone supernatants is similar; they reach a plateau about 24 hr after exposure to allogeneic cells or Con A (51, 71). TCGFactivity, on the other hand, declines rapidly after a peak at aroundthe sametime. It seems likely that this decrease, which does not depend on the Lyt 2 phenotype of the producer clone, is due to absorption and consumption by the producing cells themselves (122). Comparisonof the kinetics of factor accumulation with that of proliferation of TCGF-independentclones in response to stimulator cells suggests that factor production ceases one to several days before proliferation stops. At present we find it difficult to suggest what the biological significance of the bewildering multitude of T-cell phenotypes that emergesfrom these studies might be. Is it a culture-induced artifact? Doesit reflect a further splitting up of the T-cell lineages into sublineages? Or do the different phenotypesrepresent different maturation stages of the samelineage? So far there is little evidencefor changesin the phenotypeof a clone, but the data are not yet very extensive. It seems likely that several of the mediators producedby lymphocyteswill be purified, and their genes will be cloned in the next few years. Thus, the most promising way to approach the questions raised above may be to use such cloned probes to analyze the mechanisms by which the expression of the corresponding genes is regulated.

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CELLSURFACE MARKERS In spite of very intensive screenings in several laboratories for CTL-specificmonoclonal antibodies, no such reagents have been reported to date. Like most other T cells, murineCTLexpress the so-called lymphocytefunctional antigen-1 (LFA-1)(140), and probably all CTLas well as their precursors belong to the population of T cells expressing the Lyt 2 and Lyt 3 determinants(for review, see 104). This includes CTLspecific for/-region determinantsor for non-MHC antigens (102). Theonly reported cases of Lyt 2--specific CTLconcernantigen loss variants(35, 49, 107)or, in onecase, a cell populationmaintainedin culture for several years beforeits Lyt 2 expressionwastested (142)(see p. 306). Cerottini et al (28) found, however,that a fraction of alloreactive Lyt clones obtainedat limiting dilutions of normalcells wasable to induce5~ Cr release from targets whenConA or phytohemagglutininwas added to the assay, but it is not clear whetheror not the mechanism by whichisotope release is induced by these cells is the same as for Lyt 2+ CTL(44). Antibodies against Lyt 2, Lyt 3, and LFA-1can block CTLactivity, and wediscuss themin a later section on the lytic mechanism. In 1978, Kimura& Wigzell (73) reported that CTLcould be distinguished from CTL-Pand other non-CTLactivated in a MLCby their expressionof a glycoprotein, T145.A lectin from Vicia villosa (VV) was shownto havea preferential affinity for this moleculewhenit wasderived from Con A-activated T cell blasts (74), and W-affinity columnswere claimedto retain preferentially the CTLin a T cell populationactivated by in vitro stimulation. Other groupshavereported results that seemcontradictory in that they found no difference betweenthe W-binding capacities of CTLand of non-cytolytic blasts activated in a MLC (90), and that not all types of CTLpopulations express T145(70). Comparison of CTL-B with T lymphoma lines (31) showedthat only the former boundand were sensitive to growthinhibition by W,and that they express a glycoproteinwith approximatelythe samemolecularweight as T145.Lymphoma cells, on the other hand, expressed a glycoprotein resembling T130that is found on thymocytesand is probably structurally related to T145.The numberof W-molecules boundper CTL-B cell wasabout 107, and affinity chromatography of cell lysates showedthat Wboundnot only the T145-1ikemolecules but manyother surface glycoproteinsfromthese cells. VV-resistant mutants of a CTL-Bline that boundreducedor nondeteetable amountsof the leetin werestill cytolytically active andTCGF dependent,but they also still expresseda T145-1ikemolecule(31). Onthe basis of these results, Conzelmann (29) has suggestedthat in somebut not in all situations CTL activation is correlated with a changingpattern of glycosyl-transferase activities that results in an increased density of terminal o-linked r~-Nacetyl galactosamineresidues on O-linked carbohydrates of manyglyco-

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CYTOLYTICT LYMPHOCYTES 289 proteins. Themost prominentresult of this changewouldbe the altered lectin affinity and the changedapparent molecularweightof T130,which becomesT145.Similar alterations affecting other, less abundantmolecules maybe difficult to detect on ConAblasts, but they are obviouson CTL-B cells. Thefact that the intensive searchesfor CTL-specificmonoclonal antibodies have remainedunsuccessfulso far, evenwhenthe screening wasbased on inhibition of CTLactivity, does not meanthat CTL-specific cell surface structures do not exist. The relative frequencies with whichhybridomas that secrete monoclonal antibodiesagainst different cell surfaceantigensare obtained dependson their antigenicity and relative abundanceon the immunogenand, probably, on the immunizationschedule; therefore, CTLspecific hybridomas maynot yet havebeenisolated becausetheir incidence wastoo low. It is possible, on the other hand,that CTLuse the samesurface molecules as other cells, but with different effects because,for example,they interact with different moleculeson the cytoplasmicside of the cell membrane. Thus, maybeweshould look there for CTL-specificmolecules. Of course, technicallythis is moredifficult. Not surprisingly, our criteria for distinguishing CTL-Pfrom other T lymphocytesare even moreshaky than the ones applicable to CTL.About all weknowis that mostof thembelongto the Lyt 2+3+ subpopulation(21, 94). Weassume that CTL-Phave the same specificity as their mature progeny,but this has still not been proved. Theseobservations at least indicate that CTL.Pdo exist as cells committedto becomingCTL,and that they do not have the alternative of giving rise to T helper cells whose precursors are mainlyLyt 2-3-. The Cytolytic Mechanism Followingthe first demonstration of cell-mediatedtarget cell destructionit gradually emergedthat several different types of leukocytes could lyse target cells (for review,see 116): these include macrophages, granulocytes, and three types of lymphoidcells~CTL,natural killer cells, and Kcells that lyse antibody-coatedtarget cells. Theontogeneticrelationships betweenthe lymphoidcytotoxic cells is not dear, and to somedegree these relationships mayoverlap. It is not knownwhetheror not their effector mechanisms are different, nor has it been ruled out that CTLcan induce ~lCr release by several different mechanisms. The studies on these mechanismshave been coveredby several recent reviews(12, 92), so weonly summarizethe findings concerningCTL.Soon after their discoveryit wasshownthat CTLkill autonomously: i.e. they do not requirethe assistanceof other cells or productsto inflict lethal damage.

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Thelytic event requires contact betweenthe CTLand its target, andinnocent bystandersare not affected. This includesthe CTLitself, whichis able to interact sequentiallywith several targets: CTLrecycle. Butthey are not immuneto lysis by other CTL.In an interaction betweentwo CTL,one of whichspecificallyrecognizesthe other, onlythe latter is lysed. Theseobservationsseemto rule out killing by a short-lived, locally acting toxin. Inhibitor studies haveallowedthe dissection of the CTL-targetinteraction into three stages: bindingor conjugation,lethal hit or programming for lysis, andtarget cell lysis or zeiosis. Butthe natureof the lethal hit is still entirely mysterious.It is g~nerallyassumedto be a cell surface-mediated event as it does not affect CTL,and it does not necessarily lead to the immediatedestruction of the target. Theapproachesin this field, which includes morphologicalstudies of CTL-targctconjugates, use of metabolic inhibitors, and antibodieshas not changedgreatly duringthe last 10 years. Althoughthe availability of clonedCTLhelps to clarify someof the hanging issues, they do not as yet provide a direct approachto the problem, unless somebody invents a methodfor the isolation of non-cytolyticvariants byselection for this very deficiency. Thereis no evidencethat the different stages of the lyric interaction involve molecularstructures, whichwouldform a lytic machineryat the surface of CTLdistinct from the determinant-specific receptors, but the morphologicalstudies suggest that the CTL-targetinteraction is a very complexprocess. Recently,Ryseret al (128) haveobservedthat CTL-target conjugate formationis accompanied by the polarization of CTLactin and by rapid movementsof the CTLmembranein the contact area. These morphological phenomenahave not been shownto be required for 51Cr release or target cell lysis, or to be specific for the interactionof cytolytic cells with their targets. But they do suggestthat the recent modelof Berke & Clark (13) is a bit too simple. Theseauthors have suggestedthat "the impositionof whatis in effect a two-dimensional cross-linkinggrid consisting of the CTLsurface and its embedded MHC receptors creates instabilities at the target cell MHC protein bilayer lipid interface that lead to increased membrane permeability."This speculation, by itself (i.e. without ad hoe assumptions),does not explain whyCTL-Pdo not lyse targets, or whatthe differences are betweeneytolytic andnon-eytolytie T-cell clones that recognizedeterminantson the sametarget cell molecules. Oneapproachthat wouldsimplify the study of the CTL-targetinteraction wouldbe the construction of artificial targets, and wehavealready referred to the reports that CTL-containinglymphocytepopulations induced 51Crrelease from liposomescontaining MHC molecules(65). There are a numberof possible reasonsfor the apparentdiflficulty of reproducing these results in other laboratories (see 99), and this approachmaywell eventuallybe successful.

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Inasmuchas target cell lysis by CTLis mediated by cell surface interactions, the use of antibodies for the analysis of CTLactivity has been an obvious approach for a long time, and antibodies against the MHC determinants of the target cells have been helpful in establishing the nature of the target structures. Onthe other hand, the usefulness of conventional anti-effector antisera, which block killing activity, has been hampered by the impossibility of adequately clarifying the specificity of such reagents. As discussed above, the monoclonalantibodies that block CTLactivity have been directed, so far, either against the molecular complex bearing the Lyt 2 and Lyt 3 determinants or against LFA-1. The characteristics of these antigens have recently been reviewed (88, 140), so that we restrict ourselves to summarizingthe reported findings. 1. Both antigens are expressed not only on CTLbut also in other cells: Lyt 2/3 expression is restricted to T cells, CTL-P,suppressor T cells, and the majority of thymocytes. LFA-1is expressed on all lymphocytes, including B cells, and the majority of bone marrowcells, but not on cells outside the hemapoietic system. 2. Antibodies against both antigens inhibit not only CTLactivity, but also other functions of antigen reactive cells: Auti-Lyt 2/3 antibodies can both block the proliferation of antigen-responsive CTLclones, and can inhibit the release of lymphokines.AUti-LFA-1 reagents seem to block all T cell-dependent immuneresponses. 3. Antibodies against either antigen block conjugate formation and do not inhibit the so-called programming-for-lysisstep. Their blocking activity can be overcome by addition of Con A or phytohemagglutinins. 4. Expression of Lyt 2/3 is not an absolute requirement for specific CTL activity: certain highly active CTLpopulations cannot be inhibited, and some CTLlines or CTLhybrids do not, or no longer, express Lyt 2/3, but still are able to lyse targets specifically. Comparing the effects of controlled trypsin treatment on Lyt 2 expression, and of anti-Lyt 2 antibodies on the CTLactivity of a number of different CTLclones, MacDonaldet al (88) have arrived at the conclusion that the dependenceof the lyric interaction on Lyt 2/3, at least in an in vitro situation, probablydependson the affinity of the CTLfor its target. The strongest support in favor of this interpretation comes from experiments showing that the activity of several clones against their specific targets does not dependon Lyt 2/3, whereasactivity of the sameclones against a cross-reacting target does. Similar studies with regard to LFA-1have not yet been reported. 5. The molecule bearing LFA-1determinants consists of two noncovalently associated subunits, one of which is apparently shared with another antigen, Mac-l, expressed not on T cells but on macrophages, monocytes, and granulocytes. Anti-Mac-1antibodies inhibit the binding of erythrocyte-

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immunoglobulinM-complementcomplexes to such cells, probably by blocking their receptors for complement component3. Thesefindings emphasizethe possibility, mentionedabove,that different cell types mayuse the sameor similar moleculesfor different functions. CTL Induction Leukocytepopulations from normalanimals have no demonstrablespecific cytolytic activity prior to stimulation. Mostof us haveinterpreted this to meanthat there are non-eytolytieCTL-P that haveto receive certain stimuli to becomecytolytically competent.But mostprotocols for the generation of CTLactivity inducea strongproliferation(i.e., enrichment of the specific cells), and the only experimentsthat dearly indicate that inactive CTL-P indeedexist are those in whichactivity is inducedwhile DNA synthesis is prevented (89). Wehave chosen to call this process induction and not differentiation(or maturation)for the followingreasons.Webelievethat the termdifferentiationshouldbe appliedto irreversible or virtually irreversible events by whicha cell becomescommittedto a certain lineage or stage. The molecularbases for such a commitment are probablyheritable events at the level of the genome itself, e.g. demethylation of specificsites in certain genes (see 154for review).Although as yet there is very little evidenceconcerning the mechanismsby which CTL-PbecomeCTL,there is somereason to believe that this process may,to a large extent, be reversible and may resemblemorethe inductionof the synthesis of specific enzymes in differentiated cells of a particulartissue. Morefor a priori reasonsthan becauseof experimentalevidence,it seems probablethat the first step in the induction of CTL-P is their interaction with antigen. Thehypothesis that antigen induces the expression of TCGF receptors on CTL-Pand renders them responsive to the.growth factor is based largely on an analogywith the experiments(discussed above)that showthat ConA has these effects: short-term exposure(4 hr) to Con certainly renders a purified T cell population TCGF-responsive (81).But these experimentshave not been done with single calls and pure TCGF. Thus,they do not rule out that this effect of exposureto ConA(or antigen) requiresyet other cellular interactionsor other factors in the medium o~"the TCGFpreparation. Recent expedment~from Mescher’s laboratory (62) haveshownthat in vivo-primedspleen cells from whichadherentcells had been removedand that were exposed for 12 hr at 37°Cwith liposomes containingpurified H-2molecules,and then cultured for 4 days, developed strong CTLactivity. This wasfour times higher (in terms of lyric units) whenthe mediumduring the second culture period was supplementedwith proteins from supernatants of T-cell mitogen-aetivated leukoeytes. The sameauthors showthat the efficiency with whichliposomesactivate CTL-P

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CYTOLYTICT LYMPHOCYTES 293 is enhancedif they contain detergent-insolublemembrane matrix elements. Theseresults are promising,but they do not provethat antigenic liposomes exert their effect directly by their interaction with CTL-P,andthey certainly do not yet shed any light on the mechanism by whichantigen or Con Aactivate cells. The modelsconcerning the role of TCOFin CTLgeneration are, to a large part, based on experimentswith crude culture supernatants or ammonium sulfate-precipitated material as sourcesof biological activity, and it is not clear whetheror not their effects on CTL-generation are dueto a single factor. Thepurification of TCGF wasbasedon its capacity to maintain proliferation of CTLlines, and it shares manyfunctional properties with other growthfactors, such as epidermalgrowthfactor and platdetderived growthfactor, in that it seemsto be required for the passageof dependentceils from G1 (or GO)into S-phase (132). Other polypeptide hormonesand growthfactors possess multiple biological activities, and TCGFmaynot only act as a growth factor but also, for example,as an inducer of the expressionof eytolytie activity in CTL-P.InducedCTLdo not seem to require TCGF to remain cytolytic: cloned CTLlines can be maintainedfor several days in the absenceof exogenousTCGF without a decreaseof the lyric activity per cell (131, 132). Butwecannotyet rule out that these lines produce small amountsof TCGF,insufficient to allow growthbut enoughto maintain their cytolytic activity. This possibility seemsnot at all unlikely given the finding that someCTLclones do produce TCGF after antigenic stimulation. Recent evidence from several laboratories indicates that TCGFand stimulator ceils are not sutticient to induce CTL-P to becomeeytolytie. Raulet & Bevan(120) have shownthat whenspleen cells rendered TCGFresponsiveby incubation with ConAare cultured in supernatant fromCon A-stimulatedspleen cells (CS), they acquire strong cytolytic activity. But whensupernatantfrom a TCGF-producing cell line wasused instead of CS, no lytic activity wasinduced. Further experimentssuggest that the CTLinducingactivity in CSis a protein, different fromlymphocyte activating factor or TCGF.Althoughthe authors (120) showthat CSfrom whichthe growth-promoting activity, i.e. TCGF,has beenabsorbedare still capable of inducinggeneration of CTLactivity, they cannot rule out that TCGF is also required, becauseit is probablyproducedby the ConA-treated cells themselves. Kanagawa(manuscript in preparation) has shownthat cells treated with leukoagglutinin, whichrenders cells TCGF-responsive, but unlike ConA does not induce its production,proliferate in culture media from TCGF-producing tumorceil lines as well as in secondaryMLC supernatants, but generate CTLactivity only in response to the latter. The proportionof Lyt .2 + cells is the samein both populations. This suggests

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that mitogen-treated CTL-Pcan proliferate in response to TCC3F but require additional factors to becomecytolytic. It remainsto be shownwhether or not the sameholds for CTL-Pthat respond to an antigenic stimulus rather than a mitogenic lectin. The CTL-inducingactivity is found in supernatantsfroma fraction of alloreactive T-cell clones after stimulation with spleen cells and thus maywell be a T-cell product. CTLlines or inducedCTLpopulations maintain their cytolytic activity whenthey are grownin a source of TCGFthat does not contain detectable inducing activity. The Fate of Activated CTL After in vivo immunization with appropriate antigens (allogeneie or tumor cells, or viruses), CTLactivity can be detected only duringa short period (19), but the primedanimals showlong-lasting immunologic memory in the sensethat subsequentin vivo or in vitro challengeswith the samestimulators result in an accelerated an.d stronger CTLresponsewhichreflects, in part, an increased frequency of CTL-P(86). But whether or not these memoryCTL-Pare derived from the CTLactivated during priming, and whetheror not they havedifferent biological properties fromvirgin CTL-P,. is not clear. Activated CTLare not terminally differentiated cells: A CTLthat has killed one or moretarget cells can divide and give rise to a clone (N. Thiernesse, unpublishedresults). Togetherwith the high plating e~ciency of CTLclones, close to 1, this finding renders very improbableany models that view CTLclones as continuously differentiating systems in which alwaysonly a fraction of cells becomescytolytic duringevery cell generation. As we described above, in a MLCboth the proliferative and the CTL responseare transient. In a primaryMLC lytic activity per cell reaches a peakafter about 5 daysandthen declines fairly rapidly. Restimulationwith the samestimulator cells inducesa faster andhigher waveof CTLactivity, whichcan probablybe accountedfor entirely by an enrichmentof specific CTL-P(93). Also it is not dear in this ease whetheror not these primed CTL-Pdiffer in any wayfrom their virgin progenitors. Cell fractionation studies have unambiguouslyshownthat the CTL-P respondingto secondarystimulation are small nondividingcells derived from the blast cell population that contains the CTLat the peak of the response (87). WhenMLC-responsive clones were screened for their CTL-P content, these were found only in clones that themselveshad demonstrable CTLactivity (91). Theseresults suggest that active CTLcan revert to small inactive lymphoeytesthat are CTL-P.However,wecan not rule out that during stimulation in each clone the cells that have acquired

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CYTOLYTICT LYMPHOCYTES 295 CTLactivity die, possiblybecauseTCGF becomeslimiting, while blast cells that givc rise to the secondaryCTLresponsedo not becomecytolytically active. In CTLclones, whetherthey belongto the filler cell-dependent(CTL-A) type or to the independent(CTL-B) type, the lytic activity per cell remains constant, even whengrowth is stopped by removalof TCGF(131, 132): they do not revert to CTL-P,and they do not Survivefor prolongedperiods in the absenceof TCGF-containing supernatants,i.e. withoutproliferation, whereasin vitro-primedCTL-P can survive for several weeksin the absence of any detectable TCGF.If the primed CTL-Pgenerated in a MLCare indeed derived fromthe CTLactivated during the primaryresponse, then wemustconcludethat this reversion is not spontaneousbut is inducedby signals producedin a MLC, athoughnot in the conditionsused for culturing CTLclones.

THE ONTOGENYOF CTL-P In the adult mouse,CTL-Pcan be detected in the thymusas well as in peripheral lymphoidtissues such as spleen, lymphnode, and blood (86). Duringontogeny,CTL-P activity can first be detected in the thymusat or shortly after birth (24, 153). CTL-P in the spleenare not detecteduntil 4-5 days of age. Frequencystudies of CTL-P in the developingneonatal thymus providestrong (althoughindirect) evidencethat in situ differentiation these cells is occurring. Thus,thymicCTL-P frequenciesincrease by 10 to 40-fold in the 24 to 48-hr period followingbirth in C57BL/6 mice(153). Clearly, a synchronousimmigrationof these cells from someother organ cannot be excluded; however, more recent studies with organ-cultured thymicrudiments(25) providedirect evidencethat CTL-Parise within the thymusitself. Thelatter systemshouldprovidea useful modelfor detailed analysis of the intrathymic differentiation pathwayof CTL-P. Althoughthere is goodexperimental evidencethat CTL-Parise in the thymusduring ontogeny,the absolute necessity of this organ for CTL-P differentiation can be called into question. Recentstudies fromseveral laboratories (66, 95) have shownthat CTL-Pdo developin congenitally athymic(nu/nu) mice, albeit at a muchslower rate than in their normal (nu/+) counterparts. Surface phenotypeanalysis of CTL-Pin nu/nu mice has shownfurther that these cells are not of maternalorigin (66) andthat they express the Thy-1and Lyt-2 antigens characteristic of cells of the CTL-P lineage in normalanimals(94). However,somedifferences in antigenic specificity of CTL-Phave been observed in nu/nu versus nu/+ animals(66). Thesedata provide direct evidencefor the existence of extrathymicdifferentiation pathwayfor CTL-P and henceraise the possibil-

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ity that the thymusmayhavea quantitative rather than qualitative role in CTL-Pdevelopment. Theconversionof immatureT cells into CTL-Pis a process that almost certainly wouldfall underour dctinition of differentiation, i.e. it probably reflects heritable changesin the state of a set of genes, the expressionof whichis required for the manifestationof the CTLphenotypc.Thusgenetic analysis of the differences betweenCTLand cell lines representing immature T cells shouldallowus to identify suchgenes,andthe results of recent experiments involving crosses between routine thymomaand CTLlines suggestthis is indeedthe case (30, 32, 108). Therelevant observationshave been that (a) CTLlines differ from AKR-thymomas with regard to the followingcharacteristics--they arc TCGF-dcpcndent, VV-scnsitivc,glucocorticoid-rcsistant, andcytolyticallyactive; (b) hybridsbetweenthe twocell types behavelike either one or the other parent; and (c) circumstantial evidence suggests that thymoma-likehybrids arc derived from CTL-likc ones by loss of a CTLchromosome, but this hypothesis has not yet been proved.Wehavesuggestedthat the correlation of the expressionof several CTLcharacteristics in the hybridsreflects the existenceof one, or several, linked genes that control these traits and are expressedin CTLbut not in the AKR-thymomas, and that heritable activation of this (these) gene(s) maybe one of the steps in the differentiation of immatureT cells into CTL-P. Oneof the assumptionsin this speculation is that AKR-thymomas indeed represent an immaturestage of T-cell differentiation (and not, for example,anothertype of matureT cells). If, as is generallybelieved, AKRthymomas are the results of a retrovirus-mediatedactivation or introduction of a transacting oncogene,then our speculation predicts that the immatureprogenitor cells of such thymomas are not dependenton TCGF, but that the transformationevent abolishes someother growthrequirement. Unfortunately,as yet no convincing set of criteria exist by whichT-cell lines can be classified, but recent evidencesuggeststhat the stage of maturation representedby lymphoidtumorcell lines is correlated with the presenceof stage-specific transforminggenesin such cells (79). Confirmation of these results mayprovideus with a veryclear-cut systemfor T cell-line classification. In addition, analogywith other systemssuggeststhe possibility that the transforminggenes characteristic for a certain stage maybe derived fromcellular genescontrolling its expression(14). THE PHYSIOLOGICAL

SIGNIFICANCE

OF CTL

In recent years, numerousstudies haveaddressedthemselvesto the potential physiological role of CTL.AlthoughCTLwere originally discovered (and are still mostfrequentlystudied)in allograft reactions(26), it is clear

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CYTOLYTICT LYMPHOCYTES 297 that this recent experimentalinvention cannot be the selective mechanism responsiblefor the maintenance of these cells throughoutvertebrate evolution. Thediscoverythat CTLwerealso generatedin the course of immune responsesto pathogenicviruses (161)providesa moreplausible explanation (in evolutionaryterms) for the existenceof these potentially destructive cells. As discussedin detail previously, CTLrespondingto viruses behave in a MHC-restricted fashion in the sense that they preferentially lyse infected target cells bearing compatible(self) MHC antigens (160). Although this degreeof specificity wouldseemto be at oddswith the alloreactivity exhibited by CTL,recent studies of CTLclones have demonstrateda considerable degreeof cross-reactivity betweensyngeneicvirus-infectedtarget cells andallogeneicuninfectedtarget cells (17, 28). Thus,it is possiblethat all CTLdo potentially recognizevirus-modifiedsyngeneictarget cells, and that allogeneic cells represent a universe of antigenic determinantsthat resemblethese modifiedself determinants. Althoughthe presence of CTLcan be demonstratedin viral infections andallograft responses,andevenat the site of rejection of certain allogeneie or virus-inducedsyngeneictumors(58), it has not beena simplematter demonstratethat CTLare directly involvedin the pathologyof these phenomena. At least twomajordifficulties are encountered in the interpretation of experimentsin whichsomeaspect of cellular immune function (skin graft or tumorgraft rejection, delayedtype hypersensitivity,lethal graft-versushost disease,etc) is assessedin vivofollowingtransfer of puritied subpopulations of T lymphocytes. First, the large number of cells that mustordinarily be transferred, together with the long ~imeperiod required to observethe effect of the transferred calls, combine to makeit difficult to rule out the possibility that activity is mediatedby rare contaminantsselected for in vivo. This problemcan be overcome,at least in theory, by the utilization of clonedpopulationsof T lymphocytes;however,at the present time, there are fewsuccessful reports of systemicimmunitymediatedby clonedT cells (vide infra). Asecondditiieulty, whichis not solvedby the use of homogeneous T-cell populations, is the inherent complexityof cellular immune responses in vivo. Thus,graft rejection and other inflammatory-typereactions involve the participation of non-lymphoid cells (suchas neutrophilsandcells of the monocyte/macrophage series), as wall as T lymphocytes.The underlying mechanisms of such reactions remainpoorly understood(see 58), despite (or perhaps becauseof) the impressive array of lymphokinesshownto released frommixturesof sensitized T cells and appropriate antigen-presenting cells. Furthermore,it has provedextremelydifficult (and maybe impossible)to eliminatethe participation of endogenous (host-derived)cells in the inflammatory processby irradiation or drugtreatment.In viewof this complexity, the only conclusion that can be firmly derived from a cell

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transfer experimentwouldbe that a particular subclass (or clone) of lymphocytesis capable of provokinga particular immune reaction in vivo. Theparticipation of other (host-derived) cells in this reaction cannot ruled out, neither (moreimportantly)can it necessarily be excludedthat another T-cell subclass (or clone) mightbe capable of mediatingthe same phenomenon. In consideringthe evidencefor a participation of CTLin graft or tumor rejection in vivo, it is clear that the aboveproblemshavefrequently been ignored. Thus,investigators haveconcludedthat CTLdo or do not mediate a particular in vivo function simplyon the basis of whetherthe test population transferred did or did not havedetectable CTLactivity. Studies of T cell subpopulations andtheir potential role in graft rejection-aretoo voluminousto reviewhere. Interested readers are advised to consult a recent volumeon this topic (43). Insofar as data with cloned CTLpopulationsare concerned,several workershave beensuccessful in eliminating allogeneie or syngeneic(virus-infected) tumorcells by injecting the clonedCTLSimultaneouslyat the site of tumorchallengein a Winntype assay (47). Clearly, such tests do not assess the ability of the CTLto recirculate and hometo the site of tumorchallengein vivo. Morerecently, Engerset al (40) have obtainedevidencethat certain CTLclones are able to provokethe elimination of allogeneictumorcells implantedat a distant site. In their studies, C57BL/6anti-DBA/2CTLclones were injected intravenously into sublethally irradiated syngeneicanimalsthat received 125IUdR-labeled P-815 mastocytoma (DBA/2)cells intrapedtoneally. Tumorcell elimination was followedkinetically with a whole-bodycounting procedure. The results indicated that completeeliminationof allogeneie tumorcells (and concomitant survival) could be achievedwith someCTLclones, whereasno effect wasseen with other clonesof similar specificity. At present, the basis for this heterogeneityin the protective effects in vivo is not known,although preliminarystudies suggestthat it maybe correlatedwith the ability of some CTLclones to proliferate speeitically in responseto antigen in the absence of exogenouslyadded TCGF.Furthermore, as emphasizedabove, the actual mechanism wherebygraft rejection occurs in such systems(i.e. via direct cytotoxicity or via indirect activation of other non-lymphoid cells) mayprovedifficult to assess, since clonedCTLare capable of releasing a variety of lymphokines uponexposureto appropriate antigens (51). In any event, manymore data concerningthe recirculation and homingpatterns of CTLclones are required before any rational approachto immunotherapy with these cells can be devised.

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Literature Cited 1. Ahman, G. B., Naeller, P. I., Biukraut, A., Hodes,R. J. 1979.T cell recognition in the mixedlymphocyteresponse. I. Non-T,radiation-resistant spleen adherent cells are the predominant stimulators in the routine mixedlymphocyte reaction. Z Immunol. 123: 903-9 2. Ahman, G. B., Naeller, P. I., Binkraut, A., Hodes,R. J. 1981.T cell recognition in the mixedlymphocyteresponse. II. Ia-jpositivesplenicadherentcells are required for non-I region-inducedstimulation. J. Immunol.127:2308-13 3. Allison, A.C. 1974.Interactionsof antibodies, complementcomponentsand various cell types in immunityagainst viruses and pyrogeniebacteria. Transplan~ Rev. 19:3-55 4. Alter, B. J., Sehendel,D. J., Bach,M. L., Bach,F. H., Klein,J., Stimpfling,J. 1973. Cell-mediatedlympholysis. Im~po. trance of serologieally definedH-2reons. Z Exp. Med. 137:1303-9 5. Altman,A., Cohen,I. R. 1975.Cell-free media of mixed lymphocytecultures augmentingsensitization in vitro of mouseT lymphoeytes against allogeneic fibroblasts. Eur. Z Immunol. 5:437-44 6. Andrew,M. E., Braciale, T. J. 1981. Antigen-dependentproliferation of cloned continuous lines of. H-2restricted iniIuenzavirus-specifictyrotoxic T lymphocytes. Z lmmunol. 127:1201--4 7. Asantila, T., Vahala,J., Toivanen,P. 1974. Responseof humanfetal lymphoeytesin xenogeneicmixedleukocyte culture: Phylogeneticand ontogenetic aspects, lmmunogenetics1:272-90 8. Bach, F. H., Amos,D. B. 1967. HU-I: Majorhistoeompatibilitylocus in man. Science 156:1506-8 9. Bach, F. H,, Widmer,M. B., Bach,M. L, Klein, J. 1972.Serologicallydefined and lymphocyte-definedcomponentsof the majorhistocompatibilitycomplexin the mouse. Z Exp. Med. 136:1420-44 10. Bain, B., Vaz, M. R., Loweustein,L. 1964. Thedevelopmentof large immature mononuclearcells in mixedlymphocyte cultures. Blood23:108-16 11. Baker,P. E., G-illis, S., Smith,K. A. 1979.Monoelonal ¢ytolytic T-cell lines. d.. Exp. Med.149:273-78 12. Berke,G. 1980.Interaction ofcytolytic T lymphocytesand target cells. Prog. Allergy 27:69-133 13. Berke, G., Clark, W.R. 1982. T lymphocyte-mediatedeytolysis--a eompre-

heusive theory. I. The mechanismof CTL-mediated cytolysis. Adv. Exp. Med.Biol. 146:57-68 14. Beug, H., Palmied, S., Freudenstein, Zentgraf, H., Graf, T. 1982. Hormonedependent terminaldifferentiationin vitro of chicken erythroleukemiacells transformedby ts mutantsof avian erythroblastosis virus. Cell 28:907-19 15. Bevan,M.J. 1975.Interaction antigens detected by eytotoxic T cells with the major histocompatibility complexas modifier. Nature256:419-21 16. Bevan,M.J. 1977. In a radiation chimera, host H-2 antigens determineimmuneresponsivenessof donorcytotoxic cells. Nature269:417-18 17. Braciale,T. J., Andrew, M.E., Braciale, V. L. 1981.Heterogeneityand specificity of clonedlines of influenza-virusspecific cytotoxic T lymphocytes. £ Exp. Med. 153:910-23 18. Brondz,B. D. 1972. Lymphocyte receptors and mechanisms of in vitro cellmediated immunereactions. Transplant. Rev. 10:112-51 19. Brunner,K. T., Cerottini, J.-C. 1971. oC~tceotoxic Lymphocytes as effector cells II mediated immunity. In Progress in Immunology,ed. B. Amos,pp. 38598. NewYork: Academic 20. Brunner,K. T., Mauel,J., Ccrottini, J.-C., Chapuis,B. 1968.Quantitativeassay of the lytic action of immune lymphoid cells on S~Cr-hbeledalloseneic target cells in vitro; inhibitionby lsoantibody and by drugs. Immunology14: 181-96 21. Cantor, H., Boyse,E. A. 1975. Functional subclassesofT lymphocytes bearing different Lyantigens.I. Thegeneration of functionallydistinct T cell subclasses is a differentiative process independentof antigen. £ Exp. Med. 141:1376-89 22. Cantor, H., Boyse,E. A. 1975. Functional subclassesofTlymphoeytes bearing different Ly antigens. II. Cooperation betweensubclassesof Ly+cells in the generation of killer activity. J. Exp. Med. 141:1390-99 23. Ceppelini, R. 1971. Old and newfacts and speculations about transplantation antigens of man. In Progress in Immunology,ed. B. Amos,pp. 973-1025. NewYork: Academic 24. Ceredi~, R. 1979. Frequencyof alloreactlve cytotoxicT cell precursorsin the mousethymus and spleen during ontogeny.Transplantation28:377-81

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25. Ceredig, R., Jenkinson, E. J., MacDonald, H. R., Owen,J. J. T. 1982. Development of cytolytic T lymphocyte precursors in organ-culturedmouseembryonic thymus rudiments. J. Exp. Med. 155:617-22 26. Cerottini, J.-C., Bruuner,K. T. 1974. Cell-mediated cytotoxicity,allograft rejection and tumor munol. 18:67-132 immunity.Adv. Im-

tional in cytolysis. J. Exp. Mec~ 153:595-604 36. Dutton,R. W.1966.Spleencell proliferation in responseto homologous antigens studied in congenic resistant strains of mice. Z Exp. Med. 123: 665-71 37. Dutton,R. W.,Falkoff,R., Hirst, J. A., Hoffman,M., Kappler, J. W., Kettman, J. R., Lesley, J. F., Vaun,D. 1971.Is there evidencefor a non-antigenspecific 27. Cerottini, J.-C., MacDonald,H. R. 1981.Limitingdilution analysis of aldiifusable chemicalmediatorfrom the thymus-derived cell in the initiation of loantigen-reactive T lymphocytes.V. Lyt phenotype of cytolytic T lymthe immuneresponse? In Progress in phocyteprecursorsreactive against norImmunology,ed. B. Amos,pp. 355-68. real and mutant H-2antigens. J. ImNewYork: Academic munoL126:490-96 38. Eijsvoogel,V. P., DuBois,M.J. G. J., Meinesz,A., Bierhorst-Eijlander, J., 28. Cerottini, J.-C., MacDonald,H. R., Brunner,K. T. 1982. Alloreaetivity of Zeylemaker,W.P., Schellekens,P. Th. self H-2 restricted cytolytic T lymA. 1973.Thespecificity andthe activaphocytes.Behr. Inst. Mitt. 70:53-58 tion mefhanismof cell-mediated lymA. 1982. Somaticcell ge29. Conzelmann, pholysis (CML)in man. Transplant. netic analysisof cytolytic T-lymphocytex Proc. 5:1675-78 PhDthesis. Univ. Lausanne,Switzer39. Eijsvoogel,V. P., DuBois,M.J. G., Meland fief, C. J. M., De Groot-Kooy,M.L., 30. Conzelmann,A., Corth~sy, P., NabKoning, C., Van Rood, J. J., Van holz, M. 1982. Correlation between Leeuwea,A., DuToit, E., Schellekens, cytolytic activity, growthfactor depenP. Th. A. 1972. Position of a locus dedenceandlectin resistance in cytolyfic terminiag mixed lymphocytereaction T-cell hybrids.In Isolation, Characteri(MLR),distinct from the knownHL-A zation anit Utilization of T Lymphocyte loci, andits relation to ceil-mediated lympholysis(CML).Histocompatibility Clones, ed, C. G. Fatlunan, F, W. Fitch, pp. 205-15. NewYork: AcaTesting 1972, Munskgaard,pp. 501-8 40. Engers, H. D., Glasebrook,A. L., Sodemic 31. Conzelmann,A., Pink, R., Acuto, O., renson, G. D. 1982. Allogeneic tumor rejection inducedby the intravenousinMach,J. P., Dolivo, $., Nabholz,M. 1980. Presenceof T145on cytolytic Tjection of Lyt-2+ cytolytic T lymcell lines andtheir lectin-resistant muphocyte clones. J. F, xp. Med. 156: tants. Eur. J. ImmunoL 10:860-68 1280-85 32. Conzelmann, A., Silva, A., Cianfriglia, 41. Engers, H. D., MacDonald, H. R. 1976. M., Tougne,C., Sekaly,R. P., Nabholz, Generationof cytolytic T lymphocytes M. 1982. Correlated expression of in vitro. Contemp.Topics Immunobiol. 5:145-90 TCGF.dependence, sensitivity to Vicia Villosaandcytolytic activity in hybrids 42. Engers, H. D., Thomas,K., Cerottini, betweencytolytic T cells and T lymJ.-C., Brunner,K. T. 1975. Generation phomas.Z Exp. "Med.In press of cytotoxic T lymphocytes in vitro. V. Responseof normal and immunemouse 33. David,C. $., Shreitter, D. C., Frelinger, spleen cells115:356-60 to subeellularalloantigen.Z J. A. 1973. Newlymphocyte antigen Immunol. system(Lna)controlledby the Ir region of the ScL mouse H-2 complex.Proc. NatL 43. Fefer, A., Goldstein,A. L., eds. 1982. Acad. USA 70:2509-14 Thepotential role of T cells in cancer 34. Degiovanni, (3., Cerottini, J.-C., Bruntherapy. Progr. Cancer Rex Therapy ner, K. T. 1980.Generationof cytolytic 22:1-297 T lymphocytesin vitro. XI. Accessory 44. Fischer Lindahl, K., .Nordin, A. A., cell requirementin secondaryresponses Sfhreier, M.H. 1982.Lectin-dependent cytolytic andcytolymicT helper clones to particulate alloantigen. Fur. J. Imand hybddomas. Curr. Top. MicrobioL munot 10:40-45 Immunol. 100:1-10 35. Dialynas, D. P., Loken,H. R., Glasebrook, A. L., Fitch, F. W.1981. Lyt 45. Forman,J., Klein, J. 1975. Analysisof H-2 mutants: Evidence for multiple 2-/Lyt 3- variants of a clonedcytolytic CML target specificities controlled by T cell line lackan antigenreceptorrune-

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~ym

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NOTEADDEDIN PROOF(see

p. 288)

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It is nowclear that Lyt-2- CTLclones specific for I-region determinants do exist, as well as their humancounterparts (ie OKT8-CTLclones specific for HLA-DR determinants). The frequency of such clones has not yet been determined, and we therefore do not know what proportion of I-regionspecific CTL-Pis of this phenotype.

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AnnRev. Immuno£1983. 1:307-33 Copyright©1983by AnnualReviewsInc. All rights reserved

REGULATION OF B-CELL GROWTH AND DIFFERENTIATION BY SOLUBLE FACTORS Maureen Howard and William

E. Paul

Laboratory of Immunology, NationalInstitute of AllergyandInfectious Diseases,NationalInstitutes of Health,Bethesda,Maryland 20205 INTRODUCTION Interleukins are a familyof moleculesthat transmit growthanddifferentiation signals betweenvarious types of leukocytesand thus presumablyare major effeetors of immune regulation. Cell biologists and immunologists havedirected muchattention to the nature of interleukins that operate on T lymphocytes(84, 85) and macrophages (15, 106). Recently, it has become clear that suchantigen-nonspecificcofactors also operate on B lymphocytes (5, 27, 49, 51, 52, 56, 63, 73, 97, 99, 104,110, 129,135, 141,142). However, as is common in an emergingfield, an overall understandingof the involvementof soluble factors in B-cell responseshas beenhamperedby competing nomenclatures and by the use of differing operationalcriteria to identify, describe, enumerate,and characterizethe various B cell-specific interleukins. Herewereviewa variety of observationsderivedfromboth single and multicellular in vitro assays, using both mouseand humanlymphocytesand numerous sourcesof interleukins, in an effort to derive a unified conceptof factor-mediatedB-cell devdopment. Suchan understandingwill require an appreciationof the substantial functionalheterogeneityof B cells, e.g. subsets, lineages,differentiationstages, andindividualspecificity. Thus,it is desirable to preface our reviewwith a brief description of the physiology of this system. Splenic B lymphocytesof the mousemaybe divided into two major subpopulationsof approximatelyequal size that differ fromone anotherin the membraneantigens they express, in the immunogens to which they 307 0732-0582/83/0410-0307502.00

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respond, and in their sensitivity to distinct regulatory mechanisms.The appreciationthat suchsubpopulations exist is based,to a very large extent, on studies of mice that have an immunologic defect determinedby the xid gene (1, 22, 53, 54, 89, 100, 117, 128). This X chromosome gene, when present in the hemizygousor homozygous state, leads to a series of Blymphocyteabnormalities, amongwhichare unresponsivenessto soluble polysaccharidesand other type II antigens (89), depressedserumIgMand IgG3concentrations(100), andfailure of B lymphocytes to proliferate upon stimulation with anti-immunoglobulin (Ig) antibodies (117). Micewith xid defect lack B lymphoeytesthat bear the Lyb3(54), Lyb5(1), (128), and Ia.W39(53) antigens. Lymphocytes bearingthese differentiation + B lymphocytes.Lyb5+ B lymphocytesare antigens are referred to as Lyb5 foundin all normalstrains and the defects of xid micecan be accounted for by the absenceof these cells. Thepropertiesassignedto these twomajor + and Lyb5-)are basedon studies either of B cell subpopulations(i.e. Lyb5 the responsivenessof B cells from xid mice, or of normalLyb5-B cells prepared by treating B cells from normalmice with anti-Lyb5 antiserum and complement. Of particular interest for this review,a majorfeature that distinguishes + and LybS-B cells is their requirementsfor activation andfor prolifLyb5 erative responsesto antigen. Accordingto the workof Singer, Hodesand + B cells are readily responsiveto the combination colleagues(8, 120), Lyb5 of antigen andsoluble helper factors. In contrast, LybS-B cells require a contactual major histocompatibility complex(MHC)-restricted(cognate) interaction with helper T cells. Similar conclusionshave recently been reachedby Takatsuand colleagues (134, 138). Usingeither normalmice whichthe ability to respondto a soluble T cell-derived helper factor had been suppressed via neonatal treatment with an alloantiserum directed againstthe acceptorsite for this factor (134)or, alternatively, utilizing the mutant mousestrain DBA/2Ha,which is unresponsive to this soluble helper factor (138), they clearly demonstrated a distinction betweenB cells that are responsive to factors alone and those subject to cognateT-cell interactions. Bothsubsets appearto require soluble differentiation factors [T cell replacingfactors (TRFs)]to drive proliferatingceils into Ig-secreting cells (ISC). These concepts are summarizedschematically in Figure Basedon such a compartmentalization of B-cell function, this reviewfocuses on three mainareas of factor regulation: (a) the nature of soluble + B cells for initial activation andproliferahelper factors utilized by Lyb5 tive events; (b) the nature of differentiation factors that drive proliferating B cells into ISC;and(c) a considerationof the role of solublefactors in the B-cell-responses dependentuponcognate interactions with helper T calls.

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INTERLEUKINSFOR B LYMPHOCYTES 309 RESTING EXCITED CYCLING ANTIBODY CELL B LYMPHOCYTE B LYMPHOCYTE B LYMPHOCYTESECRETING

1. FACTOR DEPENDENT ACTIVATION (LybS+)

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HELPER FACTORS

2. MHC ACTIVATION (LybS-)

~

HELPER T CELL

Figure ] Schematic depiction of thetwopathways of B-cellgrowth anddifferentiation. MHC-restricted activationmayalsobe designated cognate interaction. MHC-UNRESTRICTED B-CELL TRIGGERING Evidence for Unique B-Cell Proliferation Cofactors Accordingto the basic tenets of clonal selection (16), immune responses involve the activation of a small numberof precursor cells committedin specificity to the invadingantigen, whichresults in their proliferation and differentiation into a populationof functionaleffector cells. Earlyexperimentalevidenceindicatingthat B-cell responsesincludeda significant proliferative response in addition to obvious maturation was obtained by demonstratingautoradiographically that most antibody-secreting cells (ASC)had incorporated tfitiated thymidineinto their DNA whenexposed to the labeled nucleotide 1 or 2 days precedingthe assay (42) and by the demonstrationthat bromodeoxyuridine and light markedlydiminishedthe numberof plaque-formingcells appearingin primaryin vitro responsesto sheeperythrocytes(28). Morerecently, sophisticatedB-cell cloningassays havebeendevelopedthat not only demonstratespecific antigen-initiated B cell proliferation, but that actually measurethe numberof ASCderived froma single precursor, thus providinga minimum estimate of the degree of antigen-drivenproliferation of precursorsof ASC(2, 80, 94, 95). Withthe ultimate goal of regulating B-cell function, considerableattention has beenfocusedon the nature of signals governingB-cell proliferation and differentiation. Thediscoverythat discrete TRFswereinvolvedin the production of ASC(27, 110) seemedadequately to accommodate the wallrecognizednonspecificrole of T cells in B-cell immunity.This, together with reports that antigenaloneinducedB-celi proliferation(25, 26) andthat TRFfunctioned whenaddedafter B-cell exposureto antigen (110), led the proposal of a modelin whichnonspecificTRFinducedantibody secretion by acting on B cells already proliferating in responseto antigen (26,

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111). Datafrom a numberof laboratories wouldnowsuggest this modelis oversimplified. As is outlined in moredetail below, Andersson,Melehers and their colleagueswereamongthe first to proposethat the occupancyof membrane Ig by antigen and/or cognate T-cell interactions involving the Ia antigensof resting B cells resulted in functionalexpressionof receptors for soluble factors and that it wasthese factors that in turn stimulatedB cells to replicate andthen to differentiate (5, 6). Althoughthe expression of growth-factorreceptors on B cells has yet to be demonstratedexperimentally, the general notion is supportedby numeroussubsequentreports, A particularly valuablesystemfor addressingeventsrelated to B-cellproliferation has beenthe use of purified anti-Ig antibodyas a polyclonalanalogue for antigen, first developedin the rabbit systemby Sell &Gell (115) and adapted to mouselymphocytesby Parker (96) and Weineret al (143). Anti-Ig combinesthe advantagesthat it acts uponthe receptors for antigen of virtually all Bcells andthat the resultant activationis restricted to DNA synthesis without developmentof ISC(63, 99). Usingthis system, Parker showedthat soluble T cell-derived factors, whichhadno apparenteffect on resting B ~ells, enhancedanti-Ig-inducedB-cell proliferation particularly whenrelatively low concentrationsof anti-Ig wereused (97, 98). This was the first clear demonstrationof soluble factors that could influence the proliferative component of a B-cell response.Morerecently, definitive evidencethat antigen-inducedB-cell proliferation requires soluble cofactors has been providedby Nossal and colleagues (101, 141). Usingthe elegant single cell cloning assay of Wetzd& Kettman(144), they cultured single aff~ty-purified hapten-specific B cells and showedthat antigen-specific B-cell proliferation wasonly obtainedin the presenceof exogenously supplied cofactor-richsupernatants. Amajorproblemin precisely identifyingfactors involvedin B-cell proliferation has beenthe lack of simplefunctional assays in whiehcontaminating accessory cells capable of mediating secondary effects have been excluded. EvensomeB-cell cloning assays do not escape this dilemmaas they often dependon filler call supplements.In an attemptto overcome this problem,wehavedevelopeda short-term B-cell costimulatorassay (49) whichhighly purified B cells are cultured at lowcell density, thus greatly reducingthe possibility of accessorycell contamination.Eneottragedby the observationsof Parker(97, 98), wesdectedati~nity-pudfiedanti-IgMantibodies as a polyclonal analoguefor antigen. Underappropriate culture conditions, anti-IgMcauses approximately50%of resting splenic B cells to synthesize DNA (22). As mentionedabove, these responsive cells are + populaabsent from xid mice and they appear to correspondto the Lyb5 tion of normalB cells (1). Thus,the use of anti-IgMantibodies seemed appropriatechoicefor defining the solubleproliferation cofactorsshownin Figure1.

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INTERLEUKINSFOR B LYMPHOCYTES 311 Using this low-cell-density B-cell costimulator assay, and measuring DNA synthesis via [3H]-thymidineuptake after 2.5 days of culture, we + subset found that three separate stimuli were required to cause the Lyb5 of small B lymphocytesto enter S phase: a signal delivered by antibodies specific for the Ig receptor expressed on the B-cell membrane;a signal delivered by a T call-derived factor, whichwe designatedB-cell growth factor (BCGF);and a signal ddivered by the macrophage-defivedfactor intedeukin1 (ILl)(49, 52). BCGF and ILl acted in an antigen-nonspecific, synergistic manner.Therequirementfor BCGF and ILl had not previously been observed whenB cells were cultured at high density, presumably reflecting accessorycell contaminationand endogenous factor production at suchcell densities. Full details of our currentprogressin characterization of BCGF are providedin the followingsection. Identification of the macrophageproduct involved in anti-Ig-inducedB-cell proliferation as ILl is basedon correlation of B cell andthymocyte costimulatingactivities following a seres of biochemicalpurification procedures(52). Both murineILl purified to apparenthomogeneityby Mizel(83) and humanILl purified high specific activity by Lachman (65, 67) showedexcellent B-cell costimulatingactivity in our assaysystem.Indeed,in the final step in the mouse ILl purification scheme,B-cell and thymocytecostimulatoryactivity migrated identically. Althoughour data provide compellingsupport for the notion that BCGF and ILl are the proliferation cofactors illustrated in Figure 1, wewish to emphasizethat ultimate proof that these agents act directly on B cells will await reagents capable of demonstratingspecific factor-receptor binding. Ourresults are consistent with manyprevious observations documented in the literature andhelp resolvesomeareas of conflict. In particular, the involvementof accessorycells in anti-IgM-induced B-cell proliferation has previously been a matter of controversy. It had been proposedby some laboratories that anti-Ig-inducedproliferation proceededindependentlyof T cells and other accessorycells, as vigorousdepletion of such cell types failed to prevent activation (96, 117). However,involvementof macrophageshad beenreported by Monginiet al (86) and Parker’s morerecent workon T cell-derived factors suggesteda needfor T cells (97). It would nowseemthat failure to observethe needfor oneor both cell types reflects the use of assay conditionsin whichsmall numbersof oneor both accessory cells are present with consequentendogenousfactor production. Ourown experiments showa need for both exogenousBCGF and ILl only at low cell densities (2 X104 cells/well and below),despite the rigorous B-cell purification procedurewehave employed.Thus, it wouldappear that very small numbersof accessorycells can provide B cell-stimulatory cofactors. Theproposalthat ILl is involvedin B-cell function is not a newconcept (34, 46-48, 76, 112, 148-150). However,whereasothers have previously

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suggested such a role for IL 1, their experiments measuredASCresponses in high cell density culture systems and thus failed to distinguish a direct action on B cells versus secondaryactions via other call types. A particular problem in this respect is that ILl appears responsible for the generation of at least someT cell-derived lymphokines,e.g. intedeukin 2 (IL2) (35, 71, 122, 123) and ~lony-stimulating factor (88). With the recent identification of several distinct B cell-specific lymphokines (5, 27, 49, 51, 52, 56, 63, 73, 97, 99, 104, l l0, 129, 135, 141, 142), one could certainly envisage ILl-mediated factor cascades in manybiological assays for B-cell function. Although such objections mayalso be leveled at the experiments from our laboratory outlined above, we believe they are not likely to apply here for the following reasons: (a) Wehave used very highly purified B cells cultured at low cell densities, thus greatly reducing the possibility of accessory cell contamination; (b) as is discussed in the following section, we showILl maybe added quite late in the G1 phase of the B-cell cycle, thus providing little opportunity for a factor cascade to develop; (c) we have shownthat several other T cell.derived B cell-specific lymphokines, e.g. BCGF,and two distinct TRFs [EL-TRFand B15-TRF(see 93 for nomenclature)] fail to replace the need for ILl. Again, however, ultimate proof that ILl acts directly on B cells will await reagents capable of demonstrating specific binding.

Characteristics of BCGF In cultures of mouse B lymphocytes, the term BCGFis currently used to designate the T cell-derived B cell costimulator that synergizes with altinitypurified anti-IgM antibodies to induce polyclonal B-cell proliferation (49). AlthoughILl is not generally added to these cultures, the conditions usually employedfor assays of B-cell activation probably allow its endogenous production. BCGFis found in culture supernatants of a subline of the thymoma,EL4, which has been stimulated with phorbol mydstate acetate (49) and in antigen-induced supernatants from normal T-cell helper lines (50) propagated in long-term culture, as described by Kimoto & Fathman (60). Surprisingly, it is not detected in supernatants of spleen cells cultured for 24 hr with concanavalin A or phytohemagglutinin. BCGFhas little or no apparent action on resting B cells and its effect on anti-IgM-activated B cells is polyclonal, not MHC-restricted, and seemingly is limited to DNA synthesis without the production of ASC(49). It also acts as a proliferation costimulant for some, but probably not all, B cells activated with lipopolysaceharide. Whencultures are supplementedwith additional differentiation cofactors, BCGFis found to be an essential componentof both antigenspecific (49) and polyclonal anti-Ig-induced (93) ASCresponses (see Unrestricted B-Cell Differentiation). Preliminary experiments indicate that

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INTERLEUKINSFOR B LYMPHOCYTES 313 BCGF is requiredfor antigen-specificproliferation of affinity-purifiedhapten-specifie B calls stimulated by hapten conjugatedto the semisynthetic polysaccharideFicoll (52a). BCGF can be separated fromIL2 by several different methods,including gel filtration, phenylsepharosechromatography,sodiumdodecylsulfate (SDS)-polyacrylamidegel dectrophoresis (PAGE),tds(hydroxymethyl)aminomethane (Tds)-glycinate-PAGE, and isoelectric focusing, as well as cellular absorption(31, 33, 49). BCGF can be distinguishedboth functionally andby molecularweightfromother previouslydescribedT-cell factors, namelyTRF(27, 109, 110, 142), colony-stimulatingfactor (15), intedeukin 3 (44), B cell replicationandmaturationfactor (5), andB cell differentiation factor (BCDF~) (56). It does not cause proliferation of phytohemagglutinin-stimulatedthymocytes,indicatingthat it is also distinct fromILl (67). Theproportionof B calls responsiveto BCGF has not yet beenestablished. However, the levels of proliferation obtainedin B-cell costimulatorexperimentsare consistent with involvementof the entire anti-IgM-responsive B-cell pool, a pool recently shrwnto consist of approximately50%of normalB cells (22). Splenic B cells fromxid mice fail to synthesize DNA in response to the combinationof anti-lgM and BCGF (50). Independent confirmationof the existence of murineBCGF with physical and biological properties similar to those outlined abovehas recently been providedby Leandersonet al (72). Farrar et al (31, 33, 49) havestudied the physicochemical properties BCGF.Theyhaveshownthat BCGF is a polypeptideby virtue of its trypsin sensitivity. BCGF producedby phorbolmyristate acetate-inducedEIAcells in serum-containing mediais readily separated fromthe bulk of proteins in such culture supernatants by ammonium sulfate precipitation. Morethan 90%of the BCGF activity is precipitated between80 and 100%saturated ammonium sulfate; only 5-9%of the total protein is foundin this fraction. Byconventionalgel filtration on ACA 54 in neutral solution, BCGF elutes as a broad peak between13,000 and 20,000daltons. Analysis of BCGF by SDS-PAGE reveals two componentsthat exhibit biologic activity after extraction from the gels and dialysis. Onehas a monomeric Mr of 11,000, the other 14,000. A degree of microheterogeneityis also observedwhen BCGFis examinedby electrofusing. Twopeaks of BCGFactivity are observedmigratingwith a pI of 6.4-6.7 and7.4-7.6, respectively. BCGF can be completely separated from IL2 by a combinationof ammonium sulfate precipitation, phenylsepharosecolumnchromatography,and Tds-glyeine gel electrophoresis. Surprisingly, BCGF displays a lower electrophoretic mobilitythan IL2on Tris-glycinegels, despite its lowermolecularweight; this is probablydue to the alkaline pI’s of BCGF. AlthoughBCGF has not been purified to homogeneity,it has been brought to very high specific activity by a sequential purification procedureinvolvingammonium sulfate

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precipitation, phenylsepharosechromatography,Tris-glycinate PAGE,and SDS-PAGE (33). Fourgroupshave isolated humanB-cell growthfactors (17, 37, 64, 75, 90, 91, 126 152). Theexperimentalsystemsused in these laboratories vary, andit is not yet clear whetherthe samefactor is beinginvestigatedby each of the groups, or whether the humanBCGFs are analogous to the mouse factor. Nevertheless, someuniformconclusions regarding the nature of humanBCGF(s)have emerged. HumanBCGFis currently used to designate either a material that costimulateswith anti-Ig or other polyclonal B-cell activators to cause DNA synthesis by normalor leukemichumanB cells (64, 90, 91, 152),or alternativelya materialthat maintainsthe proliferation of activated purified humanB lymphocytes in suspensionculture (37, 75, 126). Obviously,these two functions maywell be mediatedby different moieties. HumanBCGF defined by both assays can be found in supernatants of lectin-activated normalT lymphocytes (37, 90, 126, 152) and also in T-cell hybridomasupernatants (17, 64). HumanBCGF can be distinguishedfrom humanIL2 by its relatively delayedappearanceafter lectin stimulation of humanT cells and by cellular absorptionstudies with either normalhumanT-cell blasts or long-termIL2-dependent T-cell lines (37, 64, 91, 152). Such cells removeIL2 but do not diminish BCGF activity in culture supernatants. The existence of T hybridomasthat secrete BCGF and not IL2 (17, 64), and of conditioned mediathat lack one or other activity (107), verify this distinction. Biochemical characterizationof humanBCGF has commenced in three laboratories; by standard gel filtration procedures,material with BCGF activity in 72-hr lectin-stimulated peripheral blood lymphocyte-conditioned mediahas been shownto have approximate Mrof 17,000-20,000(17, 152) and 12,000-13,000(75). Twoof these groups have reported the isoelectric point of humanBCGF to be approximately 6.5-6.9 (37, 152). As is the case with IL2, humanBCGFgives excellent responses in the mouseBCGF assay, but mouseBCGFis not detected in humanBCGF assays (52b). Direct binding of humanBCGF B-cell blasts has beendemonstratedvia its depletion followingabsorption on humanleukemicB cells activated with anti-Ig or anti-idiotypic antibodies (64, 152). Recently, Kishimotoand colleagues havereported a seconddistinct humanBCGF producedby an IL2-dependentallospecifie helper T-cell doneuponstimulationwith cells bearingthe alloantigenfor whichthe T cells are specific (152). This factor stimulatedproliferation of normalB eeds cultured with anti-Ig or of leukemicB cells cultured with anti-idiotype antibody.It had a Mrof 50,000by gel filtration. In 4 Murea, its Mrfell to 19,1300. Both size estimates distinguished it from the humanBCGF the samegrouphad describedin supernatantsof peripheral bloodT cells stimu-

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315

lated with phytohemagglutinin. This BCGFshowed a Mr of 17,000 by conventional gel filtration and of 12,000-14,000in 4 Murea. NormalT cells stimulated with the combination of phytohemaggiutinin and phorbol myristate acetate produced both the 17,000 and 50,000 BCGFs.Most interestingly, these two BCGFsshoweda synergistic effect on the proliferation of anti-Ig-stimulated B cells. This suggests that the larger BCOF is not simply an aggregated form of the smaller.

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B-Cell ~4ctivation: Two-Signal ModelRevisited The understanding of B-lymphocyteactivation has bccn a tantalizing prospect for both immunologistsand cell biologists as these cells havethe unique characteristics of possessing a well-characterized receptor for, and displaying a clear inducible response to, a specific stimulant. Despite this apparent simplicity of B-lymphocytesystems, progress in understanding their activation has been relatively slow. Some10 years ago, three major models to explain antigen-specific B-cell activation were proposed: (a) Activation occurs solely as a result of the binding of antigen to membrane(m)Ig (24, 117); (b) activation occurs via an unrelated nonspecific receptor, with serving specifically to focus molecules capable of binding to this receptor (20); and (c) activation requires both a signal resulting from the binding antigen to the mlg receptor and a signal delivered by an interaction at another, antigen-nonspecific receptor on the B-cell membrane(13, 14). The recent explosion of information on B-cell proliferation cofactors outlined in the precedingsections promisesto clarify this question, although it must be recognized that the events of DNAsynthesis and mitosis may be far removedfrom those signals operating at the resting (Go) lymphocyte level. For this reason, somegroups have attempted to devise experimental systems to analyze the activation events that precede entry into the S phase of the cell cycle. One useful approach has been the monitoring of size enlargement following lectin or antigen stimulation of B lymphocytes. Andersson ct al (4) initially probed early activation events by measuringboth size enlargement and DNAsynthesis in response to a variety of B-cell mitogcns. Of particular interest, they found that purified protein derivative of tuberculin (PPD) was capable of driving blasts that had been prepared by lipopolysaccharidc stimulation through subsequent rounds of replication, but that PPDcould not induce Go B cells to complete the first round of replication. This was the first indication that the activation requirements of Go B cells and of "activated" B cells were different. More recently, this theme has been developed and extended by DeFranco ct al (22). Theyfound that culture of purified GoB cells with anti-IgM for as brief a time as 1 hr led to a distinct enlargement of these cells. This enlargement was progressive and quite synchronous for the first 24 hr of

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culture provided the anti-IgM was present continuously. It appeared to reflect entry of resting B calls into and transition through G1 phase as it correlated well with both increased RNAsynthesis and acquisition of the ability to enter S phase upon receipt of the appropriate additional signal. At 20-24 hr, a second signal provided by lipopolysaccharide or high concentrations of anti-IgM antibody was required for the completion of G1 and for the entry of cells into S phase some 6-10 hr later. The concept that distinct mechanismscontrol entry of Goceils into G1 and late G1 cells into S was strongly supported by experiments involving the immune-deficient xid mice. Although anti-IgM fails to stimulate DNAsynthesis by xid B cells, it quite effectively transfers these cells fromGoto G1as monitoredby size enlargement. The studies of DeFrancoet al prompted speculation that one or both of the soluble helper factors we had shownto be critical to anti-IgM stimulation of DNAsynthesis (i.e. BCGFand ILl) might regulate the second control point, i.e. the transition of late G~B cells to S phase. Careful kinetic experiments were designed to test this hypothesis (52). Anti-IgMand one cofactor were added to B cells at the initiation of the culture, with the second cofactor being added at various times over the course of 30 hr. In light of the findings of DeFrancoet al (22), tritiated thymidine was added at 30 hr and cultures were terminated at 42 hr so that most of the thymidine uptake wouldrepresent B cells that had entered S phase for the first time. The somewhatsurprising results are illustrated in Figure 2. The cofactor ILl could be added16--20 hr after initiation of culture without any significant decrease in final thymidineuptake; it thus seemslikely that this factor corresponds to the second control agent suggested by DeFranco’s studies. The surprising result was obtained when the time of addition of BCGFwas varied. Its effectiveness as a costimulator declined rapidly and linearly as its addition was delayed. If its addition was delayed beyond10 hr, it was essentially without effect for thymidine uptake between 30-42 hr. This suggests that BCGF acted either on Go B cells or during the early (excitation) portion of the 1 phase o f t he cell c ycle. T he t ime of a ction a nd biological function of BCGF thus makesit a prime candidate for the second signal of the original Bretscher/Cohn hypothesis (24, 87). Whether BCGF acts directly on the resting B cell or on the early G~B cell, its precise role in B-cell activation and its possible role in B-cell tolerance are the subjects of current investigation.

Triggering Lyb5"/- B Cells: Summary Based on the various experiments described above, we propose the following model, summarizedschematically in Figure 3, for the activation and proliferation of Lyb5+ B cells. Resting B cells that bind anti-IgM through their

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INTERLEUKINSFOR B LYMPHOCYTES 317 mlgMenter the G I phaseof the cell cycle. This process presumably requires cross-linking of mIg; multivalent antigens wouldbe expected to have the sameeffect on those resting B cells that possess receptorsspecific for that antigen. BC(3Facts either together with anti-IgMto cause the (30 ~ (31 transition, or acts uponthe early G1cell. It is knownthat anti-IgM is required continuously during early G1 (22); whetherBCGF is also needed continuouslyor acts at a single point in early GI (or Go) is not known. a result of these two stimulants, the B cell progressesthroughearly G~and reaches a point at about 16-20 hr whenit requires ILl. The presenceof ILl allows the cell to continueto progressthrough131 andthen to enter S phase. Thefirst calls to enter S phasedo so at about30 hr after initial stimulation. Fromthe workof DeFrancoet al (22), it appears that anti-IgM is not requiredfor cells to progressthroughlate G~andto enter S phase. Theneed for BCGF in late G1has not been established. It seemslikely that the time of actions of the BC(3Fand ILl correlate with the time that receptors for themare expressedon the B cell surface, just as T cell sensitivity to IL2 appears to correlate with the expression of receptors for IL2 on T cells stimulated with lectins or antigen (105). Weemphasize,however, that direct evidence for receptors for BC(3For ILl on activated mouseB cells has yet been obtained. BCGF IL1 24

x. ~2 E 3H--thy

3H--thy

4

0

0

10

20

30

TIME OFCULTURE (hrs)

40

0 I

0

I

10

I 20

I 30

TIME OFCULTURE (hrs)

Figure 2 Time of requirements of BCGFand ILl in responses of purified

B cells

to anti-#

In the left panel (titled BCGF)B cells were cultured at 5 X 104 cells per 0.2 ml; anti-/~ (10 ug/ml) and a source of ILl were added at the initiation of the culture. BCGFwas added at various times, as indicated, and all cultures received 1 /zCi of tritiated thymidine 30 hr after initiation of culture. Uptake of tritiated thymidine was measured at 42 hr. The experiment shown in the right panel (titled ILl) was carried out similarly except that cells were cultured at 2 X 104 cells per 0.2 ml and anti-pt and BCGFwere added at the beginning of the culture. ILl was added at various times in the course of the culture.

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+ B cells is similar to Thus,the overall conceptfor activation of Lyb5 modelsproposedfor T-cell activation(3, 69, 70). Lafferty,in particular, has emphasizedthat antigen plus one costimulator acts on the resting T lymphocyteto inducean excited or receptive state, andthat this excited lymphocyteproliferates whenit receives a secondeostimulatorsignal (68, 69).

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Long-TermB-Cell Lines B-cell lymphomas,myelomas,and hybridomashave provided invaluable resourcesfor the studyof antibodydiversity, idiotypy, and immunoglobulin gene rearrangementand expression (82, 103, 108). Thelimitation of such tumorlines is that they haverelatively little immunocompetence and so do not allow the analysis of manyof the cellular and biochemicalmechanisms related to B-call activation, isotype switching,idiotypeselection, and immunoregulation.Therecent development of long-term cultures of antigenspecific immunocompetent T-call clones (11, 36, 39, 92, 127) has raised hopes that the sametechnologymightbe possible for B lymphocytes.Indeed, muchof the impetusfor defining B-cell proliferation eofaetors as outlined abovehas beento obtain a growthfactor(s) capableof maintaining continuousin vitro growthof normalB cells, just as IL2maintainscontinuous in vitro growthof somenormalT cells. In manyrespects, the developmentof B-cell clones wouldseeman easier endeavorthan that of T-cell clones. Thetechnologies for markedenrichmentof antigen-specific B lymphocytes (43) and for isolation of individual immunocompetent antigenspecific clones (102) have been established for someyears. The one remainingdifficulty in producinglong-termclonedlines of antigen-specific immunocompetent B lymphocyteslies in the continuous propagation of these cells. Althoughit has beenpossible to propagateindividual B-cell clones in vivo for periods of up to 10 months(9, 147), early attempts culture B lymphocytesfor periods longer than 4 weekswere unsuccessful (81). Indeed, the continuouscultivation of B lymphocytesis still some EARLY Gr

Go

RESTING B CELL

LATE G1

BCGF ..~"’~OA

~ 2-4 HR Figure 3 Schematic depiction

~

~16-18 HR of the regulation

G= + M

S

DNA

~ 10 HR of B-cell activation

~

~ 12 HR and replication.

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distance from being an established procedure. However,activity in this area has recently flourished, with the emergenceof five independent reports of long-term B-cell lines (18, 37, 51, 75, 126, 146). Althoughthese procedures are clearly tedious and difficult to perform on a reproducible and routine basis, it is hoped that they represent a foundation for greater successes ahead.

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MHC-UNRESTRICTED B-CELL Several Distinct TRFs

DIFFERENTIATION

It is generally agre~ that onceB cells are proliferating, their differentiation into ISC requires one or more soluble T cell-derived products, generally designated TRFs. This was first demonstrated in the pioneering studies of Dutton et al (26, 27) and Schimpl & Wecker (110, 111). Both groups observed that the response of T cell-depleted spleen cells to sheep erythrocyte antigens required a soluble factor that could be found in mixed lymphocytereaction culture supernatants and in supernatants produced by mitogen- or antigen-activated T cells. The picture became more complicated when other antigens were investigated. In general, nonspeeifie T cell-derived factors are inefficient in supporting primary antibody responses of T cell-depleted populations to soluble proteins and hapten-protein conjugates; the latter generally require either antigen-specific factors and/or direct cognate interactions with antigen-specific MHC-restricted helper T cells. The initial concern that the action of TRFmight be unique to the rare erythroeyte antigen system was soon dispelled, however, by two ensuing sets of observations. Firstly, nonspecific soluble factors were found to drive the response of T-depleted hapten-primed B cells into ASC(61, 133, 136). Secondly, with anti-Ig antibodies as a polyclonal analog for antigen, it was shownthat the subset of B cells responsive to this reagent could develop into ISC only if T cell-derived soluble factors were additionally provided (63, 98). As the pool of anti-Ig-responsive B cells has recently been quantitated as 50%of normalsplenic B cells (22), this latter observation estabfishes the extensiveness of TRFphenomenology. To date, little definitive biochemical data on the nature of TRFshave been obtained. Twogroups have used standard biochemical procedures to identify in concanavalin A-induced spleen cell supernatants a factor of Mr 30,000-40,000 which supports the ASCresponse of spleen cells from congenitally athymic donors to sheep erythroeyte antigens (109, 142). However, an increasing number of laboratories are now reporting that ASC responses involve syngergism between two or more factors (34, 48, 73, 93, 129). Therefore, the biochemicaldefinition of a single TRFis subject to the concerns that one has identified the chromatographic re#on where two or more synergizing components overlap. Thus, before accurate molecular

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characterization is possible, the number of factors required in the assays of TRF function must be elucidated. Once this is achieved, the characterization and purification of TRF should be greatly aided by the recent advent of T-cell clones and hybridomas that secrete B-cell differentiation factors (23, 129, 135). It must be remembered, however, that although such clones represent monoclonal sources of the factors, they are not necessarily sources of single factors. Indeed, evidence from several laboratories suggests that T-cell lines, hybridomas, and tumors are capable of synthesizing numerous lymphokines (23, 31, 41, 56, 113). One obvious problem to contend with is the recent discovery that the proliferative component of an ASC response requires its own separate cofactors (see Figure 3). Most reports of synergizing factors involved in ASC responses do not distinguish whether this simply reflects a requirement for proliferation and differentiation cofactors or whether in fact there are several differentiation cofactors. In an attempt to clarify this issue, our laboratory has again utilized the anti-IgM polyclonal activation system as an effectivemeans of separating proliferation and differentiation events, and hence the cofactors involved. As outlined above, the proliferation cofactors involved in this system (BCGF and IL1; see Figure 3) support excellent proliferation, but do not lead to production of ISC. Recently, Nakanishi in our laboratory has demonstrated (93) that two additional T cell-derived products must be provided for development of ISC to ensue: these factors are currently designated B151K12-TRF(B15-TRF) and EL-TRF. They are found in supernatants from B 151K 12 T hybridoma cells (1 35) and phorbol myristate acetate-induced EL-4 cells (32), respectively. They are further distinguished by kinetic studies that clearly demonstrate their independent roles in ISC development: B15-TRF is required before 48 hr in a 4-day culture, whereas EL-TRF appears to function in the final 24 hr. The actions of B15-TRF and EL-TRF are apparently antigen nonspecific and are dependent on the presence or prior action of BCGF. The cycling status of cells responsive to B15-TRF and EL-TRF, and the biochemical characteristics of these factors, are currently unknown. Figure 4 presents a model for proliferation and differentiation of LybV B cells based on the concepts developed in our laboratory with the anti-IgM polyclonal activation system. A major outcome of the studies of Nakanishi et a1 (93) is the identification of two distinct factors that may be appropriately classified as T cell-replacing (or differentiation)factors. The existence of two TRFs would obviously help resolve current conflicts in the literature concerning the biochemical and biological nature of this factor. Although further information is required to relate B15-TRF and EL-TRF to differentiation factors described in other laboratories, our results are consistent with the following suggestions. On the basis of factor source alone it would appear that the TRF

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described by Takatsu et a1 (135) in B15 1K12 supernatant corresponds to B15-TRF, and that BCDFp described by Pure et a1 (104) may correspond to EL-TRF. As the TRF in concanavalin A-induced supernatants described by Schimpl & Wecker (110, 111) is absent from EL-4 supernatant and functions at 24-48 hr of a 5-day culture, it may very well correspond to B15-TRF. The TRFs observed in other concanavalin A-induced supernatants (45, 98, 99, 142), in the supernatant of the C.C3.11.75 cell line (129), and in supernatant from antigen-stimulated T helper cell lines ( 5 ) may well include both B15-TRF and EL-TRF. Numerous reports suggest that T cells are not only responsible for differentiation of activated B cells into ASC, but in fact influence the immunoglobulin isotype expressed in a particular immune response (12, 87, 121,137,139). This may be achieved either by expansion of B cells precommitted to a particular isotype, or alternatively through the induction of a “switch” in isotype expression in individual B cells. Although the precise mechanism of this phenomenon remains elusive, several groups have accumulated data that strongly suggest the existence of isotype-specific helper T cells that act in an antigen nonspecific, MHC-unrestricted manner (10, 29, 61, 78, 107). The existence of such cells prompts speculation regarding their secretion of class-specificsoluble helper factors, i.e. the existence of a family of TRFs, each of which causes the expression of a particular Ig isotype. There are currently two separate reports in support of this contention. Kishimoto & Ishizaka were first to demonstrate that carrier-specific T helper cells involved in IgE antibody responses were different from those involved in IgG antibody responses to the same antigen (61). Furthermore, the two types of helper cells were found to release different soluble factors that triggered release of the respective Ig class from hapten-primed precursor cells (62, 63). More recently, Vitetta and colleagues have reported a RESTING B LYMPHOCYTE

EXCITED

B LYMPHOCYTE

CYCLING B LYMPHOCYTE

+E15 TRF

ANTIBODY SECRETING CELL

+EL TRF

Figure 4 The factor dependent pathway of B-cell activation, proliferationand differentiation.

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soluble factor(s) in supernatants produced by certain T-cell hybridomas and T-cell lines that selectively enhances the expression of IgGl secretion by lipopolysaccharide-induced blasts (56). This factor can be distinguished from numerous other lymphokines, including both BCGF and the TRFs of Takatsu et a1 (Le., B151K12 supernatant) and Swain et al (C.C3.11.75 supernatant), which appear mainly to induce IgM secretion (104). These reports imply a level of factor complexity skillfully camoflaged by the simple term “T-cell replacing factor” (Figure 4).

Are TRFs Also Proliferation Cofactors? One aspect of factor regulation of B-cell function still requiring significant clarification is whether or not any or all of the T-cell differentiation factors described in the preceding section are additionally capable of stimulating proliferation. There are marginal data both for and against this notion. who in generating Against are the observations of Kishimoto et a1 (a), factor-secreting human T hybridomas obtained lines that secrete BCGF or TRF, but none that secrete a product(s) that supports both proliferation and differentiation. On the other hand, several laboratories have provided evidence in favor of a role for TRF in stimulating proliferation. Firstly, there is the report of Anderson & Melchers that a 30,000-Mr component released by antigen-activated T-cell lines served as both a replication and maturation factor (5). However, further evidence is required to eliminate the possibility of a separate TRF and BCGF being co-purified in these experiments. Perhaps more persuasive are recent reports by Swain et al (130) and Nakanishi et a1 (93) that BCGF-free preparations of DL-TRF and/or B15-TRF produce a small but significant level of proliferation in purified B cells (130) or alternatively enhance the proliferation obtained with anti-IgM and BCGF (93). Nakanishi has additionally demonstrated that B 15-TRFimproves final cell yields in such experiments. These observations prompt speculation that Bl 5-TRF is the mouse analogue of the 50,000-Mr human BCGF recently reported by Kishimoto and colleagues (see Characteristics of BCGF) (see also 152). Although some of the biological characteristics of B15-TRF and 50,000-Mr human BCGF seem disparate, both factors synergize with low-molecular-weight BCGF to cause extensive proliferation of activated B cells. Finally, in considering the possible function of TRF as a proliferation cofactor, one must consider an elegant study recently reported by Wetzel et a1 (145). Using a cloning assay in which the responses of single isolated B cells were followed, they showed that DL-TRF increased the frequency of B cells that undergo clonal proliferation. However, it was not clear in this assay whether DL-TRF was acting as a growth factor per se or whether it was activating otherwise resting B cells to respond to the polyclonal B-cell

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activators lipopolysaccharide and dextran sulfate, which were also present in the culture.

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Involvement of lL2 in B-Cell Responses The preceding discussions on soluble factors that regulate B-cell function have until now avoided a controversial issue in this area, namely whether or not the growth factor for T lymphocytes, IL2, is capable of exerting a direct effect on B lymphocytes. Those proponents of direct action of IL2 on B cells have provided the following lines of evidence to support this concept: depletion of IL2 from cofactor-richsupernatants by absorption on IL2-dependent T-cell lines also removes the B-cell costimulating factor (98) and/or B-cell differentiation factor (73, 76) in these sources; when various cofactor-rich supernatants are analyzed, there is strict correlation between levels of IL2 and of one of two B cell-specific factors required in plaqueforming cell responses (73, 76, 129, 131); this same B cell-specific factor cannot be distinguished from IL2 in terms of molecular weight (73, 76). Although the possibility that IL2 acts directly on B cells cannot yet be formally excluded, we believe this to be unlikely in view of the following considerations. ( a ) Excellent reagents for detecting IL2 receptors on cell surfaces are now available (105) and these have failed to demonstrate such receptors on resting or activated B cells. (6) Attempts to absorb IL2 from cofactor-rich supernatants with B cells and B-cell blasts have failed (73). ( c ) Both mouse (31, 33, 49), and human (17, 37, 64, 75, 90, 91, 126, 152) BCGFs, and the TRFs described by Melchers & Anderson (9,Howard et al(49), Nakanishi et a1 (93), Schimpl et a1 (109, 110), Takatsu et al(135, 136), Swain et al(129, 131), Kishimoto et a1 (63), and Pure, Isakson et al. (56, 104) have been distinguished from IL2 either by biochemical separation, by difference in source, by cellular absorption, or by a combination of these procedures. (d) A recent report by Pike et a1 (101) demonstrates that clonal expansion and differentiation of individual hapten-specific B cells into ASC requires antigen together with one or more soluble factors distinct from IL2. Several explanations can be offered to accommodate the observations of those proposing a direct role for IL2 in B-cell function. Firstly, it is now clear that the IL2-containing supernatant generally used in these laboratories (the supernatant of FS6-14.13) also contains BCGF (M. Howard, unpublished data) and possibly one or more of the TRFs that have been described. It would not be surprising if these factors were nonspecifically absorbed by the “sticky” activated target cells used for IL2 depletion. Alternatively, although these laboratoriesemploy vigorous purification procedures to obtain T cell-free B-cell populations for their analyses, the lengthy in vitro assays required for B-cell function provide time for pre-T

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cells to differentiate into mature lymphocytes, which could then be effectively expanded by IL2 into a significant pool of lymphokine-secreting T cells. Indeed, in support of this view, several laboratories have shown that T-cell lines can be established from nude mouse cells with the combination of antigen or mitogen activation and IL2 (40, 55). Thus although the arguments outlined above challenge the concept that IL2 acts directly on resting or activated B cells, it is quite probable that IL2, added to culture or produced endogenously, may have profound indirect effects on B-cell proliferation and differentiation.

SOLUBLE FACTORS IN MHC-RESTRICTED B-CELL RESPONSES The previous sections of this review have concentrated on B-cell activation, proliferation, and differentiation stimulated by cross-linkage of membrane Ig together with a series of soluble factors derived from T cells and macrophages. It is now clear that a second principal pathway of B-cell activation exists that depends upon the interaction of a helper T (TH) cell with a B cell in an antigen-specific, histocompatibility-restricted manner. More precisely, this pathway of B cell stimulation depends upon the capacity of the TH cell, or its product(s), to recognize both antigen and a class I1 MHC molecule on the surface of the B cell. Indeed, there had been considerable controversy for several years as to the importance of MHC-restricted TH-B cell interactions. Several authors had pointed out that much of the available data interpreted as supporting such interactions could actually have reflected MHC-restricted interactions between antigen-presenting cells (APC) and TH cells, with the ensuing production of soluble factors acting upon B cells activated as a result of receptor cross-linkage that resulted from the binding of antigen (30, 79, 116, 118, 119). This seemed particularly plausible since, in most experiments, APC and B cells were derived from the same donors. However, careful experiments with both in vivo (124, 125) and in vitro (132, 151) systems now make it clear that MHC-restricted TH-B cell interactions leading to antibody synthesis do occur and are responsible for a significant component of the total antibody response. There is mounting evidence, mainly derived from the work of Singer, Hodes and their colleagues (120), that MHC-restricted cellular interactions are mainly a feature of the subset of B lymphocytes found in mice with the xid-determined immune defect. That is, Lyb5- B cells can respond to “thymus-dependent” antigens only through MHC-restricted TH-Bcell interactions. Moreover, it appears that the very same TH cells can participate in both the MHC-restricted TH-B cell interaction and in the factor-mediated activation of Lyb5+ B cells described in previous portions

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of this review, although the conditions required to activate TH cells for these two types of interactions appear to be quite different. Low concentrations of antigen appear to lead to MHC-restricted TH-B-cellinteractions whereas high concentrations mainly result in interactions controlled by nonspecific soluble factors (7). The control of B-cell responses in MHC-restricted TH-B-cellinteractions is still a matter of considerable controversy. Perhaps the most uncertain issue is the need for antigen binding to the mIg receptors of the responding B cells for activation, proliferation, and differentiation to occur. In a study of B-cell proliferation in response to stimulated TH cells, Tse et a1 (140) showed that B cells from nonprimed donors could be stimulated to proliferate by TH cells from donors primed to the terpolymer of G l ~ ~ A l a ~ 0 T y r ~ ~ (GAT) in the presence of GAT. This TH-B-Cell response appears to be restricted by products of the I-region of the MHC, since primed T cells from (B10 X BIO.S)FI donors could cause proliferation by B10 but not BIO.S B cells, even if both B cell types were present in the same culture. The response to GAT is controlled by an I-A b-encodedIr gene; B 10 mice are responders and BIO.S mice are nonresponders. Thus, F1 T cells contain clones that recognize B10 B cells that have bound GAT to their membranes but lack clones that recognize B1O.S B cells with bound GAT. Furthermore, the B cells participating in this interaction may be drawn from donors that have not been primed to GAT; and B cells from primed donors do not react to any greater degree than do unprimed B cells. Thus, it seems most likely that the bulk of B cells that respond do not have mIg receptors specific for GAT. Rather, they appear to have simply bound GAT to their membranes and to present it with I-A -encoded class I1 gene products to T cells in a manner analogous to that used by APC. Thus, B cell activation for proliferation mediated by a MHC-restricted interaction with TH cells does not appear to require binding of antigen to B cell receptors. These findings have been confirmed with resting B cells and cloned long-term T cell lines both for size enlargement (Go-+ GI transition) (21) and for proliferation (58). Similarly, Coutinho and colleagues have reported that a TH line specific for a minor histocompatibility antigen expressed on the B cell surface can stimulate the appearance of very substantial numbers of ISC despite the fact that no antigen is provided to cross-link receptors of the vast majority of cells (19, 77). Groups that have studied antibody responses to specific antigens in MHC-restricted TH-B cell interactions have, by contrast, emphasized the need for the binding of receptors of precursor cells to obtain measureable responses (6, 59, 153, 154). Indeed, one group has reported that even the polyclonal generation of ISC in MHC-restricted interactions between lines of TH cells specific for minor histocompatibility antigens, and B cells that

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expressthose antigens is dependentuponthe presenceof anti-Ig antibodies, presumablyto provide the analog of the antigen-dependentstimulation mediatedby receptor cross-linkage (59). Apossible explanationto resolve these apparentdifferences is that an initial round of MHC-dependent B-cell proliferation can be initiated by interactionsof B cells with Trt cells in the absenceof receptorcross-linkage. Subsequentdivisions of precursor cells, whichare neededto expandthe B cell done, mayrequire receptor cross-linkage. Theneedto use Tn cells to obtain MHC-restricted TR-Bcell interactions tends to obscure any need for lymphokinesin this response since the Tn ceils, in addition to participating directly in TH-Binteractions, mayalso producethe soluble factors need~xlfor the response. Somehave reported that Tn cells can producesoluble antigen-specific, MHC-restdeted factors capable of acting in place of the Tn cell itself (5, 57, 74). A detailed discussionof this importantbut controversial subject is beyondthe scope of this review,whichis mainlyconcernedwith nonspecificsoluble factors. Twogroupshaveparticularly emphasized a role for soluble factors in the MHC-restrieted Tn-Bcall interactions that lead to the development of ISC (5, 114,153, 154). In general, it has beenproposedthat these solublefactors act uponexcitedB cells (or B cell blasts) andcausetheir further proliferation and differentiation. Relatively little has beenreported on the number of factors required and on the physicochemical properties of these factors. Suchfactors appear to be present in the supernatant of somelong-term T-cell lines, in the supernatant of the thymoma cell line, EL-4, and in supernatants from secondaryin vitro mixedlymphocytecultures. Andersson & Melchers(5) have reported a factor with a r of 3 0,000 (p30) isolated fromthe supernatantof a TI-I cell line specific for horse erythrocytes. This appearedto be the nonspecificfactor active in stimulation of B cells activatedby an antigen-specifichelper factor. This p30factor has been designated by themas B-cell replication and maturation factor. Whetherit is active in all MHC-restdcted interactions and whetherit acts in a manneranalogousto BCGF or to the early acting B 15-TRFdiscussed earlier has not yet beendetermined.Theinvolvementof other differentiation factors such as EL-TRF or BCDFbt in this process is also a matter of uncertainty. It can be anticipated that rapid progressin the understanding of the role of nonspecificlymphokines on B cells activated as a result of MHC-restdcted TH-Bcell interactions will be madein the next few years. SUMMARY The B-lymphocytefamily of cells presents one of the most remarkable opportunitiesfor the detailed studyof regulationof growthanddifferentia-

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tion. Somemembersof this cell population have the property that they may be stimulated by ligand-receptor interactions, together with the sequential action of a series of lymphokines,to progress from the resting state, through several rounds of proliferation, and then to differentiate to immunoglobulin secretion. Other cells in this group participate in cognate cellular interactions with helper T cells in whichthe recognition of both antigen and a class II MHC molecule on the B-cell surface is key to activation. The differentiation of these cells is also controlled by soluble products. Wehave reviewed our developing knowledge of the biochemistry and modeof action of the lympholdnes that act upon B cells. These include distinct growth and differentiation factors. Amongthese are the BCGFs of mice and humans and the various TRFs, which include molecules often described as differentiation factors. The next several years should witness major progress in understanding the physicochemicalproperties of the B cell-specific factors, their time and nature of action, and the nature of their receptors. In addition, we can anticipate a major effort to understand the intracellular events that flow from the action of specific growthand differentiation factors that act upon B cells. Such information should lead to a new physiologically-based pharmacologyfor manipulation of antibody responses in humandisease and in responses to vaccines. In addition, the fuller understanding of the nature and modeof action of the various growth and differentiation factors should makelong-term growth of cloned B cells a procedure that can be routinely used in immunologicallaboratories_for the precise study of the biology of responses by homogeneouspopulations of B lymphoeytes. LiteratureCited 1. Ahmed, A., Scher,I., Sharrow,S. O., that distinguishes in its actionresting, Smith,A.H., Paul,W.E., et al. 1977. small B cells fromactivated B-cell B-lymphocyte heterogeneity:Developblasts. Z Exp.Med.150:1339-50 meritandcharacterization of an alloan- 5. Andersson,J., Melehers,F. 1981.T tiserum whichdistinguishes B-lymcell-dependent activationof restingB phocyte differentiation alloantigens.J. cells: Requirement for bothnonspecifie Exp. Med~145:101-10 unrestrictedandantigen-specificIa2. Andersson,J., Coutinho,A., Lernrestrictedsolublefactors.Proc.Natl. hardt, W.,Melchers,F. 1977.Clonal 8c£ USA78:2497-501 growth and maturation to immuno- 6. .4cad. Andersson, J., Schreier,M.H., Melchglobulinsecretionin vitro of every ers, F. 1980.T-cell-dependent B-cell growth-inducibleB lymphocyte.Cell stimulation is H-2restrictedandanti10:27-34 gendependent onlyat the restingB-cell 3. Andersson, J., Gronvik,K., Larsson, level. Proc. Natl. .4cad. ScL USA E., Coutinho,A. 1979.S~tudieson T77:1612-16 lymphocyte activation. I. Requirement for the mitogenic-dependent production 7. Asano,Y., Shigeta, M., Fathman,C. G., +Singer, A., Hodes,R. J. 1982. of T-cell growthfactors, gur. Z ImLyb5andLybS-B cells differ in their raunol.9:581-87 requirements for restricted activation 4. Andersson, J., Lernhardt,W.,Melehby clonedhelper T cells. FedProc. ers, F. 1_979..Thepurifiedproteindedvalive ot tuo~rculln.AB-cellmitogen 41:721

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immunoglobulin. Z Exp. Med. 155: 1523-36 23. Dermert,G., Weiss,S., Warner,J. 1981. T cells mayexpressmultipleactivities: Specificallohelp, eytolysis anddelayed type hypersensitivityare expressedby a cloneT cell line. Prac. Natl. Acad.Sc£ USA78;4540-3 24. Diener, E., Feldman,M. 1972. Relationship betweenantigen and antibodyinduced suppression of immunity. Transplant Rev. 8:76-94 25. Dutton,R. W.1974.T cell factors in the regulationof the Bcell response.In The ImmuneSystem, Genes,Receptors, Signals, ed. E. E. Sercarz, A. R. Williamson, C. F. Fox, pp. 485-96.NewYork: Academic 26. Dutton, R. W.1975. Separate signals for the initiation of proliferation and differentiationin the Bcell responseto Antigen. Transplant. Rev. 23:66-77 27. Dutton,R. W.,Falkoff,R., Hirst, J. A., Hoffman, M., Kappler,J., et al. 1971.Is there evidencefor a non-antigenspecific diffusable chemicalmediatorfrom the thymus-derived cell in the initiation of the immuneresponse? Pragr. Immunol. 1:355 28. Dutton, R., Mishell, R. 1967. Cellular events in the immune response. The in vitro responseof normalspleenceils to erythrocyte antigens. ColdSpringHarbor Syrup. Quant.Biol. 32:407-14 29. Elson, C. O., Hcik,J. A., Strober, W. 1979. T cell regulation of routine IgA synthesis. J. Exp. Med.149:632-43 30. Erb, P., Meier,B., Matunaga,T., Feldmann,M.1979. Nature ofT-cell macrophageinteraction in helper-cell induction in vitro. II. Twostagesof T-helpercell differentiation analyzed in irradiation and allophenic chimeras.Z Exp. Med. 149:686-701 31. Farrar, J., Benjamin, W.,I-Iilfiker, M., Howard,M., Farrar, W., Fuller-Farrar, J. 1982.Thebiochemistry,biology,and role of IL-2in the inductionofeytotoxic T cells andantibody-forming Bcell responses, lmmunol.Rev. 63:129-66 32. Farrar, L, Fuller-Farrar,J., Simon,P., Hilflker, M., Stradler, B., Farrar, W. 20. Coutinho,A., Moiler, 13. 1974. Immune 1980. Thymoma production of T cell activation of B cells: Evidencefor one growth factor (intedeukin 2). J. Imnon-specifictriggeringsignal not delivmunol. 125:2555-58 ered by the Ig receptors. Scand.J. Im33. Farrar, J., Howard,M., Fuller-Farrar, muno~3:133-46 J., Paul, W.E. Submittedfor publica21. DeFranco,A., Ashwell,J., Schwartz, tion R. H., Paul, W.E. Manuscriptin prepaW., Fuller-Bonar, ration 34. Farrar, J., Koopman, 22. DeFranco,A., Raveehe, E., Asofsky, J. 1977.Identificationandpartial purification of two synergistically acting R., Paul, W.E. 1982. Frequencyof B lymphocytes responsive to antihelper mediators in human mixed 8. Asano,Y., Singer, A., Hodes, R. J. 1981. Role of the majorhistocompatibllity complex in T cell activation of B cell subpopulations. MHC restricted and unrestricted B cell responsesare mediatedby distinct B cell subpopulafions. J. Exp. Med.154:1100-15 9. Askonas, B. A., Williamson, A. Wright,B. E. G. 1970. Selection of a single antibody-formingcell alone and its propagationin syngeneicmice.Proc. Natl. Aca~SoL USA67:1398-403 10. Augustin, A. A., Coutinho, A. 1980. Specific T Helpercells that activate B cells polyelonally.In vitro enrichment andcooperativefunction. J. Exp. Med. 151:587-601 11. Augnstin, A. A., Julius, M. H., Cosenza, H. 1979. Antigen-specific stimulation and trans-stimulation of T cells in long-termculture. Fur. Z lmmunol. 9:665-70 12. Braley-Mullen, H. 1974. Regulatory role of T cells in IgGantibodyformation and immunememoryto type III pneumococcalpolysaccharide. J. lmmunol. 113:1909-20 13. Bretseher, P. 1975. The two signal modelfor B cell induction. Transplant Rev. 23:37-48 14. Bretscher, P., Coim,M. 1970.A theory of self-nonself discrimination.Science 169:1042-49 15. Burgess,A., Metcalf, D. 1980. Thenature and action of granulocyte-macrophagecolonystimulating factors. Blood 56:947-58 16. Burner, F. M. 1959. TheClonal Selection Theory of Acquired Immunity. London:CambridgeUniv. 17. Butler, J., Muraguchi,A., Lane, C., Fauei, A. 1983. Developmentof a humanT-Tcell hybddoma secreting B cell growth factor. J. Exp. MecL157:60-8 18. Coutinho, A. Personal communication 19. Coutinho, A., Augustin, A. A. 1980. Major histoeompatibility complexrestricted and unrestricted T helper cells recognizingminorhistocompatibility antigens orbcell surfaces.F, ur. J. lramunol. 10:535-41

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MEDIATORS OF INFLAMMATION Gary L. Larsen

and Peter

M. Henson

Departmentof Pediatrics, National Jewish Hospital and Research Center/National AsthmaCenter, and Departmentsof Pediatrics, Pathology, and Medicine,University of ColoradoSchool of Medicine,Denver, Colorado80206 INTRODUCTION Inflammationis a term used to describe a series of responses of vascularized tissues of the body to injury. The clinical signs of this phenomenon can now be related to increased flow in local blood vessels (calor and rubor), increased vascular permeability and/or cellular infiltration (tumor), and release of a variety of materials at the site of inflammationthat induce pain (dolor). Loss of function was later considered an additional cardinal sign of inflammation. However, mechanisms by which the function of a given vascularized tissue is impaired depend greatly on the nature of the tissue and on the detailed processes that contribute to the inflammatoryreaction. The subject of this review is the mediators that producethese alterations. However,it is important to indicate at the outset that the inflammatory process is both varied and extremely complex. Weemphasize the broad outlines of (a) alterations in hemodynamicsand vessel permeability, and (b) infiltration by inflammatory cells as representing a common theme for the process. However,each site and stimulus mayresult in a different mix of these elements, a different time course, and a different outcome. The inflammatory process is also influenced by the nutritional and hormonal status of the individual, as well as by genetic factors. In addition, the processes themselves represent an extensive network of interacting mechanisms, mediators, and cells (85). This represents a high degree of redundancy in the system, which provides a mechanismboth for amplification and for preservation of the response if one componentof the system is deficient or inactivated. For example, we present an argumentthat biologically active fragmentsof C5 are critical initiators of the acute inflammatory 335 0732-0582/83/0410-0335502.00

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reaction. Nevertheless, genetic C5deficiency is not a major impairment, suggestingthat collateral mechanisms can compensate(58). Bycontrast, certain elementsof the inflammatory processare critical. Thus,deficiencies in neutrophil function result in defective inflammatoryresponsesand insufficient protection against infection and, without aggressivechemotherapy, represent lethal mutations(48). Theseobservationssuggestthat inflammationis a beneficial reaction of tissues to injury. In fact, it normallyleads to removalof the inciting agent and repair of the injured site. This mayinvolve temporarydiscomfortand loss of function,but in the endit is protective.Nevertheless,a large number of humandiseases represent uncontrolled inflammatoryreactions that may continue unchecked(rheumatoidarthritis), mayinduce permanenttissue destruction (emphysema), or mayheal, but only by the inappropriate deposition of collagen(pulmonaryor hepatic fibrosis). The immunesystem has long been linked with inflammation. Both may be seen as protective mechanisms.However,major elementsof the inflammatoryreaction precede the immunesystem in evolution. In fact, a key elementin the former, chemotaxisof wanderingcells, was presumablya propertyof the earliest eukaryotes.Theimmune systemconfers specificity on the reaction and the inflammatoryprocess maybe seen to represent an effeetor arm of the immunesystem. Thus, antibody-antigen complexes readily initiate generationandrelease of manyof the mediatorsof inflammation. In addition, antigen-stimulated lymphocytesprovide a wealth of mediator functions themselves, as well as modulatethe immuneresponse (98). Ashort review of a subject as broad and complexas mediators of the inflammatoryprocess will be incompleteand will contain material biased towardsthe authors’ general viewson the subject. In addition, the choice of reference material cannot be comprehensive.This workis no exception. Ourgoal is, thus, to give an overview,stressing the complexityof this process by pointing out manypotential interactions of the mediatorsdescribed, while providingreferencesof both a general anda specific nature for the readers wanting more information. Conceptswill receive more emphasisthan details of any onemediator.For reasonsof simplicity, space, and amountof available information, the acute inflammatoryreaction is emphasized.This is not to belittle the importanceof the morechronic reactions, especially those associated with cell-mediatedimmunity.Rather, it is a reflection of the complexityof the latter subjectandthe recognition that weare just at the brink of moleculardefinition of the mediatorsinvolved. Thus, the field of study of lymphokines,interleukins, other "growth"factors, and macrophage-derived "activating" factors is just entering a newand exciting growthperiod, but one that requires a separate review(s)of itself.

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THE INFLAMMATORY

RESPONSE

As indicated above, inflammationis a nonspecificresponse of tissues to diversestimuli or insults. However, despite this complexityandvariability, the commonthemes seen in the process argue for commongroups of mediators.In particular, weshouldlook for moleculesthat enhanceblood flow, increase vessel permeability, and induce emigrationof inflammatory cells fromthe blood.Oncethey havereachedthe site of injury, the cells are stimulatedto phagocytosebacteria and debris, and also to secrete manyof their preformedor newlysynthesized constituents. Moleculespromoting these eventsmaybe classified as "mediators."In addition, the reactionsare controlled throughoutby positive and negative feedbackreactions, again involving "mediators"and "inhibitors." Inflammatoryreactions generally resolve. Moleculespromotingresolution and healing maynot be, strictly speaking,mediators,but nonethe less they are of critical importanceto the overall process. The hemodynamic changes associated with inflammationare often the first to be manifested.Theyare of critical importance.Cohnheim’s dictum "without vessels no inflammation"was later parodied by Metchnikoffas "there is no inflammationwithout phagocytes"(69). Vasodilation, increasedflow, and permeabilityincreases are key elementsin inflammation. It maybe presumedthat their "purpose"is to allow maximum opportunity to recruit inflammatorycells and to bring plasmaproteins to the site of injury. Theselatter couldcontributeto the initiation or perpetuationof the response(complement fragments)or the resolution of the response(plasma protease inhibitors). Howthese vascular alterations are achieved,however, is very poorlyunderstood(71, 88, 123). In part, this reflects the dynamic nature of the systems,in part the inability to study readily the processes involvedin vitro, andin part the considerablevariationfromsite to site in the bodyin the involvementof the microcireulationin inflammation.For example,in skin (44), mesentery(65), hamstercheek pouch(72), and chamber(28), the events occur predominately in the postcapillary venules. In the pulmonary vasculature, however,the capillary maybe the majorsite of leak andemigration(17, 41, 94). Whether this reflects physical effects, differencesin endothelialceils, or different types of mediatorsis not yet known. Thehistologic changesassociated with a circumscribedacute inflammatory reaction in the lung are shownin Figure1. Theresponsewasinduced by instillation into the airwaysof a phlogistic fragmentfromthe fifth componentof complement,C5adesArg(55). This same type of response maybe seen in other tissues after exposureto C5adesArg,or in the same tissue (lung) with other stimuli such as immunecomplexes(57, 90) bacterial infection (56, 86). This simple exampleis thus meantto demon-

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Figure I The inflammatory response produced by instillation of C5a desArg into rabbit airways is displayed. In the normal lung (A), resident alveolar macrophages are present in some alveoli, but neutrophils are not seen. Six hours after administration of C5a desArg (B), granulocytes (predominantly neutrophils) and protein-rich fluid accumulate in the airspaces. By 24 to 48 hr (0,the neutrophilic infiltration is replaced by mononuclear cells (macrophages). Over several days, the alveoli clear and the alveolar walls return to a normal thickness and cellularity (hematoxylin and eosin stain; X400).

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MEDIATORSOF INFLAMMATION 339 strate a normalinflammatoryresponse. Thesequenceof permeability, neutrophil accumulation,and mononuelearphagocyte(macrophage)infiltration that occursbefore resolution is apparent. In Figure 2 maybe seen a similar inflammatoryreaction in one capillary examinedultrastructurally at the time of changefroma granulocyticto a monocyticinfiltrate. Extensive tissue damagehas occurred. For this site to be repaired, the mononuclear influx of newcells mustbe halted, injurious oxygenradicals and proteasesmustbe inactivated, fluids and proteins mustbe removedor reabsorbed,inflammatorycalls and debris mustbe removed,and damaged cells (in this case epithelial cells) must replaced.Thislast results fromthe inductionof replicationof the epithelial cells and laying downof newbasementmembrane material. In other circumstances,tissue fibroblasts maybe similarly inducedto divide andsecrete connectivetissue elementssuchas collagen. Thesesubjects, andthe mediators that inducethem,are poorlystudied to date and are just beginningto receivespecific emphasis. MEDIATORS

OF INFLAMMATION

Definition of Mediators In simplest terms, an inflammatorymediatorcan be thoughtof as a chemical messengerthat will act on bloodvessels and/or ceils to contribute to an inflammatoryresponse. As is discussed below, a multitude of agents fulfill this definition. Themediatormaybe distinguishedfroma neurotransmitter on the one hand, and a hormoneon the other, largely by the extent of its sphere of influence (neither at a nerve ending nor alternatively throughout the body). Douglashas introduced the term "autacoids" (20) to refer to manyof the substancesto be discussedhere. As he defined the word,it refers to a variety of substancesof intense pharmacologic activity that are normallypresent in the bodyand cannot be convenientlyclassed as neurohumorsor hormones.This is very similar to the use of the term mediatorindicated above, whichis used throughoutthis review. Sources of Mediators Themediators of the inflammatoryresponse can be classified in several ways.Oneof the mosthelpful subdivisionsis source. First, the chemical messengermaybe an exogenousmediator (comingfrom outside the body) or an endogenousmediator. Although this review concentrates on the latter, it is importantto acknowledge the existence and importanceof the former, whichinclude bacterial productsand toxins (53, 115). Whenconsidering endogenous mediators, several other classifications accordingto source can be made.First, is the origin of the mediatorthe plasma,blood

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Figure 2 An electron micrograph of a normal alveolar capillary unit ( A ) is shown. By comparison (B), an inflammatory reaction in a capillary unit at the time of emigration of a monocyte through the capillary walls exhibits extensive damage, which occurred during the prior neutrophil emigration. Evidence of activation of the coagulation system is manifest by fibrin strands in the extravascular space. Top, X11,500, bottom, X8335)

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MEDIATORS OF INFLAMMATION 341 cells, or tissues? Theplasmacontains three majormediator-producing systems(kinin, coagulation, complement),whichinteract in defined manners to generate phlogistic compounds.Other mediators are cell derived and, withinthe cells of origin, maybe preformedand stored in granules(histaminein mastcells, cationic proteins in neutrophils)or maybe newlysynthesized bythe cells (interleukin1, leukotrienes,platelet-activatingfactor). The importance of these distinctionslies in part in the rapidity of release of the molecules,but also in therapeutic approachesthat maybe taken to modify their effects. Manypeptide mediators are generated as a consequenceof multiple enzymaticsteps involving sequential activation of moleculesby limited proteolysis (complementand coagulation systems). The small fragments derived fromsuch steps often exhibit biological activity and maybe extremely importantmediators(CSa,C3a). Depending on one’s point of view, they could be seen as byproductsof complement activation or as key derivatives of the proteolytic sequence. Lipidmediatorsare generallysynthesizedde novowithincells whenthese cells are activated. Little is known as yet aboutthe mechanisms of secretion, but the biochemicalsynthetic pathwaysof a numberof importantmolecules (the eicosanoidsandplatelet-activating factor) are beginningto be understood. Arachidonicacid is the precursor of a wide variety of molecules collectively termedeicosanoids. It is derivedfrommembrane phospholipids by the direct action of phospholipase A2 (PLA2)or indirectly followingthe effect of phospholipaseC. Thearachidonicacid maythen be convertedinto prostaglandins and thromboxanesvia cyclooxygenaseand subsequentenzymesor into hydroperoxyand hydroxyeicosatetraenoic acids (HPETEs and HETEs)by lipoxygenaseenzymes.Leukotrienes C4, D4, and E4 (slow reacting substances)as well as B4 (a potent chemotacticagent) are also derivatives of the lipoxygenasepathways.Thesemetabolicevents appearto be of great importancein the inflammatoryprocess. For example,our most potent anti-inflammatorydrugs are knownto inhibit someor all of these events. For considerationof this fast-movingfield, the reader is referred to recent reviews by Goetzl, Parker, and Samuelssonand co-workers(31, 79, 89). The Mediators Examplesof someof the mediatorsof inflammationare listed in Table 1. Thelist is not exhaustivein termsof the mediatorslisted, andthe actions given for any chemicalor group of compounds are incomplete. The table does provide, however,an overviewof the types (chemicalnature), sources, and actions of somecentral compounds.

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EFFECT OF MEDIATORS ON THE INFLAMMATORY PROCESS The above paragraphs have dealt with the inflammatory process and mediators of inflammation in general terms. Wenow address the individual componentsof this process and look specifically at the actions of the mediators in producing these effects. It is important to point out that several mediators produce the same effect, and the relative importance of one mediator versus another is often ditficult to assess. In addition, it is also probable that mediators with similar actions have different levels of importance depending on the tissue involved and the stimulus for injury. To complicate the picture further, certain mediators can be responsible for morethan one of the vascular or cellular changescentral to the process (see Table 1).

Vasodilation and Hyperemia Of the features associated with the inflammatoryprocess, vasodilation has been the least studied. Despite this fact, it is generally consideredof central importance in the development of acute inflammatory reactions because local blood flow determines in large degree the amountof exudate produced (88). It is knownthat the flare after histamineinjection is at least partially under central nervous system control (1), but the subject of involvement the neuronal system in inflammatory processes is underdeveloped. A number of mediators, including histamine and various eicosanoids, are also clearly involved in regulation of blood flow, even if the mechanismsare not well understood. Injection of phospholipase A2 into the skin induces a hyperemiavery similar to that seen in inflammation (105). Since PLA2may be expected to cause release of arachidonic acid, products of cyclooxygenase or lipoxygenase pathways of arachidonate metabolism are implicated. In fact, prostaglandins are turning out to be important mediators of inflammation and mayexert their effects in part by modulating blood flow. It is interesting to note that although prostaglandins were first described approximately one-half century ago by yon Euler in 1934 (110) and Goldblatt 1935 (34), it was not until the last decade that they were proposed inflammatory mediators (107, 129, 130). Even now, their exact contribution to the process is poorly understood. In large measure this is because the administration of prostaglandins does not induce inflammation directly, and indeed, at someconcentrations, some of the compounds,such as PGEI (which is not madein mammals)or PGI2, actually inhibit the function inflammatory cells (11, 61, 134). However,it has recently becomeapparent that prostaglandins such as PGE2and PGI2 can have a marked proinflammatory effect as potentiators of the effect of other mediators. The evidence supporting a role for this group of compounds is threefold.

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First, PGE2and PGI2 injections induce vasodilation, presumably by an action on cells of the blood vessel wall (54). Second,prostaglandins, especially PGE2,have been detected in inflammatory exudates (125, 130). Their accumulation is usually 6 to 24 hr later than histamine or bradykinin. Third, drugs with knownanti-inflammatory properties such as aspirin inhibit the production of prostaglandins by acting on the enzymecyclooxygenase (107, 108). At physiologic concentrations, vasodilator prostaglandins have been reported to enhancethe permeability effects of histamine and bradykinin (128). A similar potentiating effect of vasodilator prostaglandins has been noted with the permeability-inducing effects of LTB4, C5 fragments, and Nformyl-methionyl-leucyl-phenylalanine (FMLP)(12, 117, 127). These all chemotactic agents, and the effect has been attributed to an interaction between neutrophils, ehemoattractants, and prostaglandin. Local vasodilation is suggested to enhance the effect of neutrophils on the blood vessel wall, and their emigration. In fact, Williams (126) has showna similar potentiation of permeability by ehemoattractants using an unrelated vasodilator, vasoactive intestinal peptide (VIP) (see Table 1). Nevertheless, the prostaglandins might also affect other componentsof these systems, including the inflammatory cells, the presentation of chemotactic factors, local shear stresses, or the leukocytes adhering to the endotheliumor endothelial junctions. This area requires moreinvestigation. It is of critical importance, not only because it is an integral part of the inflammatory process, but also because of the widespreaduse of nonsteroidal anti-inflammatory drugs that prevent prostaglandin production. It should be noted that other products of the cyelooxygenase system (thromboxaneA2) have vasoconstricting activity (4). Critical here is determine the overall effect of such mediators on the blood flow through the different portions of the vascular bed. Moreover,prostaglandins have been reported to inhibit leukocyte and mast cell secretion by increasing intracellular cAMP (11, 61, 134), thus preventing inflammatory reactions. Indeed, stable forms of PGE1have been used experimentally to prevent inflammation (23). Althoughthese reports wouldsuggest an anti-inflammatory effect of the prostaglandins, Tauber and co-workers (102) noted low concentrations of prostaglandins (PGE1or PGF2~)enhancedantigen-induced release of histamine from lung tissue whereashigh concentrations had the opposite effect. These results underscore the complexity of these interactions and point out that quantity of mediator present maydetermine if the effect is pro- or anti-inflammatory. In addition, the actions of the various prostanoids mayvary amongdifferent vascular, beds. Moreover, different cell types, including those lining vessel wails, producea different mix of prostanoids both intrinsically and after exposureto different stimuli. This complexity argues for a careful examinationof each tissue to determinethe fine

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tuning of the regulatory events involved. Despite these conflicting effects, the overall response and the meansto achieve it are similar in kind from tissue to tissue. Vasopermeability Whenconsidering various mediators that maycause increased permeability, it is important to rememberthat these alterations occur at various times after the injury. In manytypes of tissue injury, increased permeability has been noted to occur in at least two phases: an early, transient phase, occurring almost immediatelyafter an injury; and a late or second phase, beginning after a variable latent period, but then persisting for hours or days. As discussed by Wilhelmin his review of these and other patterns (123), the types of response in terms of the time course or pattern of permeability noted are functions of the type and intensity of the injury as well as the species of animal being studied. There is good evidence that the early, transient permeability seen with certain types of injury (heat) are due release of histamine, in that the early permeability, as measuredby skin bluing, maybe suppressed by antihistamines (99, 124). Mediation of the delayed phase of exudation is more controversial and complexand has been attributed in part to kinins (123), prostaglandins (19), neutrophils (74, and, more recently, lipoxygenase products of arachidonic acid metabolism (54). (See also the discussion of eicosanoid and neutrophil effects noted above.) The mediators to be discussed mayact together or sequentially in an inflammatory reaction to alter permeability. Histamine is the one mediator most often associated with the early increase in permeability after various types of injury. The physiologic and pharmacologic properties (contraction of smooth muscle and vasodilation) of this vasoaetive aminewere first described by Daleand associates (15, 16). Histamineis widely distributed, but that contained in mast cells and basophil leukocytes (plus platelets in the rabbit) provides the main source acute inflammation. Tissue mast cells are commonlylocated around blood vessels, suggesting the possibility of extremely local effects. The stimuli causing release of histamine from mast cells are many,including a variety of low-molecular-weight substances, compound48/80, physical agents ineluding heat and trauma, various drugs, IgE and other immunologicreactions, and anaphylatoxin (C3a and CSa). The mechanisms of histamine release and regulation have been well studied (60-62). Recently, it has been suggested that arachidonie acid metabolism within the cell is important in both basophil and mast cell secretion. A lipoxygenase product(s) may required for histamine release from humanbasophils and may blunt endogenous control mechanisms (66). For example, 5-hydroperoxyeicosa tetraenoie acid (5-HPETE)has been shownto induce both a dose-dependent enhancementof histamine release and a reversal of the inhibition of

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MEDIATORSOF INFLAMMATION 347 histamine release causedby agonists acting via adenylate cyclase (82). Peters et al (82) also report that inhibitors of phospholipase A2activation and inhibitors of arachidonic acid metabolismthrough the lipoxygcnase pathwayact not only on IgE-mediatedrelease of histamine but also on release initiated by anaphylatoxins,f-met peptides, and ionophoreA23187. Thus,these data suggestcell membrane phospholipidprocessesarc essential final common pathwaysleading to secretion of histamine. However,it shouldbe emphasizedthat althoughstudies of cell functions with eicosanoid metabolisminhibitors are widespread,they must be interpreted with caution. Theinhibitors are not very specific, the intracellular biochemical events are not understood,.and in mostcases one cannot determinewhether the arachidonatederivative acts intracellularly as a true messengeror is acting extracellularly on the cell of origin or other cells in the area of the reaction. It has beensuggestedthat histamineincreases vascular permeabilityby inducingcontraction of the endothelial cells of the postcapillary venule, thus allowing the passage of fluid and proteins through the openingof interendothelial junctions (44, 64). However, this viewhas beenchallenged (36) andthe question of endothelial contractibility is controversial. Althoughgapsin the endotheliumcan certainly be seen, the exact site of the barriers to protein transudation in nonglomerular vessels is not yet fully understoodand the effect of vasopermeabilityagents requires morestudy. Chargeeffects maybe of critical importancein the passageof proteins through blood vessels. Whenleukocytes are involved, the impact of proteases on the endotheliumand the basementmembrane must also be considered. Serotonin,like histamine,is a vasoactiveaminethat will contract smooth muscleand increase vascular permeability, althoughits importancemaybe moreas a neurotransmitter.It has beenimplicated,by the use of inhibitors, as an inflammatory mediatorin the early phaseof increasedvascular permeability in certain inflammatory responsesin miceandrats (70, 73). Its role as an inflammatoryagent in manis unclear. Theactions of bradykinininclude its ability to increase vascularpermeability (122). The permeabilityincrease of kinins has been attributed endothelial cell separation with gap formationin postcapillary venules (101). Thegenerationof this mediatoris complex andinvolvesseveral steps and pathways.First, Hageman Factor (factor XII of the clotting system) is activated by contact with negativelychargedsurfaces (glass) or contact with a variety of biological material (14). This enzymethen activates (and is activated by) plasmakallikrein. Thekinin is reaved fromkininogenby this kallikrein (133)or kallikrein fromtissues (122), as well as possibly other proteases such as plasmin(35). Kinins, like most potent inflammatorymediators, are rapidly broken

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downin the plasmaand tissues by kininases and within the circulation undergoalmost completeinactivation during one passage throughthe pulmonarycirculation (27). However,several pathophysiologicchangesmay alter these normalcontrol mechanisms.For example,hypoxiamaydecrease pulmonaryangiotensin-converting enzymeactivity, leading to impaired clearanceof bradykinin(whichis cleavedby this enzyme)in the lung (100). Alternatively,hypereapnic acidosis can lead to activation of the kallikreinkinin system, with subsequentbradykinin production (78). Whenthe animals(sheep) used for these studies wereboth acidotic and hypoxic, both pulmonaryartery and aortic bradykinin concentrationswere elevated, and pulmonaryvascular permeability was increased. This exampleserves to illustrate someof the general control mechanisms for inflammatorymediators and howdisease states mayreadily alter these--leading to enhanced pathologicevents. Platelet-activating factor (PAF)or acetyl glyceryl ether phosphorylcholine (AGEPC) is another recently described mediator that can cause increasesin vascular permeability.First describedin terms of its biological effects on platelets (6, 38) followingearlier studies of leukocyte-platelet interactions, this moleculehas nowbeenshownto havemultiple effects and to be derived from neutrophils and mononuclearphagocytes, as well as fromrabbit basophils (83). It nowmustbe consideredas a broadspectrum mediatorof inflammatory reactions. Definitionof its chemicalstructure (7, 10, 18, 37) has alloweda moredetailedinvestigationof its phlogisticproperties (83). PAFproducesa whealand flare reaction in manthat, on a molar basis, is 100 to 1000times morepotent than histamine. It also induces non-histamine-dependentincreased vascular permeability in the skin of various species. Thequestion of whetheror not the mediatoracts directly on someother moleculeis as yet unanswered.However,it is noteworthy that the permeability inducedby PAFin isolated perfused rat lungs may be dependenton release of leukotriene C4and D4(109). Manyother molecules have been shownto induce increased vascular permeability.Theyincludefibrinopeptides(88), fibrin degradationproducts (5), lymphokines,(22, 63), and anaphylatoxins(C3a and C5a)(42). last probablyact indirectly via histaminerelease and/or leukocyteattraction. This point emphasizes the needfor (a) careful studies of the direct indirect effects of these mediators,(b) determinationof the relative importance of a givenmediator,as well as (c) definedstructural identification the moleculesinvolved. Finally, a group of moleculesderived fromleukocytes should be mentioned.Fourcationic vasoactive peptides from rabbit neutrophils received considerableattention in the 1960s(84, 93) but have largely beenignoredfor the last 10 years. Asinterest in neutrophil-mediated vascular permeability is revived, most emphasishas been on leukocytederivedtoxic oxygenradicals (whichcertainly can alter endothelialpermea-

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bility) or leukotrienes and PAF. However, it would seem important to reinvestigate the role of these cationic peptides and determine whether or not analoguesare present in the leukocytes of other species. Three of these proteins were suggested to act directly (this wouldhave to be re-evaluated in the light of today’s knowledgeof lipid mediators) and one via mast cell degranulation.

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Leukocyte Emigration and Chemotaxis As displayed in Figures 1 and 2, one of the most markedhistologic changes noted in an inflammatory response is the accumulation of cells within the tissues. Early in the reaction the cellular infiltrate is predominatelyneutrophils, later followed by monocytes. The exact molecular mechanismsby which leukocytes migrate out of blood vessels are poorly understood. Much emphasis has been placed on the phenomenonof chemotaxis, which probably represents only a small part of the wholeprocess of emigration, in vivo. Nevertheless, molecules chemoattractive in vitro can induce leukocyte accumulationin tissues. Someof the most potent of these for neutrophils are C5 fragments. These fragments are low molecular weight factors produced through cleavage of C5 by a variety of endopeptidases. C5 convertases derived from classical or alternative complementpathways cleave a 74 aminoacid terminal fragment termed CSa. Other proteases including plasmin, trypsin, kallikrein, and bacterial proteases maycleave at the same or different sites on C5, (112, 113, 121), but generate fragments with similar biological activities. In addition, enzymesthat cleave C5 have been found in lysosomesof neutrophils (114) and platelets (119). The CSa, like C3a, cause release of histamine from mast cells, can alter vascular permeability, and is capable of constricting smoothmuscle.In addition to these activities, however, the molecule is also chemotactic for neutrophils and will induce oxygen radical generation as well as neutrophil granule exocytosis (42). HumanCSa desArg, the fragment derived from CSa after the serum enzyme carboxypeptidase N removes the C-terminal arginine, is poorly active in terms of increasing vascular permeability and as a spasmogen, or as a stimulus for neutrophils in vitro, but will cause secretion from mononuclear phagocytes (67, 116). By contrast, studies in vivo have shownhumanCSa desArg to be as or more active at inducing inflammationthan CSa (55). The explanation for this observation mayrelate to a serum helper cofactor for CSadesArg (26, 81,132) which wouldbe present in the in vivo experiments. Alternatively, differential binding of CSa and CSa desArgto tissue structures maybe important. It is interesting in this regard that CSa and CSa desArgfrom pig and guinea pig are moreequivalent in activity and also lack a carbohydrate moiety (42). The relative importance of C5 and its fragments as mediators of neutrophil influx have been examinedin various types of experiments. Snyderman

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and co-workers(96) found serumfromCS-deficient micegave no chemotactic response whentreated with immunecomplexesor endotoxin, but found the activity wasrestored by addingpurified C5. Intraperitonealinjection of endotoxininto CS-deficientmiceresulted in little chemotacticactivity and a markeddepressionof the early neutrophil accumulation.TheC5deficient micealso exhibit a delay or abrogationof the neutrophil accumulationinto the lung following intratracheal challenge with immunecomplexes(57), Pseudornonas aeroginosa(56), or exposureto hyperoxia(80). Injection CS-sufficient plasmarestores the response. It is importantto point out, however,that eventhoughone can argue that the presence of C5is important for early neutrophil influx in these systems, neutrophil influx does occurin its absence,thus pointingto the fact that other factors are present that are also neutrophil chemotoxins.Withinthe lung, chemotacticfactors producedby alveolar macrophages after various challengesalmost certainly act in concert with the complementsystem to mounta full inflammatory response(43, 52). This mayalso be true elsewherein the bodybut is more difficult to prove. Thealveolar macrophages synthesizeandsecrete a small molecularweightprotein chemoattractant(68) and a low molecularweight lipid (106). Withan organismlike Pseudomonas aeroginosa,another factor that can cause neutrophil emigrationmustbe considered:chemotacticfactors derived from the bacterium (53, 115). Someof these mayrepresent productsof normalprokaryoticprotein synthesis: n-formylatedmethionyl peptides (91). Suchpeptides havereceived enormousattention recently definite probesof neutrophil function. Receptorshavebeenwell characterized on neutrophilsfor these peptidesandthey are provingvaluablein basic studies of chemotaxis,stimulus-secretion coupling, receptor downregulation, and cellular oxidative metabolism. C5fragmentsare also chemotacticin vitro for monocytes,(97) eosinophils (49, 51), andbasophils(50). This raises twoimportantpoints. First, the demonstration that a moleculeis attractive to a cell in the in vitro test systemis far fromdemonstrating its efficacy in vivo. In fact, fewmolecules havebeendefinitively shownto havesignificant chemotacticeffects in vivo. Evenwith the C5fragmentsit is not yet clear whetherthis is a direct effect or an action via someother, as yet undefined,mediator. Second,wemustconsiderthe specificity of chemotacticfactors. Instillation of CSadesArginto the lung results first in an accumulation of neutrophils and then an accumulationof monocytes.Is the latter due to a slower responseof the monocyteto CSadesArgitself or due to someother factor possiblyderivedfromneutrophils (114)?Dueto relative potenciesin vivo, moleculessuch as CSamayexhibit increased cell specificity. Fewhighly specific chemoattractantsfor inflammatorycells have been described. Among the most interesting are lymphokinesthat are chemoattractants in vitro for neutrophils, monocytes,eosinophils,and basophils,

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and also to lymphocytes themselves (87). Recent studies with T cell hybddomassuggest segregation of different cell-specific chemotactic lymphokines maybe possible; that is, each mayattract a separate cell type. Other sources of interest are connective tissue breakdownproducts. Thus, fibronectin fragments may specifically attract monocytes (77). A variety mast-cell-derived eosinophilotactic molecules are known(4) and, recently, a mast-cell-associated neutrophil chemotacticfactor has been described (see Table 1) (2, 4, 59). Lipid mediators mayalso be chemotactic. Muchthe most potent lipid chemotactic for humanpolymorphonuclear leukocytes so far studied has been leukotriene B4 (LTB4) (29, 32). Mono-HETEs are less active (33). Injection of PAF(AGEPC)into the skin has been noted to cause a neutrophil infiltrate, and the moleculealso activates neutrophils in vitro, although rather high concentrations are required (83).

Tissue Damage Damageto tissue in an inflammatory reaction can be by several mechanisms. Twoof the more prominent reasons for tissue injury are the release of enzymesfrom the granules and lysosomesof infiltrating cells as well as the production of oxygen radicals by these cells. As reviewed above, the central feature of inflammatoryprocesses is the infiltration of cells into the injured tissues. In a simplified acute inflammatorylesion, the most prominent cell types include polymorphonuclearleukocytes at early time points followed by mononuclearphagocytesas the lesion evolves. Both of these cell types carry within them lysosomes with manypotential inflammatory mediators. The evidence for the participation of the lysosomalconstituents in inflammatory processes include the observation of increased lysosomal activity in inflamed tissues. For example, degranulation of polymorphonuclear leukocytes has been demonstratedin injured tissues along with release of degradative proteases (40). In addition, as first pointed out by Thomas (103), extracts of lysosomes are capable of producing an inflammatory response. Lysosomalenzymesaad proteases are secreted during phagocytosis (118) or whensuch cells are exposedto stimuli on surfaces too large ingest (39). The mechanismof secretion of lysosomal acid hydrolases and neutral proteases is a subject of muchinvestigation, but is beyondthe scope of this review. It is of interest, however,that in addition to phagoeytosable stimuli, the same molecules that induce chemotaxis can also induce secretion of enzymesand oxygen radicals. The contribution of lysosomal components to the inflammatory process has been discussed in several recent reviews (30, 88, 131). Manyof these enzymesare only optimally active at acid pH. Whetherthe pH in inflammatory lesions gets low enough for a significant contribution of the acid hydrolases is as yet unclear. This has led to the suggestion that neutral

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proteases, enzymesactive at neutral andalkaline pHs, are of mostimportance in terms of modulatingthe inflammatoryreaction and possibly contributing to tissue destruction, whereasacid proteases mayfunction primarily within lysosomalvacuolesto digest phagocytosedmaterial (46). Theseneutral proteasesinclude collagenases,a serine elastase (neutrophils and monocytes)and a metallo-elastase (macrophages)(120), and cathepsin G. Weand others have suggested that not only are they important in the tissue damageof inflammation,but they also allow leukocytesto emigrate throughthe basementmembrane into the lesions in the first place. Adegree of "injury" maytherefore be a requirementfor initiation of inflammation. In addition, these proteases can generate a host of inflammatory mediators by limited proteolysis of precursorproteins, as noted above.Theeflieient inhibition of the enzymes,therefore, becomesa critical control mechanism to prevent unbridleddigestion of the tissues. It maybe arguedthat a key "purpose"for the vasopermeabilityin inflammationis to flood the lesion with such inhibitors. Oxygenradicals are derived from reduction of molecular oxygen by inflammatorycells. Theyare highly bacteriocidal and are also extremely toxic for cells, and in addition they maymodify(inactivate) a key protease inhibitor, alpha 1 antitrypsin (13). Muchof the early damagein inflammation as well as someof the vascular permeabilitymaybe attributed to this groupof compounds, and there is great current interest in this field (25). However,these moleculescannot readily be classed as mediators. For this review, it is perhaps more pertinent to emphasizeonce again that many moleculesdiscussed under "Chemotaxis" can cause release of oxygenradicals fromphagocytes.

Resolution Thefactors that orchestrate the resolution of an inflammatory responseare poorly understoodand are just nowbeginning to receive attention. The delineation of normalrepair mechanisms mayhelp define basic abnormalities in diseasestates wherepersistent inflammation leads to tissue destruction. The paragraphs belowcite examplesof recent experimental work dealing with resolution of inflammation. Asmentionedearlier, for repair to beginat an inflammatory site, several events haveto occur. First, influx of cells mustbe halted. This probably occursin various ways,dependingon the insult leading to the inflammation andthe tissues involved.For example,chemotacticfactor inactivator (8, 24) activity foundin humanserumirreversibly inhibits C5fragmentand other chemotacticactivity and can inhibit cell infiltration in immune complex lung disease(47). Oncethis influx of cells has abated, andantiproteasesandoxygenscavengers fromthe serumhavearrived at the site, inflammatory cells and other

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MEDIATORSOF INFLAMMATION 353 debris must be removed. Newman and co-workers (76) recently showed that matureinflammatorymacrophages,but not freshly isolated monocytes or resident alveolar macrophages, are able to phagocytose agedneutrophils. Thepresenceof serumwasnot required for neutrophil aging or this ingestion, and wasdemonstratedin autologousas well as heterologoussystems employing rabbit and human cells. It is suggestedthat in a normallyevolving inflammatoryreaction, incomingmonocytesdevelopinto inflammatory macrophages capable of removingnot only bacteria and cellular debris, but also effete neutrophils. Thestimuli (mediators)that inducethis monocyte maturationare yet to be defined. Duringthe resolution process, the connectivetissue responseto inflammatorystimuli maydetermineif the injured tissue will return to a normal functional state, or whetherfibrosis mayresult becauseof an expandedor hypersecretingpopulationof fibroblasts. Recently,Schmidtand co-workers (92) reported that interleukin 1, a macrophage-derived growthfactor that augmentsreplication of certain lymphocytes,maybe one factor regulating fibroblast proliferation, andspeculatedthat local productionof this mediator by infiltrating mononuclear cells maycontributeto the fibrosis observed in chronic inflammatorydisease states. Bittermanand co-workers(9) have also recently describedanothergrowthfactor for fibroblasts that is produced by humanalveolar macrophageswhenthose macrophagesare stimulated by particulates and immunecomplexes.Thefactor had an apparent molecularweightof 18,000and appeareddistinct fromother characterized growthfactors including interleukin 1. Overproduction of this factor could also be importantin the pathogenesisof fibrotic lung disease. Bothof these observationspoint out that cells that producemediatorsthat help initiate an inflammatoryresponsealso producesubstancesthat can either help or hinderin the repair of the tissue. Delineationof the factors that determine this outcomewill shed importantlight on the inflammatoryprocess.

CONCLUSIONS Thestudy of inflammatory mediatorsis in a state of transition. Many of the moleculesinvolvedhaveonly just beenstructurally characterized and many otherscanstill be identifiedonlybytheir activity. Beforewecan really piece together the complexinteractions betweenthese mediators and the cells uponwhichthey act, such molecularcharacterization is essential. Also neededis detailed studyof the interaction of the mediatorswith their target cells. In addition, it is no longer enoughto demonstratein vitro that one morecompound is chemotactic,or induces leukocytesecretion, or contracts smoothmuscle.It is time to determinewhichof these mediatorsactually participate in inflammatoryreactions in vivo. Thesestudies are immensely difficult and require not only precise assays for the mediatorsor their

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breakdownproducts in biologic fluids, but also assessmentof their effect on cells or tissues. Eventheir presence does not prove significant function, so highly specific inhibitors are also required. Most useful in this regard are molecular analogues of the molecule in question, or monospecific antibodies. Genetic ddetions of the material or its precursor (as for C5) are also useful. Thus, an interplay between in vitro and in vivo experiments, and betweenbiochemistry, cell biology, physiology, and pathology will be necessary for the further pursuit of this critically importantand fascinating field. ACKNOWLEDGMENTS

Wewish to thank Georgia Wheeler, Lydia Titus, and Jaunita Graves for their assistance in the preparation of this manuscript. This work was supported by Grants No. HL-21565, HL-27063, HL-28510, and GM-24834 from the National Institutes of Health. Literature Cited 1. Appenzeller, O., McAndrews, E. J. 1966. The influence of the central nervous system on the triple response of Lewis. J. Nerv. Ment. Dis. 143:190-94 2. Atkins, P. C., Norman,M., Weiner, H., Zweiman, B. 1977. Release of neutrophil chemotaetic activity during immediate hypersensitivity reactions in humans. Ann- Intern- Meal 86:415-18 3. Austen, K. F. 1979. Biological implications of the structural and functional characteristics of the chemical mediators of immediate-typehypersensitivity. Harvey LecL 73:93-161 4. Bach, M. K. 1982. Mediators of anaphylaxis and hypersensitivity. Ann. Rev. MicrobioL 36:371-413 5. Iklew, M., Gerdin, B., Porath, J., Saldeen, T. 1978. Isolation of vasoactive peptides from humanfibrin and fibrinogen degraded by plasmin. Thromb. Re~ 13:983-94 6. Benveniste, J., Henson, P. M., Cochrane, C. G. 1972. Leukocyte-dependent histamine release from rabbit platelets. The role of IgE, basophils, and a platelet-activating factor. J. Exp. Med. 136:1356-77 7. Benveniste, J., Tence, M., Varenne, P., Bidanlt, J., Boullet, C., Polonsky, J. 1979. Semisynthese et structure proposee du facteur activant los plaquettes (P.A.F.): PAF-acether, un alkyl ether analogue de la lysophosphatidylcholine. CR. Acad. Sci. (Paris) 289:1037-40 8. Bcrenbcrg, J. L., Ward, P. A. 1973. Chemotaetie factor inactivator in nor-

mal human serum. J. Clin. lnvest. 52:120~6 9. Bitterman, P. B., Rennard, S. I., Hunninghake, G. W., Crystal, R. G. 1982. Human alveolar macrophage growth factor for fibroblasts, d. Clin. lnvest. 70:806-22 10. Blank, M. L., Snyder, F., Byers, L. W., Brooks, B., Muirhead, E. E. 1979. Antihypertensive activity of an alkyl ether analog of phosphatidylcholine. Bioche~ Biophy~ Res. Commun. 90: 1194-200 11. Bourne, H. R., Lehrer, R. I., Cline, M. J., Melmon,K. L. 1971. Cyclic 3’, 5’adenosine monophosphate in the hnmanleukocyte: synthesis, degradation, and effects on neutrophil candidacidal activity. £ Clin. Invest. 50:920-29 12. Bray, M. A., Cunningham,F. M., FordHutchinson, A. W., Smith, M. J. H. 1981. Leukotriene B4: a mediator of vascular permeability. Br. £ Pharmacol. 72:483-86 13. Carp, H., Janoff, A. 1979. In vitro suppression of sernm-elastase-inhibitory capacity by reactive oxygenspecies generated by phagocytosing polymorphonudear leukocytes, d. Clin. Invest. 63:793-97 14. Coehrane, C. G., Revak, S. D., Wuepper, K. D., Johnston, A., Morrison, D. C., Uleviteh, R. 1974. Soluble mediators of injury of the microvasculature: Hagemanfactor and the kinin forming, intrinsic dotting and the fibrinolytic systems. Microvasc. Rez 8:112-21

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38. Henson,P. M.1970. Releaseof vasoactive aminesfrom rabbit platelets inducedby sensitized mononuclear leukocytes and antigen. J. Exp. Med.131: 287-306 39. Henson,P. M. 1971. The immunologic release of constituentsfromneutrophil leukocytes.I. Therole of antibodyand complementon nonphagocytosablesurfaces or 107:1535-46 phagocytosable particles. J. lmmunol. 40. Henson,P. M. 1972. Pathologic mechanisms in neutrophil-mediatedinjury. Am. £ Pathol. 68:593-612 41. Henson,P. M., Larsen, G. L., Webster, R. O., Mitchell, B. C., Goins, A. J., Henson, J. E. 1982. Pulmonarymicrovascular alterations and injury induced by complementfragments: synergistic effect of complement activation, neutrophil sequestration, and prostaglandins. Ann. NYAcad. ScL 384:287300 42. Hugli, T. E., Muller-Eberhard,H. J. 1978. Anaphylatoxins: C3a and CSa. .4dv. Immunol.26:1-53 43. Hunninghake,G. W., Gallin, J. I., Fauci, A. S. 1978. Immunologic reactivity of the lung:the in vivoandin vitro generationof a neutrophilic chemotactic factor by alveolar macrophages. Am. Rev. Respir. Dis. 117:15-23 44. Hurley,J. ¥. 1963.Anelectron microscopic study of leukocyric emigration andvascular permeabilityin rat skin. Aust. J. Exp. BioLMed.Sci. 41:171-86 45. Issekutz, A. C. 1981.Effect of vasoactive agents on polym.orphonuelear leukocyteemigrationin vwo.Lab. Invest 45:234-40 46. Janoff, A. 1972.Neutrophilproteasesin inflammation. Ann. Rev. Med. 23: 177-90 47. Johnson,K. J., Anderson,T. P., Ward, P. A. 1977. Suppression of immune complex-inducedinflammation by the chemotacticfactor inactivator. J. Clin. lnvest. 59:951-58 48. Johnston, R. B. Jr., Newman,S. L. 1977. Chronicgranulomatousdisease. Pediatr. Clim North An~24:365-76 49. Kay,A. B. 1970.Studies on eosinophil leukocytemigration.II. Factorsspecifically ehemotaetiefor eosinophils and neutrophils generated fromguinea-pig serumby antigen-antibody complexes. Clin- Exp. Immunol.7:723-37 50. Kay, A. B., Austen, K. F. 1972. Chemotaxisof humanbasophil leukocytes. Clin- Exp. lmmunol.11:557-63 51. Kay,A. B., Stechschulte,D. J., Austen, K. F. 1971. Aneosinophil leukocyte

chemotacticfactor of anaphylaxis. J. Exp. Med. 133:602-19 52. Kazmierowski, J. A., Gallin, J. I., Reynolds, H. Y. 1977. Mechanism for the inflammatory response in palmate lungs: demonstrationand partial characterization of an alveolar macrophagederivedchemotacticfactor with preferential activity for polymorphonuclear leukocytes.J. Clin. lnvest. 59:273-81 53. Keller, H. U., Sorkin, E. 1967. Onthe chemotactic effect of bacteria.Int. Arch. Allergy AppLImmunol.31:505-17 54. Kuehl,F. A., Egan,R. W.1980. Prostaglandins,arachidonicacid, and inflammarion. Science 210:978-84 55. Larsen, G. L., McCarthy,K., Webster, R. O., Henson,J., Henson,P. M.1980. Adifferential effect of C5aandC5ades Argin the induction of pulmonaryinflammation.,4m. ,I. Pathol. 100:179-92 56. Larsen,G. L., Mitchell, B. C., Harper, T. B., Henson,P. M.1982. Thepulmonaryresponseof C5sufficient anddeficient mice to Pseudomonas aeruginosa. .4m. Rev. Respir. Diz 126:306-11 57. Larsen,G. L., Mitchell, B. C., Henson, P. M. 1981. Thepulmonaryresponse of C5sufficient anddeficient miceto immunecomplexes.A~r~Rev. Respir. Dis. 123:434-39 58. Larsen,G. L., Parrish, D. A., Henson, P. M. 1983. Lungdefense: the paradox of inflammation.Chest. In press 59. Lee, T. H., Nagy,L., Nagakura, T., Walport,M.J., Kay,A. B. 1982.Identification and partial characterizationof an exercise-inducedneutrophil ehemotactic factor in bronchial asthma. J. Clin~ lnvest 69:889-99 60. Lichtenstein, L. M.1969. Characteristics of leukocytichistaminerelease by antigen and by anti-immunoglobulin andanti-cellular antibodies.In Cellular and [-lumoral Mechanisms in Anaphylaxis andAllerg~,ed. H. Z. Movat, pp. 176-86.Basel: Karger. 288 pp. 61. Lichtenstein, L. M., DeBernardo,R. 1971.Theimmediateallergic response: in vitro action of cyclic AMP-active and other drugson the twostages of histaminerelease. J. lmmunol.107:1131-36 62. Lichtenstein,L. M., Margolis,S. 1968. Histamine release in vitro: inhibitionby catecholamines and methylxanthines. Science 161:902-3 63. Maillard, J. L., Pick, E., Turk, J. L. 1972. Interaction between"sensitized lymphocytes"and antigen in vitro. V. Vascularpermeabilityinducedby skinreactive factor, lnt Arch. Allergy 42:50-68

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86. Rehm,S. R., 13ross, 13. N., Pierce, A. K. 1980.Early bacterial clearancefrom mudnelungs. Species-dependentphagocyteresponse. J. Clin. Invest. 66: 194-99 87. Rocklin, R. E., Bendtzen, K., 13reineder, D. 1980. Mediatorsof immunity: lymphoklnesand monokines. Adv. ImmunoL29:55-136 88. Ryan, 13. B., Majno,G. 1977. Acute inflammation,a review.Am.J. Pathol. 86:185-276 89. Samuelsson, B., Hammarstrom,S., Borgeat, P. 1979. Pathwaysof arachidonic acid metabolism.In Advancesin InflammationResearch, ed. G. Weissman,B. Samuelsson, R. Paoletti, 1:40512. NewYork: Raven. 640 pp. 90. Scherzer, H., Ward,P. A. 1978. Lung and dermalvascular injury producedby preformed immune complexes. Am. Rev. Respir. Dis. 117:551-57 91. Schiffmann,E., Aswanikumar, S., Venkatasubramanian,K., Corcoran,B. A., Pert, C. B., et al. 1980.Some characteristics of the neutrophil receptor for chemotactic peptides. FEBSLett. 117:1-7 92. Schmidt,J. A., Mizel,S. B., Cohen,D., Green,I. 1982.Interleukin 1, a potential regulator128:2177-82 of fibroblastproliferation. J. lmmunoL 93. Seegers,W., Janoff, A. 1966.Mediators of inflammation in leukocytelysosomes. VI. Partial purification andcharacterization of a mastcell rupturing component. J. Exp. MetL124:833-49 94. Shaw,J. O. 1980.Leukocytesin ehemotactic-fragment-induced lung inflammarion.Vascularemigrationandalveolar surface migration. Am.J. Pathol. 101:283-302 95. Simon,P. L., Willoughby,W.F. 1982. Biochemicalandbiological characterizatiun of rabbit interleukin-I(IL-1). Lymphokines6, Lymphokinesin Antibodyand CytotoxicResponses,ed. S. B. Mizd, 47-63. NewYork: Academic. ~ pp. 96. Snyderman, R., Phillips, J. L., Mergenhagen,S. E. 1971.Biologicalactivity of complement in viva. Role of C5 in the accumulation of polymorphonuclear leukoeytesin inflammatory exudates.J. Exp. Med. 134:1131-43 97. Snyderman,R., Shin, H. S., Hausman, M. H. 1971. A chemotactic factor for mononuclearleukoeytes. Proc. Sac. Exp. Biol. Med.138:387-90 98. Sonnenfeld,G. 1980. Modulationof immunity by interferon. In Lymphokine Reports Led. E. Pick, M. Landy, pp.

113-31. NewYork: Academic.259 pp. 99. Spector, W.13., Willoughby, D. A. 1959. Experimentalsuppressionof the acute inflammatorychangesof thermal injury. J.. PathoLBacterial. 78:121-32 100. Stalcup,S. A., Lipset, J. S., Legant,P. M., Leuenberger,P. J., Mellins, R. B. 1979. Inhibition of converting enzyme activity by acute hypoxiain dogs. Z AppLPhysiol. Resp. Environ. Exercise Physiol. 46:227-34 101. Svensjo, E., Arfors, K.-E., Raymond, R. M., Grega, G. J. 1979. Morphological and physiological correlation of bradykinin-induced macromolecular efflux. Am.J. Physiol. 236:H600-6 102. Tauber, A. I., Kaliner, M., Stechschulte, D. J., Austen,K. F. 1973. Immunologic release of histamine and slow reacting substanceof anaphylaxis from human lung. Z lmmunol. 111:27-32 103. Thomas,L. 1964.Possiblerole of leukocyte granules in the Schwartzman and Arthusreactions. P}’oc.Sac. Exp.Biol. Med. 115:235-40 104. Uriuhara, T., Movat,H. Z. 1967. Role of PMNqeukocyte lysosomesin tissue injury, inflammation and hypersensitivity. V.Partial suppressionin leukopenie rabbits of vascular hyperpermeability dueto thermalinjury. Proc. Sac. Exp. Biol. Med.124:279-84 105. Vadas,P., Wasi,S., Movat,H. Z., Hay, J. B. 1981. Extracellular phospholipase A2mediates inflammatoryhyperaemia. Nature 293:583-85 106. Valone,F. H., Franklin,M., Sun,F. F., 13oetzl, E. J. 1980. Alveolar macropha~gelipoxygenaseproductsof arachidomeacid: isolation and recognitionas the predominant constituents of the neutrophil ehemotaetieactivity elaborated by54:390-401 alveolar macrophages. Celllmmunol. 107. Vane,J. R. 1971.Inhibition of prostaglandinsynthesis as a mechanism of action for aspirin-like drugs. NatureNew Biol. 231:232-35 108. Vane,J. R. 1976. Themodeof action of aspirin and similar compounds. J. Allergy Clin. lmmunol.58:691-712 109. Voelkel,N. F., Worthen,S., Reeves,J. T., Henson,P. M., Murphy,R. C. 1982. Nonimmunological production of leukotrienes induced by plateletactivating factor. Science 218:286-88 110. van Euler, U.S. 1934. Zur kenntnis der pharmakologisehenwirkungenvan nativsekreten und extracten maunlicher accessodschergeschlechtsdrusen.Arch. Exp. Pathol. Pharmakol.175:78-84

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111. Ward, P. A. 1968. Chemotaxis of ity factors in mildthermalinjury. Br. Z mononuclearcells. £ Exp. Med.128: Exp. Pathol. 41:487-506 1201-21 125. Williams, T. J. 1977. Oedemaand 112. Ward,P. A. 1971. Leukotacticfactors vasodilatationin inflammation. Therelin health and disease. Am.J. Pathol. evance of prostaglandins. Postgrad. 64:521-30 Med. Z 53:660-62 113. Ward,P. A., Chapitis, J., Conroy,M. 126. Williams,T. J. 1982.Vasoactive intestiC., Lepow,I. H. 1973. Generationby nal polypeptide is more potent than bacterial proteinasesof leukotacticfacprostaglandinEa as a vasodilator and tors from human serumand C3 and C5. oedema potentiatorin rabbit skin. Br. £ J. ImmunoL 110:1003-9 PharmacoL77:505-9 114. Ward, P. A., Hill, J. H. 1970. C5 127. Williams,T. J., Jose, P. J. 1981.Mediachemotacticfragmentsproducedby an tion of increasedvascular permeability enzymein lysosomalgranules of neuafter complementactivation. Histatrophils. J. Immunol.104:535-43 mine-independent action of rabbit CSa. J. Exp. Med.153:136-53 115. Ward,P. A., Lepow,I. H., Newman, L. J. 1968. Bacterial factors chemotactic 128.Williams,T. J., Peck, M.J. 1977.Role for polymorphonuclear of prostagiandin-mediated vasodilation J. PathoL 52:725-36 leukocytes..4~ in Inflammation.Nature270:530-32 116. Webster,R. O., Hong,S. R., Johnston, 129.Willis, A.L. 1969.Releaseof histamine, R. B., Henson,P. M.1980. Biological kinin and prosta$1andindurinl~ careffects of the humancomplement tragrageenin-inducedinflammationIn the ments CSaand CSades Argon neutrorat. In Prostaglandins, Peptides and phil function. Immunopharmacology Amines,ed. P. Mantegazza,E. W.Hor2:201-19 ton, pp. 31-38. NewYork: Academic. 117. Wedmore, 191pp. C. V., Williams,T. J. 1981. Control of vascular permeability by 130. Willis, A. L. 1970. Identification of prostaglandin E~ in rat intlammatory polymorphonuclearleukocytes in inflammation. Nature289:646-50 exudate. Pharmac.Rex Comrr~2:297118. Weissmann,G., Zurier, R. B., Hoff3O4 stein, S. 1972.Leukocyticproteasesand 131. Wintroub, B. U. 1982. Neutrophilthe immunologicrelease of lysosomal dependentgenerationof biologicallyacenzymes.Am.J. Pathol. 68:539-64 tive peptides. In Advancesin Inflamma119. Weksler, B. B., Coupal, C. E. 1973. tion Research, ed. G. Weissmann, 4:131-45. NewYork: Raven. 200 pp. Platelet-de~pendent generation of chemotact~c activity in serum.J. Exp. 132. Wissler,J. H., Stecher,V.J., Sorkin,E. Med. 137:1419-30 1972.Chemistry andbiologyof the ana120. Werb,Z., Banda,M. J., McKerrow, J. phylatoxin related serumpeptide sysH., Sandhans,R. A. 1982.Elastasesand tem. III. Evaluationof lencotactic acelastin degradation.J. Invest. Dermatol. tivity as a property of a newpeptide 79:154s-59s systemwithclassical anaphylatoxinand 121. Wiggins,R. C., Giclas, P. C., Henson, cocytotaxin as components.Eur. J. Immunol. 2:90-96 P. M.1981.Chemotactic activity generated from the fifth componentof com- 133. Wuepper,K. D., Cochrane,C. G. 1972. plementby plasmakallikrein of the rabPlasmaprekallikrein: isolation, characbit. J. Exp. Med.153:1391-1404 terization, and mechanism of activa122. Wilhelm,D. L. 1971. Kinins in human tion. J. Exp. Med.135:1-20 disease. Ann. Rev. Med.22:63-84 134.Zurier, R. B., Weissmann,G., Hoff123. Wilhelm,D. L. 1973. Mechanismsrestein, S., Kammerman, S., Tai, H. H. 1974. Mechanism of lysosomal enzyme sponsiblefor increasedvascularpermeability in acute inflammation,agents release from humanleukoeytes. II. Actions 3:297-306 Effect of cAMPand eGMP,autonomic 124. Wilhelm,D. L., Mason,B. 1960. Vascuagonists, and agents whichaffect milar permeability changesin inflammacrotubule function. Z Clin. lnvest. tion: the role of endogenous permeabil53:297-309

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THE ROLE OF CELL-MEDIATED IMMUNERESPONSES IN RESISTANCE TO MALARIA, WITH SPECIAL REFERENCETO OXIDANT STRESS Anthony C. Allison

and Elsie

M. Eugui

Institute of BiologicalSciences,SyntexResearch,PaloAlto, California94304 SUMMARY Asexualbloodformsof malariaparasites are microaerophilicandsensitive to oxidant stress. Plasmodium falciparumand someother species of malaria parasites undergoschizogony attachedto endothelialcells of postcapillary venules, whereoxygentensions are low. Acquiredimmuneresponses to all forms of malaria parasites so far investigated are thymusdependent.Animalsdeprived of T lymphocytesdo not recover from the infections and cannot be immunized against malaria parasites. In contrast, animalsunableto makeantibodies recovernormally from someprimary infections, e.g. with Plasmodium chabaudi, and when rescued by chemotherapyfrom other species of malaria parasite develop lasting, nonsterile immunity.Immunityto malaria can be transferred in mice by T lymphocytesof the Lyl+ phenotype, but transfer of B lymphocytestogether with this T-cell subset increases the effectivenessof immunityto Plasmodium yoelii. Thus, antibodies facilitate recovery from someprimarymalaria infections and increase the effectiveness of cellmediatedimmuneresponsesin these infections. Miceof the A strain are highly susceptible to malaria and are unable to increase the numberof mononuclear calls in the spleen duringthe courseof the infections. It is postulated that T lymphocytesrespondingto parasite antigens release factors that stimulatethe proliferation of effector cell precursorsand 361 0732-0582/83/0410-0361 $02.00

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their recruitmentinto the red pulp of the spleen.In this site, the liver and probablythe peripheral circulation, effector cells bind to the surface of parasitized erythrocytesand are activated to release superoxide(02-). The consequentexposureto oxidantstress can lead to degenerationof parasites in erythrocytes. This effect on the parasites can be preventedby agents chelating metals, whichsuggeststhat iron-catalyzedlipid peroxidationand consequentK+ loss, or inactivation of metal-containingenzymes,maybe the mechanism by whichoxidant stress kills the intracellular parasites. Antibodieson the surface of schizont-infectedcells could facilitate binding of effector cells andtrigger O~release, therebyacting synergisticallywith cell-mediated immunity. Inherited traits, such as abnormalhemoglobinsand G-6-PDdeficiency, and acquired cell-mediated immunityboth subject malaria parasites to oxidant stress and mayreinforce one another, increasing the chancesof survival of childrenbearingthese traits duringthe dangerousyears of first exposureto malaria in areas wherethe disease is endemic. INTRODUCTION Therelationships betweenplasmodiumparasites, anophelinevectors, and mammalian hosts are complexand varied (6). Consideringhumanmalarias, the situation differs in variouscountriesandin geographicalregionsof the samecountry. WherePlasmodiurnfalciparurn is holoendemic,the greatest effects of malariaare manifestedin children betweenthe ages of 6 months and 5 years, after whichthe disease becomesprogressively less severe. Resistanceto malaria developsin stages. First, the severity of clinical symptoms and later levels of parasitemia fall and splenomegalydecreases (91, 92). Bythe time they are adults, mostpersonsliving in endemicareas showlittle parasitemia, although they are frequently bitten by infected mosquitoes;but after as little as 6 monthsabroad,or after eliminationof parasites by chemotherapy,they mayagain becomesusceptible to severe malaria (94). Theseobservationssuggest gradually acquired, adaptive immunity dependenton frequent antigenic stimulation and persistence of parasites. Such nonsterilizing immunityto parasites has been termed premunition(135). As discussedbelow,nonsterilizing immunityis observed in experimentalmalarias whenthe immune responseis purely cell mediated, whichis one reasonthat type of responseis of interest. Thenumberof children dyingdirectly or indirectly from malaria is not accurately known.Certainly the majority survive to school age, by which time parasite rates andcountshavefallen as a result of acquiredimmunity. Untreatedinfections with the sameparasites in Europeanvisitors are often lethal. Severalfactors probablycontributeto the relative resistance of Afri-

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CELL-MEDIATED IMMUNERESPONSESTO MALARIA 363 can children to falciparummalaria. Someof these, such as HbSand G-6-PD deficiency, have been investigated in detail and provide informationof general interest fromthe point of viewof host-parasite relationships (6). This review is confined to mechanismsof acquired immunityto malaria parasites, whichare mosteasily studied in experimentalanimals,especially mice, in whichit is possible to suppress either T-lymphocyteor B-lymphocyteresponsesand to characterize the isotypes of antibodiesand phenotypes of cells transferring immunity.Furthermore,the availability of mice with other selective defects of immune function, such as T cell-dependent recruitmentof mononuclear cells, is useful. Mousemalarias are also convenient experimentally. Theyare divided into two groups: one includes Plasmodium berghei and Plasmodium yoelii, and the other, Plasraodiumvinckei and Plasrnodiurnchabaudi.The similarities within and distinctions betweenthe twogroupsare manifestin the structure andbehaviorof blood-stageparasites, serology,patterns of cross protection, and isoenzymetypes (21). Withineach group it is possible obtain infections from whichmost strains of micerecover and ate fully immune,so that after homologous challenge no parasitemia is detectable (e.g.P.y. yoelii 17XNL, P.c. chabaudi,andP. vinckei petteri), or strains that are lethal in unprotectedmice(e.g.P.y. yoelii 17XL or P.v. vinckei). Immunization against the asexualbloodstages of the latter can be achieved by infectionandtreatment(36), byinfection with a nonlethalparasite in the samegroup (e.g.P.y. yoelii 17XNL protecting against P.y. yoelii 17XL,or P.c. chabaudiprotecting against P.v. vinckei), or by immunizationwith homologous crude vaccines (114) or purified parasite antigens (71). availability of cross-reactingvirulent andavirulentstrains of parasites allows experimental analysis of mechanisms of immunity.As described below, protective immunity to P. chabaudi appears to be antibody independent, whereasrecovery from primaryinfections with P. yoelii is antibody dependent.Thus, different mechanisms of immunitycan be illustrated by mousemaladas,although care must be taken whenattempting to generalize from these results to humanmalarias. Acquiredimmunityis stage specific. Miceinfected with X-irradiated sporozoitesof P. bergheiare resistant to sporozoiteinfections but not to challenge with asexual bloodstages (109). Selective immunitycan also achievedby immunizationwith gametocytes(22) and can developwithout anyeffect on asexualbloodstages, being manifestedby lack of infectivity of parasitized blood for mosquitoes. In Plasmodium knowlesi infections of Macacamulatta, classical antigenie variation, demonstrableby the sequential appearanceof antibodies agglutinating schizont-infectedcells, has beendemonstrated(16). Therepeated P. falciparuminfections observedin African children over several

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years have been attributed to the presence of many antigenic variants, the existence of which can be shown by precipitation in gels (157). However, the relationship between such soluble antigens and those on the surface of infected schizonts, which are presumed targets of protective immunity (74), remains to be established.

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ACQUIRED IMMUNITY TO MALARIA Role of Antibodies It has long been believed that antibodies opsonize malaria-parasitized erythrocytes for phagocytosis by macrophages (140). More direct evidence for a role of antibodies in protection came from observations by Cohen et a1 (33) that administration of gamma-globulin fractions from pools of adult African sera accelerated the recovery of African children from falcipamm malaria. IgG fractions of immune serum were likewise found to protect rats from l? berghei infections (40, 41). More recently, monoclonal antibodies have been used to d e h e antigens specific for developmental stages of the parasite (sporozoites, asexual blood forms, and gametocytes) and to analyze mechanisms of humoral immunity (56, 115, 120). Only a few examples can be mentioned in this paper. A monoclonal antibody against a protein of molecular weight 42,000 in the membrane of l? berghei sporozoites has been shown to protect mice against mosquito-transmitted infection (1 15) and prevent the entry of sporozoites into cultured cells (72). This antibody does not protect against blood-transmittedinfection. However, monoclonal antibodies that recognize antigens of the asexual blood forms of P.y. yoeIii can protect mice against blood-transmitted infection (56). Holder & Freeman (71) have used monoclonal antibodies to identify and purify antigens of l?y. yoelii membranes. They found that mice immunized with either a 235,000-molecular-weight merozoite-specilic protein or a 230,000-molecular-weight schizont protein and its derivatives were protected against infection with asexual blood forms of P.y. yoelii. These observations show that antibodies against stage-specificantigens of the parasite can have a protective role, although this does not appear to be exclusive. For example, mice with humoral responses suppressed by neonatal injection of anti-p serum can be immunized with irradiated sporozoites against mosquito challenge (23).

Thymus Dependence of Immunity to Malaria Parasites The first observations showing the importance of thymus-dependent (T) lymphocytes in recovery from malaria infections were made by Brown et a1 (14). Rats inoculated with P. berghei when 13 weeks of age recovered from the infections, but in neonatally thymectomized rats we found higher

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and more prolonged parasitemias and an appreciable mortality (14). These observations were independantly confirmed (138). With the availability of mice congenitally deprived of mature T-lymphocytes (nude or n u h u mice), and a strain of P. yoelii producing infections from which intact mice recover, the problem was taken up again (27). In nude mice infected with P. yoelii, escalating parasitemias were observed. This parasite can invade reticulocytes, and in infected mice there was a progressive increase in susceptiblecells, until with a parasitemia of 7040% the animals all succumbed. The extreme susceptibility of n u h u mice to P. yoelii infections was confirmed (122, 152); moreover, it was shown that parasitemia recrudesced in mice after termination of the acute disease by treatment with clindamycin (122). Recrudescencewas not observed in n u h u mice that had been grafted with thymic tissue. Intact CBA mice recovered from P. chubaudi infections, whereas nu/nu mice on a CBA background developed lethal infections (46) (Figure 1). The consistent aggravation of nonlethal malaria infections by deprivation of T cells should be contrasted with the delayed mortality observed in T cell-deprived mice infected with one strain of P. berghei (44), which is explicable by thymus-dependent immunopathology.

An tibody-Independent Resistance of Experimental Animals to Malaria Rank & Weidanz (1 19) made chickens agammaglobulinemicby combined chemical bursectomy (treatment with testosterone and cyclophosphamide).

601

0

4 6 8 10 12 14 16 18 2022 Days after infection

Figure I Parasitemias in CBA nu/nu (open circles) and CBA nu/+ (closed circles) mice at different intervals after intraperitoneal inoculation of los erythrocytes infected with P. chabaudi AS. In each group, five female mice aged 6 to 10 weeks were used. All nu/nu mice had died by day 12-13 after infection. (From 46.)

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When such chickens were infected with Plasmodium gallinaceurn they died with fulminating parasitemias. However, when rescued from otherwise fatal infections by chloroquine therapy, the infection was controlled and the B cell-deficient chickens resisted challenge infection with the same parasite. Thus, the B cell-deficient chickens could produce a protective immune response, although their parasites were not completely cleared. In mice made B cell deficient by neonatal injections of anti-p serum, infections with the normally avirulent P.yoelli were found to produce lethal disease (123). However, antimalarial chemotherapy of these mice led to a nonsterilizing protective immunity (124). With another murine parasite, P. chabaudi adami, infection of B cell-deficient mice was found in the absence of chemotherapy to activate a T cell-dependent immune mechanism that terminated the malaria in a manner similar to that seen in immunologically intact mice (65) (Figure 2). The immunized B cell-deficient mice were resistant to homologous challenge, as well as to infections initiated with P. vinckei, but not to challengewith P.yoelii or P. berghei. These observations emphasize the importance of thymus-dependent, antibody-independent mechanisms of immunity to rechallenge with all species of malaria parasites so far tested, and the ability of this mechanism to bring about recovery from P. chabaudi without chemotherapy. It may be relevant that P. chabaudi elicits a powerful, thymus-dependent increase in spleen cell cellularity and nonspecific cytotoxic activity (46).

Figure 2 The course of P. chabaudi infections in eight B cell-deficient immunologically intact (m) mice. (From 65.)

(0) and seven

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Further evidence for a role of T lymphocytes in immunity to blood stages of murine malaria parasites came from observations of Finerty & Krehl(50) that infections with a lethal strain of P. yoelii (17XL) could be converted into nonlethal infections by pretreatment with cyclophosphamide. The cyclophosphamide, administered 2 days before infection, increased delayedtype hypersensitivity to parasite antigens (presumably from the elimination of suppressor cells), but antibodies against blood-stage parasites were not detectable during the recovery period. It has long been recognized that immunization of rhesus monkeys against P. knowZesi requires complete Freund’s adjuvant (15, 18), which elicits cell-mediated immunity, and resistance to challenge is not well correlated with the presence of antibodies inhibiting parasite reinvasion and replication in vitro (83).

T-Lymphocyte Responses in Malaria Observations in several laboratories showed by other methods that malaria infections elicit T-lymphocyte responses. In mice carrying chromosome markers for T lymphocytes and infected with l? yoelii, proliferation of these cells in the spleen was observed (81). T lymphocytes (sensitive to anti-Thy1 serum) from spleens of mice immune to P. yoelii showed early proliferative responses when cultured with infected erythrocytes or soluble parasite antigens (1 5 1). Likewise, T lymphocytes from humans that had been infected with malaria showed proliferative responses to P. fulciparum antigens (166).

Mechanisms by Which T Lymphocytes Particeate in Recovery from Malaria Infections The first possibility that must be considered is a helper effect on antibody formation, as postulated by Brown (15). He made the ingenious suggestion that T-lymphocyte responses to common antigenic determinants accelerate the formation of protective antibodies to sequentially appearing variantspecific antigens. Mice deprived of T lymphocytes showed diminished germinal center responses and IgGl antibody formation when infected with P. yoelii (8 1). The strict thymus dependence of the formation of antibodies against l? yoelii antigens in mice and in cultured murine cells has been well documented (142). As already discussed, B cell-deficient mice are unusually susceptible to infection with this parasite (152). T cell-deficient nude mice showed recrudescence of P.yoelii infections after treatment, but this was not observed in mice given hyperimmune serum (122). A synergistic effect of T cells and antibody-forming B lymphocytes in immunity to I? yoelii 17X has been postulated by Jayawardena et a1 (80). T cells (Lyl-l- phenotype) from CBA

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mice that had recovered from P. yoelii infections transferred immunity to nonimmune recipients, but the addition of immune B lymphocytes to the Lyl cells increased their ability to transfer immunity and enhance antibody production. B cells from CBA/N mice (which are defective in producing high-affinity antibodies as well as those of the IgG3 isotype) had no such effect. In contrast to the observations with Z? yoelii, B cell-deprived mice recover from primary infections with P. chabaudi and are immune to challenge, so that antibody-independent mechanisms of immunity appear to be of major importance with this parasite (65). The thymus dependence of recovery must therefore be manifested by some function of T cells other than helper effects in antibody formation. One possibility is that the cytotoxic subset of T lymphocytes can lyse parasites or parasitized cells. Our attempts to demonstrate such a mechanism failed and are in any case inconsistent with the observation of parasites degenerating within circulating erythrocytes, as discussed below. The finding that the Ly-23 subset is not required for the effector phase of immunity to P. yoelii further supports the interpretation that cytotoxic T cells are not important defensive elements in malarial infections (80). Another possibility is that specifically sensitized T lymphocytes, stimulated by parasite antigens, release a mediator that inhibits parasite growth; for example, y-interferon or lymphotoxin are possible candidates. Finally, lymphocytes stimulated by parasite antigens may produce a factor that activates a distinct population of cells, such as macrophages or NK (natural killer) cells, which in turn release another effector molecule acting on parasites or parasitized cells. The third hypothesis seems most likely, because the effects of nonspecific immunity, which can be induced by Corynebacterium parvum even in thymus-deprived nude mice, appear to be equivalent to those of thymusdependent specific immunity. This is true of both the sensitivity of different species of Plasmodium to nonspecific immunity and its principal manifestation, death of parasites within intact, nonphagocytized erythrocytes.

NonspeciJic Immunity Powerful effects of nonspecific immunity on asexual blood forms of malaria parasites of mice were demonstrated in our laboratory (28). Mice previously inoculated intravenously with live BCG were found to develop low, transient parasitemias when infected with I? vinckei, which produces lethal infections in unprotected mice, and were subsequently shown to have lifelong immunity to challenge with this parasite. It was then found that injections of killed C. parvum produced similar protection against hemoprotozoa (29).

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Nude mice can be protected against R vinckei and R chabaudi infections by prior injection of C parvum but not of Myobacterium bovis BCG (47). This suggests that the effector phase of immunity activated by C parvum bypasses a requirement for T lymphocytes. Immunity to tumors can also be elicited by C parvum in nude mice (160). C parvum exerts some protection against P. berghei sporozoites, but there is little effect on the blood-stream forms of this parasite (108). In general, blood-stream forms of parasite strains not multiplying in reticulocytes are more sensitive to the suppressive effects of nonspecific immunity than those that can multiply in reticulocytes.

Susceptibility of Diflerent Mouse Strains to Malaria Genetic analysis of susceptibility to infections is a potent way of revealing the underlying mechanisms. For example, a single autosomal gene confers susceptibility of mice to infections with Leishmania donovani (13) and another confers susceptibility to Leishmania tropica (73). Greenberg & Kendrick (63) showed differences in the time to death of several mouse strains infected with R berghei. We observed more striking differences in the susceptibility of different mouse strains to l? chabaudi; in the A/He strain, the infections were always lethal, whereas in other strains the infections were self-limiting (46). Investigations of congenic strains suggested that susceptibility does not correlate with H-2 haplotype. The F1 offspring of susceptible A and resistant BIO.A mice proved to be resistant, and some offspring of backcrosses to the susceptible parental strain were susceptible. Our observations have been confirmed and extended (139). Segregation analysis of backcrosses and F2 progeny derived from A/J mice susceptible to P. chabaudi and resistant B1O.A parental mice suggested that host resistance is controlled by a single, dominant, non-H-2-linked gene.

Strain Direrences in Mononuclear Cell Recruitment Since macrophages recently immigrated into organs have much greater capacity than resident cells to produce 02,which is likely to be important in immunity to malaria parasites, factors affecting recruitment of these cells are relevant. During the course of malaria infections in most strains of mice, there is a rapid increase in the number of mononuclear cells in the spleen, rising to a peak (some fivefold) about the time of maximum parasitemia (46); thereafter, there is a marked increase in the number of erythropoietic precursors (55). In CBA nu/nu mice infected with R chabaudi, there is no increase in mononuclear cells in the spleen and their capacity to kill tumor cells (46), showing the thymus dependence of the phenomenon. In mice of the A/He strain infected with R chabaudi, there was no increase in the number of leukocytes in the spleen (46) (Table 1). Hence, A mice appear

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ALLISON & EUGUI Table1 Changes in the number of nucleatedcells in the spleen, andin NK activity, of differentstrains of miceinfectedwithP. chabaudi Mouse strain

a Parasitemia (%)

Spleenceils (X 107)

bNKactivity (%specificlysis)

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C57B1/6

0 5.9 (5.8-6.0) 10.9 (10.7-11.1) 13-28 22.4 (12.6-32.0) 30.2 (26.7-33.0) A/He 0 12.3 (11.3-13.4) 8.2 (7.2-9.4) 56-77 12.9 (9.6-19.4) 11.0 (9.4-14.0) a Samples weretakenat peakparasitemia,8 or 9 daysafter infection. bNKactivity wasmeasuredusing 100spleen cells to one YAC-1 target ceil, in a 51Crreleaseassay.Arithmetic meansandranges(in brackets)are taken fromEugui&Allison(46). to be defective in the production by T lymphocytesof factors stimulating the proliferation and/or recruitment of mononuclearcells or in the response to such factors. Consistent with this interpretation is the observation that strain A/J mice inoculated with BCGare not protected against P. berghei, whereas other strains are (103). In contrast, strain A/J mice can be protected against Babesia microti infections by C. parvum(158); recruitment of mononuclear cells by this agent, but not by BCG,is thymus independent. The possible relationship of the defective response of mononuclearcells in A strain mice to their low NKactivity (68) and low mononuclearcell-mediated cytotoxicity of tumorcells (2) is discussed later. Immunespleen cells implanted into the peritoneal cavity in mill;pore chamberstogether with parasitized erythrocytes were found to elicit a large macrophageinfiltrate around the chambers (7). Presumably, this was due to the release from antigen-activated lymphocytesof factors stimulating the proliferation of mononuclearcells and their recruitment. Possibly, the monocyte chemotactic factor and macrophagemigration inhibition factor described in the spleens of infected rodents (34, 162) were involved. The finding of splenomegaly during acute malaria infections in humans (94) suggests that a similar recruitment occurs. Splenomegalyis marked children under school age exposedto repeated malaria infections, and thereafter the proportion of persons with palpable spleens declines (92).

Changes in Lymphocyte Subsets During Malaria Infections Changes in proportions and absolute numbers of lymphocyte subsets in human and experimental malarias have been described. The number of circulating T cells in humanperipheral blood decreases, and the numberof null ceils (lacking B or T markers) increases, during malaria infections (64, 161); B cells may decrease (64) or remain unchanged (161). In rhesus monkeysinfected with lethal P. knowlesi, a markeddecrease in the number

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CELL-MEDIATED IMMUNERESPONSESTO MALARIA 371 of T cells, and somedecreasein B cells, wasobservedin peripheral blood (143). Complexchangesreported in rodents infected with lethal and nonlethal parasites include increases in T and B cells, granulocytes,and especially mononuclearphagocytes in spleens of mice; these are more markedin nonlethalP. yoelii than in lethal P. bergheiinfections (55, 113). Increased numbersand activity of macrophagesin the bone marrowhave been described (54), andprotective immunityis correlated with peripheral blood monocytosis (84). Theseobservationsare consistent with the interpretation that duringmalariainfectionsTcells, andto a lesser extentB ceils, migrate fromthe peripheral bloodinto the spleen andliver, wherethey proliferate as a result of antigenic stimulation. Macrophage percursorsproliferate in the bone marrow,appear as monocytesin peripheral blood, and migrate into the spleenandliver; there mayalso be local proliferationof these cells. Death of Parasites Within Circulating Erythrocytes It is well knownthat degenerateformsof malaria parasites are observed inside red cells after chemotherapy. In 1944,Taliaffero &Taliaffero(141), studying Plasmodium brasilianum in Cebuscapucinus monkeys,noted the appearanceof "crisis forms"of parasites inside circulating red cells about the time that host immunitywasbecomingestablished. B. microti produces a high parasitemia in mice, followedby natural recoveryduring whichwe observed manycondensedforms of the parasite inside circulating erythrocytes (31). Systematicelectron microscopeobservationsconfirmedthat these weredegeneratingintra-erythrocytic parasites. Similar changeswere subsequentlyobservedin mice infected with P. chabaudiand P. vinckei. Degenerateforms are rare whenthe parasitemiais rising but predominate whenit is failing during natural recovery or protection by BCGor C parvum. In the absenceof evidencefor killing of these parasites elsewherein the body,it appearsthat the infections of miceare normallyterminatedby their death withinred cells. This implies that inhibition of merozoiteattachment to erythrocytes,andopsonizationof parasitized erythrocytesfor phagocytosis, cannot be major mechanisms of immunityto these parasites in mice. Similar degeneratingintra-erythrocytic parasites are seen duringrecovery from P. berghei infections in the rat (163), and the observations on brasilianum quoted above showthat the same is true of at least some primatemalarias. Since the sensitive stages of P. falciparumoccurin erythrocytes attachedto postcapillaryvenules,majoreffects of oxidantstress wouldbe expectedlocally rather than being manifestedin circulating erythrocytes.

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Role of Phagocytosis For a long time it wasbelievedthat phagocytosisof infected cells or free parasites was the principal mechanismby whichimmunitywas effeeted (140). Macrophagesof mice with P. berghei infections showedenhanced phagocyticactiv~ity towardinfected anduninfectederythrocytesin culture (136) and in millipore chambersimplantedinto the peritoneal cavity (37). Immuneserumincreases phagocytosis by macrophagesfrom animals with malaria (37) and normalanimals (24, 145). However,recovery fromnonlethai P. berghei and P. chabaudi does not correlate with increased phagocytosis(19, 87) but is coincident with the presence of degenerate parasites in intact erythrocytes. Thus,increased phagocytosisis certainly not the only mechanism of immunityin malaria, and often it appears to be secondaryto intra-erythrocytic death of parasites or extracellular lysis of infectedcells. The Spleen as an Effector

Organ in Immunity to Malaria

Arole of the spleen in resistance to malariaparasites has long beenrecognized. Thenarrowhost range of mostspecies of Plasmodiurn is partly due to the requirementfor erythroeytereceptors, an appropriateI-Ib type, and nutritional factors (99), but can in manycases be extendedby splenectomy (164). Examplesare the transfer of P. gallinaceumfromchickens into the Norwegian rat, P. vinckei andP. chabaudifromthe Africanthicket rat into the Norwegian rat, P. berghei fromthe Africanthicket rat into the rhesus monkey,and P. falciparum, P. vivax, and P. ovale from maninto the chimpanzee (164). In other cases, infection with a parasite highly virulent to the natural host can result in brief, low parasitemiain unnaturalhosts (e.g.P. falciparumin the owl monkey,Aotus trivirgatus). Splenectomy in the unnaturalhost mayconvertthe infection to a malignantcourselike that in the natural host (164). Theseobservationssuggestthat the spleenefficiently filters out parasitized cells as soonas they can be recognizedas foreign. Rat erythrocytes parasitized by P. berghei are morerapidly removedfrom the circulation into the spleen than are uninfected cells (116, 165). A uniquestructural feature of the spleen not foundin other lymphoidorgans, the red pulp, is likely to be the site wherethis filtration occurs(155). Asshownin Figure 3, arterioles openinto cords that are connectedwith sinuses. Themacrophage is the dominantleukocyte in the cords; blood mononuclearcells entering the cords througharteriolar terminationsappearto be selectively held in the interstices of the reticular meshwork. In other words,immigrant mononuclearcells with high capacity for a respiratory burst are concentrated in the post-arteriolar region, and parasitized erythrocytesentering

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through arterioles must pass through this region in close apposition to these effector cells, which can bombardthem with O~(Figure 4). If the parasitized erythrocytes are retained in the post-arteriolar region (for example, by binding of IgG antibodies on their surface to Fc receptors on the macrophages), the dose of O~they receive will be increased. High O2tensions in the post-arteriolar region facilitate O~formation. Erythrocytes or bacteria bearing small numbers of IgG molecules are removedfrom the circulation into the spleen but not into the liver or other organs (53). Hence, cells with high-atfinity receptors for IgG are concentrated in the spleen. Furthermore, as discussed below, immunecomplexes can activate O~ production by effector cells. Thus, synergistic effects of antibodies and cell-mediated immunitymight occur. Products of activated T-lymphocytescould stimulate the proliferation and recruitment of a subset of mononuclearcells with high capacity to produce O~. Antibodies of high affinity and appropriate isotype could facilitate binding of these effectors to target ceils and activate O~formation by the effectors. Thus, the initial effector mechanismwould be of antibody-dependent cellular cytotoxicity (ADCC)rather than NKtype, although the same effector cell might involved. A role of antibodies in recovery from primary infections with P. yoeliiin the mouseand P. gallinaceum in the chicken (119, 124) has been discussed above. In other infections, such as with P. chabaudi in the mouse, cellParasitized Antibody erythrocyte~ molecules

Splenic macrophages Figure4 Thespleniceffectorsystem.Parasitizederythrocytespassingbetween macrophages in the post-arteriolarregionof the red pulpare detainedandbombarded withO~.Antibody molecules onthe surfaceof parasitizedcells bindto Fc receptorsonthe macrophages and facilitatethis interaction.

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CELL-MEDIATED IMMUNERESPONSESTO MALARIA 375 mediated immunity canperform thisfunction efficiently intheabsence of specific antibodies, perhaps because theparasitized cells bind tothesurface of thecffcctors andactivate O~production byan antibody-independent mechanism. Thesecond filter system ofthercdpulpmustalsobcmcntioncd. Blood leaving thecordenters thelumen ofthevascular sinus bypassing between endothelial cells (155). Thisconstitutes a slit-like space, andcrythrocytes passing through itmust bcpliant. Ifthedeformability ofthecrythrocytcs isreduced, aswhenthey harbor malaria parasites, their passage isdelayed anda portion of thecellcontaining a rigid body, suchas a degenerate parasite, maybreak offandremain inthecords, justoutside thesinus. "Crisis forms" ofparasites persist inthecirculation ofsplcncctomizcd animals longer thaninanimals withintact spleens (7). Mononuclear Phagocytes as Splenic Effectors "Mononuclear phagocyte"is used broadly to cover phagocyticcells other than polymorphonuclearleukocytes. VanFurth and his colleagues (148) applied the designationto a single lineageof ceils originating in the bone marrowfrom commonstem cells (CFU-GM, precursors of granulocytes and monocytes)and differentiating into promonocytesand monocytes;the latter enter the circulating bloodand then tissues, becoming Kupffercells as wall as peritoneal, alveolar, andother resident macrophages. Notall authorities acceptthis widelyheld view.From3H-labelingstudies it has been concludedthat resident macrophageshave the capacity to proliferate locally, althoughthey can be replenishedfroma special subset of bone marrowprecursors (39, 149). Daems(38) drewattention to different pattern of peroxidaseactivity in monocytes and resident macrophages (Kupffer cells and peritoneal macrophages).In monocytes,the peroxidase reaction product is observedin cytoplasmicgranules (peroxisomes),whereasin the resident cells it is observedin the nuclearmembrane and endoplasmic reticulum. Thesubstrate specificity and thermalstability of peroxidasein the two cell types are different, and enzymeactivity is inhibited by aminotriazoleonly in the resident cells. Nocells with transitional characteristics, whichwouldsuggest that monocytescan become resident maerophages,were observed. Mononuelear calls in mice or guinea pigs elicited into the peritoneal cavity or lungs by C. parvumor BCG,whenappropriately stimulated, show muchgreater chemiluminescenceand HzO 2 production than do resident cells (17, 82, 106). Activationby lymphokines of mononuclear cells, so that they can kill tumorcells, red bloodcalls, and intracellular parasites such as Leishmaniais well documented (105, 118). Mononuclear cells recently recruited into the peritoneal cavity by lymphokines are muchmorecytolytic

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for tumorcells than are resident cells (98, 106). In the killing by mononuclear phagocytesof tumorcells (1, 125) and Leishmaniapromastigotesand amastigotes (67, 104), H202 appears to be a morepotent mediator than O: since cytotoxicity can be inhibited by catalase but not superoxidedismutase (SOD).Asolublecytolytic factor, believedto be a serine esterase, can act synergistically with H202in mononuclear cell-mediatedkilling of tumor cells (1). If mononuclearcells are able to function as NKor K cells in malaria, it wouldbe expectedthat they wouldbind to infected cells and that such bindingwouldactivate a respiratory burst. Preliminaryindications that this maybe the case havebeen published(95). Mouseperitoneal cells exposed to P. berghei-infectederythrocytesor lysates sometimesrespondedby luminol-enhancedchemiluminescence, and in the presence of immuneserumthe reaction wasconsistently observedand of increased magnitude. The situation in vivo is complex(68), and complementary information can be obtainedwith cells clonedin culture. Human mononuclear cell lines HL60(153) and U937(118), treated with phorbol myristate acetate (PMA) or lymphokines, differentiated into macrophage-type cells (phagocytic,with Fc receptors) that werecytotoxic for tumorcells. Thelymphokine triggering differentiation in these cells appearedto be distinct from colonystimulating activity (whichstimulates proliferation of CFU-GIVD, and cell lines with myelomonocytic markers did not acquire NKor ADCC activity whencultured with the samelymphokine.Oneinterpretation of these observations, consistent with that of Volkman(149) and Daems& De Bakker (39) mentionedabove,is that a lineage of mononuclear cells, distinct from immediateprecursors of monocytesand granulocytes, can be triggered by productsof activated T-lymphocytes to differentiate into macrophage-like cells with NKand ADCC activity. If these cells becomecytotoxic in the circulating blood before they becomephagocytic, they mightbe a major subset of cells with NKand ADCC activity. Sequestration of Parasitized Erythrocytes Erythrocytesbearingmalariaparasites are often differentially sequestered in vascular beds. This dependson preferential adhesionto different types of endothelialcells of parasitized erythrocytesat particular stagesof development.Thus, erythrocytes infected with P falciparum, P. fragile, or P. coatneyi adhere preferentially to vessels in heart and adipose tissue, and erythrocytes bearing P. falciparumadhere to cultured humanendothelial cells (147). Youngformsof P. bergheiare sequesteredin the deepvasculature of the heart andkidneyin the rat, whereasschizontsof P. bergheiare concentrated in the bone marrowin rats and in spleen and in the bone marrowin mice, but showno preferential localization in hamsters. In

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CELL-MEDIATED IMMUNERESPONSESTO MALARIA 377 venoussinuses of the bonemarrowof mice, Weiss(154) observedreticuloeytes parasitized with P. berghei NK-65adherentto endothelial cells and to nonparasitized retieulocytes. Hesuggests that merozoites mayenter adherentreticulocytes withoutsignificant extracellular exposure.Therelease of factors fromparasitized cells or lymphoeytesmightexplain the early retieuloeytosis observedin this disease before the hematocritfalls greatly; thus, newretieuloeytes wouldbe released in a site favorable for parasitization. Bythis anatomical arrangementthe spleen is largely avoided,and the parasitized cells are resistant to oxidantstress, so that cell-mediatedimmunityis less effective than in P. chabaudi,whichshows far less predilection for the bonemarrowand multiplies in mature erythroeytes. ,~ Peripheral Effector System The spleen is not the only site in whichimmunityto hemoprotozoais manifested.Splenectomized animalscan often recover fromthese infections (7). Asecondorgan that maybe involvedis the fiver, since migration mononuclear cells into the liver occurs duringinfection (42), andpartial hepateetomycan aggravate experimental malaria (20). However,an additional possibility is that effeetor cells can recognizemalaria-parasitized erythroeytesin the peripheral circulation (Figure 5). This mechanism might be especiallyeffective whensehizontsdevelopattachedto endothelialcells in postcapillaryvenules, as in the case of P. falciparum.Parasite antigens appear on the surface of infected erythrocytes only about the time of schizogony,so that infected erythroeytes becomerecognizableas foreign only at the stage of developmentwhenthey are secluded. Smallamounts of IgGantibodies on the surface of infected erythroeytes mightprovide a recognition mechanism throughFc-IgGreceptors on effector cells, but the possible existence of recognitionsystemsnot dependenton antibodies must also be consideredin the ease of someinfections. Theeffector cells in such a systemshouldbe present in the peripheral bloodand should haveFc-IgG receptors or bind malaria-parasitizedcells in the absenceof antibodies,and such binding should trigger O~production. NKcells are candidates, and their possible role in immunity to malariais consideredin the next section. NK and ,4DCC Activity in Immunity to Malaria In 1980, wesuggested that NKcells might play a role in immunityto malaria (46). Miceof the A/Hestrain were found to be moresusceptible to P. chabaudiinfections than other strains, and wefound two defects in them.First, the usual thymus-dependent increase in mononuelear cell number in the spleen of malaria-infected mice did not occur in A mice, as already mentioned.Second,miceof the A strain havea well-known defect

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Antibody-

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erythr0cyte"~’--~-~

Endothelial cell Figure 5 Theperipbera| d~’ector system. An erythrocyte parasitized with /~. falciparum adheresto a postcapil|ary venousendothdia|cell through surface knobs. Antibodymolecules binding to the knobsinteract with F¢ receptors on an effector ce|], triggering O~formation at the site of interaction.

of NKactivity, as measuredwith several target cells (68), and we found increase in NKactivity during the course of malaria infections in A mice

(46). Wood& Clark (159) have concluded that NKcells are irrelevant resolution of infections with P. vinckeipetteri and B. microti. Increased NK activity during the infections precededthe fall in parasitemia, and infections followed a normal course in 89St-treated mice and in bg/bg mice (see also 127). However,these criticisms are irrelevant if the subset of NKcells not depleted in bg/bg mice and sensitive to 89Sr (88) is that whichfunctions as an effector against blood-stage forms of hemoprotozoa. BCGcan augment NKactivity in bg/bg mice (11) and can protect mice other than the A strain against P berghei infections (103). Thus, a subset of NKcells unaffected by the bg/bg mutation and responsive to BCG,but defective in mice of the A strain, could participate in immunityto malaria. Genetic analysis of basal NKactivity against YAC-Itarget cells in backcross and congenic mice will likewise be uninformative. What is required is information about capacity of animals to increase NKactivity against relevant targets during the course of infection. Roder et al (125) have characterized cells from humanperipheral blood with capacity to kill K562and provided evidence that release of O~ is involved in the killing mechanism.The leukocytes were depleted of monocytes and were separated on a discontinuous Percoll gradient. More than 50% of the cells in the fraction with NKactivity expressed OKMIand HNK-1markers (the latter believed to be NKspecific) and Fc-IgG. contrast, ~ 0.5% of the cells expressed the Mo2marker believed to be specific for humanmonocytes,were phagocytic, or stained for acid esterase.

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CELL-MEDIATED IMMUNERESPONSESTO MALARIA 379 In the presenceof K562,but not insensitive targets, the NKcells released considerable amountsof O~, as shownby chemiluminescenceand cytochromec reduction, and SOD(but not heated SODor catalase) inhibited their capacity to kill K562cells. Additionalevidencethat human peripheral blood leukocytes with receptors for Fc-IgGand reacting with the monoclonal antibody OKM1 mediate NKactivity has been presented, and a clonedNKcell line, derivedfromthe "null cell" fraction of human peripheral blood, lacking T3, TS, B1, and Mo2markersand able to lyse K562, has beenobtained(69). Increased NKactivity of peripheral blood cells, assayed against K562 target cells, has beenobservedin childreninfectedwith P. falciparum(111). Thesera of mostinfected children were foundto haveantiviral activity, predominantlya-interferon. Addition of exogenousinterferon increased NKactivity of peripheral bloodcells in normalchildren whereasit hadno significant effect on cells frommalaria-infected children,suggestingprior in vivoactivationof the latter. WhenNKcells have Fc receptors they mayalso display ADCC activity, and an increase in this activity of humanperipheral blood cells during malaria infections has been described (64). Increased ADCC activity has likewise been observedin the spleens of mice during the second weekof P. chabaudiinfections (90). Theproperties of humanperipheral blood NKor ADCC cells, as defined above, wouldmakethemefficient peripheral effectors in malaria, and this possibility shouldbe tested directly by culturing suchcells with synchronized P. falciparumschizont-infected humanerythrocytes in the presence and absenceof immuneIgt~ and interferon. Surface markers defined by monoclonal antibodies, and tests for capacity to generateO~after binding to infected cells, shouldestablish the lineageof the cells andtheir capacity to produceoxidant stress. Likewise,the isolation of effector cells from spleens of infected mice, and similar tests with P. chabaudi-beadng mouse erythrocytes, should establish their lineage and capacity to generate O~, therebyimposingoxidantstress on the intra-erythroeytic parasites. ClaEsson& Olsson(25) have reported NKactivity in colonies of murine bone-marrow cells growingin semisolid agar. Thecolonies were distinct morphologically from typical macrophage and mixed macrophagegranulocytecolonies. Theseresults suggestthat certain bone-marrow cells, possibly distinct from monocyte-granulocyte precursors, are cytotoxic for susceptibletarget cells suchas syngeneicfibroblasts. Aworkinghypothesis is that this distinct cell type is the precursorof NKcells lacking the NK 1.2 markerand not depleted by SgSr, becausethe precursors either are relatively radioresistant or are presentalso in extramedullary sites. Characterization of this lineage of murineNKcells, and ascertainingwhetherthey

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are able, like humanNKcells bearing OKM and HNK-1markers, to release O~followingbindingto susceptibletargets, is a high priority task for the future.

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Soluble Factors Wehave recently reviewed the role of interferon (IFN) in immunity malaria (47), and only the principal findings are summarized here. IFN observedin the serumsoonafter P. bergheiblood-stageinfections, reaching a maximum at 35 hr (75). Injections of virus-inducedIFNslightly ddayed the rise of parasitemiaanddeath in this infection (133), whereasinjection of antibodiesagainstthis typeof IFNslightly acceleratedthe rise of parasitemia and death (129). These observations suggest a modestrole for IFN (t~ or/~) in retarding the proliferation of lethal P. bergheLIFNinducedby Sendaivirus hadno effect on blood-stageinfection with P. vinckei (31, 35), and potent antibodies against a- and B-IFNhad no effect on P. vinckei petteri or P. chabaudiinfections (31, 46). Hence,it is unlikelythat or~-IFNplays a significant part in these infections, whichdoes not exclude a possible role of lymphocyte-derived~-IFN. Several IFNinducers were foundto protect micesignificantly and sometimescompletdyagainst sporozoite-inducedinfections (77, 78), the protection being greatest against the late pre-erythrocytic (liver) stage. Theseeffects mayhavebeen due IFN-mediatedactivation of NKcells or macrophages. Emphasishas been placed recently on the role of lipopolysaceharide endotoxins(LPS)in malaria infections and the release of tumor-necrosis factor (TNF)from maerophages(3, 26, 93, 113). It is highly improbable that so chemically specialized a product as LPS(knownonly in certain gram-negativebacteria) should be present in plasmodia,and LPScould not be demonstratedin sonlcates of large numbersof P. berghei blood-stage parasites (49). Conceivably,LPSof bacterial origin mightplay a role in the immunopathology of somemalarial infections, but such a chance event makesit unlikelythat LPScontributessignificantly to the regular recovery of mice from infections with nonlethal malaria parasites and of humans living in endemicareas fromP. falciparum.TNFhas beenfound to inhibit the replication of P. falciparum in cultures of humanerythrocytes (C. Haidaris, D. Haines, M. S. Meltzer, A. C. Allison, unpublishedresults). Polyamine oxidase in the presenceof sperminehas the sameeffect, with the appearanceof degenerateparasites in the cells (A. Ferrante, C. P, zepczyk, A. C. Allison, unpublishedresults). Polyamine oxidaseis foundin activated maerophages (101). TNFactivity requires 02 and can be inhibited by metal chelation (128), whichsuggests it maybe a dioxygenase.A possible commonfactor in all of these mediatorsis the impositionof oxidantstress on parasitized erythrocytes.

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PRODUCTIONAND REACTIONS OF O~ AND H202 Whenneutrophils and macrophagesare stimulated by phagocytosis, there is a burst of increased metabolism by the hexose monophosphateshunt pathway, with the production of O~ and H202 (8). At the same time, chemiluminescenceis observed and can be amplified by luminol and other compounds(3, 82). Production of O~ can also be increased by immune complexes (156). In chronic granulomatous disease, production of O~ impaired (8, 134), and patients are susceptible to infections with organisms possessing catalase (96). O~ can interact with manybiological molecules (70, 130). Of special import is the interaction with redox-active metal ions, for example(70): +. Fe(II) + O~ ~ Fe(O~) The oxidative addition of O~ to Fe(II)EDTAin aqueous solution produces a ferric peroxo complex, Fe(III)EDTA(022-) (130). The formation of such powerfully oxidant complexesis relevant to iron-catalyzed lipid peroxidation and to inactivation of metal-containing or metal-activated enzymes considered below. O~ does not oxidize unsaturated lipids such as linoleic acid; however, this reaction can be catalyzed not only by iron, but also by lipid hydroperoxide(144). Thus, chain reactions, initiated by an iron oxidant system and perpetuated by hydroperoxide, can occur. Autoxidation of polyunsaturated fatty acids mayb¢ one of the principal reactions by which free radicals produce damagein biological systems. Somelipid hydroperoxides, e.g. those produced by the lipoxygenase and cyclo-oxygenasesystems, have potent pharmacological activity and may regulate biochemical pathwaysin parasites, as they do in other cell types. Exogenoushydroperoxides, such as t-butylhydroperoxide, can impose oxidant stress on cells by consuming reduced glutathione through glutathione peroxidase (52) and thereby oxidizing NAD(P)H.An example is the effect of t-butylhydroperoxide on Ca2+ retention by mitochondria (86). The importance of the conjugate acid of O~, the perhydroxyl radical (HO~), is nowbeing appreciated. In aqueous solutions, HO~is in equilibrium with O~ (12): O~ pKa = 4.8 HO~. As a consequence of this equilibrium, nearly 1%of any O~ formed under physiological conditions (,pH near 7.0) will be present as HO~.In acidic microcnvironmcnts (such as phagocytic vacuoles or where negatively

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chargedeffector and target cell membranes are closely apposed),the proportion of HO~will be muchgreater. In manycases, HO_"reacts orders of magnitudefaster than O~or is the sole reactant with a biological compound (e.g. vitaminE) (12). SinceHO~ is uncharged,it couldreadily diffuse hydrophobicregions of lipid bilayers or through membranes, whereaspassage of O~through membranes is confined to anion channels (89). Mostmetal ions and complexesare not oxidized by O~in aqueoussolution at rates competitivewith its disproportion, evenwhensuch oxidations are thermodynamicallyfavored (130). The probable explanation is that O~must first be coordinatedand reducedin the first coordinationsphere of the metal. Oxidationsby HO~ are not subject to the samerestriction and readily occur: for example,of aqueousFe(II) (130). WhenFe(II) is already coordinated, as in heine-containing enzymesand Hb, HO~will be much morelikely than O~to oxidize Fe. Fe can also catalyze the formationof highly reactive hydroxylradicals (OH’)from O~andH202,and lipid peroxidation by this mechanismcan inhibited by the iron chelator deferoxamine(66). EFFECTS

OF OXIDANT STRESS

Erythrocytes Theprincipal manifestationsof oxidantstress on erythrocytesare reduction in their content of reducedglutathione, lipid peroxidation, Hbdenaturation, andthe formationof inclusions knownas Heinzbodies. K+ is lost from the cells (51, 150), whicheventually lyse. Erythrocytesdamaged by oxidants but not lysed are rapidly removedfromthe circulation by the spleen and liver (9). Erythrocytesdeficient in glucose-6-phosphate dehydrogenase (G-6-PD-)are more sensitive than those with a normalcontent of this enzyme(G-6-PD+).Since activity of G-6-PD,SODand other protective enzymes (57) declinesas erythrocytesage (I0, 97), their sensitivity to dant stress increases with age. Agentsknownto produce oxidant damagein erythrocytes include phenylhydrazine (79) and alloxan, whichforms a redox system with dialudc acid (48, 126). In both cases productionof O~(57) andits interaction a hydrogenatom transferred from the organic molecule could generate powerfullyoxidant radicals (130). Damage by alloxan of pancreatic islet B-cells can be prevented by SOD,and is thought to be mediatedthrough O~production (62). Baehneret al (9) incubated G-6-PD-erythrocytes with normal human leukoeytesin whichthe respiratory burst had beentriggered by phagocytosis. Theerythrocytes were lysed, or whenlabeled and injected into human

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subjects were rapidly removed in the spleen and liver. Whenchronic granulomatous disease leukocytes were incubated with G-6-PD- erythrocytes, or normal leukocytes were incubated with G-6-PD+ erythrocytes, damagewas not seen. Hence, activation of a respiratory burst in leukocytes, with concomitant production of O~ and H202, can impose oxidant stress on erythrocytes. The relevance of this mechanismto immunity against malaria parasites will be evident. Asexual Forms of Malaria Parasites in Erythrocytes P. falciparum is microaerophilic: in cultures of humanerythrocytes, it growsbest at 3 %02 and it is inhibited by higher concentrations of 02 (132). However,there is a critical level (< 0.5%) below which the parasite will not survive, in contrast to true anaerobic forms of life. Fromthese observations and the lack of a Pasteur effect, Scheibel &Adler (131) concludedthat in the malaria parasite 02 maynot play the same role in electron transport as it does in many mammaliancells, and they suggested that 02 mayact through a distinct type of metalloprotein oxidase enzymesystem. They found that the metal chelators diethyldithiocarbamate, 8-hydroxyquinoline, and 2-mercapto-pyridine-N-oxideinhibit P. falciparum replication in culture (131). This effect was attributed to favorable lipid-water partition coefficients and binding constants allowing inhibition of metalloprotein oxidases. The importance of metalloenzymesin protozoan parasites is illustrated by the iron-sulfur ferredoxin system of anaerobe Tritrichomonas, which confers sensitivity to oxidant damageas well as to drugs such as metronidazole (102). The existence of such an enzymesystem in Leishmania has been postulated on the basis of inhibitory effects of redox-active dyes (117). Although suggestions that hemoprotozoa and leishmania have metalloprotein oxidases are stimulating, their existence is at present conjectural. Metal chelators could have other effects: for example, hemeis boundto at least three soluble proteins in P. falciparum(51). In addition to functioning as the active site of enzymesand O2-combiningsites of Hb and myoglobin, heine is knownto have major regulatory effects, including activation of a factor (eIF2cr) required for the initiation of protein synthesis (110) and guanylate cyclase (76). Thus, hemecould participate in the regulation metabolism in Plasmodiumparasites. Chloroquine is thought to function as a heine chelator (51), and other metal chelators mayalso bind heine. Etkin & Eaton (45) presented evidence that in the erythrocytes of mice infected with P. berghe£H202is generated and that the ability of infected cells to prevent or repair oxidant damageis decreased. They observed in P. berghei-infected mice an increase in steady-state metHbconcentration

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directly proportionalto parasitemia. Catalasein infected erythrocyteswas inhibited by 3-amino-l,2,4-tfiazole (an inhibition requiring the presence of H202),and infected erythrocytes accumulatedmore metHbthan uninfected cells whenexposedto oxidants generatedby ascorbic acid. In vitaminE-deficient mice, P. berghei infections were found to developmore slowlythan in normalmice(43). In the vitaminE-deficient mice, parasites were found only in reticulocytes and anemiawas marked,whichsuggests protection was mediated by premature lysis of oxidant-sensitive erythrocytes. Inhibition of catalase by aminotriazolehas also been used by Friedman (61) as a measureof H202production by P. falciparumcultured in human crythrocytcs. Thecatalasc inhibition wasfound to be proportional to the partial pressure of 02 in the culture mediumover the range 10-30%. Thesensitivity of malariaparasites to oxidantstress is emphasized by the damagingeffect on intra-erythrocytic parasites of agents that exert such stress on erythrocytes.In 1950,Rigdonct al (121)reported that administration of phenylhydrazincto rhesus monkeysinfected with P. knowlesi resulted in the appearance of degenerate forms of the parasite within circulating erythrocytes.His finding attracted little attention andremained withoutexplanationfor 30 years. Ourinterest in this subject arose fromthe observationthat incubationof mousecrythrocytesinfected with P. chabaudiin the presenceof a dioxygcnase enzymesystem generating H202and other products (bovine serum polyamincoxidaseand spermine)abolishedtheir infectivity (100). Clark Hunt(30) injected alloxan intravenouslyinto miceinfected with P. vinckei and observeda fall in parasitcmiaand the appearanceof degenerateparasites within circulating crythrocytes; this effect waspreventedby prior injection of the selective iron chelator deferoxamine,or the less selective metal chelator dicthyldithiocarbamatc (DDC). Oxidanteffects of t-butylhydroperoxideon erythrocytcs have been described(35, 137, 146). Wehavefoundthat injection of alloxan or t-bntylhydroperoxidcproducesa dramaticfall in parasitemiain miceinfected with P. chabaudior a lethal strain of P. yoelii (17XL),with the appearance crisis formsof parasites in circulating erythrocytes(Figure 6). Thetreatmentdid not kill all parasites, and transient parasitemiarecurred afterwards; however,most animals recoveredand wereresistant to challenge, showingthat exposureto t-butylhydroperoxidedid not interfere with the inductionof protective immunity against the parasite. Again,prior administration of deferoxamineor DDC preventedthe effect of t-butylhydroperoxide on the parasite. Whyasexual formsof malaria parasites in erythrocytes are sensitive to oxidant stress is at present unknown.Conceivably,the primarymetabolic effect of oxidantsis on the host erythrocyte,with secondaryeffects on the

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CELL-MEDIATED IMMUNERESPONSESTO MALARIA 385 ÷

+

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LU 4(:

rr ~-

20

4

12

8

DAYS

AFTER

16

20

24

INFECTION

Figure 6 Effect of t-butylhydroperoxide on the course of P. yoelii 17XLinfections in male C57B1/6mice. In control animals (. l, parasitemia increased progressively and all died. Followinginjections of t-butylhydroperoxide (0.2 ml of a 1%solution), parasitemia decreased. In treated animals with low initial parasitemia (O), a moderateparasitemia recurred, but all six recovered. In animals with higher initial parasitemia, severe parasitemia recurred after treatment, and three out of five mice died (1).

parasite. For example,oxidant drugs produceloss of K+ fromerythrocytes (48, 150), and this could reducetheir capacity to supportparasite multiplieation (60). However,the inhibition by DDC as well as deferoxamine oxidantdamageto plasmodiaraises the interesting possibility that a metalloenzyme (e.g. Fe-Cuor Fe-Mo)is irreversibly damaged by strong oxidants; reversible binding of deferoxamineor DDCduring the period of exposure to a strong oxidant could protect the enzymefrom permanentdamage. Whatever the explanation,oxidanteffects are likely to be greater on parasites multiplyingin matureerythrocytesthan in reticulocytes. Thedegenerationof parasites in circulating erythrocytesafter exposure to oxidantsis closely analogousto the crisis formsobservedduringrecovery frommalariainfections as a result of cell-mediatedimmunity.A reasonable conclusion is that a major etfector mechanism of cell-mediated immunity is the impositionof oxidantstress on parasitized cells.

WORKINGHYPOTHESIS Fromavailable evidence, it is possible to formulatea workinghypothesis about immunityto asexual blood forms of malaria parasites, bearing in

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mindthat this is a framework for future research rather than a definitive statement(Figure 7). Parasite antigens e~ciently stimulate a subset of lymphocytes(Lyl+ in the mouse)¯Presumably,the antigens are presented to T cells by In-bearing cells such as splenic dendritic cells (107) and Langerhanscells¯ Clonesof T cells with receptors for parasite antigens proliferate in the spleen andother lymphoidorgans, andcirculating T cells are diverted to the spleen andliver. T cells contribute to immunity against malariaparasites in two ways.The formationof antibodies against parasite antigens requires T cell help, and such antibodies are necessary for recovery fromprimaryinfections with someparasites, e.g.P, yoelii in the mouseand P gallinaceum in the chicken. Second,activated T lymphocytesrelease factors stimulating the proliferation of mononuclearcells with high capacity to synthesize O~. Proliferation of the samesubset of cells can be stimulated by C parvum independentlyof T-lymphocytes.Whenthese cells are released into the peripheral blood they havenot yet acquiredphagocyticcapacity, but have Fc-IgGreceptors and can be triggered to produceO~by binding to target cells, thereby exerting NKand ADCC activity. Onesuch target is erythrocytes bearing P. faleiparumschizonts attached to postcapillary endothelial cells; antibodiescombining with parasite antigens on the surface of infectederythrocytesfacilitate suchbindingandactivationof effector cells. Thesamesubset of T-lymphocytes activated by parasite antigens releases a factor chemotacticfor the O~-producing subset of mononuclear cells. The latter migrateselectivelyinto sites suchas the post-arteriolar regionof the red pulp of the spleen, wherethey comeinto close contact with malariaparasitized erythrocytes, bind to their surfaces, and release O~. Malariaparasites in erythrocytesare sensitive to oxidantstress. Theyare relatively resistant to H202,whichcan actually be producedin parasitized cells¯ Theyare moresensitive to O~, perhapsin the form of HO’, 2 and are highly sensitive to radicals producedby interaction of O: and organic compoundssuch as phenylhydrazine. Oxidants produce K~ leakage from cells, whichis unfavorablefor parasite growth,and the parasites mayhave a metalloproteinelectron transport system that is inactivated by strong oxidants. It followsthat cells that exert cytotoxiceffects throughrelease of O~,such as NKcells; maybe moreefficient as effector cells against malaria-parasitized erythrocytes than are monocytes,whichrelease H202.Subsets of NK cells resistant to the bone-seeking isotope sgSr and unaffectedby the beige mutation in mice, and bearing the HNK-1 and OMK1 markersin humans, could be importantin immunity to malaria. Miceof the Astrain are highly susceptible to malaria and are unable to recruit the mononuclearcells required as effectors of cell-mediatedimmunity.Aspeculative interpretation is that a distinct lineage of macrophage precursorsin the bonemarrow

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can be seededout to the spleen, peritonealcavity, andother sites. In these locations they are still able to respondto lymphokines and other stimuli by proliferation, so that they are not bone marrowdependent(and therefore insensitive to 89Sr). Promacrophages of this lineage in the circulation are termedNKcells. Whenthey settle downin the spleen and other sites they becomephagocytic,this differentiation being promotedby lymphokines(as in the U937cell line). This lineageof cells, characterizedby peroxidasein the endoplasmicreticulum and high O~production, is an importantmediator mcell-medmted~mmumty to malaria and other mfect~ons,whereascells of the promonocyte-monocyte lineage, with peroxidase in peroxisomes,are not. Malaria-parasitizederythrocytesadhereto endothelialcells in characteristic patterns. Schizontsof P. berghei in the mouseadhere to sinus endothdial cells in bonemarrowand merozoitesenter reticulocytes adherentto schizonts, whichmakesthemrelatively resistant to immuneattack. P. falciparurn schizonts completetheir replication attached to postcapillary endothelial cells, whereoxygentensions are low. Underthese conditions, P. falciparumdoes not multiply well in human erythrocytes bearing HbS(58, 112), whichmayexplain the relative resistance of carders of this trait to this parasite (4, 5). P. falciparumgrows poorly in G-6-PD-and ~-thalassemicerythrocytes whenexposedto oxidant stress (59). Suchstress wouldbe minimalwhenparasite replication completedin postcapillary venules, but wouldbe increased if adherent effector cells released O~. Thus, resistance due to inherited erythroeyte defects could be reinforced by cell-mediated immunity,increasing the chancesof survival of childrenbearingthese traits duringthe critical years of first exposureto malaria. Theexperimentsreviewedsuggest that immunitybased solely on T cell and macrophage activation maynot be sterile. Presumably,someparasites escapefromoxidantdamage.As the parasitemiafalls, the T cell andmacrophageactivation decline. If the parasitemiarises (frompersistent parasites or reinfection), memory T-lymphocytesare able to respond quickly and immunityis reactivated. This is the basis of premunition(135), which observedin infections wherecell-mediated immunityplays a predominant role. Human malaria infections mayfall into this category, although interactions of cell-mediated and humoralimmunityare likely to promote survival of hosts undernatural conditions. Parasite survival dependson the suppressionof immune responsesagainst antigens essential for its replication, such as receptors on merozoitesfor erythroeytes.Thatis the pdneipal reason whyimmunityto malaria is not moreeffective, requiring many infections over a period of several years, and rather easily lost following chemotherapyor residence in a non-malariousregion.

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ACKNOWLEDGMENTS We are indebted to J. Grun and W. Weidanz, and to L. Weiss, for permission to reproduce Figures 2 and 3, respectively.

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24. Chow,J. S., Kreier, J. P. 1972. Exp. ParasitoL31:13-18 25. Clai~sson,M.H., Oh;sen,L. 1980. Nature 283:578-80 26. Clark, I. A. 1978. Lancetii:75-76 27. Clark,I. A., Allison,A. C. 1974.Nature 252:328-29 28. Clark,I. A., Allison, A. C., Cox,F. E. G. 1976. Nature 259:309-11 29. Clark, I. A., COx,F. E. G., Allison, A. C. 1977. Parasitology74:9-17 30. Clark, I. A., Hunt,N. H. 1983. lnfect lmmutt 39:1-6 31. Clark,I. A., Richmond, J. E., Willis, E. J., Allison, A. C. 1977. Parasitology 75:189-96 32. Clark,I. A., Virelizier,J. L., Carswell, E. A., Wood,P. A. 1981. lnfect. 1mmug 32:1058-66 33. Cohen,S., McGregor, I. A., Carrigton, S. 1961. Nature192:733-37 34. Coleman,R. M., Bruce, A., Renerieea, N. J. 1976.J. Parasitol. 62:137-46 35. Corry, W.D., Meiselman,H. J., Hoehstein, P. 1980. Biochim.BiophyxActa 597:224-34 36. Cox,H. W.1964.Z Parasitol. 50:23--29 B. S., Butler, W.T., Rossen, 12. Bielsky, N. 1983. In SuperoxideandSu- 37. Criswell, R. D., Knight, V. 1971. J. Immuno£ peroxide Dismutase,ed. G. Cohen,In 107:212-21 press. NewYork: Raven 13. Bradley,D.J., Taylor,B. A., Blaekwell, 38. Daems,W.T. 1980. In TheReticuloen. dothelial System, A Comprehensive J., Evans,E. P., Freeman, J. 1979.Clin. Treatise, ed. I. Cart, W. T. Daems, Exp. lmmunol. 37:7-14 1:57-75. NewYork: Plenum 14. Brown, I. hi., Allison,A. C., Taylor,R. 39. Daems,W.T., De Bakker, J. M.1982. B. 1968. Nature 219:292-93 lmmunobiology161:204-11 15. Brown,K. N. 1971. Nature 30:163-65 40. Diggs,C. L., Osier, A. G. 1969./. lm16. Brown, K. N. 1977. lmmun. Parasig munol. 102:298-305 Dig Colloq. 1NSERM Paris 72:46-58 41. Diggs,C. L., Osier, A. G. 1975. Z Im17. Bryant,S. M., Lynch,R. E., Hill, H. R. munoZ114:1243-47 1982. Cell lmmuno£69:46-58 18. Butcher,G. A., Mitchell,G. H., Cohen, 42. Doekrell,H. M., DeSouza,J. B., Playfair, J. H. L. 1980. Immunology41: S. 1978. Immunology34:77-86 421-30 19. Cantrell, W.,Elko, E. E., Hopff,B. M. 43. Eaton,J. W.,Eckman, J. R., Berger,E., 1970.F, xp. ParasitoL28:291-97 Jacob, H. S. 1976. Nature 264:758-60 20. Cantrell, W.,Moss,W.G. 1963.,/.. ln44. Eling, W.M. C. 1982. In lmmuneReac~c~ Dig 112:67-71 tions to Parasites, ed. W.Frank, pp. 21. Cat, r, R., Diggs,C. L. 1977.In Para141-56.Stuttgart: GustavFischer sitic Protozoa,ed. J. P. Kreier,I 11:359466. lqew York: Academic 45. Etkin, N. L., Eaton,J. W.1975.In Ery22. Carter, R., Gwadz,R. W., Green, I. throcyte Structureand Function,ed. G. 1979. Exp. ParasitoL47:194-208 J. Brewer,pp. 219-34.NewYork: Liss 23. Chen, D. E., Tigdar, R. E., Weinbaum, 46. Eugui,E. M., Allison,A. C. 1980.ParaF. I. 1977. ~. ImmunoL118:1322-27 site ImmunoL2:277-92

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ters, J. H., Davies,A.H., Pearson,F. A. 1956. BrR Med. Z 2:686-92 93. MacGregor,R. R., Sheagren, J. N., Wolff, S. M. 1969. J. lmmunol. 102:131-38 94. Maegraith,B. G. 1974. In Medicinein the Tropics, ed. A. W.Woodruff,pp. 27-73. Edinburgh: Churchill Livingstone 95. Makimura,S., Brunkmann,V., Mossmann,H., Fischer, H. 1982. lnfec~ lmmun. 37:800-4 96. Mandell, G. L., Hook,G. W. 1969. Z Bacteriol. 100:531-32 97. Marks,P. A., Johnson,A. B., Hirsehberg, E. 1958. Proc. NatLdcad. Sc£ USA44:529-32 98. Meltzer, M.S., Ruco,L. P., Boraschi, D., Naey, C.26:403-15 A. 1979. Z Reticuloendothel. Soc. 99. Miller, L. H., Carter, R. 1976. Exp. ParasitoL40:132-46 100. Morgan,D. M. L., Christensen,J. R., Allison, A. C. 1981. Biochem. Soc. Tram 9:563--64 101. Morgan, D. M.L., Ferluga,J., Allison, A. C. 1980. In Polyaminesin Biomedical Research,ed. J. Gaugas,pp. 303-8. NewYork: John Wiley 102. Miiller, M.1981.Scand.Z Infec~ Dis. 26 (Suppl.):31-41 103. Murphy,J. R. 1981. lnfect, lmmun. 33:199-211 104. Murray, H. W. 1982. Z Immunol. 129:351-54 105. Murray,H. W., Masur,H., Keithly, J. S. 1982. J. Immunol.129:344-50 106. Nathan, C. F., Root, R. K. 1977. Z Exp. Med. 146:1648-62 107, Nusscnzw¢ig,M.D., St¢inman,R. M., Gutehinkov,B., Colin, E. A. 1980. J. Exp. Med. 152:1070-84 108. Nussenzweig, R. S. 1967. Exp. Parasitol. 21:224-31 109. Nusscnzweig,R. S., Vanderberg,J., Most, H., Orton, C. 1969. Nature 222:488-89 110. Oehoa,S. 1981. Eur. J. Cell. Biol. 26:212-16 111. Ojo-Amaize,E. A., Salimonu,L. S., Willimns,A. I. O., Akinwolere,O. A. O., Shabo,R., Aim,G. V., Wigzell,H. 1981. Z Immunol.127:2296-300 112. Pasvol,G., Wetherall,D. J., Wilson,R. J. M. 1978. Nature274:701-3 113. Playfair, J. H, L. 1982.BrRMed~Bull. 38:153-59 114. Playfalr, J. H. L., DeSouza,J. B., Cottrell, B. J. 1977. Immunology32: 681-87 115. Potocnjak, P., Yoshida, N., Nussen-

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zweig,R. S., Nussenzweig, V. 1980. J. Exp. Med. 151:1504-13 116.Quinn,T. C., Wyler,D.J. 1979.J. Clin. lnvest. 63:1187-94 117. Rabinovitch,M., Dedet, J. P., Ryter, A., Robineaux,R., Topper,G., Brunet, E. 1982. J. Exp. Med.155:415-31 118. Ralph, P., Williams,N., Moore,M.A. S., Liteofsky, P. B. 1982. Cell. lmmunoL71:215-23 119. Rank, R. G., Weidanz, W. P. 1976. Proc. Soc. Exp. BioL Med. 151:257-59 120. Rener, J., Carter, R., Rosenberg,Y., Miller, L. H. 1980. Proc.Natl. ,4cad. ScL USA77:6797-99 121. Rigdon,R. H., Micks,D. W., Breslin, D. 1950. Am. Z Hyg. 52:308-22 122. Roberts, D. W., Rank,R. G., Weidanz, W.P., Finerty, J. F. 1977.lnfec~ lmmun. 16:821-26 123. Roberts, D. W., Weidanz,W.P. 1978. Infect. lmmun.20:728-31 124. Roberts, D. W., Weidanz,W.P. 1979. Am. Z Trop. Med. Hyg. 28:1-3 125. Roder, J. C., Helfand,S. L., Werkmeister, J., McGarry, R., Beaumont, T. J., Duwe,A. 1982. Nature 298:569-72 126. Rose, C. S., Gytirgy, P. 1952. Am;J. Physiol. 168:414-20 127. Ruebush,M.J., Burgess,D. E. 1982.In NKCells and Other Natural Effector Cells, ed. R. Herberman,pp. 1483-89. NewYork: Academic 128. Ruff, M, R., Gifford, G. E. 1981. In Lymphokines,ed. E. Pick, 2:235-72. NewYork: Academic 129. Sauvagier, F., Fauconnier, B. 1978. Biomedicine29:184-87 130. Sawyer,D. T., Valentine, J. S. 1981. Acc. Chem;Rez 14:393--400 131. Scheibel, L. W., Adler, A. 1980. Mol. Pharmacol.18:320-25 132. Scheibel,L. W., Ashton,S. H., Trager, W.1979. Exp. Parasitol. 47:410-18 133. Schultz, W.W., Huang,K. Y., Gordon, F. B. 1968. Nature 220:709-10 134. Segal, A. W., Allison, A. C. 1979. In OxygenFree Radicals and Tissue Damage, Ciba Found. Symp., 65:205-19. Amsterdam:Exerpta Medica 135. Sergent, E. 1963.In ImmunitytoProtozoa, ed. P. C. C. Garnlmm,A. E. Pierce, I. M.Roitt, pp. 39-47.Oxford: BlaekwellSci. 136. Shear, H. L., Nussenzweig, R. S., Bianco, C. 1979. J. Exp. Med. 149: 1288-98 137. Srivastava,S. K., Awasthi,Y. C., Beutler, E. 1974. Biochem.J. 139:289-95 138. Steehschulte,D. J. 1969.Proc.$oc. Exp. BioL Med. 131:748-52

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lanr, R. E. 1976. ,L Immuno£117: 139. Stevenson, M. M., Lyang,J. J., Ska1999-2005 mene, E. 1983. Z ImmunoLIn press 153. Weinberg,J. B. 1981. Science 213: 140. Taliaferro, W. H. 1929. The Im655-57 munologyof Parasite Infectiong New 154. Weiss,L. 1983.J. Parasito£In press York: Century 155. Weiss,L. P. 1979.In Role of the Spleen 141.Taliafcrro, W.H., Taliaferro, L. in the Immunology of ParasiticDiseases, 1944. J. Infec~ Dig 75:1-17 ed. G. Torrigiani, pp. 6-20. Basel: 142. Taylor, D. W.,Bever,C. T., Rollwagen, Schwabe F. Evans,128:1854-59 C. B., Asovsky,R. 1982. 156. Weiss, S. J., Ward,P. A. 1982. Z ImJ. M., ImmunoL munol. 129:309-13 143. Taylor, D. W., Crum,S. R., Kramer,K. 157. Wilson, R. J. M. 1977. In Immunityin J., Siddiqui, W.A. 1980. lnfecL ImParasitic Diseases, Colloq. INSERM mun. 28:502-7 Paris 72:87-101 144. Thomas,M. J., Mehl,K. S., Pryor, W. 158. Wood,P. R., Clark, I. A. 1982. lnfecg A. 1982. J. Bio£ Chem.257:8343-47 Immun~35:52-57 P. R., Clark, I. A. 1982.Parasite 145. Tosta, C. E., Wedderburn,lq. 1980. 159. Wood, Clin. Exp. ImmunoL42:114-20 Immuno£4:319-27 146. Trotta, R. J., Sutlivan,S. G., Stern, A. 160. Woodruff, M. F. A., Warner, N. L. 1982. Biochem.J. 204:405-15 1977, I. Natl. Cancerlns~58:111-16 147. Udeinya,I. J., Schmidt,J. A., Aikawa, 161. Wyler,D. J. 1976. Clin. Exp. lmmunoL 23:471-76 M., Miller, L. H., Green, I. 1981. 162. Wyler,D. J., Gallin, J. I. 1977.J. ImScience 213:555-57 munoL118:478-84 148. VanFurth, A., Cohn,Z. A., I-Iirseh, G., Humphrey,J. H., Speeto_r,,,W.~ 163. Wyler,D. J., Miller, L. H., Sehmidt,L. H. 1977. J. InfecL Dig 135:86-93 Langevoont, H. L. 1973. But~ Hlth. Org. 46:845-50 164. Wyler,D. J., Oster, C. N., Quinn,T. C. 1979, In Role of the Spleenin the Im149. Volkman,A. 1976. J. Reticuloendotl~ munologyof Parasitic Diseases, e~.. Soc. 19:249-68 G. Torrigiani, pp. 183-204. Basel: 150. Weed,R. I. 1961. J. Clin. lnvest. Schwabe 40:140-43 D. J., Quinn,T. C., Chert, L. T. F. I., Evans, C. B., Tige- 165. Wyler, 151. Weinbaum, 1981. J. Clin. Inves~67:1400-4 laar, R. E. 1976. J. lmmunol. 116: F. I., Herrod, 166. Wyler,D. J., Weinbaum, 1280-83 H. R. 1979. J. lnfect Dis. 1~0:215-21 F. L, Evans, C. B., Tige152. Weinbaum,

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BIOSYNTHESIS AND REGULATION OF IMMUNOGLOBULINS Randolph Wall MolecularBiology Institute and Departmentof Microbiologyand Immunology, University of California Schoolof Medicine,Los Angeles,California 90024

Michael Kuehl National Cancer Institute-Naval MedicalOncologyBranch, National Naval Medical Center, Tower1, Room415, Bethesda, Maryland20814

INTRODUCTION Immunoglobulingene expression exhibits a numberof intriguing regulatory features. Immunoglobulin genes undergo dynamic DNArearrangements and somatic mutations. The expression of immunoglobulin genes exhibits allelic exclusion and isotypic exclusion. The developmentalregulation of immunoglobulingene expression during B cell differentiation is characterized by (a) the differential onset of heavy and light chain production, (b) great changesin the level of expression, and (c) transition from the insertion of immunoglobulinas antigen receptors in the lymphocytemembraneto its active secretion. Darnell (40, 41) and Brown(27) have recently reviewed the variety transcriptional, post-transcriptional (e.g. RNAprocessing), and translational mechanismsemployedin the regulation of eukaryotic gene expression. This review summarizes recent progress towards understanding the molecular mechanisms in immunoglobulin gene expression and regulation in the context of the developmentsin other eukaryotic genes. The complex array of lymphoid cell and lymphokinefactor interactions that affect immunoglobulin gene expression are not considered here. 393 0732-0582/83/0410-0393502.00

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IMMUNOGLOBULINSTRUCTURE Most immunoglobulinmolecules consist of two identical light (L) chains and two identical heavy (H) chains (74). ImmunoglobulinL and H chains are encodedby three unlinked gene families--one each for K and ~ L chains, and one for H chains--which are present on mousechromosomes6, 16, and 12, respectively (reviewed in 43). Each chain is composedof a series homologousdomains, the NH2-terminal domain is the variable (V) region and the remainder form the constant (C) region. The combination of H and L-chain V regions are responsible for antigen binding and may include thousands of different sequences. In contrast to V regions, the C regions have only a few alternative sequences. The various classes (IgM, IgD, IgG, IgE, IgA) and subclasses (e.g. IgGi, IgG2, IgG3, IgG4) of immunoglobulins with different biological functions are distinguished by different heavy chains (~, ~, "g, e, tz) defined by their C regions mC8, C~,, C~ , C~). Al classes of immunoglobulincontain light chains of either r or ~ isotype (containing C~ or Cx regions). The immunoglobulin(Ig) molecule can exist in two very different environments: in the lymphocytecell membraneas a surface antigen receptor, and in the circulation as a secreted antibody. For most immunoglobulin classes, the Ig molecules are secreted as four-chain disulfide-linked monomers (H2L2). Howeverfor IgM and IgA, the heavy chains carry a short COOH-terminalsequence, which permits polymerization of the monomers. These polymeric immunoglobulin molecules are covalently linked by disulfide bonds from their H-chain COOH-terminalsegments to a single J (or joining) chain molecule per IgMor IgA polymer (85, 100). Thus, is secreted as a pentamer (/ZELE)sJ and IgA can be secreted as a monomer (a2L2), dimer (a2L2)2J, or trimer (cr2L2)3J. Immunologistshave long knownthat the proliferation and maturation of B lymphocytes in response to antigen (and perhaps other external signals such as anti-idiotypic antibody) is triggered by binding of antigen to membrane Ig displayed on the surfaces of B cells. It is likely that all classes and subclasses of Ig can exist in a membraneform as well as a secreted form. In contrast to secreted Ig molecules, all membraneIg molecules, including IgM and IgA, are present as monomericstructures (H2L2) (110). Studies from a number of laboratories have demonstrated that membrane ~ (/zm) and secreted /z (/.tO chains have different molecular properties. wouldbe expected for integral membraneproteins, /.t m chains exhibit hydrophobic behavior (123, 176). Protein and nucleic acid structural studies demonstrate identical sequences up to their COOH-terminalsegments, but unique COOH-terminalsequences for both/-ts and #mH chains (2, 48, 77, 136, 196). Morerecent studies have showndifferent membraneand secreted

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species for murineY3, Yl, Y2a, "~2b, 8, and a H chains (58, 82, 88, 97, 102, 112, 116, 121, 159, 188). The COOH-terminalsequences of the membrane forms of y3, y~, Y2a, Y2b,8, and t~ H chains have been inferred from nucleic acid sequences of eDNAor genomic DNAclones (33, 125, 140, 171, 189, 193). The COOH-terminalsequence of the membraneforms of H chains includes (a) an acidic segmentof 12-25 aminoacids, which has a net charge of- 6; (b) a highly conserved sequence of 26 aminoacids, which is designated the hydrophobic segment and is thought to anchor the H chain into the plasma membrane;and (c) an intracellular segment of variable length, which begins with the residues Lys-Val-Lys. The transmembrane peptide sequences of all H chain M exons show an unexpectedly high level of sequence conservation. This sequence conservation is the basis of a twochain model for transmembraneinsertion of surface immunoglobulins(136, 140). The short, positively charged intracellular sequence constitutes the complete intracellular segmentof/.t and ~ H chains, whereas2’1, )’2b, and 72a (and presumably~/3) H chains have conserved intracellular segments 28 residues, which include the Lys-Val-Lyssequence. It is not knownif the different intracellular segmentsexert different activation functions in cells expressing membraneIgG or IgA as compared to cells expressing membrane IgM or IgD.

DIFFERENTIAL IMMUNOGLOBULIN EXPRESSION DURING B-LYMPHOCYTE DEVELOPMENT The developmental stages in B-lymphocyte development into Ig-secreting plasma cells are distinguished by differences in the organization of H and L-chain genes, the kinds of Ig chains expressed, and the levels of Ig gene expression (see Figure 1). It appears that a pluripotential hematopoietic stem cell generates lymphoid stem cells that subsequently generate pre-B cells, i.e. the first stage of B cell development in whichcells express immunoglobulin chains (29, 95, 154). As a lymphoidstem cell differentiates to pre-B cell, there is/z H chain gene rearrangement, transcription, translation, and expression of cytoplasmic # H chains, but no expression of L chains. Early pre-B cells have not rearranged their L-chain genes, but are proliferating cells presumedeventually to rearrange and express different L-chain genes together with the H chain to which the pre-B cell is already committed(29, 95, 134b). A possible late pre-B cell developmentalstage suggested by several recent examplesof pre-B-cell lines that have functionally rearranged r L chain genes, but do not transcribe L chain mRNA until the cells are stimulated by bacterial lipopolysaccharide (101a, 128, 142a). This presumedlate pre-B-cell developmentalstage (i.e., with L chain DNA rearrangementbut no L chain expression) has not been clearly identified in

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normalcell populations and mayrepresent a transient stage of normalB cell development. Asthe pre-Bcell developsto an immatureB cell, L chains are expressed and IgMmonomers(H2L2)are displayed on the cell surface as antigen receptors (177). TheimmatureB cell maturesto a B cell that co-expresses IgMand IgD on the B lymphocytesurface (57). Co-expressed ~ and chains in IgMand IgD, respectively, have the sameV region, and their expressionexhibits alldic exclusion (36, 64, 133). Upto this stage, lymphocytedevelopmentis antigen independent,although immatureB cells that express surface IgMare presumablycapable of being affected by contact with antigen or anti-idiotypic antibody. Maturationbeyondthe B cell stage is thoughtto require activation by mitogensor an appropriatecombination of antigen, T cells or T cell factors, andmacrophages (100a, 109a). Theactivated B cell that secretes significant amountsof IgMmaturesinto either a memory B cell that continues to express surface Ia antigens but expresses a newisotype of surface immunoglobulin (Ig~, IgE, or IgA), a terminallydifferentiated plasmacell that no longer expressessurface Ia antigens but expresseslarge quantities of secreted IgM,IgG, IgE, or IgA with little if anysurface Ig (190, 122). Antigenicstimulation of memory cells generates plasmacells secreting IgG, IgE, or IgA. Either memory B cells or plasmacells can express immunoglobulin isotypes other than IgMor IgD. Theexpressionof different H chain C regions (Cr, C,, or Ca) occurs with the sameV region associated with C~in IgM on the surfaceof immature B cells andis a result of a processcalled Hchain "class switching."Heavychainclass switchingis thoughtto be an antigenand T cell-dependent event mediatedby DNA rearrangementsthat delete the Ct, regionand bring the H-chainVregion next to a Cr, C~, or Caregion gene segment(42, 71, 191, 192). In additionto differential expressionof HandL chains, there is differential expressionof J chainduringB-cell development (100). Thereis little

STEM CELL Ii’~IUIqOGLOBtlLINS Igla ANTIGEMS la-

PRE-BCELLIM~ATURE B CELLB CELL ~ ONLY la-

MEMORY B CELLPLASiSA CELL

MEMBRANE Igl~ MEMBRANE IQM÷ MEMBRANE IgG, SECRETED MEMBPO~NE ~ IgE ORIgA IgG, IQEOR + -+ + la lala Ia

Figure I Asimplilied scheme of B-celldifferentiation showing stages in immunoglobulin and la antigen geae expression (lOOa).

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IMMUNOGLOBULINS 397 no J chain expressionin the pre-Bcell, immatureB cell, or B cell stages of development, but J chain expressionis prominentin all plasmacells and apparentlyoccurs in all B cells that secrete immunoglobulins (100, 105a, 134). Similar to the situation for Hchain and L chain expression,J chain expressionis elevatedsubstantially (ca 10- to 100-fold)if onecompares cell lines that represent early andlate stages of B call development. Tumorsof the B cell lineage correspondingto various developmental stages haveprovento be of great value in the analysis of immunoglobulin gene expression. The pre-Bcell is representedby the carcinogen-induced 70Zcell line (122a), as well as by Abelsonvirus-transformedlymphoidcell lines (6, 142a). ImmatureB cells, B cells, and memory B cells are represented by B cell lymphomas (57, 92, 100a, 159, 180, 188). Plasmacells are represented by plasmacytomas (myeloma)tumors(132). Needlessto say, is critical to study normalcells fromdifferent B lymphocyte developmental stages to verify that cell lines and tumorsrepresent valid modelsfor B lymphocytedevelopment.Althoughsomestudies of normalcells have been possible, isolation of pure populationsof calls representinga particular B lymphocytedevelopmentalstage previously has been dit~cult (155, 164). Therecent developmentof continuouscloned lines of normalmouselymphoidcells andtheir precursorsis an extremelyimportantbreakthrough for future studies on B cell development (72, 183). STRUCTURAL FEAT~URES IN IMMUNOGLOBULIN GENE CONTROL Regulation of lmmunoglobulin Gene Formation: Allelic and lsotypic Exclusion FunctionalH chain andL chain expressionis subject to allelie exclusion, in both normalB cells andB-cell tumorsor tumorcell lines. Allelic exclusion is reflected in the random expressionwithina single cell of either the paternal or the maternal allele for each H or L chain. Althoughrare exceptionsthat mayviolate allelic exclusionhavebeenreported for B-cell tumors,it is likely that allelie exclusionapplies to greater than 99%of B cells. Allelic exclusionhas not beendescribedfor other eukaryoticautosomalgenes.In additionto allelic exclusion,functional L chainexpressionin normalB cells andB-celltumorsor cell lines is subjectto isotypicexclusion, so that a single cell generally expresseseither x or h L chains. Although somecloned murineB cell lines simultaneouslyexpress K and k L chains, it remainsto be determinedif both kinds of L chain are functional (3, 46, 92). Asa result of allelic exclusionand L chain isotypic exclusion, single B cell usually expresses functional immunoglobulinmolecules (comprisedof associated Hchains and L chains) with only one combining site specificity.

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As a consequence of the Dreyer &Bennett (45) hypothesis that immunoglobulin genes are created somatically by joining V and C gene segments, it was proposed that the creation of a joined V + C gene maygenerate a signal (perhaps the immunoglobulinchain itself) that inhibits subsequent joining of V and C genes in the same family (45, 141). The finding that cloned B cells can simultaneously express both normal and abnormal L chains, apparently encoded by two rearranged genes, required modification of this idea so that the signal that prevents further joining events is not activated by an abnormaljoining event but only by a normal joining event. A large numberof studies from manylaboratories have resulted in substantial support for this modifiedhypothesisto explain allelic exclusion for both H and L chain as well as L-chain isotypic exclusion. Analysis of L-chain genes in normal B cells, hybridomas, and B-cell tumorsor cell lines representing different stages of B cell developmentcan be briefly summarized:(a) In approximately two thirds of normal or transformedB cells producing K L chains, one K allele is functionally rearranged and one allel~ remains in a germline configuration; (b) in approximatelyone third of normal or transformed B cells, one K allele is functionally rearranged and one allele is abe~rar~ly (see below) rearranged or deleted; (c) with one exception, all K alleles a~ deleted or aberrantly rearranged in normal or transformed B ceils that express-functional h L chains but no functional ~ L chain; (d) ~ alleles remainin a ge~’m-lineconfiguration in cells that express a functional r L chain; and (e) h alleles can be aberrantly rearranged in B cells that express a functional h L chain (3, 37, 46, 84, 118). Aberrant rearrangements of ~ (or ~) alleles can be of several types: (a) joining of a normal V gene segment to a non-J or pseudo-J segment: (b) joining of a pseudo-V or non-V segment to a normal J segment: and (c)joining of a normal V segment to a normal J segment so that codons are deleted from the VJ junction or an out-of-phase reading frame is established beyondthe VJ junction (3, 7, 17, 22, 34, 46, 87, 90, 108, 129, 148-150). In addition, several possible examples (grouped with aberrant rearrangements above) of B cells express a normal ~ (or h) L chain and also express an abnormal second L chain despite an apparently correct VJ joining event (22, 148). Aberrantly rearranged L chain genes maybe transcribed and translated, but they do not encode functional L chains (i.e. chains that el~ciently assemble to H chains for expression as membraneor secreted immunoglobulin). These data on L-chain gene expression have led to two current hypotheses regarding L chain isotypic and allelic exclusion. First, it has been proposed that there is a hierarchy of L-chain gene formation in which r alleles are rearranged before h alleles. However,morecomplicatedpossibilities cannot be ruled out with certainty (e.g., r genesare specifically deleted

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or can undergoaberrant rearrangements in B cells that express a functional ~ L chain). Second, Alt et al (3) have proposed that further L-chain gene rearrangements are actively prevented when a functional L chain (see above) is expressed. However,the possibility that a functional VJ junctional DNAor RNAsequence somehowprovides the signal to prevent further L chain rearrangements cannot be rigorously disproved at present. Although less is knownabout allelie exclusion of H-chain expression, somedifferences and similarities to L chain are apparent: (a) In 90% more of normal or transformed B cells representing a variety of B cell developmental stages expressing H chains, both H-chain alleles are rearranged (presumably one functionally and the other aberrantly); (b) in 90%or more of Abelson virus-transformed null lymphoid cell lines, all detectable H chain alleles are rearranged (presumably aberrantly) deleted, even though no functional H chain is expressed; (c) an Abelson virus-transformed lymphoid cell line that has undergone a potentially functional D-JHjoining event generates subclones with different function VH-D-JHcombinations; (d) L-chain genes are never present in rearranged form unless at least one H chain gene is rearranged; and (e) as noted for L-chain genes, transcription and translation of some aberrantly rearranged H-chain genes can occur (1, 5, 6, 29, 131, 140, 179, 193). Theseresults have led to the following proposals for regulation of H chain gene formation. First, H-chain genes are rearranged at an earlier stage of B-cell developmentthan are L-chain genes. Second, formation of functional H-chain genes is muchless efficient than is formation of functional L chain genes, possibly as a consequenceof the more complexevents involved (i.e. D --> JH plus VH ---> D joining, with the possibility of D -> D joining and somatic mutation as well). A significant population of null B lymphoidcells seems to result from the inefficient formation of functional H-chain genes (6). Third, functional H-chain gene formation seems to be necessary before further B-cell development and L-chain rearrangement can occur. Fourth, allelic exclusion of H chain maybe an active process in which expression of a functional H chain prevents additional H-chain gene rearrangements. The possiblity that functional Va-D-J~t junctional DNAor RNAsequences provide a signal for allelic exclusion seemsunlikely on the basis of a recent report (5). In summary, although the nature and hierarchy of H and L chain gene rearrangements are relatively well established, more direct proof of the hiearchy of K and ~ L-chain gene rearrangements and of allelic exclusion of immunoglobulin gene expression is needed. In addition, much remains to be learned about the molecular mechanismsand events that control these processes.

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Transcriptional

Control Signals in Immunoglobulin Genes

Theinitiation sites for both light andheavychaintranscriptionunits exhibit "promoter"elementssimilar to these in other eukaryotic genes (reviewed in 25) located 5’ to the leader codingsequencein V-regiongenesegments (Figure 2). These sequences function in phasing correct initiation transcription (25). Thesesequencesin immunoglobulin genes are not altered by somatic mutation, even though nearby V regions showup to 5%base changes (35). Thus, somatic mutations of V regions is apparently not a mechanism used in controlling levels of immunoglobulin gene transcription. Enhancersequencesare a newlydiscovered class of control elements, whichappearto affect the efficiencyor level of transcription andwhichare located several hundrednucleotides upstream(i.e. to the 5’ side) of the "TATA box" and cap site in the few viral and cellular genes wherethey havebeenidentified (10, 11, 44, 61, 62, 94, 111, 165, 166). Theseso-called activator or enhancer sequences maybe involved in RNApolymeraseII recognition of eukaryotic transcription units in chromosomes, and appear to affect the efficiencyof transcription(I0, I I, 25, 44, 61, 62, 94,I I I, 165, 166). Twofeatures also suggest that these regions maybe importantfor specific generegulation. Thefirst is the finding that these enhancersequencesexhibit no significant sequencehomology,as wouldbe expectedfor specific rather than universal genecontrol regions such as the TATA box. Second,in virus systemsthese enhancersequencesare functionally interchangeable(10, 44, 94) and their activity is correlated with host range restriction and the specificity of viral geneexpression.PolyomaandSV40 mutantswith altered host range fromthe wild-type virus have minorsequencealterationsexclusivelyrestricted to these specific regions(54, 63, 76, 152). Theenhancersequencesin viral genomesand in cellular histone genes are located somedistance 5’ of the TATA boxand cap site (61, 62). Several lines of evidenceraise the intriguing possibility that enhancersequencesin immunoglobulin genesmightnot be conventionallylocated in the flanking DNA 5’ to the TATA box but instead are associated with the C region. First, immunoglobulin germline Vregions contain 5’ flanking sequences with all of the general signals (TATA box, cap site) neededfor correct initiation of transcription (13, 18, 21, 75, 79, 108, 167), becausespecific initiation of transcription is observedin vitro andin Xenopusoocytes(13). Nonetheless, unrearrangedV regions are not transcribed in vivo (106), whereasrearrangedV + C genes are actively transcribed in myeloma cells (56, 104). Second,unrearrangedC~regions are transcribedin myeloma cells at levels comparableto active rearrangedlight chain genes (173). Tran-

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IMMUNOGLOBULINS SOURCE ~

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A

401

TRANSCRIPTION~I~RANSU~TiON_~

C

-29

+ 64

-29

+1

+64

-28

+l

+26

-34

+1

+34

HPCM2V H

T~ HKI01

Vxll

Figure2 Comparison of the 5’ flankingsequencesof representativeimmunogiobulin V region gene segments.Selected immunoglobulin light andheavychain sequencesare aligned to indicatethe conservedplacement of presumed promoter sequencesin relationto the consensus sequenceof Breathnach &Chambon (25). Experimentally determined transcriptioninitiation sites (i.e. capsites) are denoted by asterisks. Theotherimmunoglobulin sequences shown are alignedonthe basis of homology withexperimentally established transcription initiation sites. References for V region 5’ flanking sequences are MCI01Vn and MOPC141Vn (75), MOPCI67VH and HPCM2VH (35), T V, (21), HK101(13), MOPCI73V, (108), (79), Vxi(18), V~u(167), C~(173). scripts of the unrearranged C~-gene segment begin near a sequence strongly resembling a TATAbox. This strongly suggests that the C~ (and possibly other immunoglobulin C regions) may contain an enhancer-like sequence. Finally, sequence comparisons of mouse and humanJ + C~ germline gene

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segments have revealed a region of strikingly conserved sequence homology approximately0.7 kb 5’ to the C~region (68). This is striking because the only other areas of sequence conserved between the mouseand humangenes are the J and C~coding regions. Interestingly, we have noted that this highly conserved intron sequence 5’ to the C~ region contains short sequences related to the SV40enhancer sequences and to the specific polyomaenhancer sequences from polyoma host range variants (R. Deans, R. Wall, personal communication). This conserved intron sequence is present in an aberrantly rearranged K light chain gene (from MPC11), which is the smallest active immunoglobulintranscription unit known. This truncated r gene is as actively transcribed as the normally rearranged V + C~ gene in MPC11 cells (34, 150). Heavychains also appear to contain control sequencesin the JrI ~ CIt intron sequence(5, 35, 80, 81). Interestingly, large deletions in this intron region in 18-81 pre-B cell lines are correlated with the loss of/.t expression, whichis restored by LPSstimulation (4). If this novel prediction is confirmed, then the V region (with the general promoter elements essential for correct transcript initiation) and the C region (with enhancer function) both contribute important functions for activating immunoglobulin genes.

Methylation and GeneExpression IMMUNOGLOBULINGENES The demethylation of methylated cytosines in chromosomalDNAis closely correlated with gene expression as in manyeukaryotic gene systems (reviewed in 134a, 139, 185). Most of the methylation in mammalianDNA is 5-methylation of cytosines in the symmetrical dinucleotide C-G, and most of the C-G sequences contain 5-methylcytosine (Cm-G). The sequence C-C-G-G is cut by restriction enzymes Hpa II and Msp I, whereas C-Cm-G-G is cut only by MspI. Therefore, parallel Southern blots (161) of chromosomal DNAcut with HpalI or Msp I can be used to show whether these restriction sites are methylated or not. Methylation patterns have been analyzed in mouse heavy chain genes (CmC8, and Cyl, Cy2b ) that are activated by different mechanisms. The C~, gene is expressed as a result of the DNArearrangements that join the VI~, DR, and JH gene segments (V region formation) (reviewed in 46). C8 gene is expressed by transcription through from the rearranged C~, gene, in a VDJn-Ct~-C8complex transcription unit (33, 102, 112). The Crl and C~,2b genes are expressed by means of DNArearrangements, which bring the VDJHclose to either the C~,1 or C~,2b gene segment (class switching) (reviewed in 46, 103).

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The HpalI/MspI restriction mapping technique has been used to determine the methylation of immunoglobulin heavy chain genes. The C~,, C8, Cyl, C~,2b, and Ca regions all are methylatedin cells that do not express them but that are demethylated whenthey are expressed (39, 139, 190). particular, the ~ gene remains methylated, and thus presumably untranscribed, in a lymphomacell line that probably represents an early stage of B cell differentiation and produces only # heavy chains (139). Because/~ and ~ RNAsare co-transcribed from a single complex transcription unit at a later stage of B-cell differentiation, this finding suggeststhat the ~-plus8-complextranscription unit is of variable length regulated by C8 methylation. These results showcomplete correlation between demethylation and expression of immunoglobulinCn genes, in agreement with results in numerous other eukaryotic gene systems (reviewed in 134a, 139, 185, 190). Selective demethylation of one heavy chain C-region allele represents one possible molecular mechanism for allelic exclusion. However, in the B cell lymphomaW279, which makes only a single allotype of ~ chain (153a, 180), both alleles of the t, gene are d emethylated. Furthermore, both Ce~ regions are also fully demethylated in IgG-secreting P3Kmyelomacells (139). If these findings in established tumor cell lines are relevant to regulation in normal B cells, it wouldappear that selective demethylation of one chromosomeis not the mechanism of allelic exclusion. J-CHANGING

Secreted pentameric IgM (and multimeric IgA) is assembled by covalent linkage to a small protein called the J chain. J-chain synthesis is induced in B cells after antigenic stimulation (85, 100). Its production is initiated by the onset of J-chain gene transcription (105a). J-chain genes are not rearranged when germline DNAis compared to DNAfrom lymphoid cells expressing J chain. The J-chain gene is methylated in tumor cell lines representative of l~re-B cells; immature and mature B-lymphocytesdo not express J chain. However, the J-chain gene is demethylated in immunoglobulin-secreting cell lines representative of plasma cells (190). J-chain expression is thus closely correlated with demethylation, just as are immunoglobulin genes. It is not clear whether or not demethylation precedes immunoglobulin gene and J-chain gene transcription and is a mechanismthat initiates expression. However,recent studies showing that estrogen induction causes demethylation of the 5’-flanking regions of hen vitellogenin (185) and that in vitro methylationof a single HpalIsite in SV40inhibits late but not early

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SV40expression(53) strongly supportthe likelihood that demethylation(at least in 5~ control regions)precedesthe onset of transcription. Chromatin Structure in lmmunoglobulin Gene Expression As first demonstratedby Weintraub& Groudine(18 I), genes in chromosomesthat are expressed(i.e. active chromatin)are preferentially digested by treatment of nuclei with the sequencenonspecific nuclease DNaseI. Subsequentstudies in a numberof eukaryotic genesystemshaveconfirmed that transcribed regions, as well as considerableflanking sequencesof expressedgenes, are moresensitive to nucleasedigestion than are unexpressed genes or bulk chromatin (reviewed in 99, 107). Becauseimmunoglobulin genes undergoboth productive and aberrant DNA rearrangements,it is of interest to compareIg gene expression and chromatin configuration in germlineand variousrearrangedstates in different lymphoidcells. Storbet al (163) have examinedDNaseI sensitivity of light-chain nuclear V~and C~gene segmentsin nuclei from myelomacells (r+), B-lymphoma cells (r+), and liver cells (~-). Theyfoundthat C~genesare sensitive to DNase I in both rearranged and germlineconfigurations in myelomatumors and a B lymphoma.UnrearrangedV~regions are insensitive to DNaseI, but becomeDNaseI-sensitive whenrearranged. No difference in DNaseI sensitivity could be detected betweenthe two rearranged C~ regions of MOPC-21. In this cell line, one C~ gene is productively rearranged and makesfunctional ~ light chains; the other CKgeneis aberrantly rearranged with a misalignmentof V-J, whichleads to a shift in translation reading frame (177a). This nonproductiveC~gene is transcribed into a nonfunctional K mRNA. Restriction enzymedigestion of nuclei has shownthat rearranged expressedCKregions are relatively moreaccessible to enzymethan are germline C~regions(.130). Many expressedeukaryoticgenesin nuclei also contain sites (often in the flanking sequences5’ to the TATA box) that inhibit nuclease sensitivity 10-fold greater than transcribed genesequences,whichin turn are 10-fold moresensitive than is bulk chromatin(reviewed in 49, 99, 107). These so-called hypersensitivesites are apparentlynucleosome-free in viral minichromosomes, and include the enhancercontrol regions importantin regulating transcription (63, 73, 145, 174, 175). Furthermore,nuclease-hypersensitive sites in certain eukaryotic chromosomal genescontain DNA sites whosemethylationpattern is correlated with tissue-specific geneexpression (70, 99). Chromatinfrom T lymphocytesfrom thymusexhibits a DNaseI hypersensitivity in the sequencesbetweenC~, and JrI (162). This maybe related to the widespreadoccurrenceof aberrant kt RNA species in T cells. These /x RNAsdo not contain Vn regions and appear to initiate 5’ to the

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region, where Clarke et al (35) have evidence for a transcription origin unrearranged # genes from certain myelomacells. Parslow & Granner 024) have recently determined the DNaseI hypersensitivity patterns of the CKregions in the pre-B-cell line, 70Z(124). The 70Z cells only synthesize detectable K mRNA with LPS (128), even though they contain a productively rearranged V + C~ gene (in addition to an unrearranged CKregion) (101a). LPS activation of K mRNA transcription was associated with the appearanceof a DNaseI hypersensitive site approximately 0.7 kb 5’ to the C~ region exon (124). This sequence exhibits the hypersensitivity of knownenhancer regions and coincides with the highly conserved sequence in the J ~ C~ introns of mouseand humanC~ regions (68). This study found no evidence of hypersensitive sites 5’ to the V region in the LPS-activated light chain gene. This interesting result further supports the proposal that enhancer sequences in immunoglobulingenes might be adjacent to the C regions. Weischetet al (182) have reported that DNase I hypersensitive sites are present in the 5’ sequences upstream of both transcribed and untranscribed rearranged light chain genes in a myeloma cell.

CONTROL OF IMMUNOGLOBULIN GENE EXPRESSION BY RNA PROCESSING Membraneand Secreted Heavy Chains Membraneand secreted immunoglobulin heavy chains are coded by heavy chain mRNA species with different 3’ ends generated by RNAprocessing mechanisms (reviewed in 140, 177). This was first shown for fi mRNA. Various B lymphomacells and myelomacells were found to contain two prominent fi mRNA’s at 2.7 and 2.4 kb, which coded for membrane(fim) and secreted (fis) chains (2, 80, 128, 136, 158). Analysis of fi eDNA clones of both fi mRNA species revealed that the fis and fire mRNA’swere identical through the end of the C#4 constant region coding sequence, but thereafter contained very different COOH-terminalcoding segments and 3’-untranslated regions (3’-UT) (2, 136). The fis COOH-terminalsequence encoded a 20-residue hydrophilic segment identical to the fis COOH-terminal amino acid sequence determined by Kehry et al (1978). The firn COOHterminal sequence encodes 41 residues (designated the Mor membrane region) with the properties of a transmembraneprotein (48, 136). The locations of the COOH-terminalsegments of fim or fit s mRNA’s were established by R-loop electron microscopy and nucleotide sequencing of the cloned fi gene (48, 136). The secreted COOH-terminuscoding sequence and the 3~ untranslated region of fis mRNA is encoded contiguous with 3’ end of the C~4 domain (Figure 2). The firn COOH-terminusand 3’-UT region is encoded in two exons (the M exons) located ~ t o t he

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C~4 domain. These two Mexons are joined to the C~4 domain by two RNA splices that replace the pt s COOH-terminus and 3’-UT region sequences to generate ptm mRNA.The chromosomal gene codons at the 3’ end of the C~4 domain where the Mexons are spliced have the sequences G/GTAAA encoding Gly-Lys. This sequence in the /z chromosomalgene (30, 48) identical to the consensus sequence for an "upstream" RNAsplicing site, G/GTAAG (93, 138, 151). The underlined GThas been universally found at the exon/intron juncture of "upstream"splicing sites in eukaryotic genes (reviewed in 25). Because all immunoglobulin heavy chains contain the sequence Gly-Lys at the end of the last domainencodedby a potential site for RNAsplicing (G/GTAAA or G/GTAAG),Rogers et al (136) predicted that all bther immunoglobulin heavy chain genes would also contain Mgene segments and would generate membranemRNA species by RNAsplicing. It has now been confirmed that all B-cell lines that producedeither 71, ~2a, ’~2b, or ~3 secreted heavy chains contained both 7s mRNAand a minor ~ mRNA species O’m) with a spliced structure analogous to that of the ptm mRNA (135, 137, 140, 171). Mappingand sequencing of the "Yl, YEa, ~2b, and "~3 Mgene segments (136, 171, 193) have located the Mexons in these heavy chain genes. Membraneand secreted forms of a heavy chain mRNA have recently been reported (82, 189). The Mexon of the a heavy chain gene segment has now been determined 089). The C~gene is organized differently from other heavychain genes in that it contains an extended hinge and lacks an internal Cri2 domain(98, 169). Unlike all other heavy chain genes where the secreted C-terminal sequences are continuous with the final C region domain, the 8 gene has both its secreted and membraneexons in separate noncontiguous gene segments 3’ to the C~3domain(33, 98, 102, 112, 169, 170). The Mexon sequences (33) of ~mchains also exhibit a relatively high degree of homologyto the Mexon sequences of other heavy chains (140). MouseB lymphomacells co-expressing membranept and ~ chains contain both #mand ~t s mRNA,as well as two ~mmRNA species (102, 112, 116). Both these ~im mRNA species apparently contain identical coding sequences but terminate at different poly(A) sitcs and therefore have different 3’-UT sequences (33, 102). An analogous situation has been reported for two H-chain am mRNA species for a heavy chains (189) and dihydrofolate reductase (153). There is no evidence for functional differences between these two forms of ~ and a mRNA’s.

Heavy Chain Genes are Complex Transcription Units Most eukaryotic genes now studied (including immunoglobulinlight chain genes) (127, 178) are simple transcription units that code for a single mRNA (reviewed in 40, 41). Simple ~ranscription units contain a single

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poly(A) addition site, and the pattern of RNA splicing is invariant except in unusualcases wherealterations in splicing sites generate aberrant splicing patterns. Complextranscription units contain multiple poly(A) addition sites and/or alternative RNA-splicing sites, which are used to generate multiple mRNA species from a single gene (40, 41, 197). Complextranscription units have been found in adenovirus, SV40,polyomavirus (40, 41, 197), and retroviruses (165, 166). The mappingof membraneand secreted mRNAs established immunoglobulin heavy chain genes as the first-known nonviral complextranscription units. The genes for yeast invertase (31) and rat calcitonin (8) have recently been shownto be complextranscription units. Immunoglobulinheavy chain complextranscription units contain at least two poly(A) addition sites. Whennuclear RNAprecursors have poly(A) added at the end of the secreted COOH-terminalsequence, RNA splicing then acts on all exons to yield a secreted heavy chain mRNA (Figure 3). Nuclear RNAprecursors that have poly(A) added at the of the Mexons undergo two additional RNAsplices that delete the secreted C-terminal sequence and connect the final C region domainto the Mexons to generate membrane heavy chain mRNA’s. Even though membrane heavy chain mRNAsare produced by two RNA splicing events more than are required for secreted heavy chain mRNAs, it is believed that the processing pathwaysleading to secreted or membrane heavy chain mRNA’s are determined by the choice of poly(A) addition sites (48, 136, 140, 178). This proposal is based on the general finding that polyadenylation precedes splicing under normal conditions (reviewed in 40, 41). REARRANGED v HEAVYCHAIN GENE

P V DJ

C~I C~2

C~3 Cv4 Z

M1 M2

SECRETED POLY(A) SITE

MEMBRANE POLY{A)SITE

MEMBRANE p mRNA PRECURSOR

SECRETED # mRNA PRECURSOR

AMPUTATED TRANSCRIPT

Figure3 RNA speciesandsplicing eventsin the generationof membrane andsecretedheavy chainmRNA. Theupperline depictsthe structureestablishedfor the ~ gene(30, 48). Boxes are exonsanduntranslated regionsare shaded.Thelowertwolines depictthe twopolyadenylated alternativeprimary transcriptsmade froma singlegene,withtheir splicingpatterns.The amputated transcript is a novelpolyadenylated RNA speciesgeneratedin the additionof poly(A)to the secretedmRNA precursor.Although shown herefor the/x gen¢,all heavychain geneshavea similar organizationencodingmembrane andsecretedmRNA species.

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Several studies have reported large nuclear RNAprecursors of sizes expected for primarytranscripts and nuclear RNAprocessing intermediates to secreted and membraneheavy chain mRNA’s (35, 104, 128, 140, 147). However,becauseof the low levels of the presumptiveprimarytranscripts anddifficulties in reproduciblydetectingsplicing intermediates,the differential processing pathwaysleading to membrane and secreted heavy chain mRNA’s have not been resolved. However,Rogers& Wall (137, 140) have obtained another line of evidence in support of the proposal that membrane and secreted heavy chain mRNA’s are processed from the transcripts of a complextranscription unit. Asa general rule, it appearsthat poly(A)addition occurs at points of cleavage in nuclear RNAmolecules rather than through termination of transcription (40, 69, 117). Indeed, transcription apparently continues somedistance past poly(A)addition sites (several kilobases) before cleavageof the nascent transcript occurs. Assuming that poly(A)addition occurs at points of cleavage in immunoglobulin heavychain gene transcripts, it wasreasonedthat transcription past the poly(A)addition site for secreted mRNA precursors wouldproducea novel polyadenylatedspecies called the "amputatedtranscript." This predicted polyadenylated RNAspecies should contain intron sequences betweenthe secreted poly(A)site and the membrane exons (Figure 2). Since the M1and M2exons are separated by completesplicing signals in the amputatedtranscript, it is likely that this intron wouldbe spliced out. Finally, the amputatedtranscript should have a 5’ terminus beginningin the sequencefollowingthe secreted poly(A)addition site and should not contain any heavy chain V or Cr~ region exons. Discrete nuclear RNA species with precisely these predicted properties havenowbeendetected and characterized in both ~- and y2b-producingcells (137, 140). Theconfirmation of the amputatedtranscript as predicted establishes that heavy chain genesare complextranscription units, and reaffirms that poly(A) added by a mechanisminvolving RNAcleavage.

Co-Expressionof Different HeavyChainClasses It waspredicted that the co-expressed/.tand 8 heavychains in surface IgM and IgDwouldbe generatedfroma single large complextranscription unit by RNA processing mechanisms (55, 178). This prediction wasbased on the findings that co-expressed kt and 8 chains appeared to have the same variable region (reviewedin 57), exhibited allelic exclusion,and wereencodedon the samechromosome (23, 64). TheC~and C8regions are closely linked. TheC8geneis separated fromthe/.tin exonsonly by approximately 2 kb of DNA (33, 98, 102, 112, 169). If/z and 8 are co-expressedfroma single gene, then the 8 geneshould not be rearranged in IgM+ IgD-producinglymphocytes.This prediction

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IMMUNOGLOBULINS 409 wasconfirmedby mappingstudies that showedthat the C8genewasin the germlineconfigurationin pt + ~-producingcells (83, 102, 112). Thesedata clearly indicated that IgDexpressionin pt + ~ B cells did not involve a Vnto C8DNAclass-switch DNA rearrangement. Accordingly, the simultaneous expression of Ct, and C~with a single Vii region appears to be mediatedby alternative routes of RNA processingof a very large primary nuclear transcript that contains the Vn, Cm and C8 gene segments(reviewedin 102, 140). Thept + 8 complextranscription unit is approximately25 kb long, with five knownalternative polyadenylationsites and morethan a dozenpotential exons for splicing. Large nuclear RNA species approachingthe 25-kb size estimatedfor the # + ~ primarytranscript haveeluded direct detection. TheseRNAs are likely to be present in <10copies/cell, and this low abundancemaypreclude their direct detection. A completeprimarytranscript for the major late adenovirnstranscription unit (32 kb long) was never detected directly (40, 41, 197). Instead, confirmationof this major transcription unit wasobtainedby indirect means,including pulse-labeling studies and UVtranscript mapping(40, 41). Suchindirect meansappear likely to be required for confirmingthe large pt + 8 complextranscription unit. As in heavy chain transcription units for membraneand secreted mRNA’s, the choice of poly(A) sites in the # + ~ transcription unit presumedto determine the pattern of RNAsplicing. The RNAsplicing events in this complexsystemare morecomplicatedthan in membrane and secreted heavy chain mRNA processing whereall complete RNAsplicing sites are used. Whenthe Vii region is spliced to the C~region to make~ mRNA, RNAsplice sites in the Cpt exons are apparently ignored. HowC~or C8exons are chosen for splicing remainsa mystery. A model for RNAsplicing has been proposed in which U-l, a ubiquitous small nuclear RNA in eukaryoticcells, base-pairs with both endsof an intron and aligns thempreciselyin register for cutting andsplicing (93, 138). Yang al (195) haveobtainedprdiminaryevidencein support of this model.However, this modeldoes not explain how# exonsplice sites that function in making/.t mRNA’s are ignored in splicing 8 mRNA’s. It seemsplausible that the secondarystructure of the nuclear RNA moleculemightdirect the courseof RNA splicing, but this hypothesisis not yet amenableto experimentaltesting. IgD-secreting myelomacells contain a single prominent8s mRNA species (52, 102, 112, 116). Rareinstances of IgDsecretion by myeloma cells in mice apparently do not occur through RNA splicing, but rather appear to result from a DNArearrangement that brings the rearranged VDJii region into proximity to the C8gene with the deletion of the C~, gene segments(102, 112). Furthermore, the 8s mRNA is not detectable in

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lymphomas,making/zs, ptm, and the two ~mmRNA species. These findings indicate that two different mechanismsare employedin ~ gene expression and the production of IgD. RNAprocessing alterations in B ceils produce membrane~ mRNA’salong with/.t mRNA’sfrom a single large complex transcription unit, whereas a rare DNArearrangement in mouse plasma cells produces secreted ~ mRNA. Rare lymphoid cells have been reported that apparently contain two classes of surface immunoglobulinsother than IgMand IgD. These cells are of considerable interest because they mayrepresent memoryB cell transitional stages in the process of class switching, leading to antibody secretion (reviewed in 103). Such cells have recently been isolated and their immunoglobulin gene organization has been analyzed. Perlmutter &Gilbert (125) have shown that splenic B lymphocytes exhibiting surface IgM and IgG1 contain two Cv genes and C~,1 genes that show no evidence of rearrangement. Similarly, #t+¢+ splenic B lymphocytes isolated by fluorescenceactivated cell sorting show no evidence of DNArearrangements between JH and the C~ gene segment (194). These two reports conclude that the co-expression of different H chain mRNA’s involves extremely large nuclear RNAprecursors. Both these reports are based on the presumption that the isolated B lymphocytes exhibiting two different heavy chains by staining are simultaneouslysynthesizing both chains. This is partially satisfied for the kt+¢+ cells, but this point needs to be rigorously confirmed. This reservation is not confronted in other studies on cloned B lymphocyte lines derived from an Abelson virus-transformed pre-B cell line called 18-81, which express two H chain classes. As characteristic for a pre-B cell, primary 18-81 subclones only express low levels of # mRNA but no light chain mRNA.Long-term tissue-culture-adapted subclones of 18-81 (18-81 A-2) apparently undergoa switch from C~, to C~,2b expression (4, 28). The level of Y2b mRNA appears to be inducible with a B cell mitogen, lipopolysaccharide. Furthermore, pt chain expression is undetectable without LPSbut is restored by LPStreatment of these cells. Thesecells contain C~, regions. Deletion events occurring within the JH-C# intron are correlated with the loss of constitutive kt gene expression and the /z --, )’2b switch (4). The JH->-C# intron has been shownto contain repetitive DNAsequences that readily undergodeletion events (46, 103). These experiments have not shownthat the ~, Y3, and Yt gene segments are present in their germline context. If this is the case, the # ~ Y2bswitch in 18-81 A-2 cells mayresult from the differential RNAsplicing of a large (>100 kb) multi-CH-genetranscript (4). Given the large sizes of the predicted /~ 71, # + ~, or /z + ’~2b nuclear RNAprecursors (100-200 kb), and the experimental inability to detect even the 25-kb # + 8 co-transcript, experimental confirmation of these large precursors will require ingenious approaches. It also remains to be established that such cells represent

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IMMUNOGLOBULINS 411 transitional stages in heavy chain class switching. Immunoglobulin gene introduction into lymphoidcells together with definedconditionsthat induceclass switching(mitogens,T cell factors, etc) shouldprovidefurther insights into the molecularmechanisms and dynamicsof class switching.

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QUANTITATIVE CHANGES IN LEVEL OF IMMUNOGLOBULIN EXPRESSION DURING DEVELOPMENT Asnotedabove,there is differential expressionof H, L, andJ chainsduring development. In addition, the expressionof all three productsis amplified substantially during B cell maturation. In general, whenboth H and L chains are expressed(i.e. in immatureB cells and later stages of development), they are synthesizedat roughlyequimolarlevels evenas the. levels of expressionare increasedseveral orders of magnitude (86, 101, 105a,113, 128,134,153b,154).In contrast, the level of J chainexpression is not tightly coordinatedwith the level of HandL chainexpression,despite the fact that in plasmaceils, J chain is expressedat levels roughlycomparableto the levels of Hand L chain expression(85, 100, 134). Clonedmurinepre-B-cell lines and someimmatureB-cell lines maysynthesizeas little as 0.01-0.1% of their protein as # only or as # plus L chains (101, 153b,154), whereas murinemyeloma cells maysynthesize as muchas 20-30%of their protein as H and L chains (86, 113). Similar amplification of H and L chain expressionin late stages of B lymphocyte development is also seen in studies of normalcells (109, 155,). Thelevels of HandL chainexpressionin most immatureB-cell, B-cell, and memoryB-cell lines appear to be roughly intermediate betweenthe extremescited above, although there are few critical measurements for the intermediate stages of B-cell development (100, 134, 188). VariousB-cell lines and normalB cells havebeenshownto amplifyboth H and L chain impressionapproximatelytwo to ten times whenstimulated in vitro with mitogens(e.g. lipopolysaccharide),anti-immunoglobulins, appropriate combinationsof antigen and lymphocytesupernatants (24, 60, 101, 109, 122b, 154). Anumberof pre-B cell lines stimulated with LPS beginto expressL chainat levels comparable to Hchain, with little stimulation of the level of Hchain synthesis(101, 122b,142a). In contrast, after LPSstimulation, several Abelsonvirus-transformedpre-B-cell lines are stimulated to synthesize H chains at an approximately10-fold increased level but still do not expressL chains (4, 5). It has beennoted for these Abelsonvirus-transformedpre-B-cell lines that LPSstimulation of H-chain synthesis occurs only in sublines or clones that spontaneouslyhave decreased the level of H-chainsynthesis substantially (about 10-fold). The

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decreasein the unstimulatedlevel of H-chainsynthesis is correlated with a substantial deletion in the Jn-C~,intron 5’ of the/z switchregion. Based on these results, it has been suggested that LPSstimulation somehow compensatesfor the loss of a DNA sequencethat has a positive effect on H-chain production. Myeloma cells with smaller deletions in the JH-Ct, intron seemto be unaffected in their levd of H-chainexpression. Somaticcell hybrids betweenmousemyelomacells and mouseor human pre-B cells, immatureB cells, B cells, or memory B cells co-dominantly express H and L chains at approximatelythe samehigh level of expression observedin the myeloma fusion partner. In all reported studies of this type, the overall phenotypeof immunoglobulin expression(i.e. quantity of Hand L chains and secreted versus membrane form of H chain) is determinedby the moredifferentiated myeloma cell fusion partner (50, 91, 96, 134, 134b, 188). A numberof studies with murinecell lines have demonstratedthat the qualitative andquantitativelevels of H, L, andJ chainexpressionat different stages of B cell development are determinedlargely, if not entirely, by the amountof cytoplasmicH, L, and J chain mRNA’s, respectively (38, 56, 104, 128, 134, 147). Thefew studies on normalB lymphocytes are consistent with this conclusion(168). The rates of in vivo H and L chain RNAsynthesis and processing has been studied extensively in MOPC 21 and MPC11 murine myelomacell lines by the laboratories of Walland Perry, respectively (56, 104, 147). Thesestudies, together with other studies on the turnover of cytoplasmic mRNA in MOPC 21 myelomacells (38), provide insight into howa single functional H- or L-chain gene can account for as muchas 10%of the protein synthesized by a rapidly growingmyelomacell. The following conclusionsapplyto both myeloma cell lines: (a) Therate of transcription is greater than 20-30 transcripts per minute(whichis comparableto the apparent maximalrates of transcription observed for ribosomal RNA); (b) processing of the putative primarytranscripts into mRNA and transo port of mRNA from the nucleus to cytoplasm is rapid and essentially quantitative; (c) cytoplasmicmRNA is very stable, so that it is minimally metabolizedduring the 20-hr cell generation time; and (d) the fraction cellular mRNA encodingH and L chains is comparableto the fraction of cellular protein synthesis comprisedby Hand L chains. Thus, it appears that the level of H- and L-chaingeneexpressionin myeloma cells maywell approachthe maximum level possible for a higher eukaryoticcell, with an approximate20-hr generation time. Weknowmuchless about regulatory steps that result in a 10- to 1000fold lower expression of H and L chain mRNA (and protein) in B cells representing earlier stages of B lymphocytedevelopment.However,some preliminary informationis available from a study comparingsteady-state

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IMMUNOGLOBULINS 413 nuclear and cytoplasmic H chain mRNA contents of MPC11 murine myelomacells and the 70Z murine pre-B cell line (128). MPC11 cells contain about 30,000 copies of Hchain mRNA or about 300 times as much cytoplasmicH-chainmRNA as 70Zcells, consistent with the fact that H chain comprises about 10-12%and 0.02-0.10%of protein synthesis in MPC 11 and 70Zcells, respectively (86, 101). In contrast to the large difference in cytoplasmicH-chainmRNA content, only six times as much nuclear H-chain mRNA occurs in MPC11 myelomacells as in 70Zcells. The50-fold higher ratio of nuclear to cytoplasmicH-chainmRNA in 70Z cells comparedto myeloma cells is probablya consequenceof differences in one or moreof the followingpost-transcriptional events: (a) rate H-chain nuclear RNAprecursor processing; (b) extent of intranuclear H-chain mRNA degradation; (c) rate of transport of mature H-chain mRNA from the nucleus to the cytoplasm; and (d) rate of cytoplasmic chainmRNA turnover.It is of interest that the ratio of nuclearto cytoplasmic nonimmunoglobulinpoly(A)-containing RNAin either 70Z or MPC 11 myeloma cells is essentially the sameas the ratio of nuclearto cytoplasmic H-chainmRNA in the 70Zcells. In view of the apparent differential post-transcriptional events specifically affecting H-chainmRNA metabolism, it seemsunlikely that differencesin transcription rates are fully responsible for the vastly different levels of cytoplasmicH-chainmRNA in these two cell lines. Obviouslymorestudies of the transcription rates and metabolismof H- and L-chain mRNA’s are required to understand howthe levels of H- and L-chain mRNA (and thus H and L chains) are regulated during B lymphocytedevelopment. TRANSLATIONAL AND POST-TRANSLATIONAL REGULATION IMMUNOGLOBULIN EXPRESSION

OF

Initiation of translation of H- and L-chain mRNA’s appears to be much moreefficient than the averagecell mRNA in that Hand L chains represent a larger fraction of the newlysynthesizedprotein whenthere is a nonspecific decreasein initiation of translation(e.g. mitosis,starvation,viral infection) (119, 120, 160). However,there is no convincingevidencefor regulation the efficiency of mRNA translation in B cells of different developmental stages. Microsomal localization of H, L, and J chain mRNA’s to the rough endoplasmicreticulum and translocation of H, L, and J chains into the cisterna of the roughendoplasmicreticulumseemstq be determinedby the aminoterminal signal sequence,whichis proteolytically removedfromthe primarytranslation productprior to chainterminationand release fromthe ribosome(reviewedin 86). Thesignal sequencefunctions normallyeven

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an unusual situation where the signal sequence is contiguous with the CK sequence rather than a V~ sequence, as found for normal r L-chain mRNA’s (51, 141). There are no knownexamples of either cell variants or normal regulatory events that alter binding of mRNA to the rough endoplasmic reticulum or the vectorial transport of immunoglobulinchains into the cisterna. Glycosylation of all normal H chains as well as L chains that contain the Asn-X-Thr(or Ser) tripeptide N-glycosylation acceptor site in the amino terminal V region domainusually occurs by transfer of the core oligosaccharide (glucose3-N acetyl-glucosamineE-mannosetl)from a lipid carrier to a nascent polypeptide (15, 16). The transfer of the core oligosaccharide to a growing nascent H chain apparently slows polypeptide elongation sufficiently so that the net rate of H chain synthesis is decreased 10-25%, a phenomenonthat maycontribute to the slight imbalance of H and L chain synthesis observed in both normal and malignant B lymphocytes(9, 16, 86). Parameters that affect the efficiency of N-glycosylationor potential tripeptide acceptor sites of immunoglobulinchains are reviewed elsewhere (16). There is no evidence indicating that core glycosylation is regulated differentially in B cells of different developmentalstages or in different physiological states. Most processing of the core oligosaccharide, as well as addition of terminal sugars, is thought to occur during the brief period the glycosylated immunoglobulinchain traverses the Golgi apparatus (16, 164). The decision process for generating simple versus complex asparagine-linked oligosaccharides is poorly understood. The best exampleof possible regulation of glycosylation involves a block in converting a core oligosaccharide to a complexoligosaccharide on the secreted H chains in less differentiated B cells (see belowfor a morecompletediscussion of this interesting regulatory event). Intramolecular folding, including covalent disulfide bond formation, occurs to a significant extent on nascent L chains, and presumablyon nascent H chains as well (14). Intermolecular assembly of immunoglobulinchains also begins on nascent H chains, with some classes of immunoglobulin forming H-Hdisulfide bonds and other classes of immunoglobulinforming H-L disulfide bonds on nascent H chains (15, 146). However,most assembly occurs between completed chains, with the pathwayof assembly dependent on the class of Ig synthesized by the cell (19, 86, 146). Assemblyof chains is restricted to the chains of the sameclass, so .that cells expressing /.t and T H chains, for example, do not form covalent /.t- y heterodimers (105). The co-expression of secretory and membraneforms of H chain (e.g. ~mand /.ts) in various ratios in the same cell at different B lymphocyte developmentalstages raises the question as to whether or not the secretory

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IMMUNOGLOBULINS 415 and membraneforms of H chains form heterodimers. The best studies to address this issue involve B lymphomas and myeloma cells, whichsimultaneously express 7mand Ts. In two different murine B lymphomasthat express~2amand 72as in roughlyequivalentamounts,no "Y2am’T2as heterodimerswereseen (121, 188). Analysisof labded cell surface Ig of MPC myeloma cells revealed no detectable T2bschains expressed on the cell surface (88). Theseresults then suggest that membrane and secreted chainheterodimercovalentassemblyis rare or non-existentin B cells where the membrane and secreted chains are co-expressed. Lackof significant heterodimerassemblyof these chains could be due to one or moreof the following: (a) preferential assemblyof Hchains synthesizedby the ribosomeson the same mRNA molecule; (b) preferential assemblyof H chains selectively partitioned into the membrane (Hm)instead of into the cisterna (Hs) of the rough endoplasmicreticulum; and (c) preferential assembly homodimers due to intrinsic properties of the Hchains. In contrast to the results cited above,recent worksuggests that covalentheterodimerformation and membrane insertion of ~ls and )’lm H chains occurs in MOPC 21 myeloma cells, a cell line in which),1~ is synthesizedin a vast molarexcess compared to 7~rn(58). This result mustbe questionedin viewof the results in B lymphomaand other myelomacells cited above. The putative chains in labeled surface Ig of MOPC 21 cells could, for example,represent partially degraded~/~mchains. Differential intracellular degradation of mutant and normalH and L chains represents a well-documented exampleof post-translational regulation. For example, MOPC 173 and MOPC 21 myelomalines synthesize a molar excess of L chains over H chains, but degrade the unassembledL chains (9). AMOPC 21 variant that synthesizes L chains but not H chains does not secrete the L chains, but quantitatively degradesthemwithin the cell (184). Fusionof this MOPC 21 L chain-producing variant to a different myeloma cell permits assemblyof the MOPC 21 L chain to a heterologous H chain. This assembly prevents the MOPC 21 L chain from being degradedintracellulady (184). However,in most lymphoidcells, unassembled L chains are not degradedintracellularly (9). Variantor normalcells that express H chain in the absence of L chain expression mayor maynot rapidly degradethe Hchains within the cell (113, 154). Variant myeloma cells that express L chains and J chains, but not Hchains, degradethe J chains(113-115).Finally, there is very little informationregardingregulation of H, L, or J chain degradationin ceils representing different Blymphocytedevelopmentalstages. Thefinal step in expressionof Ig by B lymphocytes is cell surface insertion of membrane Ig or secretion of secreted Ig. In general, free L chains are secreted, whereasnormalHchains are not secreted unless linked to L

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chains (86, 113). Thus,pre-Bcells generallydo not secrete their Hchains (101, 122b,142, 154). Asingle report of human pre-Bcells activelysecreting H chain in the absenceof L chain (95) needs to be rigorously documented by further experiments. In contrast, there are a numberof examplesof lymphoidcells that secrete mutant H chains in the absence of L chain expression (113). As noted above, unassembledMOPC 21 L chains are not secreted, but are degradedintracellularly. However,translation of MOPC 21 L chain mRNA in Xenopusoocytes results in an L chain that is neither secreted nor degraded (172). However,the MOPC 21 L chain is secreted if MOPC 21 H chain mRNA is simultaneously translated in oocytes (172). If another L chain MRNA is co-injected into oocytes with MOPC 21 mRNA, only the non-MOPC 21 L chain is secreted (172). Thus, it appears that the lack of secretion of free MOPC 21 L chain from MOPC 21 myeloma cells maynot result from the rapid intracellular degradationof this L chain. Severalvariant myeloma cell lines synthesizemutantL chains or Hchains that are not secreted evenwhenassemblednormally(113, 115). Themutationsare not localized to a uniqueregion. This suggeststhat these mutations maycause conformational changes in immunoglobulinchains that are incompatiblewith secretion. Expressionof membrane Ig seemsto require simultaneousexpressionand co-assemblyof Hand L chains. Pre-B cells generally express cytoplasmic bt but no surfaceor secretedbt chains(95, 101, 122b,153c, 154). Thereare several recent reports of murineand humanpre-B lymphomas that express #mon the external call surface in the absenceof L chain expression(58a, 122a). Themurinecell line (70Z)usually expressescytoplasmic/z,but expresscell surface/z spontaneously or after dextransulfate stimulation. If this result can be validatedfor normalB cells, it has importantimplications for potential regulationof pre-Bcells by anti-idiotypic antibodyor antigen. Aplethora of studies has attemptedto determinewhetherglycosylation of normalor variant Hchainsis necessaryfor cell surface expressionand/or secretion of Ig. In general, glycosylationof Hchains is not necessaryfor cell surface expression(67, 153b). Theresults are morecomplicatedfor secretion of nonglycosylated Ig: (a) Secretionis blockedor markedlyinhibited for tunicamycin-treatedmyelomas that synthesize IgM,IgE, and IgA, but a tunicam.ycin-treatedhybridomasecreted a different IgAmoleculeat apparently normalrates; (b) secretion from tunicamycin-treatedmyelomas is little affected for IgG or IgD; (c) a B lymphoma (WEHI279) secretes lowlevels of IgMat only a marginallyslowerrate after tunicamycintreatment(20, 65, 66, 82, 113, 153b,155, 156, 157, 187). Someof the results obtainedabovedo not dependon the cells per se, since the samehybridcell is shownto secrete nonglycosylatedIgGbut not to secrete nonglycosylated IgM.In sum,whetheror not nonglycosylatedIg is secreted dependson the class of.Ig, the amountof Ig synthesized,and possiblythe VHgenesegment

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IMMUNOGLOBULINS 417 in the H chain. Asnoted above,there is no evidencefor regulation of core glycosylation in B lymphocytes. Themostintriguing exampleof differential post-translational regulation of Ig at different stages of B cell developmentinvolves expressionof H chains in secreted and surface Ig. As noted previously, B lymphocytes that synthesize H chain mRNA contain both membraneand secreted H-chain mRNA’s.The ratio of secreted/membranemRNA increases during B-cell development (140). Fromlimited data, it appearsthat the ratio of intracellular membrane andsecreted polypeptidechains reflects the ratio of the two H-chain mRNA’s.Approximately20-50%of the H chain is kq in pre-B or immatureB cell lines and small resting B lymphocytesfromanimals(2, 4, 5, 48, 101,136, 122b,153b).Yetthese cells secrete little (or none)of /zs polypeptidetranslation product (101, 122b,155). In contrast, the translation product is quantitatively secreted frommyeloma cells, B-cell lines and normalresting B cells stimulatedwith mitogensor T cell factors (86, 113, 122b). Thelack of secretion of Ps at early B cell developmental stages is not dueto a lack of core glyeosylation,since the unseeretedkts chains contain core oligosaccharide(but lack terminal sugars). Neither it due to a block in H and L chain assembly,since IgMwith Ps chains is formedin these cells. Similar results havebeenfoundfor a B cell line that expressesboth as and am(82, 159), althoughthe lack of secretion here less well documented than the examplescited above. Themechanism of this specific post-translational regulation of secretion is unclear. It mightbe due to any or a combinationof the following:(a) differential oligosaccharideprocessingand terminal glycosylation of the core oligosaccharide; (b) failure of transport into the Golgi apparatus, whichwouldexplainthe glycosylationdeficiencies; (c) lack of J chain(100). Theapparentlack of quantitative secretion of as froma B lymphoma argues against a role for J chainsince IgAcan be secretedas a monomer (82, 159). Similarly, the normalsecretion of nonglycosylatedIgMfroma B lymphoma cell line (153b)arguesthat glycosylationdoesnot explainthis situation. Studieson certain lines of 70 Z and18-81cells suggestthat the expression of surface IgMmayrequire morethan the expression of Pmand L chains (26, 122a, 142). The molecularmechanisms that control IgMsecretion early B cells represent interesting questionsyet to be resolved. FUTURE

DIRECTIONS

The studies reviewedhere dearly provide considerable insights into the events in immunoglobulin gene expression and regulation. Nonetheless, manyof the molecularmechanisms affecting the changingpatterns of immunoglobulin geneexpressionin B-cell development remainto be resolved.

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The powerful resolution of recombinant DNAcloning and sequencing will continue to provide insights into immunoglobulin gene structure. Several exciting recent developments provide molecular immunologists even more penetrating approaches for dissecting immunoglobulin gene control. These include (a) the establishment of cloned normal pre-B cell and B cell lines that undergo developmental changes in culture (183a), (b) the successful introduction and expression of cloned immunoglobulin genes transfected into lymphoid cells (42a), and (c) the increasing refinement of culture conditions and factors that stimulate B cell growth and affect changes in immunoglobulin gene expression (55a, 93a, 124a). ACKNOWLEDGMENTS Michael Kuehl has been supported by USPHSgrants AI 12525, AI 17748, and AI 00293 at the University of Virginia Medical School, Department of Microbiology. Randolph Wall has been supported by USPHS grants AI 13410 and CA 12800, and by NSF grant PCM79-24876. Literature Cited 1. ~tcad. Alt, F., ScL Baltimore, D. 1982.Proc.Natl. USA79:4118-22 2, Alt, F. W., Bothwell,A. L M., Knapp, M., Siden,E., Mather,E., et al. 1980. Cell 20:293-301 3. Alt, F., Enea, V., Bothwell,A. L. M., Baltimore,D. 1980. Cell 21:1-12 4. Alt, F. W., Rosenberg,N., Casanova, R. J., Thomas,E., Baltimore,D. 1982. Nature 296:325-31 5. Alt, F. W., Rosenberg,N., Enea, V., Siden, E., Baltimore, D. 1982. Mol. Cell. Biol. 2:386-400 6. Alt, F. W., Rosenberg,N., Lewis, S., Thomas,E., Baltimore, D. 1981. Cell 27:391-400 7. Altenburger, W., Steinmetz, M., Zachau, H. G. 1980. Nature 287:603-7 8. Amara,S., Jonas, V., Rosenfeld,M.G., Ong,E. S., Evans, R. M. 1982. Nature 298:240-44 9. Baumal, Scharff,M.D. 1973.J.. Immunol. R., 111:448-56 10. Banerji, J., Rusconi,S., Schaffner,W. 1981. Cell 27:299-308 11. Benoist, C., Chambon, P. 1981. Nature 290:304-10 12. Bentley,D.L., Farrell, P. J., Rabbitts, T. H. 1982. Nucl. Acids Rez 10: 1841-56 13. Bentley, D. L., Rabbitts, T. H. 1980. Nature 288:730-33 W.,254:8869-76 Kuehl, W. M. 1979. 14. Bergman, J. Biol,. L. Chem.

15. Bergrnan, L. W., Kuehl, W.M. 1979. J. Supramol.Struct. 11:9-24 16. Bergman,L. W., Kuehl, W.M. 1982, In The Glycoconjugates, 3:81-98. New York: Academic 17. Bernard, O., Gough,N. M., Adams,J. M. 1981. Proc. Natl. Acad. Sci. USA 78:5812-16 18. Bernard, O., Hozumi,N., Tonegawa, S. 1978. Cell 15:1133-44 19. Bevan,M., Parkhouse,R. M. E., Williamson, A. R., Askonas,B. A. 1972. Prog. BiophyzMol. Biol. 25:131-62 20. Blatt, C., 11:65-66 Haimovich,J. 1981. Eur. J. lmmunol. 21. Bodary, S., Math, B. 1983. EMBO Journal6:In press 22. Bothwell, A. L. M., Paskind, M., Schwartz, R. C., Sonenshein, G. E., Gefter, M.L., Baltimore,D. 1981. Nature 290:65-67 23. Bourgois,A., Kitajima,K., Hunter, I. R., Askonas,B. A. 1977. Eur. J. lmmunol. 7:151-56 24. Boyd,A. W.,Goding,J. W., Shrader,J. W.1981. Z lmmunol..126:2451-65 25. Breathnach, R., Chambon,P. 1981. An~Rev. Biochem. 50:349-83 26. Brock, E. J., Kuehl, W. M. Unpublished observation 27. Brown,D. 1981. Science 211:667-74 28. Burrows,P. D., Beck,G. B., Wabl,M. R. 1981. Proc. Natl. Acad. ScL USA 78:564--68

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78. Kehry, M., Sibley, C., Fuhrman,J., Schilling, J., Hood,L. E. 1979. Proc. Natl. Acad. ScL USA76:2932-36 C., Perry, R. 79. Kelley,D.E., Coleclough, P. 1982. Cell 29:681-89 80. Kemp,D. J., Harris, A. W., Adams,J. M. 1980. Proc. Natl. Acad. ScL USA 77:7400-4 81. Kemp,D. J., Harris, A. W., Cory, S., Adams,J. M. 1980. Proc. Natl. ~lcad. ScL USA77:2876-80 82. Kikutani, H., Sitia, R., Good,R. A., Stavnezer, J. 1981. Proc. Nail Acad. Sci. USA78:6436-40 83. Knapp, M. R., Liu, C., Newell, N., Ward, B., Tucker, et 79:2996al. 1982. Proc.R. Natl. Acad. P. ScLW., USA 30OO 84. Korsmeyer, S., Hieter, P., Ravetch,J., Poplack, D., Waldmann, T., Leder, P. 1981. Proc. Natl. Acad. Sci. USA 78:7096-100 85. Koshland,M. E. 1975. Adv. Imtnunol. 20:41-69 86. Kuehl, W.M. 1977. Curr. Topics Microbiol. ImmunoL76:1-47 87. Kuehl, M. 1981. Trends Biochem.Sci. 6:206-8 88. Kuehl,M. W., Word,C. J. Unpublished results 89. Kurosawa,Y., Tonegawa,W.1982. J. Exp. Med. 155:201-18 90. Kwan,$.-P., Max,E. E., Seidman,J. G., Leder,P., $charif, M.D. 1981.Cell 26:57-66 91. Laskov, R., Kim,J. K., Asofsky, R. 1979. Proc. Natl. Acad. Sc~ USA 76:915-19 92. Laskov,R., Kim,J. K., Woods,V. L., McKeever,P. E., Asofsky, R. 1981. Eur. £ Immunol. 11:462-68 93. Lerner, M. R., Boyle,J. A., Mount,S. M., Wolin,S. L., Steitz, J. A.1980.Nature 283:220-24 93a. Lernhardt,W., Corbel, C., Wall, R., Melchers,F. 1982. Nature. 300:355-7 94. Levinson, B., Khoury, G., Vande Woude,G., Gruss, P. 1982. Nature 295:568-72 95. Levitt, D., Cooper,M. D. 1980. Cell 19:617-25 96. Levy,R., Dilley, J. 1978. Proc.Natl. Acad. ScL USA75:2411-15 97. Lifter, J., Higgins,G., Choi,Y. S. 1980. Mol. Iramunol. 17:483-90 98. Liu, C., Tucker,P. W., Mushinski,J. F., Blattner, F. R. 1980. Science 209:1348-52 99. McGee,J. D., Wood,W.I., Dolan,M., Engel, J. D., Felsenfeld,G. 1981. Cell 27:45-55

100. McHugh,Y., Yagi, M., Koshland, M. E. 1981. In B Lymphocytesin the ImmuneResponse: Functional. Developmental,and Interactive Properties,ed. N. Klinman,D. Mosier, I. Sher, E. Vitetta, pp. 467-74.NewYork:Elsevier 100a.Immunol. McKenzie, I., Potter, T. 1979. Adv. 27:179-338 101. Mains,P. E., Sibley, C. H. 1982.J. lmmunol. 128:1664-70 101a. Maid, R., Kearney, J., Paige, C., Tonegawa, S. 1980. Science 209: 1366-69 102. Maki,R., Roeder, W., Traunecker,A., Sidman,C., Wabi,M., et al. 1981. Cell 24:353-65 103. Marcu,K. B. 1982. Cell 29:719-21 104. Mareu,K. B., Sehibler, U., Perry, R. P. 1978. J. Mol.Biol. 129:381-400 105. Margulies,D., Kuehl, W.M., Schatff, M. D. 1976. Cell 6:405-415 105a.Mather,E. L., Alt, F. W.,Bothwell,A. L. M., Baltimore, D., Koshland,M. E. 1981. Cell 23:369-78 106. Mather,E., Perry, R. 1981. NucLAcids Res, 9:6855-67 107. Mathis, D., Oudet, P., Chambon,P. 1980. Prog. Nucl. Acids Rez MoLBioL 24:1-55 108. Max,E. E., Seidman,J. G., Miller, H., Leder, P. 1980. Cell 21:793-99 109. Melehers, F., 4:181-88 Andersson,J. 1974.Eur. J. lmmunoL 109a. Melchers, F., Andersson,J., Lernhardt, Rev. W., Sehreier, M. H. 1980. lmmunol. 52:89-114 110. Melchers,U., Eidels, L., Uhr, J. W. 1975. Nature 258:434-35 111. Mellon, P., Parker, V., Gluzman,Y., Maniatis, T. 1981. Cell 27:279-88 112. Moore,K. W., Rogers,J., Hunkpiller, T., Early, P., Nottenburg,C., et al. 1981. Proc. Natl. Acad. ScL USA 78:1800-4 113. Morrison,S. L., Scharff, M.D. 1981. CRCCrit. Rev. lmmunoL3:1-22 114. Mosmann, T. R., Gravel, Y., Williamson, A. R., Baumal,R. 1978. Eur. Z Immunol. 8:94-101 115. Mosmann,T. R., Williamson, A. R. 1980. Cell 20:283-92 116. Mushinski,J. F., Blattner, F. R., Owens, J. D., Finkelman, F. D., Kessler,S. W., et al. 1980. Proc.Natl. Acad.Sci. USA77:7405-9 117. Nevins,J. R., Darnell, J. E. 1978.Cell 15:1477-93 118. Nottenberg, Weissman, I. 78:484-88 L. 1981. Proc. Natl. C., Acad. Sci. USA 119. Nuss, D. L., Koch,(3. 1976. Z ViroL 19:572-78

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An~Rev. lmrauno£ 1983. 1:423-38 Copyright ©1983by AnnualReviewsInc All rightsreserved

THE BIOCHEMISTRY OF ANTIGEN-SPECIFIC T-CELL FACTORS D. R. Webb Roche Institute

of Molecular Biology, Nutley, NewJersey 07110

Judith A. Kapp and Carl W. Pierce Department of Pathology and Laboratory of Medicine, The Jewish Hospital of St. Louis, and the Departments of Microbiology and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

INTRODUCTION Soluble factors producedby immunoeompetent cells in response to antigenie stimulation represent one communications link by whichthe immune systemregulatesits activities. Thesefactors maybe spedtic for the inducing antigen or maybe non-antigen-speeitic.Otherchapters in this volumediscuss in detail the cellular aspects of immune regulation. This communication focuseson the biochemicalandserological nature of antigen-specific regulatory molecules.Thesemoleculesare exclusively the products of Tcells (immunoglobulins areobviously antigcn-spccilic andmayalsoserve regulatory function, buttheyarcdiscussed elsewhere). Theymaybedivided into twocategories: helper factors (Tr~F), which helpT cffector cells or cells torespond (e.g. become cytotoxic ormakeantibody); andsuppressor factors (TsF), which suppress T and/or B cellresponses. These factors are extremely potent interms oftheir specific activity, andarepresent in picogram quantities inlymphoid tissues. Forthese reasons, studies onthe biochemical nature of these substances haveprogressed veryslowly. A limited numbcr ofthcsc factors havc been purified toanysignificant degree andonlya fewlaboratories havereported purilication tohomogeneity (II, 21).This state ofaffairs haschanged recently with application ofsomatic 423 0732-0582/83/1M I0-0423502.00

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call hybidizationmethodology to T cells (3) and the use of microanalytical protein purification andanalysis to characterizethe hybddoma T-cell produet (16, 21, 49, 50). SomaticT-cell hybrids provide reasonably stable, rapidly growingcell lines, whichconstituitively produceT-cell factors. PrimaryT-cell clones havealso beenproduced,but so far havebeen less useful for the extensivebiochemicalcharacterizationof T-cell factors, due to culture limitations and stability (12). Mostof the studies discussedhere havenot beencarried out on fully puritied factors. Thus,conclusionsregarding the precise biochemicalproperties of most of these substances should be regarded as tentative. The reason for such caution becomes apparentwith the serological evidenceimplicatingIgVH region genesas the sourceof a portionof antigen-specificT-cell factors. Whatfollows,then, is in the nature of a progress report on whatis knownabout antigen-specific factors, both their structure and their genetic origin. Weclose with a consideration of what questions need to be addressed in the immediate future. ANTIGEN-SPECIFIC

HELPER FACTORS (THF)

In 1980, several very excellent reviews appeared, whichdiscussed the progress that had been madeon understandingthe biology, genetics, and biochemistryof antigen-specificfactors (1, 37, 45). In the ensuing2 years it is safe to say the progresshas beenuneven.This is particularly true of the antigen-specific helper factors. Despite the developmentof T-cell somatic hybrid technology, it is still di~cult to makeantigen-specific THF-producing T-cell hybrids. Becauseof this, and the fact that those hybridomasthat have been madestill produce relatively small amounts (micrograms/liter) of THF,biochemicalcharacterization has proceeded slowly. TheTnFmostextensively characterized are presented in Table 1. Wehavenot attemptedto list all of the THFthat havebeenreported;rather, wehavetried to include those factors for whichthere are moredata than wereavailablein 1980(1, 37, 45). Asmaybe seen, the list is short. However, somegeneralities about structure andfunction are beginningto emerge,and someconclusionsreached fromearlier studies seemto be holding up. First, those factors that havebeentested react with an antisera specific for Vn-framework determinants; manyTBFalso react with anti-idiotypespecific antisera (Id+). This impliesthat T cells use VHgenesor a portion of Vii genesto specify TnF.Recentresults fromseveral laboratories raise somedoubtabout the validity of this conclusion. Followingthe observations of Tonegawa and his colleagues (32), as well as others (33), mature B cells contain rearranged VLand Vri genes, several groups analyzed T-cell DNA (7, 19). Theresults havebeenquite consistent. T cells

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Z

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Z

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mayrearrange D-region and J-region sequences, but no T cell has yet been found that has a productively rearranged (e.g. functional) VRor VLgene. This leads to the conclusion that whatever T cells use to make antigenspecific proteins, they do not need rearranged VH or VL genes. Furthermore, molecular probes that represent the gene sequences specifying certain idiotypes have bcen utilized in studies of RNAand DNAfrom s¢rologically idiotype-positive (Id +) T cells. Using these probes, the two groups conducting these studies have failed to detect functional rearrangements of the VH genes in Id+ T cells; neither have they been able to showthe existence of mRNA coding for Id + proteins in the serologically Id + T-cell lines studied so far (27; E. Kraig, personal communication).This wouldsuggest that the Id + T cells maybear a determinant that cross-reacts with antiidiotype antisera but is not encoded by the gene specifying the original idiotype. A fascinating result is the observation by Lambet al (22) that the streptococcal antigen (SA)-specific THFfrom monkey and mouse react with xenoantisera against human//2 microglobulin. Since//2 microglobulin is homologouswith constant region domains, once again there is a suggestion of commongenetic origin for these T-cell factors and Ig (not to mention Thyl, H-2, etc). It is also possible that in the case of the SA-specific TriF, //2 microglob.ulin constitutes part of the molecule. In the case of mouse SA-specific THF,it reacts with chicken, rat, and guinea pig anti-//2 microglobulin but not monoclonal anti-human//2 microglobulin; monkeySAspecific Tt.IF react with chicken, rabbit, and rat anti-//2 microglobulin, as well as with monoclonalanti-human f12 microglobulin (22, 53). These data cannot discriminate between the possibility that the SA-specific THFare associated with//2 microglobulin or that they have sequence homologywith //2 microglobulin,although Lambet al favor the latter possibility. All of the factors tested react with antisera specific for H-2loci. This raises the issue as to whether or not the mixing of putative gene products of chromosome 12 with those of chromosome17 can occur. Since anti-Id antisera may not be detecting a chromosome12 gene product, there may be no problem at all. Also, if somefactors contain two chains, then one can easily concoct a two-gene two-polypeptide chain hypothesis to explain the results. This maybe the case for one class of two-chain polypeptide suppressor factors (see below). One-chain THF, containing apparently both VHand H-2 determinants, would present the biggest problem, but no THFof this type have yet been reported. The solution is simply to wait until more data become available concerning the nature of the genes that specify these molecules. It is notable that none of the studies summarizedin Table 1 has assigned a specific activity to the factor being examined. This is unfortunate. The measurementof biological activity by dilution analysis, coupled with careful estimates of protein concentration, give one. of the best measures of

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427

relative purity whenone calculates activity/unit weight (e.g. milligrams or micrograms).Purity, or its lack, represents the biggest obstacle to clear-cut interpretations of data. This is particularly important whendrawingconclusions about molecular weight or structural composition. For this reason, the data summarizedin Table 1 must be regarded as tentative, pending more complete purification.

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ANTIGEN-SPECIFIC

SUPPRESSOR

FACTORS

In contrast to the antigen-specific helper factors, more progress has occurred in the biochemical characterization of antigen-specific suppressor factors. Reports are beginning to appear on purification and concommitant biochemicalanalyses of the isolated factors (11, 21). In addition, at least two groups have begun studies aimed at the cloning of the C-DNA derived from the mRNA that codes for TsF, which would for the first time allow us to look directly at the genes responsible for these factors (44, 51). A large number of TsF have been described, and new reports continue to appear (1, 37, 45). The progress has been made possible both by the continued improvementin our capacity to grow cloned T cells that maintain their function and by the use of hybridomatechnology (6). Most reports factor-producing hybridomas that have appeared since 1980 are concerned with suppressor factors rather than antigen-specific helper factors (6). Whether this is related to the almost exclusive reliance on BW5147 as a fusion partner or to other reasons is not clear. In addition to these techniques, the successful production of radiation leukemia virus-transformed T-cell lines with suppressor function has been presented recently (30, 31). The fact that such lines produce large amountsof antigen-specific TsF, are easy to grow, and have apparently a normal chromosomecomplement may makethem increasingly attractive to workers in this field. Since other authors in this volumecover the regulatory aspects of suppressor and helper factors, we confine ourselves, as with TnF, to a consideration of the biochemistry of TsF. It is known that antigen-specific suppressor factors could be elaborated to manykinds of antigens. For example, several different synthetic polypeptides, the immuneresponse to which is knownto be under Ir gene control, induce suppressor cells. In manycases these cells have been shownto release suppressor factors that block the appearanceof antibody-formingcells (18, 20, 34, 48). Suppressor factors that block the humoral immuneresponse to natural proteins such as KLHor HGGhave also been reported (39, 40). Cellular immuneresponses to alloantigens or tumor antigens maybe shownto be regulated by suppressor cells and TsF (15, 38). Also, haptens, either on proteins or autologous cells, mayinduce specific TsF (14, 54). It maybe concludedthat virtually every type of immuneresponse results in the generation of suppres-

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sor cells that mayrelease antigen-or determinant-specificTsF.It wasalso shownthat such factors could be idiotype specific rather than antigen specific and still be able to induceantigen-specificsuppression(36). Many of the factors reactedwith both anti-Vregion antisera (underwhichheading for convenienceweinclude anti-idiotype) and anti-I-region-specific antisera; andnonehad Ig constant-regionmarkers,as they are definedserologitally (1, 37, 45). Rather than present a completelisting of all of the suppressorfactors describedsince 1980,onceagain wefocus on those factors for whichwenow have substantial biochemicalinformation. Acompilationof these factors and their characteristics is listed in Table 2. There are four groups of investigators whohave been carrying out the most extensive biochemical analyses. Theyinclude(a) our group, whichis studyingfactors specific for the synthetic copolymersGAT,GA,and GT(16, 21, 49); (b) Cantor his associates, studyinga SRBC-specific factor (10-12); (c) Taniguehi Tadaand their co-workers,studying KLH-specificsuppressor factors (4143, 46, 47); and (d) Dorf, Greene,Benacerraf, and their associates, havedoneextensive serological studies and perhapshavethe mostcomplete set of suppressorfactors specific for NP(26, 28, 29). Manyother investigators have performedstudies on suppressor factors, but they have emphasized the morebiological aspects of these agents andfor that reasonthese other factors are not listed here (1, 37, 45). Fresnoet al have studied TsF obtainedfrom a cloned T-cell line using radiolabeled TsF. TheTsF in Table 2 have beenshownto suppress primary humoralimmuneresponses, secondaryhumoralimmuneresponses, or cellmediatedresponses. All of the factors react with antisera specific for one or another determinant on antibody Vr~ chains. All but one factor, the glycophorinTsF, react with H-2Iregion-specific antisera. Wherestudy has beenpossible, manyof the factors showMHC restriction or Igh restriction, or both. In terms of molecular weights, these molecules range between 19,000-80,000and maybe composed of one or two chains. Oneof the more interestingfeaturesof the table is the listing of specificactivities. Of the four research groups,only two havemadeestimates of specific activity. In our opinion, such estimates are crucial to any biochemicalstudy, since such estimates represent the only wayof assessing the degree of purity. Verylittle is knownconcerningstructure-function relationships of the majority of the TsF. This is no doubt largely becausebiochemicalstudies are quite recent. Fresnoet al (10) have reported that the glycophodnTsF maybe dissociated into an antigen-binding peptide of 25,000-MW and a 45,000-MW protein that retains the capacity to be suppressive; however,it nowlacks antigenic specificity. Thestudies of MHC and immunoglobulin

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allotype restriction suggest that both H-2 determinants and Igh determinants present on TsF are required for function. In this regard, the recent results of Sorensen et al are of interest (17). They have obtained two-chain GAT-TsFfrom a hybridoma (372D6.5) in which they have confirmed the observations of Taniguchi et al (41-43, 46, 47). That is, one chain carries the antigenic specificity and one chain bears an I-J determinant(I-Jb). When the two chains are dissociated by reduction and alkylation, they maystill be shownto have suppressive activity when added together. What is most surprising, the antigen-binding chain maybe added to spleen cell cultures given antigen; 24-48 hrs later, the I-J-boring chain may be added and suppression of the anti-GATresponse is still observed (17). This result suggests that in tissue culture, at least, antigen-binding chains mayfunction in the absence of I-J chain and the I-I chain may perform its function possibly without the antigen-binding chain. That such complex structurefunction relationships are the normrather than the exception is also suggested by recent data reported by Gershonand his associates (9, 52), who propose that suppressor-inducer factors may form molecular associations with "schlepper" or carder moieties which help to direct them to the proper cellular receptor. Anotheraspect of structure-function is specificity. In the synthetic polypeptide system (GAT,GA,or GT), it maybe seen that hybridomas derived from fusions with GATas the immunizing, antigen may be either GAT/{3A- or GAT/GT-specific. Of interest are the two hybridomas derived from fusions with H-2s splenic T cells. Whether the immunizing antigen was GT or GAT,the binding specificity to GAT-, GA-, or GT-Sepharose is the same: both 342.B1.11 and 395A4.4 TsF bind GATor GT. In fact, 395A4.4 may be analyzed using GAT-MBSA-pdmed cells with identical results to GT-MBSA-primed cells. The amino terminal sequences currently available through residue 30 show few differences between GAT-or GTspecific TsF (D. Webb,manuscript in preparation). Wherethe specificitydetermining regions are in these molecules remains to be determined. The SRBC-induced TsF-producing clone C1Ly23/4 was shown by Fresno et al (11) to have specificity for glycophodn.Interestingly, these TsF can discriminate (apparently) glycophorins from different species red blood cells (e.g. sheep versus burro). To date, no studies have been F. performed to determine the precise specificity of the KLH-Ts In addition to the results of studies shownin Table 2, two groups have also characterized to a similar extent the mRNA that codes for their respective suppressors. A summaryof these results is presented in Table 3. Wieder, in our laboratory (50, partially purified polyA+ RNAfrom hybddoma258CA.4by subjecting it to sucrose density gradient centrifugation. Fractions were collected and were translated in a rabbit reticulocyte lysate

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translation system, and the translated products were assayed for OATspecific suppression. RNAthat had a sedimentation coefficient of 16S was able to code for GAT-TsF.The molecular properties of this translated product indicate that it is similar if not identical to supernatant OATTsF from 258C4.4. Other investigators in our group (35; K. Krupen, unpublished data; E. Oerassi, unpublisheddata; C. Healy, submitted for publi+ cation; C. Sorensen, manuscript in preparation) have translated polyA RNAfrom T-cell hybridomasand have obtained similar results. Anintriguing feature in somehybridomasis the appearancein the cell-free translation products of a larger molecular weight form of TsF (80,000). Whatrelationship this has to the lower molecular weight form remains to be determined. In another study, Taniguchi and co-workers (44) isolated polyA+ RNA from a KLH-TsF-producinghybfidoma and subjected it to sucrose density centrifugation. They were able to isolate three species of RNA.Twoof these RNAs,13S and 18S, when translated in a frog oocyte system, yielded the antigen-binding chain, and the I-J-bearing chain was derived from an mRNA with a sedimentation coefficient of 11S. The properties of the translated products are similar if not identical to those of TsF obtained from culture supernatants of the hybddomas.Both our group and that of Taniguchi are involved in the molecular cloning of their respective mRNAs. This suggests that in the not-too-distant future we should be able to study directly the genes involved in the synthesis of regulatory T-cell products. Such molecular probes will undoubtedlybe useful in deciphering the nature of the antigen receptor on T cells. Having outlined the biochemical data currently available on antigenspecific TsF, it is necessaryto address the significance of these results. Some notable differences shownin Table 2 are the different molecularweights that have been determined for the different factors and the differing structures (e.g. one chain or two). A plausible reason for these differences may derived from studies in the OTsystem as well as the data obtained with the NPhapten, which suggest that several types of suppressor factors exist. Indeed, Germain&Benacerraf, in a recent review (13), have concluded that a suppressor cell "cascade" exists in which TsF serve as the links between different suppressor cell subclasses, as well as acting on nonsuppressor targets, T-helper cells, or B cells. Althoughit maybe that the details of the Germain& Benacerraf"consensus pathway" are incorrect, the general idea seemsto fit the available biochemicaldata. In the GTsystem, it is possible to show that single-chain, non-MHC-restricted, antigen-specific factors (TsF1) induce the appearance of MHC-restficted (presumably two chain) antigen-specific suppressor factors (TsF2). In the NPsystem, a cascade has been established involving an antigen-specific idiotype-positive suppressor inducer TsFl, which induces an anti-idiotype TsF2 which induces antigen-

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ANTIGEN-SPECIFIC T-CELL FACTORS 433 It is the TsF3 that directly interacts withTri or B cells. If one accepts this kind of scheme,it is possible to assign the KLH-TsF to the TsF2 class, based on homology to GAT-TsF currently being studied by C. 2 Sorensen, D. Webb,and C. Pierce (manuscriptin preparation). This TsF2 is composedof two chains, one antigen-specific and one I-J +, and is inducedby TsF~.Fresnoet al (12) havepreliminarydata that suggestthat the glyeophorinTsFcan directly affect B cells, whichwouldplace themin the TsF3 class. Notethat the TsF2 maybe either antigen-specificor idiotypespecific. Althoughthese explanationsremainhypotheticalto a large extent, they serveto illustrate the point that someof the different factors presented in Table2 almostcertainly represent different classes of TsF. Anissue rdated to molecularand structural heterogeneityis the question of serological or immunoehemieal analysis. Virtually all the serological evidencefor Igh, VH,idiotype determinants, or H-2determinantsmust be regardedas tentative. Theprincipal reasonfor makingso sweepinga statementis that for eachcase mentioned above,the antisera wereinitially raised with another antigen. Thus, reactions of TsF with these sera are crossreactive. In mostinstances it is not knownwhetherthe antisera recognize sequence-specificdeterminantsor conformationallyderived determinants. Asmentionedearlier in the discussionof TIFF,it is possible to demonstrate expressionof idiotype-positivematerialby T cells serologicallyin the absence of any detectable mRNA that can hybridize to the IgVri gene probes that encodeidiotype-positive Ig. In the case of the GAT-TsF, for example, E. Kraig (personal communication)has shownthat RNAextracted from several GAT-TsF-producing hybridomaswill not hybridize a molecularprobe that codes for idiotype-bearinganti-GATantibody (IgG). Nonetheless, the GAT-TsF will react with xenoantisera specific for this idiotype (GAT).Furthermore, preliminary sequence analysis of three GAT-TsF from three different hybridomas(258C4.4, 372B3.5, 395A4.4; Table 2) shows no homologyso far, with either anti-OATmonoelonal antibody or any knownV~. or VHregion sequences. Suchdata suggest one cannot be too cautious about drawingconclusionsfromserological data in the absenceof corroboratinginformation. In someof the cases shownin Table2, corroborationdoes exist. In the NPsystem,the existenceof I-J reactivity on the TsFand an I-J restriction at the cell target level supportsthe notion of a true I-J determinanton NPTsF. Likewise, the Igh determinants and Igh restriction imply a strong likelihoodthat an Igh-linkedgeneor genesplays a role in the structure and function of TsF. Thefact that both Tada(42) and Taniguchi(47) can by differential absorptionthe presenceof uniqueI-region geneproductsas well as Igh geneproductson T cells stronglysuggeststhat both sets of genes are importantin determiningthe structure and function of TIfF and TsF.

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specific TsF3.

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Thepossible existence of two determinantson a single polypeptide(GATTsF), eachspecified by genesthat lie on separatechromosomes, is a dit~cult problem.Onepossible solution, particularly whereanti-idiotypic antisera are used, is that the apparent presenceof the idiotype on the factor is misleading.In the case of the GAT-TsF, the anti-idiotypic antisera, anticOAT and anti-GA-1,have not beenadsorbedwith idiotype first and then tested for binding to GAT-TsF; thus, the idiotype-positive GAT-TsF may turn out either to be cross-reactiveor to reflect the limited heterogeneityof aminoacids allowedin the combiningsite for any GAT-specificprotein. Thecase for the I-J determinantreflecting a real rather than a cross-reacting epitopeis strongerin those cases whereI-J restriction has beennoted(e.g. NPand KLH).It is also moreconvincingthat the epitope is at least "I-J like" in those instances wheremonoclonal antibodies are available. This is the case for two of the GAT-TsF,the KLH-TsF,and the NP-TsFmolecules. Theexperimentswith the cell-free translation product of (3AT-TsF mRNA are also of interest in this regard. Wiederet al (51) showedthat GAT-TsF generated from mRNA translated in a rabbit reticulocyte lysate systemwereboundby anti-Iq-Sepharose.Since little if any glycosylation takes place in the microsome-free, rabbit reticulocyte lysate system,these results suggest that the I-region determinantrecognizedis not carbohydrate. These results were confirmedby Sorensenet al (35) using GATTsF (H-2b) translated in a rabbit reticulocytc lysate system, whichwas boundby monoclonalanti-I-Jb-Sepharose (,provided by C. Waltenbaugh).

THE T-CELL ANTIGEN RECEPTOR AND ANTIGEN-SPECIFIC FACTORS Part of the reason for studying TsF or TnFis the possibility that such factors will be related to the elusive T-cell receptor for antigen. Indeed, there arc manyserological similarities betweenTsFand T-cell membrane moieties. For example,both T cells and factors maybear idiotype, I-J, or Igh determinants(1, 37, 45), and Binz and Wigzclland their colleagues (3-5) haveimplicatedidiotypc-positivemoleculesas beingpart of the T-cell antigen receptor. Several factors (e.g. all of the (3AT-TsF and the KLHTsF)can be isolated from membrane extracts ofT-cell hybridomas.Indeed, Matsuzawa &Tada(37) showedthat I-J + T cells could bind soluble antigen andthat anti-I-J antisera couldblockantigen-bindingto I-J-positive cells. Theauthors interpreted these results as implicatingI-J-bearing molecules in involvement at antigen-binding sites. Amajordifficulty that arises, however, is the structural heterogeneityof T-cell factors in comparison to the Ig on B cells. Setting aside for the moment the fact that Ig mayformdimers or pcntamcrs,the basic structure of Ig is the samefromisotype to isotype,

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e.g. two light chains andtwo heavychains linked to one anotherby disulfide bridges. By contrast, amongthe antigen-specific TsF, there are single polypeptide chain moleculesof 29,000, an apparentlysingle chain of 70,000, and two-chain TsF, which mayhave disulfide bonds or not dependingon their cellular location. Do these molecules reflect the antigen receptor on the respective T cells? The question must remain unanswered.It should be noted that C. Healyet al (submittedfor publication) have foundin a GATTsF-producinghybridomaa membrane form of GAT-TsFthat exists primarily as a dimer of 60,000-70,000 molecular weight (with minorspecies of higher molecular weight polymers). Since its amino acid content is identical (in moles percent) to the 29,000-molecular-weight form, the 29,000- and 60,000-MWGAT-TsFappear to be identical. C. Sorensen (manuscriptin preparation), in a related study of GAT-TsF froma responder strain (H-2b) finds that GAT-TsF in their pure state have a strong propensity to aggregate. Thus, mulfimersof the basic 29,000-MW protein begin to appearandultimately cause the protein to precipitate fromsolution even thoughthe solution contains a hydrophobicsolute (propanol). All of the characteristics outlined aboveshouldserve as a caution to the unwarythat considerable workremains to bc done to unravel the nature of the T-cell antigenreceptor. Thebest predictionat this time suggests that such a receptorwill reflect the natureof the cell fromwhichit is derived, as well as reflecting the natureof that cell’s products.It is unlikely that different T-cell subsets use the sameantigen receptor, if we use the antigenspecific T-cell factors as a guide. Additional questions concerningT-cell productand receptor diversity, andthe extent of T-cell repertoires, the influence of haplotypeon these, and their relationship to the T-cell antigen receptor mustawait the biochemical analysis that is just beginning.It is likely in the next 5 yearsthat manyof the remainingquestions will have foundat least partial answers. Literature Cited 1. Altman, A., Katz, D. H. 1980. Production andisolation of helperandsuppressor factors. J. Immunol.Metl~ 38:9-41 2. Apte, R. N., L~wy,I., DeBaetscher, P., Mozcs,E. 1981. Establishment and characterization of continuous helper T celllines spcciiic topoly (L tyr, L glu)poly(DL ala)~poly(L lys).Z Immunol. 127:25-30 3. Binz, H., Soots, A., Nemlander, A., Wight, E., Fenner, M., et al. 1982. Induction of speeitie transplantationtolerance via immunization with donorspeeitie idiotypes. Ann. NY Acad. Sci. 392:360-74

4. Binz, H., Wigzell, H. 1976. Spccitic transplantation tolerance induced by autoimmunizationagainst the individual’s ownnaturally occurring idiotypic antigen-bindingreceptors. J. Exp. Med. 144:1438-57 5. Binz, H., Wigz¢ll, H. 1981. T-cellreceptors with all0-major histocompatability complexspcciticity fromrat andmouse. J. Exp. Med. 154:1261-78 6. Boehmer,H. V., Haas, W., KiJhler, G., Melehers, F., Zeuthcn, T., eds. 1982. T-cell hybridomas. Curr. Top. crobio£ ImmunoL 100 7. Cory, S., Adams, J. M., Kemp, D. 1980. Somatic rearrangements forming

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active immunoglobulin/z genes in B and T lymphoid lines. Proc.Natl. Acad. Sci. USA77:4943-47 8. Eshhar, Z., Woks, T., Zinger, H., Mozes,E. 1982.T-cell hybridomasproducingantigen-specificfactors express heavy chain-variable-region determinants. Curt. Topics Microbiol. Immunol. 100:103-9 9. Flood, P., Yamanchi,K., Gershon,R. 1982. Analysisof the interactions betweentwo moleculesthat are required for the expression of Ly-2 suppressor cell activity. Threedifferenttypesof focusing events maybe neededto deliver the suppressive signal. J. Exp. Med. 156:361-71 L., Can10. Fresno, M., McVay-Boudreau, tor, H. 1982. Antigen-specificT-lymphocyteclonesIII. Papainsplits purified T suppressor moleculesinto two functional domains. J. Exp. Med. 155:981-93 11. Fresno, M., McVay-Boudreau, L., Nabel, G., Cantor, H. 1981. Antigenspecific T lymphocyte clones. II. Purification and biological characterization of an antigen-specificsuppressiveprotein synthesizedby clonedT-cells. J. ¯ Exp. Med. 153:1246-59 12. Fresno, M., Nabel, G., McVay-Boudreau, L., Furthmayer,H., Cantor, H. 1981. Antigen-specific T lymphocyte clones. I. Characterizationof a T lymphocyte clone expressing antigenspecific suppressiveactivity. J. Exp. Med. 153:1260-74 13. Germain,R. N., Benacerraf,B. 1981. A single major pathwayof T lymphocyte interactions in antigen-specificimmune suppression.Scand. J. ltnraunol. 13: 1-10 14. Greene,M.I., Bach,B. A., Benacerraf, B. 1979. Mechanismsof regulation of cell mediatedimmunity.III. Thecharacterization of Azobenzenearsonatespecific suppressorT-cell derivedsuppressor factors. J. Exp. Med. 149: 1069-83 15. Greene,M. I., Fujimoto,S., Sehon,A. H. 1977. Regulationof the immune responseto tumorantigens. III. Characterization of thymicsuppressor factor(s) produced by tumorboring hosts, J. lmmunol. 119:757~i3 16. Healy, C. T., Kapp,J. A., Stein, S., Brink, L., Webb,D. R. 1983. Comparative analysis of a monoelonalantigenspecific T suppressor factor obtained from supernatant, membrane or cytosol of a T-cellhybridoma. In lr Genes."Past, PresentandFuture,ed. C. W.Pierce, S.

E. Cullen, J. A. Kapp,B. D. Schwartz, and D. C. Schretiter. Clifton, NJ: Humana.In press 17. Kapp,J. A., Araneo,B. A., Sorensen, C. M., Pierce, C. W.1982. Identification of H-2restricted suppressorT-cell -factor s~cific for L-glutamicacid~°-L tyrosine~° ~° _(GT)and L-glutamicacid -L-alanine~-L-tyrosine~(GAT).In Ir Genes:Past, Presentand Future,ed. C. W.Pierce, S. E. Cullen, J. A. Kapp,B. D. Schwartz,and D. C. Shreflter. Clifton, NJ: Humana.In press 18. Kapp,J. A., Pierce, C. W., DeLaCroix, F., Benacerraf, B. 1976. Immunosuppressive factor(s) extracted fromlymphoid cells of nonresponder mice -Pnirimedwith L-glutamic t° (GAT). acid~°-L-ala ne~°-L-tyrosine I. Activity and antigenic specificity. ,L. Immunol. 16:306-15 19. Kemp,D. J., Wilson,A., Harris, A. W., Shortman,K. 1980. The immunoglobulin fl constantregiongeneis expressed in mouse thymocytes. Nature 286: 169-70 20. Kontiainen,S., Howie,S., Maurer,P. H., Feldmann,M.1979. Suppressorcell induction in vitro. VI. Production of suppressor factors to synthetic poly peptide GATand (T,G)-A-Lfrom cells of and non-respondermice. J. responder lmmunol.122:253-59 21. Krupen, K., A_raneo,B., Brink, L., Kapp,J. A., Stein,S., et al. 1982.Purification and characterization of a monoelonal T cell suppressorfactor specific for L-glutamicacid~°-L-alanine~°-L. tyrosinet° (GAT).Proc.Natl. dcad.Sc£ USA79:1254-59 22. Lamb,J. R., Zanders,E. D., Sanderson, A. R., Ward,P. J., Feldmann, M., et al. 1981. Antigen-specifichelper factors reacts with antibodies to humanf12 microglobulin. £ ImmunoL127:231-34 23. Lonai, P., Arman,E., Bitton-Grossfield, J., Grooten,J., Hammerling, G. 1982. H-2restricted hdper hybddomas: Onelocus or twocontrol dual specificity? Curt. Top. Microbiol. 100:97-102 24. Lonai, P., Puri, J., Bitton, S., BenMeriah, Y., Givol, D., Hammeding, G. 1981.H-2restricted helper factor secreted by cloned hybridomacells, d. Exp. Med. 154:942-49 25. Lonai, P., Puri, J., Hammerling,G. 1981.H-2restricted antigen-bindingby a hybridomaclone that producesantigen-specifichelper factor. Proc. Natl. Acad. Sci. USA78:549-53 26. Minami,M., Okuda,K., Furusawa,S., Benacerraf, B., Dorf, M.E. 1981. Ana-

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ANTIGEN-SPECIFIC lysis of T-cell hybridomas. I. Characterization of H-2andIgh-restficted monoclonal suppressorfactors. J. Exp.Med. 154:1390-401 27. Nakanishi, K., Sugimura,K., Yaoita, Y., Maeda, K., KashiWamura,S., Honjo,T., KishimotoT., 1982. A T15 idiotype .positive T suppressor hybddomadoes not use the T15 VHgene segment. Proc. Natl. Acad. Sci. USA 79:6984-8 28. Okuda,K., Minami,M., Furusanra, S., Doff, M. E. 1981. Analysis of T cell hybridomas. II. Comparisonsamong three distinct types of monoclonal suppressor factors. J. Exp. Med. 154: 1838-51 29. Okuda,K., Minami,M., Sherr, D. H., Doff, M.E. 198EHapten-specificT cell responses to 4-hydroxy-3-nitrophenyl acetyl. XI. Pseudogenetic restrictions of hybddoma suppressor factors. J. Exp. Med. 154:468-79 30. Ricciardi-Castaguoli, P., Doria, G., Adorini, L. 1981. Productionof antigen-specificsuppressiveT cell factor by radiation leukemiavirus-transformed suppressorT ceil. Proc.Natl. Acad.Sc£ USA78:3804-8 31. Rieciardi-Castagnoli,P., Robliati, F., Barbanti, E., Doda,G., Adorini, L. 1982.Establishment of functional,antigen-speclfic T cell lines by RadLVinduced transformation of murine T lymphocytes. Curt. Top. Micro. Immunol. 100:89-96

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36. Sy, M-S.,Dietz, M.H., Germain,R. N., Benacerraf,B., Greene,M.I. 1980.Antigen and receptor driven regulatory + mechanisms. IV. Idiotype bearing I-J suppressorT-cell factors inducesecond ordersuppressorcells whichexpressanti-idiotype receptors. J. Exp.Med.151: 1183-95 37. Tada, T., Okumura,K. 1980. Therole of antigen-specificT cell factors in the immuneresponse. Adv. Immunol. 28: 1-87 38. Takei,F., Levy,J. G., Killburn, D. G. 1978.Characterizationof a solublefactor that specificallysuppresses the in vitro generationof cells cytotoxicfor syngeneictumorceils in mice.J. Immunol. 120:1218-24 39. Takemori,T., Tada,T. 1975.Properties of antigen-specific suppressiveT-cell factor in the regulationof antibodyresponsein the mouse.I. In vivoactivity and immunochemicalcharacterizations. J. Exp. Med.142:1241-53 40. Taniguchi,M., Miller, J. F. A. P. 1978. Specific suppressionof the immune responseby a factor obtainedfromspleen cells of micetolerant to human ,/-globuhn. J. lmmunol.120:21-26 41. Taniguchi, M., Saito, T., Takei, I., Tokuhisa,T. 1981. Presenceof interchain disulfide bondsbetweentwo gene products that composethe secreted form of an antigen-specific suppressor factor. J. Exp. Med.153:1672-77 42. Taniguchi,M., Saito, T., Tada,T. 1979. 32. Sakano, H., Maki, R., Kurosama,Y., Antigen-specificsuppressivefactor proRoeder, W., Tonegawa,S. 1980. Two duced by a transplantable I-J bearing types of somaticrecombination are neeT-cell hybridoma.Nature 278:555-58 essary for the generation of complete 43. Taniguchi,M., Takei,I., Tada,T. 1980. immunoglobulinheavy chain genes. Functional and molecularorganization Nature 286:676-83 of an antigen-specificsuppressorfactor 33. Seidman,J. G., Leder, A., Nan, M., from a T-cell hybridoma.Nature283: Norman,B., Leder, P. 1978. Antibody 227-28 diversity. Science 202:11-17 44. Taniguehi, M., Tokuhisa, T., Tanno, 34. Sorensen, C. M., Pierce, C. W.1982. M., Yaoita, Y., Shimizu,A., Honjo,T. Antigen-specificsuppressionin genetic -respondermice to L-glutamicacid~°-L 1982.Reconstitutionof antigen-specific t° suppressor activity with translation alanine~°-L-tyrosine (GAT).Characproducts of mRNA.Nature 298: terization of conventional and hybridomaderived factors producedby 172-74 45. Taussig, M.1980. Antigen-specific TsuppressorT cells frommiceinjected as cell factors. Immunology 41:759-85 neonates with syngeneic (}AT-macro46. Tokuhisa, T., Taniguchi, M. 1982a. phages. J. Exp. Med.156:1691-710 Twodistinct allotypic determinantson 35. Sorensen,C. M., Webb,D. R., Pierce, C. W.1982.Characterizationof an antithe antigen-specificsuppressorand engen-speeifiesuppressorfactor generated hancingT-cell factors that are encoded by cell-free translation. In Ir Genes." by genes linked to the immunoglobulin Past, Present and Future, ed. C. W. heavy chain locus. Z Exp. Med. 155: Pierce, S. E. Cullen, J. A. Kapp,B. D. 126-39 Schwartz,and D. C. Shretiter. Clifton, 47. Tokuhisa, T., Taniguchi, M. 1982b. NJ: Humana.In press Constant region determinants on the

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antigen-bindingchain of the suppressor tion of a biologicallyactive, antigenT-cell factor. Nature298:174-76 specific suppressorT-cell factor. Proc. 48. Waltenbough, C., Debre,P., Theze, L, Natl./lcad. Sci. USA79:3599-603 Benacerraf, B. 1977. Immunosuppres- 52. Yamauchi,K., Chao, N., Murphy,D. sive factor(s) specific for L-glutamic B., Gershon, R. K. 1982. Molecular ~° (GT).I. Production, acid~°-L-tyrosine composition of an antigen-specific,Ly-1 characterizationandlack of H-2restricT suppressorinducer factor: Onemoletion for activity in recipient strain. J. cule binds antigenandis I4-; anotheris Immunol. 118:2073-77 I-J + does not bind antigen, andimparts an Igh-vaxiableregion-linkedrestric49. Webb,D. R., Araneo,B. A., Healy,C., Kapp,J. A., Krupen,K., et al. 1982. tion. J. Exp. Med.155:655-65 Purificationand biochemical analysis of 53. Zanders, E. D., Lamb, J. R., Konantigen-specificsuppressorfactors isotiainen, S., Lehner,T. 1980. Partial lated from T-cell hybridomas.Curt. characterization of murineand monkey Top. Micro. and Immunol. 100:53-59 helper factor to a Streptococcalantigen. Immunology41:587-96 50. Webb,D. R., Araneo,B. A., Gerassi, E., Healy,C., Kapp,J. A., et al 1983. 54. Zimbala, M., Asherson, G. L 1974. T cell suppressionof contactsensitivity in Purification and analysis of antigenspecific suppressor proteins derived the mouse.II. Therole of soluble supfrom T-cell hybridomas.SecondInt. pressor factor and its interaction with Conf. on Immunopharmacol. In press macrophagcs. Euro. Z Imrnunol. 4: 794-804 51. Wieder,K. J., Araneo,B. A., Kapp,J. A., Webb,D. R. 1982.Cell-free transla-

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IMMUNOREGULATORY T-CELL PATHWAYS Douglas R. Green, Patrick M. Flood, and Richard K. Gershon Department of Pathology,TheHoward HughesMedicalInstitute for Cellular Immunology, YaleUniversitySchoolof Medicine,NewHaven,Connecticut 06510 INTRODUCTION Towardsthe end of the 18th and the beginning of the 19th century an alteration occurred in Westernthinking that allowedradical changesin manyfields of study,includingthe life sciences.1Prior to this time, the study of nature was predominantlyhermeneutic,concernedwith classification and interpretation of "signs" that wouldshowone the relationship of a plant or animalto its place in the universe(for example,its role in a medical cure). This perception of the order of reality persisted throughthe 18th century, until a newview emerged,in whichunderlying causes and processes wereexamined.Thus,nature and life movedinto the abstraction; the word biology was coined in 1800by Burdachand extendedin 1802to the study of living bodies by Treviranus and again by Lamark.It wasalso at this time (1803)that the germtheoryof disease first appeared(Med.J. 484). In 1826,Fidele Bretonneau concludedthat diseases are specific, developingthroughspecific reproducingagents. AgostinoBassi (1836)isolated a parasitic fungusas the causative agent in a silkwormdisease, and John Snow(1849) postulated similar agents operative in the communication cholera. Pasteur beganhis studies in fermentationin 1854, whichled to those studies that, together with those of Koch,resulted in the acceptance of the germtheory. Thus, disease cameto be understoodas an invasion of a healthy bodyby an infectious agent. Our understandingof immunology is firmly based upon this view. AlthoughPasteur generalizedthe vaccinationprocedureof Jenner to include ~Theviewspresentedin this introductionwerelargely influencedby Foueault(1). Other referencesto these laistodcal perspectivesare foundelsewhere(2,3).

439 0732-0582/83/0410-0439502.00

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manydiseases, it was Kitasato and von Behringwhoproposedthe humoral theory of immunity,whichwasgiven a molecular basis by Paul Ehdich. Thus, the view of immunityas recognition and response to an invasion by a foreign substancehas dominatedthe study of the immunesystem. The mechanism by whichself and non-self arc discriminatedhas only recently becomea pressing one in spite of Ehrlich’s conceptionof "horror autotoxicus," the body’s(usual) failure to react againstitself. Mostimmunologiststoday would agree with this view of immunology. Yet, science in the 20th century is undergoinganotheralteration in view. Not only can nature be unified by virtue of underlying processes, but processesthemselvescan be understoodin abstract terms (4). Information theory and cybernetics present waysof looking at complexsystems in a novel way. The immunesystem can be viewedas being in a complexhomeostasis, in whichrecognition of self (rather than non-self) is a key feature. This conceptrepresentsa paradigm shift, with resulting shifts in emphasis.Thus, the immune response can be viewedas a heuristic process in whichforeign antigenin the contextof particular self antigens perturbsequilibrium,producinga regulatory shift leading ultimately to positive responses(immunity) or negative ones (tolerance). Immune regulation emerges,then, as central area in immunology. It is the complexset of regulatoryinteractions that allows the immunesystem to "think" about its primary decision to generate a response that produces maximum destruction of a potential pathogenwith minimum damageto self tissues. Immune regulation is a youngfield of study, and as usually happensin newareas, terminologyis not yet standardized. Different groups have adopteddifferent terms for the systemsunderstudy, and attempts to reconcile all of these terms and systems(5) maybe prematureuntil they are better understood. In this review we consider only one of several outlooks on immune regulation, that of our laboratoryand perhapsa few others. It is our hope that this will aid the commendable attempts to unify immunoregulatory theory. IMMUNOREGULATORY DISSECTION

CIRCUITS

AND THEIR

Theregulation of the immune responseoccursby the interactions of different subsets of lymphocytes,especially T cells. Regulationmaybe negative (suppressive)or positive, as is discussed. EachregulatoryT-cell function stemsfrominteractions of cells that formcircuits with a consistent structure. Activationof a circuit involvesan inductionevent, usually a signal froman inducer-cellsubset. This signal is acceptedby anothersubset, which

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IMMUNOREGULATORY T-CELL PATHWAYS 441 transducesthat signal (thus "transducer"subset) into an effect, via effector cells. Functionally,then, inducercells act indirectly andthus are defined by the requirementfor transducerand/oreffector ceils for their activity to become manifest. Transducercells, in turn, are definedas those cells that acceptthe inducercell signals. This admittedlycircular definition is nonetheless useful. Effectorcells are definedby their productionof active molecules that containthe final informationsignal of the circuit. In each case, functionscan be ascribedto T cells that maycorrelate with discrete subpopulationsof cells, or whichmayrepresent multiplecapabilities of a single T-cell set. Todistinguishbetween these possibilities, it is necessaryto developmeansto separate T-cell populations and matcheach to its function. In multicellularorganisms, cells differentiateto a point wheretheir activity extendsbeyondcellular survivalto that of the overall individual. Acell achievesthis by the activationof discrete families of genesdesignedfor its specific function ("luxury"genes, becausethey are not vital to the cell’s immediatelivelihood and therefore they tend to be shut downduringcellular crisis). In the courseof this activation, a profile of molecularentities encodedby these genes mayappear on the cell surface. Thus, the surface phenotypeof a cell (such as a particular T lymphocyte) correlates with its specializedfunctionin the wholeorganism.Withthe use of specific reagents directed againstsuchsurfacelabels, uniqueprofiles of cellular antigenscan be associated with distinct functional populations. Notethat there is no requirementin such an approachfor knowledgeof whateach surface label is actually doingthere, only that it is there on onetype of cell andnot on another (of course such knowledgemaybe extremely valuable in understanding the mechanisms of a cellular activity). Examplesof surface markersthat are importantto the study of regulatory circuits in the murineimmune systemare the Lyantigens (6), particularly Ly-1 and Ly-2; the Qa antigens (7), particularly Qa-1; antigen associatedwith the I regionof of the H-2,such as I-A (8) and I-J (9); specificleetin receptors(10). Throughoutthis reviewa dominantthemeis the correlation of a unique cell surfaceprofile witha uniquefunctionto definecell subsetsin regulatory circuits. ORGANIZATION OF IMMUNOREGULATORY

CIRCUITS

In this review,regulatoryT-cell circuits havebeenorganizedinto levels of activity. This has been done acording to a simple scheme.Whenantigen perturbs homeostasis,there are cellular interactions that are necessaryand

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sufl[icient for a responseto occur(e.g. helperT-cell activationof a Bcell to producespecific antibody). In the absenceof one of these necessarycell populationsbecauseof, say, a genetic deficiency, no responsewill occur. This, then, wouldbe a failure to respondat the groundstate (level 0). Many theoriesof Ir genefunctionpostulategeneticcontrolat this level (e.g. failure in ability to presentantigen)(11). Activationby antigen perturbationraises the systemto level 1. At this level, failure to respondmaybe due to concomitantactivation of suppressor T cells (via the level 1 suppressorcircuit). This, again, agrees with many modelsof Ir gene function (in whichunresponsiveness is mediated by suppressorT cells) (12). Responsiveness can proceedin the face of active suppressorcell function via the activation of contrasuppressor T cells, whichprotect the targets of suppressorcell signals (13). Suchcontrasuppressor cell activationraises the systemto level 2. At this level, the suppressorcells mentioned above(level 1 suppressors)are relatively ineffective (14, 15). Unresponsiveness at this level is postulatedto be mediatedprincipally by an amplificationof suppression that werefer to as the level 2 suppressorcircuit. Suchan activity has beenidentified andseemsto correlate with a previouslydescribedsuppressor cell circuit. Thisis discussedin somedetail in a sectiondevotedto level 2 regulation. Theseimmunologic eigen states (anteigen states?) form the basis of broad view of immuneregulation. This quantumview suggests that energy is required to go to higher levels; informationtheory dictates that energy is similarly requiredto increaseorder. Thehigherlevels ,are those required to tune the immune responseexquisitely to protect maximallywith minimal autoimmune dysfunction.Thegreater the threat, the moremetabolicenergy will be expended in the processof this tuning. In the final sectionwediscuss someevidencethat is compatiblewith these conclusions. LEVEL 1 SUPPRESSION:

CELLULAR ASPECTS

The Level 1 Suppressor Cell Circuit (Feedback Suppression) Both the afferent and efferent componentsof the "feedback suppressor circuit" havebeendescribedextensively(16, 17). Basically, a signal produeedby an antigen-stimulatedinducercell is transducedand amplifiedby a secondpopulationof non-immune regulatory cells, resulting in the generation of effector cells of level 1 suppression(Figure1). Thenewdesignation "level 1 suppression"for the feedbacksuppressioncircuit is basedupona moreunified conceptof immunoregulation, discussed in the previous section. In the in vitro response to sheep red blood cells (SRBC),addition feedbackinducercells results in eliminationof plaque-forming cell genera-

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T-J

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[SUPPRESSION 1 ( )=Restrictions Qa-I TSIF INDUCER (VH~ ~ T-d

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I-J

I.

TRANSDUCER

+, Ly-1T inducercell with Figure1 Level1 suppression.Theinteractionof an I-J +, Qa-1 +, Ly-l,2T transducer an I-J+, Qa-1 cell resultsin an Ly-2T effectorcell (l-J-). Thelatter, togetherwitha naiveI-J+, Ly-1T cell acts to inactivatetargethelperT cells. tion (16). The activity of this cell population requires a transducer population that transfers the suppressorsignal from the inducer cell to the effector population. In cultures that lack transducer cells, the decrease in the amplitude of the response by the inducer cells is very inefficient. The functional activity of suppressor induction at this level is correlated with an Ly-1 cell (Ly-l+,2 -) with an I-J +, Qa-1+ surface profile. The Ly-1 marker is also found on other inducer T cells, such as those that activate B cells to makeantibody (18), activate macrophagesin inflammation (17), and activate cytotoxic T cells (19). In addition to the Ly-l+,2- phenotype, the above suppressor inducer cells also bear products of the I-J subregion and the Qa-1 locus, a profile that distinguishes them from the helper cell in the in vitro antibody response to SRBC(20). The population that transduces the induction signal into one of direct suppression is an Ly-l,2 cell (Ly-l+,2 +) with the I-J +, Qa-1+ phenotype (20). Although the mechanismby which these cells convert the inducing signal into suppression is not well understood, at least one modeof action maybe the differentiation of these cells into maturesuppressoreffector cells in the presence of the inducing signal (21). The interaction between the Ly-1 inducer cell and the Ly-1,2 transducer cell is antigen specific and is restricted by genes linked to the V region of the immunoglobulinheavy chain locus. That is, these populations must be matched at this locus as a minimumrequirement for the induction of

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suppression. Howthe interaction between these cells relates to idiotypeanti-idiotype regulation (22) is discussed in somedetail below and in the next section. The effector cells of the level 1 suppressorcircuit are functionally defined by their ability to suppress Ly- 1 T cells directly. In addition to exerting their effects uponhelper cells, these cells can also act uponthe feedbackinducer cells themselves and thus cause the inactivation of both new antibody synthesis (which requires T-cell help) and the recruitment of additional suppressorcell function (23). The latter fact illustrates the appropriateness of the term feedback as applied to this circuit. The level 1 suppressor effector cells bear the Ly-2 surface marker, but unlike the inducer and transducer subsets do not carry the same I-J gene product, at least in the case of the suppressor of the primary response to SRBCin vitro (14). The interaction between the suppressor effector cell and its target is restricted by genes mappingto the major histocompatibility locus, especially the I-J subregion. The molecular basis for this restriction is understood and is best explained in a subsequent section. Recentstudies have indicated that an I-J +, Ly-1 T cell is required for the action of the suppressor effector cell on its targets (24). This requirement is also best understood in molecular terms to be discussed in a subsequent section. The Nature of V~ Restriction Methylcholanthrene A (MCA)has been used to induce a large number sarcomas, all of which possess unique transplantation antigens (TSTA).Old et al (25) have managedto generate an antiserum to a unique antigen the surface of the MCAsarcoma and subsequently mapped the antigen to the short arm of chromosome12 in the mouse(26). This region also houses the immunoglobulin heavy chain complex, a fact that prompted Flood et al (27) to investigate the activity of anti-MCAsera in cellular interactions between T cells restricted to VH-linkedgenes. Antisera raised in homozygous syngeneic recipients all possessed the ability to block the interaction between the level 1 inducer and transducer cell. This ability was only dependent on the VHlOCUSof the interacting cells: if the cells camefrom mice that shared only Igh-linked genes and no other polymorphismswith Balb/c mice (the strain in which the antiserum was produced), the antiMCA serum blocked the reaction. In addition, the activity of the serum was independent of the antigen used to induce the suppressor cells. The MCAantigens were found to be present on all methylcholanthrene tumors tested. Taken together, these observations argue strongly that shared surface antigens on methylcholanthrene-induced sarcomas are identical to or highly cross-reactive with determinants found on molecules

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IMMUNOREGULATORY T-CELL PATHWAYS 445 involvedin Vri-restricted T-cell interactions. Sinceit is veryunlikelythat MCA-induced tumorscarry the entire array of"idiotypes" expressedin the mouse,one mustpostulate that these cross-reactive determinantsrepresent constant regions that are independentof the Vr~-encodedhypervariable regions used by antibodyand (probably)T-cell derived moleculesto recognize andbind antigen.This introducesthe possibility that Vii restriction is not alwaysdependenton individual recognition of a wide variety of idiotypes, but maybe accomplishedby recognition of a muchsmaller number of moleculardeterminants. Vr~restriction andthe activity of the anti-MCA antisera is considered further in somewhatmoremolecularterms in the following section. The question of whya cellular interaction moleculefor immune function should appearon a fibrosarcoma is openfor speculation(27), but certainly a closer examinationof this genetic region shouldyield fascinating information. LEVEL 1 SUPPRESSION:

MOLECULAR ASPECTS

Suppressor Factors In this section wediscuss the components andaction of twofactors believed to mediatethe functions of the inducer and effector cells of the level 1 suppressor circuit. Splenic T cells from animals hyperimmunized with xenogeneicred blood cells served as a source of these factors. Theimmunized T cells weretreated with antisera plus complement to produceactive, secreting populations. Theproduction of Ly-1 TsiF (suppressor inducer factor) involvedtreatmentof the T cells with anti-Ly-2.Alternatively,Ly-2 TsF(suppressorfactor) required treating the T cells with anti-Ly-1.In both cases, the enriched subsets were then cultured without antigen for 48 hr, after whichtime the supernatant (containing the molecularactivity) was harvested(28, 29). In addition to this procedure,a moleculewith the chemicalandbiological properties of the Ly-2 TsF was isolated from serumof hyperimmunized animals(30). This materialwasfoundto possessa uniqueidiotypic determinant (Shld) that allowedits purification. Since the preparationof the Ly-2 TsFcontains, in addition to the ShId+ material, other regulatoryfactors (to be discussed), the serummaterial has been designated Shld+ TsF. It is likely, however,that the Ly-2 TsF (level 1 suppressor factor) and the Shld+ TsFare identical. Ly-1 TsiF The Ly-1TsiF is dependentuponI-J + Ly-1T cells for its generation and (like the suppressorinducercell) requires I-J +, Ly-l,2 transducercells for suppressionto occur. This interaction is restricted by genesmappingto the

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Vregion, andthe effects are specific for the primingantigen(28). TheLy-1 TsiFmustbe addedto cultures prior to day 2 if suppressionis to be seen. Basedon such observations, it wasconcludedthat this material mediates the activity of the suppressorinducercell. The Ly-1 TsiF was found to be composedof two separate molecules (Figure 2). Onecan be removedby absorption with the appropriate antigen (hencedubbedantigen-bindingchain). Theother binds to and can be eluted from a columncoupled with a genetically appropriate anti I-J reagent (I-J + chain). TheI-J + chain has no antigen specificity and cannotbe absorbed with antigen. Both chains of the Ly-1 TsiF appear to be 68-72 kilodaltons on SDS-PAGE, have a low pI, and are hydrophobic. Recent evidencesuggeststhat each chainis producedby a different cell (or a cell in twostages of differentiation), one I-J + (producingthe I-J + chain), the other l-J- (producingthe antigen-bindingchain) (31). In experiments with I-J + chains and antigen-binding chains from Ly-1TsiF’s of different specificities andproducedby different strains of mice, it wasfoundthat althoughthe antigen-bindingchain determinedthe antigenspecificity of the factor, it wasthe 1-J+ chainthat determinedthe Vnrestriction of the induceractivity (32). This observationhelps to confirm thosedescribedin the previoussection in that the restriction has no antigen specificity (antigen specificity mightbe expectedfor an idiotype-anti-idiotype interaction). In support of these ideas, the anti-MCAantiserum described above was found to bind the I-J + chain but not the antigenbinding chain (unpublishedobservation). Thus,apparentlyone moleculeof Ly-1TsiF (I-J + chain) seemsto possess a determinant encodedby a locus on the 17th chromosome (I-J) and one on the 12th chromosome (Vn). These canffot be separated by SDS-PAGE or reduction and "alkylation (unpublishedobservations), whichraises the possibility that an excitingly novel genetic mechanism maybe at work. Either the structural genefor I-J is actually on the 12th chromosome (under control by the 17th), or else geneproductsfromtwo separate chromosomes

~

I-d

(VH Encoded Restriction) Meth A Morker

AntigenBinding (No Restriction Identified)

Figure 2 Biologically activemolecules mediating the induction of level1 suppression (Ly-1 TsiF).Seetextfordetails.

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can be linked by translocation, RNAprocessing, or a specific covalent protein interaction [such as can be observed in the complementsystem (33)]. The possibility that the MCAstructural gene is on chromosome has been ruled out by somatic cell genetics (26). These speculations are further complicated by observations of Ly-1 TsF made in F1 animals. (Balb/c X B6)FI animals (CB6F0 make an Ly-1 TsF that suppresses either Balb/c (I-J d, Igh-Va), or C57B1/6(I-Jb,Igh-Vb). Surprisingly, however, removal of the I-J b chain from the CB6Fronlyfactor removed activity for C57B1/6cells, whereas removal of the I-J d chain resulted in loss of activity on Balb/c cells (31). This indicates that no (or fewer) I-J b Chains were constructed with Igh-Va activity, neither were detectable I-J d chains produced with the Igh-Vb restriction (recall that the factors are Vri restricted, showingno H-2 restrictions). This paradox is illustrated in Figure 3.

Ly-2 TsF The Ly-2 TsF is produced by I-J-, Ly-2 T cells and is capable of directly suppressing helper T cells in the absence of Ly-1,2 T cells. The suppression is antigen specific. These observations support the idea that this factor mediates the function of levd one suppressor effector cells. The suppressor effector molecule appears to be a single chain of 68 kilodaltons. In SDS-PAGE, it has a low pI (4.9), is hydrophobic, and possesses a blocked N-terminus. It binds antigen and, in the case of SRBCspecific factor, bears a recognizable idiotype (Shld). This material had been identified in Ly-2 TsF, ShId+ material from hyperimmuneserum, and in ~ I-j

~

T-Jb i~

o Restriction WH

FOUND

b Restriction VH

~ ~ VH b Restriction

Figure 3 cis association of parental gene products from different chromosomesin Ly- 1 TsiF from an F~ animal.

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supernatants of an Ly-2 T suppressor cell clone (30;unpublished observation). In hyperimmune serum, ShId+ material can be found up to concentrations of approximately 0.1 mg/ml, and to be precipitable in high salt (28% NH3S04). This raises the possibility that some of the suppressive activity attributed to antibody in hyperimmune serum (34)may actually be due to products of suppressor T cells. As observed for level 1 suppressor effector cells, the Ly-2 TsF requires I-J+, Ly-1 T cells to effect suppression on I-J-, Ly-1 T cells (24). The Ly-2 TsF, in the presence of antigen, induces the production of a molecule from I-J+, Ly-1 cells, which represents the second functional chain of the suppressor factor. The induction of this second chain requires H-2 homology between the I-J+ Ly- 1 T cells and the Ly-2 TsF (manuscript in preparation). This induced molecule is antigen-nonspecific (and does not bind antigen) and bears an I-J subregion-encoded determinant. To function, it must genetically match the target helper T (I-J- cell) at VH. The functional interaction of the induced I-J+ chain and the Ly-2 TsF is restricted by the I-J subregion of the H-2. A minor number of anti-MCA antisera discussed above will block the suppressive activity of the Ly-2 TsF (27). A number of anti-I-J monoclonal antibodies have also been found to block Ly-2 TsF activity (35) (Figure 4).

H - 2 restricted specified antigen required

can also be provided by Ly-l TsiF preparation

TARGET L y - 1 T HELPER

Figure 4 Biologicallyactive effector moleculesmediating level 1 suppression (Ly-2 TsF). See text for details.

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An I-J+ chain that will function with the Ly-2 TsF can also be found in Ly-1 TsiF (24). This suggests either that the I-J+ molecule for both factors is interchangeable or that there is more than one type of I-J+ molecule in the Ly-1 TsiF. The latter case is supported by three lines of evidence: (a) Certain anti-MCA serum preparations are capable of blocking only Ly-1 TsiF activity and not Ly-2 TsF, and vice versa; (b) the target cell of the two factors is different, an Ly-1,2 cell for Ly-1 TsiF and an Ly-1 cell for the Ly-2 T s F and ( c ) an I-J+ induced from Ly-1 cells by the Ly-2 TsF was found to function with the Ly-2 TsF to effect suppression but not with the antigen-binding chain of the Ly-1 TsiF to induce suppression (unpublished observation). The latter observation may be due to the quantity of I-J+ chain required by each factor rather than the existence of two different I-J+ chains. Further work is required to characterize the differences in the two I-J+ molecules. Speculation on the subsequent action of the Ly-2 TsF, following binding of both chains to the helper cell target, points toward the possibility that the active molecule is internalized. Similar internalization has been observed for certain bacterial toxins (36), ribosome inhibitory proteins from plants (37), and several peptide hormones, including insulin (36), and suggests a similarity of action between these and the Ly-2 TsF. This similarity with toxin is enticing. Many sophisticated hormones that function in man have been identified as communication molecules in prokaryotes, an observation that suggests an evolutionary continuity in the development of hormones for different compartments of the physiology of higher organisms. Toxins may serve as defense mechanisms in some prokaryotes (38) and as a form of transplantation rejection against interspecies grafts in certain plants (37). With the appearance of the vertebrate immune system, the role of toxin may have inverted, becoming directed at elements of the immune system itself. If so, then the mechanism of action of Ly-2 TsF may well be the same as that of many bacterial toxins and plant ribosomeinactivating proteins (RIPS), i.e. inhibition of protein translation. TsF “toxicity” depends, like other toxins, upon targeting the active fragment (antigen-binding chain) with a binding chain (I-J+ chain) directed at the target (via VH recognition). It may be that specificity depends solely upon this targeting. This would explain why the cells that produce the active chain (Ly-2 TsF) seem to be resistant to its toxic effects. It does not appear, however, that target cells, in the case of Ly-2 TsF, are actually killed. Cantor et a1 (39) present evidence that suppressor factor inhibits the secretion of certain proteins from helper-cell clones, without affecting others. One possibility is that translation of certain luxury gene products depends upon specialized or specially compartmentalized ribosomes, and

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that it is only these ribosomesthat are suppressed.Specializedtranslational mechanisms for small numbersof inducedproteins have beendemonstrated in eukaryoteheat shock(40). Plant RIPsare knownto be increasinglytoxic as one movesawayfromthe species of origin (the ribosomesof whichare completelyresistant), to closely related species(partially susceptible), distant species (totally susceptible) (37). This modelpredicts, among other things, that whereasmurineTsF is knownto suppress murinecells without killing them(unpublished observations), humanTsF(suppressor effector factor), properlytargeted, mightbe foundto be toxic to the appropriate mousecells. HumanTsF has been shownto suppress murine responses(41). It should be a simple matter to test it upona mousehelper cell clone if the appropriate "schlepper" (I-J +, Vnrestricted) chain can interact withit.

Genetically ConstructedCompetitivelnhibitors of Ly-2 TsF Onetest of the modelof two-chaininteraction presentedaboveinvolvesthe construction of competitiveinhibitors for the Ly-2TsF. Accordingto the model, the l-J-, antigen-binding Ly-2 TsF induces the production of an I-J + moleculewith whichit interacts. This interaction is restricted by polymorphic I-J subregiongeneproducts. Thei-J + chain, in turn, interacts with its helper cell target via a VH-controlled interaction (Figure4). If one were to add another I-J + chain, bearing only one appropriate and one inappropriate restriction element, then this should act as a competitive inhibitor of Ly-2 TsF-mediatedsuppression(Figure 5). COMPETITOR

~

COMPETITOR

T-d°[~

VH b

Restr.

~,~..~~o

TARGET

~

I-dbwill not compete

Figure 5 Genetically constructed competitive inhibitorsof Ly-2TsF.

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IMMUNOREGULATORY T-CELL PATHWAYS 451 This is exactly the case (42). Witha C57B1/6Ly-2 TsF(SRBC)acting + uponC57B1/6Ly-1 T cells, suppression wasblocked by addition of I-J chain (antigen-absorbed Ly-1 TsiF) from CB20(matchedat Vn but mismatchedat H-2) or from BC9(matchedat H-2 but mismatchedat Vn). effect of a Balb/cfactor wasseen, as this is mismatched at both loci and therefore could not complete. Themodelpresented abovewas therefore employedto construct effects that otherwisewouldbe unexplainable(i.e., the inhibition of an Ly-2TsF by a genetically altered Ly-1 TsF). Althoughthis demonstrationdoes not provethe modelof Ly-2TsFaction, it does illustrate its predictivepower.

AdaptiveDifferentiation of GeneticRestrictions In the previous sections we described two, genetically restricted immunoregulatoryinteractions betweenT-cell subsets in the generation of level 1 suppression.Ly-1TsiF, undernormalcircumstances,cannot induce suppressiveactivity in the transducercells if these cells expressIgh-V-linked polymorphisms that are different from those of the Ly-1 T cell used to producethe factor. Ly-2TsFis also genetically restricted, requiring H-2 homology betweenthe Ly-2 cells that producedthe factor and the Ly-1 T calls uponwhichthe factor will act (to both induceand interact with the cooperatingI-J + chain). Thegenotypesof the factor-producing cells do not completelygovern these restrictions. Whenhomozygous("A") T cells mature in an (A B)F1 radiation chimeraor in an (A × B)F1thymusgrafted to a homozygous Anude mouse,these cells will producefactors restricted to both A and B (43, 44). This applies not only to the H-2restriction of the Ly-2TsF, but also to the Igh-Vrestriction of the Ly-1TsiF. Additionally, (A X B)F~ cells differentiating in an A-typethymusproducefactors restricted to A only andare unableto interact with B-typecells. Anextensiveliterature exists on the role of the thymusin the generation of H-2restrictions (45), but the adaptive differentiation of Igh-V-linked recognition in the thymusis novel. TheFrinto-parent chimeraexperiments makeit unlikely that the thymusis passively"armed"by circulating molecules (such as antibody) that mightact as the selection elements. Given these striking similarities in the development of recognition of Igh-Vand MHC geneproductsas self, one shouldconsider the possibility that Igh-Vlinked geneproducts, expressedby cells in the thymus,function to select for those Ly-1 TsiFproducercells that can recognizethese Igh-V-linkedcell interactionstructuresas self. Thismightthenbe addedto the list of similarities betweengenetic products of the MHC and Igh regions, whichinclude roles in graft rejection and cellular interaction, andgenetichomology (46).

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Althoughthe aboveobservations are certainly remarkable,they are not particularly ditficult to envision. Anevenmoresurprising observationwas madein examiningthe interaction of Ly-2TsFand its target cells in the types of chimeras mentionedabove. It was found that A cells that had maturedin an A XB thymushad also gained the ability to be suppressed by both Ly-1 TsiF and Ly-2 TsF madeby B animals. Thus, not only did the factor-producing cells learn a newself recognitionin the F1 thymus,but these cells "taught" their transducercells this newself specificity. This phenomenon has been called tandemadaptive differentiation. Theimplications have been discussed in detail (44). Oneparticularly extraordinary implication derives from the analysis that has shownthat the Ly-2 TsF recognizesprivate regions of I-J on the I-J + molecule.Since anti-I-J antibodiesdirected against the B-typeI-J determinantsdo not detect any alteration in I-J, the worksuggeststhat a variable (adaptable)portion of the I-J moleculeis recognizedby the Ly-2TsFbut not by antibodies(44). Attempts are nowbeing madeto produce antibodies to this adaptable region of I-J-encodedproducts.

CONTRASUPPRESSION: CELLULAR AND MOLECULARASPECTS Contrasuppressionand the regulatory T-cell interactions that produceit havebeenreviewedin depth (47, 48). Nevertheless,this activity serves a distinguishing feature betweenlevel 1 and level 2 suppression, and we believe it to be a crucial feature of immunoregulation,whichshould be discussedherein. Contrasuppression is definedas an activity that interferes with level 1 suppressionto allow dominantimmunity.Technically, then, the competitive inhibition experimentsdescribedin the last section couldbe considered as a formof contrasuppression (one that may,perhaps,give us insights into the mechanism of physiologiccontrasuppression).In this section, however, weare concernedwith an immunoregulatory T cell circuit that possesses this activity (withsyngeneicaction). Thecircuit is illustrated in Figure Thefirst cell identified as a member of this circuit is an immune I-J +, Ly-2 T cell, the contrasuppressor inducer(14). It is likely that other regulatory cells mightbe involvedin activating this cell, but this is currently only speculation. Thecontrasuppressorinducer cell producesa factor (TesiF) that bears an I-J + subregion-encoded determinantand binds antigen (49). Whetherthe TcsiF is composedof one or two chains is unknown.The antigen-bindingcapability is morecross-reactivethan that of the Ly-2TsF, such that a SRBC-induced factor can be absorbedon horse red blood cells (leaving the SRBC-specificTsFbehind).

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#/ (VH)

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EFFECTOR

LEVELZ

~

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Figure6 Contrasuppression. Theinteractionof an I-J +, +, Ly-2T inducercell andan I-J Ly-l,2T transducer cell resultsin an I-J+, Ly-1effectorcell. Thelatter acts to renderthe targetsof level1 suppression (e.g. helperTcells) resistantto suppressor cell signals. Both the contrasuppressor inducer cell and factor interact with an I-J +, Ly-l,2 T contrasuppressor transducer cell to activate contrasuppression (14, 49). Recentexperimentsindicate that this interaction is restricted by genes mappingto the Ig-VH region, and some anti-MCAantisera (discussed previously) are capable of blocking this induction (50). Theseantisera, unlike those discussed that block induction of suppression, are more commonlyproduced in FI animals, which are semi-syngeneic for the fibrosarcoma. MCAfibrosarcomas grown in FI animals have been found to express muchhigher levels of surface antigens, which absorb this contrasuppressor blocking activity, than those grownin the parental strain. Although this elevation might account for the appearance of the antibody predominantly in F1 animals, it leaves the puzzle of the elevation itself. This might be a phenomenonrelated to that previously described by Barret & Derringer (51) in which tumors appeared to acquire antigens whenpassaged F~ animals. The effector cell of contrasuppressionis an I-J +, Ly-1 T cell that adheres to the Vicia villosa lectin. This cell is present in the spleens of hyperimmune animals (15) and can be generated in cultures of spleen cells from neonatal animals (13), old animals (52), adult animals treated so as to removecertain inhibitory populations (discussed later), and animals manipulated become autoimmune (53).

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The contrasuppressor effector cell acts directly upon helper T cells to render them resistant to subsequent suppressor T-cell signals (13, 15). cell-free product of the effector cells with this activity has been identified and has been found to bear an I4 subregion-encoded determinant (52). Its mechanism of action is unknown. The I-J-encoded determinant found on all of the cells and factors of the contrasuppressor circuit can be distinguished from that on the cells and factors of the level 1 suppressor circuit by the use of monoclonalanti-I-J antibodies (54). The Physiologic Role of Contrasuppression Wehave suggested that contrasuppression exists as a sort of bridge between level 1 and level 2 immunoregulation,interferring with the former and being regulated, in turn, by the latter. At this point, therefore, we propose to examinethe role of contrasuppression in the regulation of immunity, which will also serve to underline the potential importanceof level 2 suppression (in balancing contrasuppression), for which relatively little information exists. Basically, contrasuppression is viewedas functioning in at least three areas of immunoregulation.These are (a) as a sequd to general suppression (leading to responsiveness), (b) in the localization of immuneresponses discrete regions, and (c) in the establishment of suppression-resistant states. A state of general unresponsivenessassociated with suppressor-cell activity (probably level 1 suppression) accompaniesthe earliest stages of neonatal life (55), parasitic infections (56), manytumors(57), and severe (58). In each case, recovery of immunefunction and establishment of dominant immunityoften requires activity of contrasuppressor cells (reviewed in 48). A similar contrasuppressive activity might serve to produce a local resistance to general suppression, thus focusing an immuneresponse into one discrete area. The Peyer’s patches of the gut-associated lymphoid tissue (GALT)contain cells of both the suppressor and the contrasuppressor circuits (59). Contrasuppression might permit GALTresponses to proceed against ingested antigen, whereas suppression prevents systemic immunity to any antigen that reaches the circulation. Such GALTimmunity in the face of systemic tolerance has been observed (60). The skin (61-63) bone marrow(64) are two other environments in which local activation contrasuppression mayhave an important role of maintaining immunityin the presence of suppression. Specialized antigen-presenting cells, such as Langerhans cells and dendritic cells, have been implicated in this local activation (63, 65). Other local mediators that might serve to activate contrasuppression are interferon (66) and histamine (67).

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Several immuneconditions lead to what can be observed as a suppressionresistant state. Although hyperimmune animals show no sign of suppression or unresponsiveness, it is well established that serum from such animals contains a potent suppressive substance (34) that is antigen specific and T-cell derived (30). A similar situation is seen in someforms of autoimmunity, i.e. autoimmunitymayproceed in the face of systemic nonspecific suppression. Examples are the MRLmurine model of systemic lupus erythematosis (68), old age (69), and autoimmunity associated with malaria (70). In each case of the suppression-resistant state contrasuppression has been implicated in some way (48,52), and it remains to be seen whether not a cause and effect relationship can be established.

LEVEL 2 SUPPRESSION Evidence for a Second "Level" of Suppression THE REGULATION OF CONTRASUPPRESSOR CELL GENERATION Manyinvestigators have observed the generation of potent suppressor activity uponculture of normal, adult spleen cells (71, 72). Whencells from very young animals are cultured, however, dominant contrasuppression, rather than suppression, is produced (13). A mixture of adult and neonatal cells was found to produce no eontrasuppressor cells, an effect requiring a Thy-1+, I-J +, Ly-l,2 cell in the adult population (73). The presence in the adult population of a cell that prevents the generation of contrasuppression raised the possibility that specific removalof this population wouldallow contrasuppressor cells to appear in cultures of adult spleen. A novel monoelonal anti-Thy-1 (F7D5) was employed, which at low concentrations reacted with a small population of adult spleen cells, including the cells responsible for the aboveinhibitory effects on contrasuppressorcell generation. Treated adult spleen cells were found to produce 1-J +, Ly-1+ contrasuppressor cells uponculture (73). Thus, the presence of one or more cell populations (including an I-J +, Ly- 1,2 population, but perhapsother ceils as well) prevents the spontaneous generation of contrasuppression by adult cells in culture (and allows dominantsuppressor activity). This activity appears ontogenetically with the loss of ability to producecontrasuppressor cells in untreated cell cultures (increasing from around day 8 to day 15 of life). PRODUCTION

OF DOMINANT

CONTRASUPPRESSOR

CELL

EFFECTS

WhenLy-2 TsF is produced and tested on cells of most strains of mice, suppression tends to be the dominant activity observed, even when the factor contains Ly-2 TcsiF and the cultures contain contrasuppressor trans-

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ducer cells. Exceptionsto this are the B10congenicmousestrains [notable becausethese were used to characterize the Ly-2TcsiF (49)] and the strain congenics. Anygenetically matchedLy-2factor will producedominant contrasuppression on B10cells (such that suppression mayonly be seen uponremovalof the TcsiF or the contrasuppressortransducer cell), whereasfactors producedin Astrain miceseemto be contrasuppressiveon otherwisesuppressiblerecipients (for example,an F1 recipient) (74). These effects were mimickedby treatment with FTD5. Cells from C57BL/6animals were treated with F7D5,then were used to produce an Ly-2factor. This factor suppressedcultures in the absenceof Ly-2+ cells in the recipients, or in the presenceof monoclonal anti-I-J reagents known to blockeontrasuppression,but otherwiseit producedno effects in cultures + cell fromthe Ly-2 of unfractionatedcells (73). Thus,removalof an FTD5 populationresulted in the productionof an Ly-2factor resemblingthe one madeby cells from untreated A strains. Similarly, treatment of C57BL/6 spleen ceils with a low concentration of F7D5causedthese cells to be resistant to suppressionunless the contrasuppressor circuit wasquenched.Thus, FTD5caused B6strain cells to behavelike B10strain cells (unpublishedobservations). + cells exist that function to override the contrasuppresTherefore,F7D5 sor circuit, in the absenceof whichthe induction of contrasuppressionby the Ly-2 TcsiF is dominant.

Contrasuppression Versus the Amplification of Suppression Aninteresting symmetryhas appeared betweensystems that under some conditionsare deficient in certain cellular members of the suppressorcell circuit describedby Tadaet al (75) and those with a dominantcontrasuppressive activity. Thesuppressorcell circuit involvesan Ly-2inducercell that is I-J +. This acts via an I-J +, Ly-l,2 transducerpopulation,resulting in active I-J +, Ly-2suppressoreffector cells. This interaction producesan amplificationof suppression(Figure 7). Miceof the A strain backgroundfail to producethis KLH-specificsuppressor-including factor (76). As previously discussed, Astrain mice producea suppressor factor for primaryanti-SRBCresponse, but one in whichcontrasuppressiondominates.Similarly, B10strains fail to accept this Ly-2 suppressorinducer factor and also showdominanteontrasuppression in the SRBCsystem. Experimentson absorption of the KLHsuppressor inducer factor demonstratedthat B10miceare indeeddeficient in this acceptor(transducer)cell (76). Theexaminationof the cell that accepts the inducer factor in Tada’s systemrevealed an I-J +, Ly-l,2 cell, whichbore a high density of Thy-1 antigen, as detected with a heteroantiserum(75). It is possible, therefore,

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LEVEL 1 LEVEL ~

/"

EFFECTOR

l-J

TRANSDUCER

INDUCER

ISUPPRESSION I Figure7 Level2 suppression.Thissuppressor-amplification circuit involvesan I-J +, Ly-2 +, inducercell, an I-J Ly-l,2transducer (acceptor)cell, andan I-J+, Ly-2effectorcell. that this cell (at leas0 will prove to be sensitive to a low concentration of F7D5. Observations similar to the above were madein another system. Peyer’s patch T cells from mice bearing the d allele at the H-2Dlocus will suppress the primary SRBCresponse of normal spleen cells in vitro. This suppression required the interaction of an Ly-2 cell in the Peyer’s patch population and an Ly-2+ cell (presumably Ly-l,2 cell) in the splenic population (77). Peyer’s patch T cells from non-H-2Dd animals induced a dominant contrasuppression (induced by an I-J +, Ly-2 cell), but also suppressed if the contrasuppressor circuit was severed (59). The resulting suppression was effected by an I-J-, Ly-2cell (level 1 suppressor). Thus, again, in cases where suppressor amplification did not seem to operate, contrasuppression was dominant. Whenthis contrasuppression was removed, level 1 suppression was found to produce dominant unresponsiveness. The resulting symmetry, i.e. contrasuppression dominates where the "Tada" suppressor circuit is absent, resemblesthe situation discussed in the last section. This leads to the possibility that the regulation of contrasuppression and the amplification of suppression are mediated by the same cellular circuit, i.e. the level 2 suppressorcircuit (Figure 7).

Regulatory ~lspects of Level 2 Suppression Wehave preliminary evidence that the level 2 suppressor circuit starts to appear at about day 8 post-birth in the mouse. Its appearancemayrepresent

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a control for the contrasuppressionthat appearsearlier (aroundday2) and maybe required for the maturation of normalresponsiveness(suppressed in neonatesby level 1 suppressorcells). Prematureintroduction of adult spleencells (whichcontainthe cells of the level 2 circuit) at or beforeday 8 blockednormalimmunedevelopment(52, 78). Treatmentof these adult cells with a low concentration of F7D5completelyremovedthis blocking activity (52). Thus,it seemslikely that the sequential appearanceof level 1 suppression,then eontrasuppression,then level 2 suppressionmustproteed on this schedule for normalimmunefunction to mature. PolyclonalB-cell activators generally inducesuppressionin vivo. When animalswere thymectomized on day 3 of life, however,they were found to produce normal T cell-dependent immuneresponses, but becameautoimmunein response to polyelonal B cell activators (53). Thethymectomized animalswerecapableof producinglevel 1 suppressorcells in vitro, but the autoimmune animals also possessed a potent contrasuppressive activity, sensitive to treatment with anti-I-J (79). Thus,removalof the thymusat time after the maturationof cells of the levd 1 suppressorcircuit andthe eontrasuppressorcircuit, but before the appearanceof level 2 regulatory cells, leads in maturity to a state prone to autoimmunity.Thepotential importanceof the level 2 suppressor circuit cannot be overemphasized. CONCLUSION:

LEVELS

UPON LEVELS

In the introduction weproposedthat the regulatory levels wediscussed becomeactivated at the expenseof increased metabolicenergy. Thus, the greater the threat, the greater the activation of a higher levd. This might explain the well-known(but poorly understood) dose response of the immunesystem (Figure 8, top). Generally, immuneresponses have an optimal antigen dose range. Suband supra-optimaldoses of antigen producesuppressionand unresponsiveness. Recentstudies of antigen-inducedregulatoryactivities in mouse(80) and man(81; T. Lehner, personal communication)have indicated that dominantcontrasuppressionis activated only in the optimal dose range. This contrasuppressiveactivity can overcomelow-dosesuppression,but not high dose (80). Wepostulate that low-dosesuppressionis associated with level 1, whereashigh-dosesuppressionis due to activation of the level 2 circuit (Figure 8, bottom).Experiments are in progressto test this notion. If higher antigen dose can be consideredan increased threat, then this is compatiblewith our notionof higher level activation. High-affaaityantibodyis morecross-reactive than low-atfinity antibody(82, 83), thus maximal, high-affinity responsesentail an increasedrisk of self destruction. Activationof the immune responsein the absenceof level 2 regulatorycells

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PATHWAYS

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l ANTIGEN DOSE

LEVELI SUPPRESSION

CONTRASUPPRESSION

Ly-I -\

csE

~

ANTIGEN DOSE

CST I

LEVEL 2 SUPPRESSION

\ ~ /

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Figure 8 (Top) Dose response and immunoregulation.(Bottom) Dose response and immunoregulation. I, inducer; T, transducer;E, effector; FB,feedback(level 1) suppression; CS,contrasuppression,SA,(level 2) suppressionamplification.

can lead to a potent autoimmune response(53, 79). Further studies on the relationship betweenlevel 2 regulation andautoimmunity are needed,as are studies Onthe role of contrasuppressionandlevel 2 suppressionin maturation of high-affinity responseswithout autoimmunity. The view that autoimmunitymaybe producedby a failure to control high-affinity responsesto foreign antigens properly is not a newone (84). Theschemeput forwardhere seeks to ascertain those regulatory sites at whichcontrol maygo awry.In addition, our viewplaces a greater emphasis

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on homeostasis,so that althoughantigensmayperturba defective system towardautoimmunity, disruptions other than those producedby antigen mayalso produceautoimmunity. Arethere morelevels? Perhaps so, butstudies on relatively simpleantigen challengesmayfail to reveal these additional complexities. As we increase our understanding of immune responsesto tumorsandparasitic infections,however, wemightwell find that suchlevels exist. Alternatively, it maybe that the immune system’sfailure to protectsuccessfullyagainst suchintrusions maybe linked to the absenceof higherlevels. Thus,the immune systemmaynot be capable of producingan effectively strong responsein someeases, preciselybecanseit lackssut~eientheuristicpower in its regulatorycircuitry.If so, thenby gaininginsight into the levels of regulationthat do exist, wemaybe able artificially to addour mental processesto those of the immune systemin producingtherapeuticmanipulations. Theprospectsof suchimmunoengineering are not far off. ACKNOWLEDGMENT

D. R. GreenandP. M.Floodare supported by NationalInstitutes of Health Training GrantnumberAI 07019. Literature Cited 1. Foucanlt, M. 1973. The Order of Things‘ NewYork: Vintage Books 2. OxfordEnglish Dictionary. 1971. Oxford: OxfordUniv. 3. Bynum,W.F., Browne,E. J., Porter, R., eds. 1981.Dictionaryof the History of Scienc~Princeton: Princeton Univ. 4. Weiner, N. 1948. Cybernetics, Cambridge: TechnologyPress 5. Germain¢,R. N., Benacerraf, B. 1981. A single major pathway of T-lym-

10. Kimura,A. K. 1982.Thefmespecificity of lectins applied to the study of lymphocyte membranestructure and immunologicalreactivity. Riv. Itnmunol. Immunofarm.3:117 11. Shevach,E. M., Rosenthal,A. S. 1973. Function of macrol?hagesin antigen recognition by guinea pig T-lymphocytes.II. Roleof the macrophage in the regulationof genetic control of the immuneresponse, d. Exp. Med. 138: 1213 PmunhOCyte interactionsin antigen-specific une13:1 suppression. Scand. £ Im12. Debre, P., Kapp,J. A., Dorf, M. E., munol. Benacerraf,B. 1975.Geneticcontrol of immunesuppression. II. H-2 linked 6. Cantor, H., Boyse, E. A. 1975. Functional subclassesof T lymphocytes beardominant genetic control of immune ing different Lyantigens,d.. F~xp.Med. suppression by the randomcopolymer GT. d.. Exp. Med.142:1447 141:1376 7. Stanton, T. H., Boyse, E. A. 1976. A 13. Green, D. R., Eardley, D. D. Kimura, newserologicallydefinedlocus, Qa-l, in A., Murphy, D. B., Yamauchi, K., Gershon, R. K..1981. Immunoregulathe Tin region of the mouse.Iraraunotory circuits whichmodulaterespongenetics 3:525 8. Shreflter, D. C., David,C. S. 1975.The siveness to suppressor cell signals: Characterizationof an effector cell in H-2 majorhistocompatibility complex the contrasuppressor and the I immune res~ponseregion: GeIraraunol. 11:973 circuit. Eur. d. netic variation, functionand organiza14. Gershon, R. K., Eardley, D. D., tion. Adz IraraunoL20:125 Durum,S., Green, D. R., Shen, F. W., 9. Murphy,D. B. 1978. The LJ Subregion Yamauchi,K., Cantor, H., Murphy,D. of the Murine H-2 Gene Complex. Springer Sera. Imraunopat& 1:111 B. 1981. Contrasuppression: A novel

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immunoregulatoryactivity. J. Exp. 26. Pravtcheva,D. D., DeLeo,A. B., Ruddie, F. H., Old, L. J. 1981.Chromosome Med. 153:1533 assignmentof the tumorspecific anti15. Green, D. R., Gershon, R. K. 1982. Hyperimmunity and the decision to bc gen of a 3-methylcholanthrene-induced mousesarcoma. ~. Exp. Me¢£154:964 intolerant. Ann. N. Y. Acad.Sc~ 392: 27. Flood,P. M., DeLeo,A. B., Old, L. J., 318 Gershon,R. K. 1983. The relation of 16. Eardley, D. D., Hugenberger, J., McVay-Boudreau,L., Shen, F. W., cell surface antigens on methylcholanthrene-indueedflbrosareomasto Gershort R. K., Cantor, H. 1978. Imcell Igh-Vlinked cell interaction molemunoreguiatorycirenits amongT-cell cules. Proc. Nat Acad. ScL USA. In sets. I. T-helpercells induceotherT-cell sets to exertfeedback inhibition.,/. Exp. press 28. Yamauchi,K., Murphy,D. B., Cantor, Med. 147:1106 17. Cantor, H., Gershon,R. K. 1979. ImH., Gershon,R. K. 1981. Analysisof an antigenspecificH-2restricted, cell free munological circuits: Cellular composition. Fed. Proc.38:2058 product(s) madeby "I-J" Ly-2 cells 18. Davies, A. J. S. 1969. Thethymusand (Ly-2TsF)that suppressesLy-2cell dethe cellular basis of immunity.Transpleted cell activity. Eur.~. lmmunol.spleen 11:913 plant. Rev. 1:43 19. Cantor, H., Boyse, E. A. 1977. Lym- 29. Yamauchi,K., Murphy,D. B., Cantor, phoeytes as modelsfor the study of H., Gershon,R. IC 1981. Analysis of antigenspecific, Ig restricted cell free mammalian cellular differentiation, lmmunoLRev. 33:105 material made by I-J + L~I cells (Ly-1 TsiF) that inducesLy-2 cells 20. Eardley, D. D., Murphy,D. B., Kemp, expresssuppressiveactivity. Eur.~. lmJ. D., Shen, F. W., Cantor, H., Gershon, R. K. 1980. Ly-1 inducer and munoL11:905 Ly-l,2 acceptor cells in the feedback 30. Iverson, G. M., Eardley, D. D., Janesuppressioncircuit bear an I-J subreway, C. A., Gershon,R. K. 1983. The gion controlled determinant, lmmunouse of anti-idiotype immunosorbents to genetics 11:549 isolate circulatingantigenspecificT-cell J. S., Shen, F. W., Cort, S. derived molecules from hyperimmune 21. MeDougal, P., Bard, J. 1980. FeedbacksuppresSera. Proc. Nat. Acad. Sc£ USA.In sion: Phenotypesof T cell subsets inpress volvedin the Ly-1 T-cell induced im31. Chao, N. 1981. Analysis of an antigen munoregulatorycircuit. J. lmmunol. specific, Ig restricted cell free material madeby I-~, FI Ly-1 cell~ thesis. Yale 125:1157 22. Jerne, N. 1974. Towardsa networktheUniv., NewHaven, CT ory of the immunesystem. Ant~ lra32. Yamauehi,K., Chao, N., Murphy,D. munol. 125:373 B., Gershon, R. K. 1982. Molecular 23. Green,D. R., Gershon,R. K., Eardley, composition of an antigen-specific,Ly-1 D. D. 1981. Functional deletion of T suppressorinducer factor: Onemoledifferent Ly-1T cell inducersubset acculebindsantigenandis I-J-; anotheris tivities by Ly-2 suppressor T lymI-J +, does not bindantigen, andimparts phocytes. Prec. NatL Acad. Sc~ USA an Igh-variable region linked restriction. J. Exp. Med.155:655 78:3819 24. Flood, P., Yamanehi,K., Gershon,R. 33. Hostetter, M. K., Thomas,M. L., RoK. 1982.Analysisof the interactionsbesen, F. S., Tack,B. F. 1982.Bindingof tween2 moleculesthat are requiredfor C3bproceedsby a transesterifieationrethe expressionof Ly-2suppressorcell action at the thiolester site. Nature activity; 3 different types of focusing 298:72 eventsmaybe neededto deliver the sup34. Hanghton,G., Nash, D. R. 1969. Spepressive signals. J. Exp. Med.156:361 cific immunosuppressionby minute 25. DeLeo,A. B., Shiku, H, Takakarki,T., doses of passive antibody. Transplant John, M., Old, L. J. 1977.Cell surface Proc. 1:616 antigens of chemically induced sar35. Golde, W. T., Green, D. R., Waltencomasof the mouse.I. Murineleukemia bangh, C. R., Gershon, R. K. 1982. virus-related antigens andalloantigens DifferentI-J speeifieitiesdefinedifferent on cultured fibroblasts and sarcoma T-cell regulatory pathways.Fed. Proc. cells: Descriptionof a uniqueantigenon 41:190 (ABSTR.) Balb/c MethA sarcoma.,/. Exp. Meal. 36. Middlebrook,J. L., Kohn,L. D., eds. 146:720 1981. Receptor-MediatedBinding and

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Internalization of Toxins and Hormodulateresponsivenessto suppressor mone~ NewYork: Academic cell signals: Thefailure of B10miceto 37. Stirpe, F. 1982.Onthe action of riborespond to suppressor factors can be someinactivating proteins: Are plant overcomeby quenchingthe contrasupribosomes species specific7 Biocher~ pressor circuit, d. Exp. Meal153:1547 202:279 50. Flood, P. M., DeLeo,A. B., Old, L. J., 38.Janzcn, D.1977. Whyfruits rot, seeds Gershon,R. K. 1983. Antisera against mold, andmeat spoils. Am.Naturalist tumor-associated surface antigens on 111:691 meth~,lcholanthrene-inducedsarcomas 39.Clayberger, C.,Cantor, H.1982. Geninhibit the inductionof contrasuppreseration, specificity andfunction of4sion. Mod. Trends Hun Leukemia 5: hydroxy-3-nitrophenyl (acetyl) specific In press clones. Fea~Proc. 41:1213 (ABSTR.) 51. Barter, M. K., Deringer, M. K. 1950. 40.Storti, R.V.,Scott, M.P.,Rich, A., Aninducedadaptation in a transplantaParduc, M.L.1980. Translational conble tumorof mice. d. NatZCancerInst trol ofprotein synthesis inresponse to 11:51 heatshock inD.mclanogastcr cells. 52. Green, D. R. 1981. Contrasuppresaion: Cell 22:825 an immunoreguiatory T cell activi~y. 41.Kontiaincn, S.,Feldmann, M.1979. PhDthesis. Yale Univ., NewHaven, Structural characteristics ofantigenspecific suppressor factors: Definition of 53. Smith,H. R., Green,D. R., Raveche,E. "constant" region and"variable" S., Snmthers,P. A., Oershon,R. K., giondeterminant. Thymus 1:59 Steinberg,A. D. 1983.Inductionof au42.Green, D.R.,Flood, P.M.,Gcrshon, toimmunityin normal mice by thymecR.K.Genetically constructed competitomyand administration of polyclonal tive inhibitors ofsuppressor activity. B cell activators: Associationwith conManuscript inpreparation trasuppressor function. Clin. Exp. lmK.,Flood, P.,Singer, A., 43.Yamauchi, munoLIn press Gcrshon, R.K.1983. Homologies be- 54. Yamauchi,K., Taniguehi, M., Green, tween cell-interaction molecules conD., Gershon,R. K. 1982. The use of a trolled byMHCandIgh-V linked gcnes monoclonal I-J specific antibodyto disthatT ceils useforcommunication: tinguish cells in the feedbacksuppresbothmolecules undergoadaptivc sion circuit fromthosein the contrasupdifferentiation in the thymus.Fur. pr.essor circuit. Immunogenetics 16:551 ImmunoZIn press 55. Mosier, D. E., Johnson, B. M. 1975. 44. Flood, P., Yamanchi,K., Singer, A., Ontogenyof mouselymphocytefuncGershon, R. K. 1982. Homologiesbetion. II. Development of the ability to roduce antibody is modulated by T tweencell interaction moleculescontrolled by MHC and Igh-Vlinked genes phocytes. J. Exp. Med.141:216 that T cells use for communication: 56. Jayawardena, A. N. 1981. ImmunereTandem"adaptive differentiation" of sponsesin malaria.Adv. Parasitic Di~ producer and acceptor cells. £ Exp. 1:85 MecL156:1390 57. Bluestone,J. A., et al. 1979.Suppres45. Moiler, G., ed. 1979. Acquisition of sion of the immuneresponse in tumor the T cell repertoire. ImmunoZRev. bearing mice. Z NatL Cancer Ins~ Vol. 42 63:1215 46. Steinmetz,M., Frelinger, J. G., Fisher, 58. McLoughlin,G. A., Wu,A. V., et al. 1979. Correlationbetweenanergyand a D., et al. 1981. ThreeeDNA clones encodingmousetransplantation antigens: circulating immunosuppressive factor Homologyto immunoglobulingenes. following major trauma. Ann. Surgery Cell 24:125 190:297 47. Green, D. R. 1982. Contrasuppression: 59. Green,D. R., Gold,J., St. Martin,S., Its role in immunoregulation.In The Gershon,R., Gershon,R. K. 1982. Mierocnvironmental Immunoregulation: Potential Role of T.Cells in Cancer Therapy, ed. A. Fefer, A. Goldstein. The possible role of contrasuppressor cells m maintaining immune responses NewYork: Raven in gut associated lymphoid tissues 48. Green, D. R., Gershon, R. K. Con(GALT)(immunosuppression/pcyers trasuppression. Submittedfor publicapatches). Proc. NatL Aca~LScL USA tion 79:889 49. Yamauchi,K., Green, D. R., Eardley, 60. Challacombe, $. J., Tomasi,T. B. 1980. D. D., Murphy,D. B., Gershon,R. K. Systemictolerance and secretory immu1981. Immunoregulatory circuits that

~ym

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IMMUNOREGULATORY T-CELL pity after oral immunization.J. Exp. Med. 152:1459 61. Iverson, G. M., Ptak, W.,Green,D. R., Gershon,R. K. Therole of contrasuppressionin the adoptivetransfer of immuuity.Submitttedfor publication 62. Ptak, W., Green, D. R., Durum,S. K., Kimura,A., Murphy,D. B., Gershon, R. K. 1981.Iznmunoregulatory circuits that modulateresponsivenessto suppressor cell signals: Contrasuppressor cells can convertan in vitro toleragenic signal into11:980 an immunogemic one. Fur. J. Immunol. 63. Ptak, W., Rozycka,D., Askenase,P. W., Gershon,R. K. 1980.Roleof antigen-presentingcells in the development and persistence of contact hypersensitivity. J. Exp. Med.151:362 64. Michaelson,J.D. 1982. Thecharacterization and functional analysis of immunoregulato.rffcelis in murin¢bone marrow.Medicalschool thesis. Yale Univ. NewHaven, 65. Britz, J. S., Askenase,P. W.,Ptak, W., Steinman,R. M., Gershon,R. K. 1982. Specialized antigen-presentingcells: Splenicdendritic cells, andperitoneal exudatecells inducedby mycobacteria activateeffector T cells that are resistant to suppression. J. Exp. Med. 155:1344 66. Knop,J., Stremmer,R., Neumann, C., DeMaeyer, E., Macher,E. 1982. Interferoninhibits the suppressorT cell responseof delayedtype hypersensitivity. Nature 296:757 67. Seigel, J. N., Schwartz, A., Askenase, P. W., Gershon,R. K. 1983. Suppression and contrasuppressioninducedrespectively by histamine-2and histamine-1 receptoragnpists. Proc.NatL,4cad.Sc£ USA.In press 68. Gershon, R. K., Horowitz, M., Kemp, J. D., Murphy,D. B., Murphy,D. 1978. TheCellular site of immunoregulatory breakdown in the lpr mouse.In Genetic Controlof Autoimmune Disease,ed. N. R. Rose,R. E. Bigazzi., N. L. Warner, pp. 223-27. NewYork: Elsevier 69. Makinodan,T., Kay, M. B. 1980. Age influence on the immunesystem. Adz ImmunoL29:287 70. Poeis, L. G., vanNiekerk,C. C. 1977. Plasmodinmberghei: Immunosuppression and hyperimmunoglobulinemia. Exp. ParasitoL42:235 71. Janeway,C. A.et al. 1975.T cell population with different functions. Nature 253:544 72. Hodes,R. J., Hathcock,K. S. 1976. In vitro generationof suppressorcell activ-

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ity: Suppression of in vitro inductionof cell mediatedcytotoxicity. J. lmmunoL 116:167 73. Green,D. R., Chue,B., Gershon,R. K. 1983. Twotypes of T cell suppression discriminated by action on the contrasuppressioncircuit. J. MoLCeILImmunoLIn press 74. Chue,B., Gershon,R. K., Green,D. R. Apparentfailure of A strain mice to rOduceT suppressor effector factor y-2 TsF) is due to dominant contrasuppressor T cell activity. Manuscript mpreparation 75. Tada, T., Okumura,K. 1979. The role of antigen-speeificT cell factors in the immuneresponse. ,~dv. ImmunoL28:1 76. Taniguchi,M., Tada, T., Tokuhisa,T. 1976.Propertiesof the antigen-specific T-cell factor in the regulationof antibodyresponse of the mouse.III. Dual genecontrol to the T-cell mediatedsuppression of the antibodyresponse. J. Exp. Med. 144:20 77. Green,D.R., St. Martin,S., 1983.Su~ppression and contrasuppressionin ~e regulation of gut assoc/atedimmune response. An~NYAcad. ScL In press 78. Raybourne,R., Arnold, L. W., Hanghton, G. 1981.Perturbationof early development of the immunesystem by normal adult lymphocytes. Y.Immunol. 127:1142 79. Smith,H. R., Green,D. R., Raveche,E. S., Smathers,P. A., Gershon,R. K., Steinberg, A. D. 1982. Studies of the induction of 129:2332 anti-DN& in normal mice. J.. ImmunoL 80. Schitf, C. 1980. Regulationof the murine immune responseto sheepred blood cells: The effects of educating lymphocytesin vitro with glycoproteinsiaolated fromthe sheep erythrocytesmembrane. Thesis. Yale Univ., NewHaven, CT. 81. Lehner,T. 1982. Therelationship between humanhelper and suppressor factors to a streptococcalprot~nantigen. J. ImmunoL129:1936 82. Little, J. R., Eisen,H.H.1969.Specificity of the immune responseto the 2,4dinitrophenyl and 2,4,6-trinitrophenyl groups. Ligand binding and fluorescencepropertiesof cross-reactingantibodies. J. Exp. Med.129:247 83. Underdown, B. J., Eisen, H. N. 1974. Cross-reactionsbetween2,4-dinitrophenyl and106:1431 5-acetourscil groups. J. Immunol. 84. Asherson,.G. L. 1968. Therole of micro-orgamsms in autoimmune responses. Prog. Allergy 12:192

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An~ Rev. Immuno£1983. 1:465-498 Copyright©1983by AnnualReviewsInc. All rights reserved

THE COMPLEXITY OF STRUCTURES INVOLVEDIN T-CELL ACTIVATION Joel W. Goodman Department of Microbiology

and Immunology, University

of California

Medical

School, San Francisco, California 94143

Eli E. Sercarz Departmentof Microbiology,University of California, Los Angeles, California 90024

FOREWORD Whereasthe discussion of epitope-antigen structure relating to B cells wouldnot be very different from a similar consideration a decade ago, the nature of T-cell recognition and activation by "antigen" encompasses a completely altered area of concern. Within the past 10 years, concepts of restriction to major histocompatibility-complex MHCmolecules and involvementof T cells in idiotypie interactions have revolutionized the view of the "antigen" that activates the T calls. In this article we use a special definition for the term antigen structure, whichincludes all elements shownto be recognized by the diversity of T-cell subpopulations: epitopes (from the nominalantigen), idiotopes (on secreted Ig or on other cell surfaces), and at least two types of MHC determinants, whichwill be defined later. Wedo not undertake a review of the basis and evidence for MHC restriction, or T-cell idiotypic interactions: earlier excellent reviews on the former (57, 139, 148, 186) and latter (47, 82, 83, 175) exist, together with several in the present volume.Our focus is confined to current information describing the complexity of structures involved in T-cell activation. 465 0732-0582/83/0410-0465 $02.00

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INTRODUCTION Theantigen specificity of T lymphocyteshas beenintensively investigated since their recognitionas a distinct class of cells, but a dear understanding of the subject has yet to be attained. Early in the game,it wasnatural enoughto assumethat T cells recognizedantigen using conventionalimmunoglobulins of perhapsa distinctive subclass, but that suppositionhas not stood the test of time. There are a numberof reasonsfor the obduracy of the T-cell antigen specificity problem.Until recently, homogeneous Tcell lines analogousto the myeloma cell, whichwasso crucial to the solution of antibody structure, were not available. Evennow,with the advent of antigen-specificnormaland neoplasticT-cell lines, it is still difficult to obtain antigen-specific soluble products(secreted proteins) in quantities sufficient to do the essential experiments.This has forced a dependency on assays basedon cellular responseto antigen rather than on simpleligandreceptor interaction. It is paramount,therefore, at the outset to acknowl. edgethe distinctions betweenthe specificity of ligand bindingand the consummaterequirementsfor a cellular response. Considerthe revolutionary contribution that hapten binding has madeto our understandingof antibodyspecificity. Considerfurther that haptens, althoughthey bind readily to the surface receptors of B cells of the appropriatespecificity, fail to activate the cells. Withsomenotable exceptions to be dealt with later, haptens also fail to activate T cells, so it has generally beennecessary to workwith antigens of epitypic complexity, complicating the is, sue further. Anothergraphic illustration of the non-identity betweenbinding and activation is the ostensibly equivalent binding of the mitogens coneanavalin A (ConA) and phytohem-agglutinin(PHA)by B and T cells (63), althoughactivation of eachcell type occursunderdistinctly different circumstances (7). Thebottomline is that ligand-receptorinteraction at the cell surface cannotbe equatedwith cell triggering, the latter involvinga different order of complexity. Anotherterm in the obduracyequationis the subdivisionof T cells into functionally distinct subsets with differing activation requirementsand, possibly, different antigen receptors as well. A majorunresolvedquestion is the mechanism by whichmostcells, particularly those with cytotoxic or helper functionactivity, manifesta dualspecificity for both nominalantigen and a product of the MHC such that activation takes place only whenthe T cell is presentedwith nominalantigen in the context of the appropriate MHC product (18, 90, 138, 153, 157, 188). Suppressor T cells can bind naked nominalantigen under somecircumstances and have been enriched by adherenceto antigen-coated polystyrene surfaces (109, 166), but the requirementsfor activation of this class of T cells havenot beenelucidated (but see below).

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T-CELL ACTIVATION 467 Themajorexperimentalevidencethat T-cell antigen receptors are related to immunoglobulinsrests on shared idiotypy. A body of indirect data supports the sharing of Vuregions betweenantibodymoleculesand surface receptors or soluble productsfromT cells of the sameantigen specificity. Sharedidiotypes havebeenfoundon receptors for alloantigens (19, 135), carbohydrates(20), proteins (32) and haptens(42, 131, 133, 166), as as on antigen-specifichelper andsuppressorfactors (10, 52, 59, 124, 163). Thereare twocritical limitations to the interpretation of these experiments. Thefirst is that serological cross-reactivitycannotbe extrapolatedto suggest V-regionidentity. Thesecondis that heterogeneous anti-idiotypic antisera havebeenemployed and it is possible that immunoglobulin and T-cell products are reacting with different componentsof the reagents. Amore convincingcase could be madeby demonstratingidiotypic sharing using monoclonal anti-idiotypic reagents, althougheventhis wouldnot constitute definitive proof of a common gene pool. The nature of the T-cell antigen receptor, howeverobscureandfascinating, is not the central themeof this review.Ourmissionis to summarize the bodyof evidenceidentifying structural features of moleculesimplicated in T-lymphocyte activation and to describe whatis knownabout the nature of the "activating moiety,"which, in at least mostinstances, is not simplynominalantigenitself.

MHC RESTRICTION AND T-CELL RECOGNITION OF ANTIGEN Thediscoverythat T cells respondto antigen in a genetically restricted fashion changedforever our concept of T-cell antigen recognition. The cytotoxie T cell attacks only targets that express allogeneic MHC markers coded by the K, D, or other class I genes of the MHC or targets that combinenominalantigen with self class I products(18, 153, 189). Avariety of helper (or inducer) T cells (Lyt-1+ 23- cells) respondto antigen only whenit is presentedin a modified,presumably"processed"formassociated with products of the I-region of the MHC (92, 138, 157). Presentation requires the participation of Ia-bearing antigen-presentingcells (APC), whichin most, but not all, cases possessthe properties of macrophages. The I-region wasquickly recognizedas the common denominatorbetweenantigen recognition by Lyt-1+ T cells and specific immune response(Ir) genes (16). Theobservationsthat Ir genesapparentlyfunction only in responses to thymus-dependent antigens (16) and manifestremarkablespecificity that readily distinguishesbetweenantigens with similar primarystructures (71, 136, 168, 169) promptedthe hypothesisthat the Ir gene product mightbe the T-cell receptoritself (16). Thereare compellingarguments opposingthis proposal, manyof whichhave been summarizedby Benacerraf & Germain (15). Themostpervasiveof these wouldappearto be the functional expres-

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sion of manyIr genes in macrophagesand B ceils (see 15) and evidence implicating Ia antigens, whichare found in greater abundanceon macrophageand B cells than on T cells, as the productsof Ir genes (108, 155). Especially illuminating in this regard havebeenstudies with the B6.C-H2bin12strain of mutantmice, whichpresumptivelydiffer fromthe parental strain only in the Ab chain of the I-A molecule(120, 104). The immune responsesof mutantmice to some,but not all, antigens are selectively impairedrelative to the parental H-2b strain (112, 113, 122, 100), furnishing vivid evidencefor a causal association betweenIr genes, Ia antigens, and T-cell responses. Recognition by Single or Dual Receptors A paramountquestion, then, is the mannerin which Ia molecules on macrophages and B cells, or K/Dmoleculeson target cells together with exogenous (nominal)antigen, producean activating signal for T cells. Various modelshave been proposedto address this question. Theysegregate accordingto whetherthey postulate independentrecognition of antigen and MHC by twodistinct receptors on the T cell (dual receptor hypothesis)(21, 38, 84, 96, 105, 177, 187) as opposedto recognition of an interaction product between antigen and MHC by a single receptor (modified self hypothesis)(127, 136, 189). It has beendifficult to designexperiments that can unambiguouslydistinguish betweenthe models. Perhaps the closest approachthus far has been the generation of T-cell hybddomas between cells that had different antigen/MHC specificities (92). Thus,hybridsspecific for KLH plus H-2f werefused with T-cell blasts specific for ovalbumin a. plus H-2 "Double"hybrids were obtained that recognizedboth parental combinations,but noneweredetected with either of the cross-specificities. In other words, none responded to KLHplus H-2a or to ovalbuminplus H-2f. Becausethe cells expressedreceptors for each of the two antigens and the two MHC products, the findings clearly argue against orthodoxdual recognition modelsof MHC restriction. However,the authors carefully point out that dual recognition of an interaction product betweenantigen and MHC cannot be excluded inasmuch as a given antigen might form different interaction productswith MHC moleculesof differing haplotype. Adefinitive decision will probablyrequire direct biochemicalcharacterization of T-cell receptors. STRUCTURES RECOGNIZED BY T-HELPER AND T-PROLIFERATIVE CELLS Macrophage-Derived Antigen-Ia Complexes If the T-cell receptorhas resisted all assaults on its character,the molecular entity recognizedby the receptor has been of comparableobstinacy. After

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the basic tenet of genetic restriction in macrophage:T-call interactions had been extended from the guinea pig to the mouse, it was reported that cell-free supernatants from histocompatible, and only histocompatible, macrophagespulsed with antigen were able to replace intact cells in the generation of helper T cells (50, 51). The macrophage-replacingfactor was shownto be removable by either anti-I-A, or antibodies to the antigen, suggestingthat it contains elementsof both. Its molecularweightis surprisingly uniform (approximately 50-60,000 daltons) considering that antigens of larger and smaller size have been used in its preparation. Lonai and his collaborators have described a similar Ia-antigen complex(IAC) released by antigen-fed macrophages(114, 132). IACwas boundby Lyt-1 +2- T cells in an H-2 restricted fashion, could suicide antigen-specific helper cells in vitro, and displayed much greater immunogenicity than free antigen in histocompatible recipients (132). Competitive antigen binding by enriched T and B cells with (T,G)-A-L in both free and Ia-coupled forms demonstrated muchhigher attinity by T cells, but not by B cells, for complexed antigen (114). Furthermore, C3H.SW B cells cross-reacted with a variety of other branched polypeptides, whereas T cells boundonly (T,G)-A-L and (Phe,G)-A-L, to both of which they are Ir gene-controlled high responders. The authors concluded that IACis recognized by T cells with high affinity and mayplay a major role in defining the specificity, H-2 restriction, and Ir-gene control of T cells. Moredetailed analysis of the provocative IAC complex or the genetically related factor (GRF) (50, 51) is awaited. Right now, the T-cell recognition problem, remains plagued by the complexity systems used and the paucity of analyzable material. Antigen Presentation by B Cells It has recently been found that Ia-positive B cells mayalso present antigen in a genetically restricted fashion to sensitized T cells. Chesnut&Grey(35) used normal mouse splenic B cells coated with rabbit anti-mouse Ig to stimulate rabbit IgG-primedT cells in a proliferation assay. A variety of controls minimized the likelihood that sources other than B cells might have been responsible for the observed stimulation. This represented a special case inasmuchas anti-mouse Ig will bind to the surface Ig of all B cells. Indeed, protein antigens that lack this property (e.g. ovalbumin)were not effectively presented by normalB cells. However,a series of H-2a B-cell tumor lines were capable of presenting ovalbumin, KLH,or HGGto Iregion-restricted, antigen-specific cloned T-cell hybridomas(177). For the most part, antigen presentation correlated with the expression of Ia antigens on the B call lines, although a few positive lines failed to present antigen. Functional activity could not be correlated with the stage of differentiation of the lines. All the available tumor lines were of H-2d haplotype, but the principle of B-cell antigen presentation was extended by Kappler et al (93)

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by fusing oneof the Ia+, H-2d tumorlines to T-cell-depletedspleen cells from mousestrains of other H-2 haplotypes. Someof the resultant hybfidomaspresentedvariousantigensto a groupof antigen-specific,I-regionrestricted T-cell hybridomaswith the phenotypeof the normalspleen cell partner. In a recent fascinatingstudy, antigen-presentingcell variants with lesions in particular Ia antigenic determinantshavebeeninducedby mutation and selected from hybfidomascreated by fusion of a B-lymphoma line which can present antigen, with normal B cells. Such hybridomaAPC mutantsnowfail to present certain antigens but remain able to present others (61). The availability of homogeneous rapidly growingAPCfines (includingmonocytelines) (177) shouldgreatly expedite the exploration I-region-restrictedantigen presentationto T cells. Theabovefindings, whichprovide a plausible scenario wherebyT cells mightdirectly collaboratewith B cells that havecapturedantigen, do raise questions about requirementsfor antigen processing pursuant to T-cell triggering. It mightnot be anticipated, a priori, that macrophages and B cells wouldhandleantigens identically in terms of degradationand subsequentassociation with surface Ia molecules.However, it has beensuggested that B cells that bind an exogenousantigen mightintefiorize, degrade,and process it in the mannerof macrophages (174). Final judgmentwill depend ondirect characterizationof the structuresresponsiblefor T-cell activation. Cross-Reactivity for Protein Antigens by T Cells and Antibodies Oneapproachto understandingthe antigen specificity of T lymphocyteshas beento compare the relative cross-reactivities for protein antigensin helper or delayedhypersensitivity responseswith those exhibitedby antibodies. A general consensushas not emergedfromthese studies, althoughmostof the findingsare potentially explicableon the basis of T cells recognizingsmall linear fragmentsof processednominalantigen presentedin association with MHC gene products on the surfaces of APC,as proposed by Benacerraf (14). Anextensiveanalysisof the specificity of helperT cells for a series cross-reactingserumalbuminsled to the conclusionthat the specifieities of virgin and primedhelpers weresimilar, if not identical, to eachother and to that of humoralantibody(134). Onthe other hand, several studies of the e_ross-reaetivitiesof erythroeyteantigenssuggesteda greater discriminatory powerfor antibodies than for hdperT cells, both in vivo (43, 130) and vitro (53, 74, 75), althoughthe particular antigens involvedin the assays werenot identified. However, there is a difficulty inherentin this type of comparison:since T cells respondto a morerestricted set of epitopes, discrimination on the part of any one T cell maybe as acute. Similar findings were gleaned from a comparisonof native and extensively denatured(urea plus reduction andalkylation) formsof the globular

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T-CELL ACTIVATION 471 proteins ovalbuminand bovineserumalbumin, using proliferation as the indicator of T-cell responsiveness(34). This study wenta step further formallyestablishing that the sameT cells respondedto both native and denaturedforms, an importantadjunct in that it eliminatedthe possibility that a subset of cells recognizedthe native protein exclusivelyand, hence, mightbe specific for conformationalfeatures. It is also apparent from studies with T-cell clones specific for lysozymethat certain clones can be propagated on either lysozymeor reduced, carboxymethylatedlysozyme (F. Manca,N. Shastri, A. Miller, and E. Sercarz, unpublisheddata). However, moreinteresting are the clones that will only growon one of these forms of the molecule. Protein Antigens are Recognized as Fragments by T Cells Thecomparative speeificities of T cells and antibodiesfor native anddenatured formsof the sameprotein antigen haveseeminglypointedto a broader specificity for T ceils, in agreement with the results obtainedwith red ceils mentionedabove. In retrospect, the evidencedoes not support the notion that T-cell receptorsare actually less precisethan their B-call counterparts. Thus, early experimentsdemonstratedthat guinea pig delayed hypersensitivity reactivity, in contrast to antibody,failed to distinguish between native and extensivdydenaturedprotein antigens (56). In another investigation, chemically modifiedBSAcross-reacted extensively with the unmodifiedprotein at the helperT-cell level, but scarcelyat all at the humoral antibodylevd (146). Thelist of eases of this kind is long. Othernoteworthy examplesincludedantigen E, whichin denaturedforminduceshelper T-cell responsesto the native antigen withoutserological cross-reactivity (81); antibodies to staphylococcalnuclease(142) and lysozyme(145) are highly conformation-dependentwhereasT-cell responses are not; and helper T cells primed with the unfolded VHand VLdomainsof MOPC-315 crossreacted with the native myeloma protein (87). All the findings indicate that the portion of the nominalantigenrecognizedby helper T cells is sequential and not conformationalin character. (It is, however,possible that two nonsequentialfragmentedpeptides derived fromthe native protein might remain boundto each other and be stabilized by disulfide or other local forces.) Theseobservations, in concert with the MHC-restrieted character of T-cell responses,inspired the notion that T cells recognizefragmentsof antigen followingprocessing and association of antigen to MHC products by the APCs(14). This proposalrests on the likelihood that proteins will lose their native conformationin the acidic environmentof the lysosome, resulting in generation of the samepeptide fragments from native and denaturedantigens. Indeed, eventhe very few studies that implya conformationaldependence of antigen-specific Th/Tpcell activation can be interpreted alongthese lines. Considerthe proliferative responsesof T-cell blasts from BDF1mice primed to beef apoeytoehromee and tested with the

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homologous apoproteins, the native protein, and cyanogenbromidecleavage productsfromeach protein (28). Cells maintainedin long term culture with apocytochrome c respondedmorevigorouslyin the proliferative assay to the apoantigcnand its cleavage products than to the native protein, suggestingat first glancethe possibility of conformationaldependence for T-cell recognition. However, a comparisonof responsesusing three different sets of APCs(the syngeneicand two parental types) led to the conclusion that the observeddifferenceswereassociatedwith different capacities of the APCto processand present the two formsof antigen. As the authors pointedout, absenceof T-cell cross-reactivity betweennative and denatured formsof a protein mightbe expectedfor proteins that are relatively stable in the acidic environmentof lysosomesand unlikely to unfold prior to proteolytic degradation. In such instances, the proteolytic fragmentsobtained will dependon the three-dimensionalstructure of the substrate. Thus,the differencesseen are likely to reside in the APCrather than in the antigen-recognizing properties of the T cell. Referencewasmadeearlier to the exquisite T-cell specificity manifested towardantigens underIr gene control. This has been particularly evident with small immunogenic peptides of defined structure and sets of closely related analoguesof the immunogen. Thus, guinea pig T lymphocytessensitized to the octapeptideangiotensinII could readily distinguish single aminoacid substitutions, whichhad no effect on antibody binding (169), whenthe assayusedwasthe ability to stimulateT-cell proliferation. Similar results were obtained in the samelaboratory with humanfibrinopeptide B anda series of structural analogues(168). Thesefindingsare consistentwith the idea that the epitopes recognizedby proliferating T cells are dictated primarily by aminoacid sequencerather than by conformation,of which theremightbe expectedto be little, in anycase, in peptidesof this size range. Theseinvestigators reasonedthat if such be the case, inverting the sequence of aminoacid residues in a small peptide antigenshouldmakelittle difference to the T cell, becausethe sameaminoacids are presentedin the same spatial order (166). Twoassumptionsare implicit in this reasoning. One that there is no polarity or directionality in the presentationof an immunogenic epitope to the T cell. Theother is that peptides of the dimensionof eight aminoacids are not degradedduringthe courseof processingby APC. Shouldadditional degradationoccur, then it can be readily foreseen that altering the sequenceof a peptide mightalter the course of degradation, leading to the formationof different neoantigens.Theprediction wastested with a fibdnopeptidefragmentof eight aminoacids and its inverted sequencepeptide in a guineapig T-cell proliferative assay (166). Strain guinea pig T cells sensitized to the native peptide werecompletelynon-

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cross-reactiveto the invertedpeptide; in fact, the invertedpeptidewasnot immunogenic in strain 2 animals. Theauthors interpreted this to meanthat the ordering of the aminoacids within the antigen is critical, perhaps because the antigen becomesassociated with MHC products in a nonrandomfashion. Thepossibility remainsthat degradation of even small peptide antigens is an intrinsic part of processingand will certainly be affected by aminoacid sequence. Studies with T-Cell Clones T-cell clones reactive with soluble protein andsynthetic polypeptideantigens haverecently beenpropagatedin long-termculture (73, 78, 97, 159). Someof these cloned lines havebeenshownto function as antigen-specific helpers and to obey the rules of MHC restriction. For example,clones reactive to the multichainsynthetic polypeptide(T,G)-A-Lprovidedspecific help to TNP-pdmed B cells whenchallengedin vitro with TNP-(T,G)A-Land respondedin proliferation assays in an I-region-restricted fashion (73). Anaspect of the workwith cloned, self MHC-restricted,antigenspecific T cells that has an importantbearingon the questionof the nature of T-cell specificity has been their cross-reactivity with allogeneic MHC determinants. Oneinterpretation of the high frequencyof alloreactive T cells, originally notedby Simonsen (156), has beenan overlapping of specificities of the alloreactive andnominalantigen-reactiveT-cell populations. This issue has beenaddressedin the past by searchingfor cross-stimulation of alloreactive cells by antigen and vice versa with heterogeneousT-cell populations(17, 30, 69, 107). Althoughcross-stimulationwasobserved these studies, interpretation wasdifficult becausethe possibility of nonspecific recruitmentcould not be excluded.This ambiguitycan be circumvented,or at least minimized, by usingextensivelyreclonedantigen-reactive T cells. Thus,a DNP-ovalbumin-specific clone froma B 10.Amouseproliferated in responseto addedantigenin conjunctionwith syngeneicirradiated antigen-presenting cells as well as in responseto allogeneicspleencells from B10.S mice in the absence of DNP-ovalbumin or B10.Aaccessory cells (158). Thesefindings do providecompellingevidencefor dually specific cells responsiveto both nominalantigen andalloantigens, but they do not yet permit distinction between, hypothesesconcerningthe receptors involvedin antigen recognition. Thus,the findings are compatiblewith completely independentsets of receptors for alloantigens and for nominal antigens randomlyexpressedon all T cells (181), with alloantigen recognition throughhigh affinity cross-reactivity with the anti-self MHC receptor (84) or the receptor for nominalantigen (38, 105) in dual receptor models, or byhighaffinity cross-reactivitywith a single receptorfor alteredself (17, 30). Additionalstudies with antigen-specific T-cell clones mayeventually

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distinguish betweenthe various modelsfor antigen recognitionby T cells. ClonedT cells will also be useful for settling other unresolvedissues concerning antigen recognition and MHC restriction. For example, the specificity of individualT cells for definedepitopesof antigenmolecules has beenobscuredby using heterogeneouspopulations of cells and relatively complexantigens. Theresponsivenessof T cells to the compound L-tyrosine-p azobenzenearsenate(ABA-T) furnishes a systemfor investigating the nature of T-cell antigen recognitionin whichthe antigen is reducedto its simplest possible form. Different functional types of T cells have been inducedthat are specific for ABA includinghelpers (5, 6, 62), suppressors (109, 161), killers (154), andmediatorsof delayedhypersensitivityreactions (11, 169). Veryrecently, long-termcultures of T cells specifically reactive to ABA-T were established from the lymphnode cells of A/J mice, which had been immunizedwith ABA-T,and the cells were cloned by limiting dilution (70). The MHC restriction of the antigen-inducedproliferation response of the clones wasassayed with accessorycells fromappropriate recombinantstrains of miceand monoclonalanti-Ia antibodies as blocking reagents. Fourteenof 15 clones tested provedto be I-A restricted in their response,whereasthe other clonewasI-E restricted. Thesefindingsindicate that the sameepitope (ABA-T) can be presented to T cells in the context of more than one MHC product. Although the bulk population of lymph nodecells respondedwhenaccessorycells compatibleat either the I-A or I-E loci werepresent, individualcloneswererestricted in their recognition to a single locus. Thefine antigenspecificity of the cloneswasanalyzedwith structural analoguesof ABA-T to induce proliferation in conjunctionwith syngeneieaccessorycells. Theclonescould be dividedinto three types with respect to responsivenessto ABA-histidine(ABA-H).Onetype responded about equally strongly to ABA-H and ABA-T.A second group responded to both, but significantly better to ABA-T, whereasthe third set did not respondat all to ABA-H. In all cases, the ABA-H-responsive clones discriminatedexquisitely betweenthe 2-azo and 4-azo isomersof histidine, respondingonly to the 4-azo derivative. Thus,at the level of individual clones, T cells display a remarkablespecificity for definedepitopes of the nominalantigen in situations wheredifferential processingof antigens is unlikely to be a factor, as it maybe whencomparingcomplexantigens differing in primarystructure.

The T Proliferative Responseis not Identical to the T Helper Cell Response It is often assumedthat cell populationsthat proliferate in responseto a particular epitope in the context of the appropriate MHC-APC are always helper cells. This is surely not the case. For example,in recent workin the

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T-CELL ACTIVATION 475 beta-galactosidase (GZ)system, T proliferation could be inducedin vitro by GZin most of the populations primedby individual cyanogen-bromide peptides derived from GZ(from 23-95 aminoacid residues in length). However, only two of a dozenpeptides tested could inducea T-helpereffect for the anti-hapten (FITC)response to GZ-FITC (101). The ability to inducedto proliferate by antigen maybe sharedby manyLyt-l+2-cells and conceivablyby Lyt-1-2+ cells [although in heterogeneouscell populations the suppressorT-cell proliferation mightbe missedin that the expressed activity of the Ts wouldprecludethe detectionof its target population(s).] Surely at the clonal level, manycellular membersof the enormousTs "regulatoryarmy,"as well as the Ts itself, mightproliferate in conjunction with antigen and/or IL-2. The secretion of IL-2 by a T inducer cell in responseto its activation stimulusmayrepresent a majorpathwayin Ts-cell and other T-effeetor-cell recruitment. It has beenshownthat manyof the cells recruited by the proliferating cell are B cells (172). It is possiblethat uponrecruitmentandproliferation, a subsequentdisplayof antigenby these B cells will back-recruita fewmore antigen-specific T calls. Surely, underthe usual Corradin&Chiller (40) conditions with whole, primedlymphnodes, a myriadof recruiting and proliferative events mustbe occurring. Determinant Selection on Multideterminant Antigens Theparticular choice of determinantson a potentially multideterminant antigen moleculecan be attributed to selectivity exercised by a component of the MHC.This concept of determinant selection was elaborated by Rosenthaland his colleaguesusing the modelof insulin recognition by the guinea pig and the mouse(136). Strain 2 and strain 13 guinea pig T cells eachresponded to a distinct regionof beefinsulin (137), andthis discrimination couldalso be observedin the mouse(140). In the case of insulin, and others subsequentlystudied, in whichmammals are challenged to respond to related mammalian proteins, it couldbe arguedthat, in fact, there is very little "selection": that is, whatevermechanisms purge the repertoire of responseagainst self antigens leave only a few regions that are different betweenthe two proteins, against whicha potential response might be directed. This is onereason whyresults with the bird lysozymesare noteworthy,in that as a groupthey differ by about 50 residues out of 129 from the mammalian congeners. In fact, almost all of the surface aminoacid residuesare different. Nevertheless, it is stalkingthat withall this potential "foreignness"only one to three determinantareas are selected by mice of differing haplotypesin the triggering of T-call hdper/proliferativesubpopulations (115, 116). In a sense, this reminds us of the concept of immunodominance of certain epitopes in the induction of antibody (88).

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At the T-cell level, it has beenpostulated(14) that whatmakesa particular determinantarea dominant or, rather, respondedto at all is the existence of a stretch of aminoacids on the nominalantigen that has an at~nity for a structure on the MHC restriction element. A useful nomenclaturefor consideringeachof the entities involvedin the recognitionhas recentlybeen proposed(144). Thus, the nominalantigenic entity that contacts the MHC has twodistinct moieties, at least conceptually:an epitope in contact with subsites on a T-cell receptor, and an agretope(antigen recognition), which acts as a site of attachmentto the MHC desetope(determinantselection). (Wemight suggest from the limited data available that roughly one agretope exists for every 50 aminoacid residues on a protein for Th/Tp cells.) Finally, the additional elementon ClassI or ClassII MHC molecules that is recognizedin additionto the epitope is designateda histotope. In workon pigeon cytochrome(64-66) by Schwartzand his colleagues, a potential agretope has been defined as giving the quality of immunogenicityto the C terminal peptide 81-104.Usingsynthetic peptides, it was shownthat residues 100 and 103 were important for antigenic strength, whichcould be related to interactions of the antigen and MHC structures on the APC.Grantingthe closeness of the agretope, residue 99 could be consideredpart of the T-cell contactingepitope related to T-cell memory,since a set of amidinatedC-terminalpeptides derivatized at position 99 were completelynon-cross-reactive with the native peptides but nevertheless showedthe same immunogenicity/non-immunogenicity discdmination amongstrains, whichis presumablydependenton the nature of the agretope. In the lysozymemodel,a completelyanalogousresult has been reported, in whichonly one dominant, circumscribedregion of the moleculenear aa 113-114is utilized in the B10.D2 haplotypefor presentation of the epitope at 113-114to T cells respondingto parallel sets of non-crossreactive bird lysozymes(96). A case in whichan agretope mayhave been synthetically manufactured appearedin the recent literature. As mentionedearlier, an octafibrinopeptide is immunogenic for strain 2 guineapig T cells (166). However,strain 13 animals are completelyunresponsiveto the peptide. Whena glycine residue wasaddedto the carboxylend of the octapeptide, the newnonapeptide inducedstrain 13 T-cell responses. Althoughthe mechanismof the immunogenic conversionis uncertain, a plausible interpretation is that the glycine residue formedpart of an agretopewhichcould productively associate with a strain 13 desetope. AnotherCandidatefor a critical componentof an agretopeis the side chain of tyrosine in ABA-T. Increasingthe hydrophobicityof the side chain decreasedimmunogenicity without palpably influencing specificity. Thus,

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T-CELL ACTIVATION 477 the tyrosyl end of the moleculecan be visualized as the agretopeand the ABA endthe epitope, althoughthe precise structures of these entities have not beendefined. If agretope scarcity were responsible for the very limited responseto certain antigens, the followingconsequencesmightbe expected: (a) when an agretopeis defined,a diversity ofTcells shouldbe availablefor combination with epitopes associated with that agretope; (b) the distribution agretopesrather than the repertoire diversity of ambientcells shouldbe the prime factor in determiningwhetheror not a particular antigen will be respondedto by the haplotype;and(c) the paucity of appropriateagretopes should lead to somenull peptides on a typical immunogen whichare not usedfor addressingany T-cell subpopulation.Someof these corollaries have beenobservedin the lysozymesystem(117). Thus, only a single lysozyme agretopeis presumablyavailable for H-2b strains near aa residue 91, but nevertheless,a heterogeneous array of T proliferative clonesof differing fine specificity for epitope can be detected, all of whichseeminglyutilize the unique agretope. TheC-terminalpeptide of lysozyme,aa106-129,makesno impactat all on H-2b mice, neither inducinghelper nor suppressorT cells; likewise, no other tryptic peptides than T11(aa74-96)are immunogenic reactive in H-2b strains. Agretope Limitations and Hierarchies Hints are available that the scarcity of agretopesmaysometimes be apparent rather than real. Accordingly,there maybe two or three immunogenic regions on a protein molecule,but a hierarchy of choice can obscurethe reactivity to all but a dominantagretope-desetopepair. Anexamplein the lysozymesystem (96) involves the subdominantC-terminal peptide in the B10.Astrain, reactivity to whichcannot be demonstratedafter immunization with native lysozymeand challengewith L3. However,initial in vivo immunizationwith L3 reveals strong responsivenessin this haplotype to either L3 or HELsubsequentlyused for in vitro challenge. Apparently,a competitivesituation exists in whichan N-terminalagretopenear residues 13-15 takes precedenceover two other agretopesthat are moreC-terminal, or alternatively,T cells possessgreater at~nities, in generalfor the dominant epitopes. SUPPRESSOR OF ANTIGEN

T CELLS

AND THE RECOGNITION

Does suppressor T-cell activation depend on the same concepts of desetope/agretope interaction we have just discussed for Lytl+ Th/Tp cells? DoS-agretopesexist that are distinct fromH-agretopes?Theconsid-

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erations relevant to this section hinge on the relationship of the MHC to suppressor cell induction and activity, an area that still has manyunknowns.

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T Suppressor Cell-Inducing Determinants SuppressorT cells (Ts) play an important role in the expression of the Tp/Threpertoire, in somecases obscuringa latent capacity to producea responseto the native molecule.In Ir gene-controlledresponses,it has been reported in a variety of systemsthat nonrespondermicehavethe capacity to respondto the antigen in the absenceof Ts, or to presentthe antigento syngeneicor to FI(Rx NR)T-helperceil populations(9, 90, 98). Disclosure of this latent potential mightrequire the dissociation of the suppressorT cells fromthe system.In the ease of lysozyme,this wasachievedby amputation of the suppressor-inducingepitope at the N-terminusof the protein (185). In fact, removalof as little as the terminaltripeptide lys-val-phe aminopeptidaseconverts the non-immunogenic lysozymeinto a derivative that no longer inducessuppression,but rather a helper effect, and T-cell proliferation (A. Miller, L. Wicker,M. E. Katz, and E. Sercarz, unpublished results). Bythis simple transformation, actually two newdeterminants were revealed from what would have simply been regarded as a non-immunogenic molecule for H-2b mice: a suppressor determinant and the previouslysuppressed, latent responsivenessto the helper epitope on tryptic peptide 11. Of course, the samegeneral problemof paired suppressor andhelper epitopes mayexist on any "null peptide" such as the C-terminal peptide of lysozyme,and additional experimentscan be performedto examine this possibility. Latentresponsivenesshas also beendemonstrated in the eytochromesystem(41), and to one of the cyanogen-bromide peptides from beta-galaetosidase (U. Krzych,A. Fowler, E. Sercarz, unpublisheddata). Thefact that a single suppressor-inducingdeterminantcould affect the entire response to a molecule had been recognized with lysozyme(HEL) earlier (1,152). Morerecently, it has beenpossible to induce Ts with the CNBr-derived,reduced and earboxymethylatedN-terminal dodeeapeptide from HEL(N. Shastri, A. Oki, M. Kaplan, D. Kawahara,A. Miller, and E. Sercarz, unpublishedresults). Earlier evidenceof suppressordeterminants with beta-galactosidase (150, 173) and poly-glu-ala-tyr (147) carried the implication that the suppressor- and helper-inducingdeterminants mightbe different.

Suppressor and Helper Epitopes are Non-Overlapping In all protein systemsexamined,epitopes addressed to the Lyt-l-2+ Tsp population and those activating the Lyt-l+2-Th/Tpsubpopulation are different! This wastrue in the case of the small globular lysozymemolecule

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T-CELL ACTIVATION 479 (HEL=129aa)and likewise for the large tetrameric B-galactosidase (GZ=1021an/monomer). Whenthe second cyanogen bromide peptide from the N terminus (aa3-92) of GZwasassessed for induction of Th Ts, both subpopulations were activated. Further analysis showedthat a tryptic nonapeptide,aminoacids 44 to 52, inducedTh but not Ts, and the reverse wastrue for an 1 l-amino-acidpeptide 27-37. Thusthe principal of non-overlapremainedintact (102). Similarly, in the response to myelin basic protein in the guineapig (160)it appearsthat the suppressor-inducing peptide is different fromthe encephalitogenic peptide in this species. However, the possibility that the determinantsare very close couldbe concluded fromexperimentsin whichspecific residue alteration causeda reversal of functionaleffects inducedby the peptides(68, 94). In a similar vein, Thand Ts that recognize the sameor very similar epitopes can be induced by ABA-T(109). Althoughthe picture is not complete,manyresults can be accommodated by a general modelcited belowthat suggests that functionally and chemically uniqueantigen-presentingentities (desetopes) are employedin the activation of different T-cell subpopulations.Consequently,it maybe the desetope-agretope interaction that controls whichT cells will be triggered. Interactions

Within the Suppressive Cell Circuitry

Theintereractions amongthe varied members of the regulatory T-suppressor family have illustrated somenewprinciples in T-call activation and interaction. In consideringthe productionof antibody, it is evident that membersof a single AgThclone can collaborate with the appropriately specific B cell, leadingto B-cell proliferation, differentiation andantibody formation.This is the tip of a cellular iceberg, whichhides a multitudeof competingregulatory cells, each of whichmust knowwith whichpartner cell it mustinteract. Theserules of intercellular communication, or as Tada has termedit, "immunosemiotie interaction" (162), musteventuallylead cell collaboration that is purposefulrather than haphazardand chaotic. Evidenceindicates that the T-Tinteractions that accomplishthe regulation of suppressionare restricted by three types of elements--antigen,MHC and immunoglobulin. Moreexplicitly, these are the suppressorepitopes on the antigen, MHC-presentation elements, and regulatory idiotopes, but in addition there maybe MHC-interaction elementsand T-cell receptor constant-region determinants that must be recognized. Each memberof the systemis inducedby a specific cell or a factor derivedfromit: the factors have componentsthat recognize epitopes or idiotopes, in addition to a constant region, coded for by genes on the 12th chromosome in the mouse (170, 171); on two-chainfactors a separate peptide bears the MHC element involved,whichin the suppressorcircuit is a type of I-J.

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Assuggestedin the last paragraph,one of the critical questionsthat is still undergoingexploration is whether suppressor T cells are MHC restricted at either oneof twolevels: (a) at the point of presentationof antigen or (b) in the interactionbetween regulatoryandtarget cells in the collaborative milieu.

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Effector-Cell

Induction Pathways

T cells whichwill eventuallydevelopinto effectors (Th, Ts, and To), seem to be activated by a particular inducer cell (augmenting cells, suppressor inducersandCTLhelpers, respectively). It can be predictedthat the rules of each partnership are immunosemiotieally defined to prevent anomalous induction. TheTs pathwayshavebeenmostextensively studied and will be discussedin a general, summary way,as other reviewshavedescribedtheir cellular constituentsin detail (31, 57). -, I-J+suppressorinducercells (Tsi) weredemonstrated Lyt-1+2 (45) to necessary in interacting with Ts precursor cells, usually Lyt-l+23+, to promotethemto functional effector status (9, 119). Morerecently, signiticant progresshas beenmadein assigningantigenicand idiotypic specificities to these cells or to factors derivedfromthem(46, 164, 184). In general, the restriction in interactions betweenTsi andTs cells appearsto be owing to Igh rather than to MHC structures, whereasTs effector cell interaction with Th targets is MHC-restricted. It has beensuggestedthat in all suppressorsystems, a cascadeof three sequential Ts calls (Ts 1, Ts2, andTs3) are induced,originally by the nominal antigen (58). In fact, in the systemswhichreveal eachof the three cell types, the next cell in the sequenceis usually inducedby a factor derived from the previous cell. However,antigen can also induce Ts3 directly, whichthen requires a Ts2-derivedsignal to achieveeffector status (123). appearsthat the systemwill employthe effector-cell specificity it needsto control the regulee: if it mustdown-regulate an idiotype-beadng cell, it will somehow raise idiotype-recognizingT-suppressorcells. Onsuch occasions, if a particular suppressorcell is in short supply,complex circuits involving amplifyingcells, whoserelationship to the effector cells is based upon idiotype, MHC, or antigenic recognition (31, 162) maybe resorted to. The Ease of Triggering Ts in Non-Responder Strains Certain well-characterizedcases of genetic nonresponsiveness,those concerning the terpolymer glu60-ala30-tyrl0 (GAT)and lysozyme,owetheir existenceto readily activated T-cell suppression(60, 151). In the lysozyme case, a Ts cell is inducedin H-2b miceby chickenlysozyme(HEL)but not

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T-CELL ACTIVATION 481 by anyvariant lysozymethat substitutes tyrosine for the phenylalanineat residue 3, suchas REL(4). Likewise,H-2~’ strains respondto RELbut not to HEL,at the antibody formation level. It can be asked whetheror not sometype of suppressiveelementexists in regulating the responseto REL in the H-2b mouse?If so, are these Ts simplyless readily activated by REL in the non-responder strain, or activated by a parallel but distinct mechanism? Althoughsuppressive circuitry mayoccur universally, the routes taken to achievethe suppressionmaybe quite different in the nonresponder andresponderstrains. Possibly,Ts inductionto certain epitopesis rapid and effective in the non-responderbecause of an MHC-restrictedactivation process that can proceedat a low level in the absenceof a T-suppressor inducer cell. In responderstrains, stimulation of the idiotype-bearingTs mightoccur at a later point, throughthe parallel mechanism involving a Tsi-delivered induction signal. Sucha circuit, for example,mightdepend on the appearanceof the predominantidiotype during B cell maturation, whichwouldarouse idiotype-reeognizing Tsi to stimulate suppression (103). Differences in Th and Ts Recognition of Antigen At the outset, antigen recognitionby Ts cells appearsto be quite different from that of Th or Tp cells. Evidencethat Ts can bind to native antigen has beenused to argue that nominalantigen recognition is independentof the MHC. Isolation of Ts on plates coated with antigen (109, 125, 165) indicates that Ts can recognizenative antigen. However, there are several publishedinstances (3, 39, 72, 118) in whichThhavelikewise beendemonstrated to bind to antigen or antigenic peptides. Endres and Grey (49) found that Ts induced by native or extensively denaturedovalbumindid not functionally cross-react with the alternative formof antigen, in contrastto helper T cells (34). Furthermore, the suppressor cells could be depletedon plastic dishes coatedwith the homologous but not with the heterologousantigen. Thesefindings suggesta closer kinship of Ts cells to B calls than to Th cells in terms of antigen recognition. However,in the lysozymesystem, the native enzymeor its reduced, carboxy-methylatedderivative or its fragments (including the N-terminal dodecapeptide)can each induceTs, althoughthe derivatives surely are not in the native conformation,nor are they cross-reactiveat the B-cell level. Certain authors (34) postulate that Ts cells recognize conformational determinantsandthat the differences in antigen recognitionbetweenTs and Th are related to the requirementsfor antigen processingandpresentation by accessorycells to Th; this argumentneglectsthe presentationof antigen to Ts. Theapparent lack of dependence on adherentcells in the induction of suppression(54, 80, 129)seemsto supportthe idea that antigen process-

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ing is not required for the activation of suppressorT cells. However, the existenceof special types of APCfor different T-cell subpopulations,with different adherenceproperties, has been documented (29) and wouldseem a relevant consideration. Theevidenceis still concordantwith the notion that Ts recognizeexternal epitopes on antigens, owingto the superficial placementof S-agretopes (also see below).This wouldpermitbindingto antigen alone, if the affinity of interaction were high enough,but possibly Ts activation wouldrequire an additional signal derivedvia associative recognition. MHCRestrictedness of Ts Cells Althoughthe evidence wehave sketched suggests that effector Ts maybe MHC restricted in the interactions with their inducers andtarget cells, we will nowexamineexperimentsthat convergeto suggest MHC restriction at the point of presentation of antigen and the induction of Ts precursors. In the lysozymesystem, the ability to induce Ts in the nonresponder H-2b mouseis limited to an epitope at the N-terminusof several gallinaceous lysozymes,those of hen, bobwhitequail, guinea hen and any other lysozymehavingphenylalanineat aminoacid residue 3. Interestingly, humanlysozyme(HUL)whichis almost completelynon-cross-reactive at the B cell level, apparently can induce the sameTs, bearing the predominant idiotype, IdXL,whichthen is equally active on the anti-HELand anti-HUL in vitro responses.This has a rationale in that not only pbe-3but also eight of the nine N-terminal residues are identical in HELand HUL.On the other hand, although the H-2q mousepossesses the same cross-reactive IdXL-bearingTs, it nevertheless can respond to HELwhile still being nonresponsiveto HUL.This failure to induce Ts by HELcan be attributed to an inability of HELto interaet with the appropriate MHC dementin antigen presentationto Ml-IC-restrictedTs (2). Othermoredirect evidence for MHC restriction in this systemwasobtainedin positive sdeetion of the IdXL+ Ts on HEL-pulsed, presenting-cell monolayers. Only syngeneic monolayerswere capable of antigen presentation to Ts, and this positive sdection could be inhibited by anti-IdXLserum(8). In recent workby Jan Klein andhis colleagues(12, 13), evidencehas been presentedfor restrietion of Ts induction by lactate dehydrogenase B in the eontext of an I-Ek restriction dement. Using nonresponderB10.A(2R) cells, antigen-presentation by B10.A(4R) lacking the I-Ek dement,gives rise to proliferation, but not suppression.Furthermore,in vivo treatment with monodonalanti-I-E antibody converts the B10.A(2R)into an LDH responder. Takentogether, the evidence points to MHC restriction at the time of initial antigen presentation, and suggeststhat further study of suchearly events mightreveal additional examples.

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T-CELL ACTIVATION 483

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DESETOPES AND HISTOTOPES IN CYTOTOXIC T-CELL RECOGNITION Theproblemsraised by recognition of class I and class II MHC proteins are really quite parallel. Severalclass I proteins (includingK, D, L, Mand R) havebeenimplicatedin associative recognition with nominalantigens. Just as in the case of Ia-restriction, it is apparentthat certain haplotypes utilize particular molecule(s)of the class I set for restricting their CTL responses. Oneinteresting exampleis providedby the recent workof Ciavarra andForman (36), studyingthe restriction elementsused for reactivity to vesicular stomatitis virus (VSV)in the H-2d haplotype. VSVwasonly d molecule[This was shownby coldrecognizedin the context of the H-2L dtarget inhibition experimentsas well as inhibition studies with H-2L directed monodonalantibodies. However,H-2L-restricted responses are not foundagainst trinitrophenol (111)or minorhistocompatibilityantigens (22). Wecan discuss these results by employingthe sameconventions describing the relationships betweenMHC class I moleculesand nominal antigenas wereusedearlier in conjunctionwith Ia restriction elements(see p. 476). Therequirementsfor association betweenan agretopeon the viral antigen and any particular class I desetope maybe unusuallydemanding. Therefore,eventhougha dozenor moredifferent class I "restriction sites" maybe available within a haplotype(if each is unique), a particular viral capsid antigen maynot be able to associate productivelywith mostclass I molecules.Likewise,hierarchical preferences also seemto be evident in class I T-cell activation. A related question of someimportanceis whethera distinct region (a histotope) on the class I or II moleculesis necessarilyrecognizedby the cell in addition to that desetopemakingcontact with the nominalepitope at an agretopic site. Oneexperimentalsystemthat seemsrelevant to this questionis the recognitionof haptenin conjunctionwith self-class I molecules by Te cells. Earlier workshowedthat hapten coupling to class I moleculesthemsdveswas unnecessaryin achieving a restricted response (143). Moleculesof TNP-BSA could insert into the cell membraneand TNP-selfrecognizingTc wouldstill respond. This lends somecredenceto the viewthat the histotope recognizedon a class I moleculemaybe some distance fromthe hapten. It has beenrecently pointed out that the hapten or nominalantigen could also be coupledto a self non-MHC protein (110). Studies of the sites of allogeneicrecognitionare also connectedto this question. Allogeneicrecognition by CTLmaybe limited to very few areas on the target class I molecules.In a recent report (77), cross-reactivity mutant mouseclass I proteins of knownsequencewasthe device utilized to approachthis problem.The70%cross-reactivity of b anti-a CTLwith bmltargets couldbe attributed to a tyrls~-tyrt~6 alteration of Kbm~.Since

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the identical alteration was uniquely found on the Lo molecule, it apparently accounts for the prominent cross-reactivity. Cold target inhibition studies verified the point. Whetherthe site functioning as an allogeneic target is related to a desetopeor a histotope is an importantarea of investigation. In this context, evidence provided by Weyandet al (178, 179) shows that 2 distinct polymorphicclusters, A and B, are recognized, both by CTL and monoclonalantibodies. It is interesting but perhaps circumstantial that both alloreactive H-2k-as well as H-2d-restricted self CTL’sstrongly prefer cluster B. It appears that a crystal ball is still neededto sort out howmanyseparate elements are recognized by the T cell. No experiments exist limiting the recognition decisively to a fixed numberof elements. Withinthe various cell subpopulafions examined, this could range from (a) epitope alone (b) tope plus somemixture of desetope/agretope to (c) histotope alone or (d) either (a) or (b) plus a distant and distinct histotope.

THE RECOGNITION OF IDIOTYPIC DETERMINANTS BY T CELLS During the past decade, voluminous evidence has implicated the T cell in intercellular idiotypic interactions (reviewed in 47, 82, 83, 175). Weshall consider the nature of the T-cell recognition and in what form the idiotopes are recognized (MHC-restricted, together with antigen, etc.). Although have discussed someof the Id-anti-Id interactions in the suppressive cell circuitry, we will focus here on the predominant idiotypic determinants displayed on the B-cell surface that seemto play a critical role in establishing the quality of the antibody response.

Predominant Idiotypes An idiotope on an immunoglobulin molecule can be recognized by an antigen-specific Th cell (AgTh)just as any other immunogenicepitope, the context of the MHC.Somestrains will be high responders to such idiotopes, and others will be low responders, as in any other immune response gene-control system (86). Jorgensen and Hannestadused different portions of MOPC-315 as T-cell priming agents,~ehallenging subsequently with 315 coupled to DNP. However, we would like to explore the nature of predominant idiotypes on B cells. Whyare there certain idiotopes that gain such prominenceas the T15 idiotope which appears on more than 90%of the anti-phosphocholine antibodies in the BALB/e(37) strain, and also in several others? many cases this maybe a consequence of the germ-line gene composition (55). The predominanceof certain idiotypes can be contrasted to the diver-

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T-CELL ACTIVATION 485 sity of private idiotypes that appearon individual hybridomas in manysuch systems.In several cases (3, 25, 182)it has beendemonstrated that T cells that recognizeidiotype and can be adsorbedon Id-coated petri dishes are implicated in causing the predominance.Onepossible scenario wouldinvolve a sequential activation of the B cells specific for the antigen by an AgThthat is MHC-restricted.Such AgThclones (73) have been shown activate B cells regardless of their idiotype (S. Cammisuli, personalcommunication), or isotype (149). In the absenceof an additional IdX-recognizing cell, this wouldlead to a nonselectivedistribution of idiotypes in the expressedrepertoire. Subsequently,dueto the displayof the special predominant idiotope(IdX)on the activatedB cell surface, a set of IdX-specific cells (IdXTh)wouldbe activated whichthen wouldpositively select those B cells for expansionthat display IdX. The kinetics of appearance of IdX amongthe B cells in a variety of systemssuggests that in cases wherethe antigen moleculeis ubiquitousor at least availablein the self-environment, a prior equih’brinm is established betweenIdX-recognizingTh and Id-bearing Ts whichdownregulate them. The Concept of a Regulatory Idiotope Evidenceseemsoverwhelmingly to favor the proposalby Jerne (85) that the lymphocytescomprisingthe immune systemare linked by an "’intricate web of V domains."A balance is established by the historical accidents of extrinsic exposureto immunogens and the receipt of passive maternal Ig transplacentally (180), both playingon the available genetic repertoire germ-lineidiotypes. The original suggestion by Jerne portrayed an ever-expandingnetwork of idiotype-1 (Ab-1)inducinganti-idiotype (Ab-2), whichthen influenced the expansionof both Ab-1and Ab-3anti-(anti-idiotype)-bearing cells. Ab-3resembledAb-1in idiotypybut not necessarilyantigen specificity. An alternative notion of regulatory idiotopes wasset forth by Paul and Bona (23, 128) whichdirectly implicatesa receptoron an idiotype-reeognizing (IdXTh)in interactions whichdeterminethe nature of the maturingB cells. Accordingto this concept, the networkessentially oscillates betweenidiotype-recognizingand idiotype-bearing lymphocytes. It is probablethat initial triggering of the B cell by antigen ~andAgTh is necessarybefore it will respondsuitably to ambientIdXTh:otherwise, virgin B cells wouldbe perpetually confrontedanddistracted by idiotyperecognizingT cells. Since the IdXidiotope mustbe clearly available for bindingby the IdXThor its specific effector factor, evenin the presenceof antigen, it has beenpostulated that such idiotopes mustreside outside of the antibodyactive site (128). Theseregulatory idiotopes mightrepresent a set of determinantswithin the framework that are quasi-isotypic, appearing on antibodies with a variety of paratopes.

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Starting with the seminalobservationsby Oudinand Cazenave(33, 126) that antibodiesof differentspecificitycan still shareidiotypy,this themehas reappearedand has been relined with the study of monoclonalantibodies (98, 106, 121). In these systemsit has beenshownthat monoclonal antibodies with differing peptidespecificity for the sameantigencan eachbear the predominant idiotype. This represents a slightly different aspect of Jerne’s conceptof a non-specificparallel set of antibodieswith sharedidiotypybut specificity for distinct antigens, evidencefor whichhas accumulatedrecentlyin severaldifferent anti-haptensystems(44, 46, 48, 75, 76, 183).What is morerdevant in our context is whetherthe perpetuation of antibodies bearing a predominantor partially predominantidiotope is a direct consequenceof activation of an IdXThwhichthen selects fromthe entire population, those B cells bearingparatopesof various distinct speciiicities for antigen. ~ It is probablethat the wavesof Ab-1,Ab-2,Ab-3,etc. havea role to play in regulating the response, but it remainsto be learned howT-cell control and serumantibody control contribute to the final outcome. Mostrecently, the question has beenaddressedof whetherany idiotope, even a private one, could subsumethe function of being "regulatory." Neonatalanimalswere treated with a particular silent Id, A48(141) and this idiotypesubsequentlybecame a significant portion of the anti-bacterial levan response. Thecapacity to promotea silent Id into a predominant one belongedto an idiotype-recoguizingLyt-1+ cell. The Idiotope as a Restriction

Element

Onthe basis of evidenceindicating a concurrentrequirementfor antigen in the activation of IdXTh(27), Bottomlyhas suggestedthat a receptor for antigenas well as for predominant idiotope exists on this cell (26). In such circumstances,the idiotope mayserve as a restriction dementsubstituting for the histotope on an MHC molecule. Does the idiotope resemble a histotope? Indeed, it is possible that a receptor for nominalantigen must exist on all T cells in the system, although Janewayremindsus (79) that the MHC-recoguizing Th cell can substitute an idiotope for the epitope. Self-idiotopes, as internal imagesof epitopes, can therebyplay a role in repertoire development. The existence of so manypredominantidiotypic systems suggests an evolutionaryreasonto focus on a limited numberof idiotopes, such as the need to allow coordinatedand simplified regulation of suppressorT cells (whichoften bear the predominantIdXof the B-cell pathway(67)). Thus, the involvement of B cells in the triggering of suppressorT cells mightserve to initiate the feedbackregulation of a well-establishedresponseby stimulating an idiotype-recoguizingT-suppressorinducercell (103).

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CONCLUDING

REMARKS

Perhapsthe mostintriguing unresolvedissues concerningthe antigen specificity of T cells center on: (a) the mechanism(s) underlying determinant selection; (b) the chemistryand genetics of T-cell receptors, whichshould providethe structural basis of geneticrestriction; and(c) a structural comparison of receptors fromdifferent T-ceil subsets with eachother and with immunoglobulins.For a complete understanding of the latter two, the receptors themselves(or the genescodingfor them)mustbe delineated. for determinant selection, the agretope-desetopeconcept, in whichthe agretope can be perceived as the "carder" moiety of T-cell immunogens, provides an attractive frameworkwithin whichexperimentaltests can be designed. Theenormouschangein our viewof T-cell activation in the past decade is almostentirely dueto the conceptsof MHC restriction and the idiotypic network.It is no longer possibleto considerthe problemof antigen activation of T cells in a context separate fromthe recognition of someother element, codedfor by the responder’sowngenome.Thedi~culty in achieving a completeunderstandingof T-cell activation is partly due to the tremendousdiversity of T regulatory ceils that has just recently been unearthed.In the considerationof antibodyspecificity, andB-cell activation, althoughit appearsthat there maybe increasingspecialization manifested by a large variety of B-cell subpopulations, there is nodoubtthat each member of the diverse array of B cells bears recognitionspecificity for a determinantregion on the nominalantigen. This statementcannotbe madeabout the T cell for twoseparate reasons: (a) the epitope whichis recognizedmaybe intricately associated with MHC moleculeand both moieties are simultaneouslyrecognized; (b) some interactions maybe restricted by idiotypic elementsin conjunctionwith MHC or antigenic moieties. In fact, wehave mentionedthat the feature whiehmakesT recognition so varied is the large numberof regulatory T cells each of whichmust circumscribeits interactions with surroundingcells. Todetermineits partner unambiguously, principles of simplicity can be invokedto assure coordinated control. Onesuch principle mightbe that recognitionby T cells and specific targeting by T cells can be accomplished by havingeach cell bind to a combinationof elements: an epitope plus idiotope, an epitope plus histotope, or an idiotope plus histotope. A second motif whichseemsto permeatethe relationships amongT ceils is that antigen-specific T cells throughoutthe system, whichthus bear idiotypic elementsin their receptors, will be activated by other T cells whichrecognizethese idiotypie elements.Aconsequence of this schemeis an alternation of antigen-specific, MHC-restricted cells with idiotype-specific T cells in sequential pathways.

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Therules of activation at the molecularlevel and details about "second messenger"function are boundto be the samefor manytypes of cells, and wehavenot consideredthemin this account. Wehaverestricted ourselves to attemptingto assemblethe informationaboutthe state in whichepitopes, idiotopes, and histotopes confront the T-cell machinery.If there is a rule about this confrontationwhichcan be discerned, it maybe that the choice of epitopewhichwill be available for a T-cell subpopulationwill dependon an "antigen-recognitionepitope," an agretope, whicha presenting cell (or entity such as a soluble factor) can recognize on the antigen. Thus, Hagretopes on the Ag will be recognized by Class IIIa molecules on APC presenting to Th, C-agretopeswill interact with an appropriate group or receptor on the Class I moleculefoundon the surface of cell types recognized by Tc, and S-agretopeswill associate with desetopes on MHC molecttles whichexist on APCof the type whichpresentsantigen to Ts. Thelist could be extended. Therefore, the basis for determinantsdection wouldreside in still unknownchemicalprinciples uniting desetopewith agretope: desetopeson the MHC molecules used for triggering each important T-cell subpopulation would seek out their complementaryagretopes based on a unique but non-clonal chemistry. An exampleof such a principle might be that the desetopesfor H-agretopesrecognizea particular groupingof hydrophobic aminoacids only, whereas the desetopes for S-agretopes combinewith hydrophilieaminoacids. This alignmentwouldhave utility in explaining two vexing problems. First, howcan the system maintain self-non-self discrimination in the face of the enormousexcess of self H-agretopes? Probablythese H-agretopesremain unexposeduntil antigenic fragmentation and "processing."Prior T-cell recognitionmaybe required to trigger processing: such maybe the role of augmentingor amplifier T calls which wouldthen haveto be able to recognizesuperficial residues on the antigen. Second,since S-agretopesare hydrophilicandexternal on the antigen, the nearbyepitope will be comparablysituated: this wouldexplain whyTs can often combinewith free antigen. Oneof the critical questionsthat can be approached experimentallyis the definition of agretopesfor particular haplotypes.Will any chemicalstructure associatedwith the agretopein the correct spatial relationship act as an epitope for T cells? This question is being experimentallyapproachedby synthetic chemistry, using as a starting point, for example,the putative agretope at the C-terminusof pigeon cytochromec, for the B10.Amouse strain. Other H-agretopeson L-tyr-ABAor lysozymecould also be used. It is important to realize that although wemaybe at the threshold of defining agretopesand synthesizing completeimmunogens with a particular agretopeas a universal carrier for a series of epitopes, difficulties may

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abound. Not all epitopes will be permissive for agretope function, as older observations seem to suggest for the small molecule, L-tyr-ABA (6). Finally, it would seem unlikely that a particular determinant area on any antigen could be designated as immunodominant for T cells in general. However, we may soon arrive at an understanding of the commonfeatures of attachment sites (agretopes) chosen by MHCelements for presentation to different T-cell subpopulations. Understanding the nature of specific epitope immunodominance for different T-cell subpopulations that might result from study of the features and constraints of desetope-agretope interaction should permit the logical manipulation of T-cell responsiveness. ACKNOWLEDGMENTS

This workwas supportedby NIHgrants CA-24442,AI-056Ot, and AI11183, and by grants IM-263and IM-323from the AmericanCancer Society. Literature Cited 1. Adorini, L, M. A. Harvey,A. Miller, gen recognition and the immunereandE. E. Sercarz. 1979.Thefine specisponse: humoraland cellular immune responses to small mono-andbifuncficity of regulatoryT cells. II. Suppressor and helper T cells are inducedby tional antigen molecules.J. Exp. Med. different regionsof henegg-whitelyso135:1228-1246 zyme(HEL)in a genetically nonrespon- 7. Andersson, J., G. M. Edelmun, G. der mousestrain. Z Exp. Med. 150: M6ller, and O. Sj~berg. 1972. Activa293-306 tion of B lymphocytesby locally con2. Adorini, L., M. Harvey, D. Rozyekacentrated concanavalinA. Eur. J. IraJackson,A. Miller, and E. E. Sercarz. munoL2:233-235 1980. Differential majorhistocompat8. Araneo,B. A., D. W.Metzger, R. L. bility complex related activationofidioYowelL and E. E. Sercarz. 1981. Positypic suppressor T cells. Z Exp. Med. tive selection of maj~histocompatibil152:521-531 ity complex restricted suppressorT cells 3. Adorini, L, M.Harvey,and E. E. Serbearingthe predominant idiotype in the carz. 1979.Thefine specificityof regulaimmuneresponse to lysozyme. tory T ceils. IV. Idiotypic complemenNatL Acad~Sci USA.78:499-503 tarity andantigen-bridginginteractions 9. Araneo,B. A., R. L. Yowell,E. E. Serin the anti-lysozyme carz. 1979.Ir genedefects mayreitect a Immunol. 9:906-909 response. Eur. Z regulatory imbalance.I. Helper T cell 4. Adorini, L., A. Miller, and E. E. Seractivity revealedin a strain whoselack carz. 1979.Thefine specificityof regulaof responseis controlledby suppression. tory ceils. I. Hen-~gg-white lysozymeZ Immunol. 123:961-7 inducedsuppressorT cells in a genet10. Bach,B. A., M. L Greene,B. Benacerically non-respondermousestrain do raf, and A. Nisonoff. 1979. Mechanisms not recognizea closely related immunoof regulation of cell mediatedimmugeneic lysozyme.J. ImmunoL 122:871nity. IV. Azobenzencarsunate(ABA) 877 specific suppressorfactor(s) bear cross5. Alkan,S. S., D. E. Nitecki, and J. W. reactive idiotype (CRI) determinants Goodman.1971. Antigen recognition the expressionof whichis linked to the and the immune response: the capacity heavy chain allotyp¢ linkage group of of L-tyrosine-azobenzenearsonateto genes. Z Exp. Med~149:1084-1098 serve as a carder for a macromolecular 11.Bach,B. A., L. Sherman,B. Benacerraf, hapten. J. Immunol.107:353-8 and M.I. Groene.1978. Mechanisms of 6. Alkan,S. S., E. B. Williams,D. E. Niregulation of cell-mediated immunity. tecki, and J. W.Goodman. 1972. AntiII. Induction and suppression of de-

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laycd type hypersensitivity to azobenzenearsonatecoupledspleen cells. J. ImmunoL121:1460-1468 12. Baxevanis,C. N., N. Ishii, Z. A. Nagy, and J. Klein. 1982.Role ofEk molecule in the generationof suppressorT cells in response to LDHe.Scand. J. ImmunoL 16:25-31 13. Baxevanis,C. N., N. Ishii, Z. A. Nagy, and J. Klein. 1982.H-2controlled suppression of T=cell responseto lactate dehydrogenaseB. J. Exp. Med. 156: 822-833 14. Benacerraf, B. 1978. A hypothesis to relate the specificity of T lymphocytes andthe activity of I region-specitieIr genes in mafrophagesagainst B lymphocytes. J. ImmunoL120:1809-1812 15. Benacerraf, B., and R. N. Germain. 1978. The immuneresponse genes of the majorRev. histocompatibility Immunol. 38:70-119 complex. 16. Benacerraf, B. and H. O. MeDevitt. 1972.Thehistocompatibilitylinked immuneresponsegenes. Science. 175:273279 17. Bevan,M.J. 1977.Killer cells reactive to altered-self antigenscan also be alloreactive. Prcn: Nat~Acad. ScL USA 74:2094-2098 18. Bevan,M.J. 1975. Themajorhistocompatibility complexdeterminessusceptibility to cytotoxic T cells directed against minorhistocompatibilityantigens. J. Exp. Meal142:1349-1364 19. Binz, H., and H. Wigzell.1977. Antigen-binding, idiotypic T-lymphocyte receptors. Contemp.Top. ImmunobioL 7:113-177 20. Black, S. J., G. J. Hammerling,C. Berek, K. Rajewsky,and K. Eichmann. 1976.Idiotypic analysis of lymphocytes in vitro. I. Specificityandheterogeneity of B and T lymphocytesreactive with anti-idiotypic antibody. J. Exp. Med. 143:846-859 21. Blanden,R. V., and G. L. Ada.1978. A dual recognition modelof cytotoxic T cells basedon thymicselection of precursorswithlowaffinity for self-H-2antigens. ScandJ.. Immuno£7:181-190 22. Blanden, R. V., and U. Kees. 1978. Cytotoxic T-cell responses showmore restricted specificity for self than for non-self H-2Dcodedantigens. J. Exp. Meal 147:1661-70 23. Bona,C. A., E. Heber-Katz,and W.E. Paul. 1981.Idiotype-anti-idiotyperegulation. I. Immunizationwith a levanbinding myelomaprotein leads to the appearanceof auto-anti-(anti-idiotype)

antibodiesandto the activationof silent clones. J. Exp. Med.153:951-967 24. Bona,C., P. K. A. Mongini,K. E. Stein, and W.E. Paul. 1980. Anti-immunogiobulin antibodies. I. Expressionof cross-reactive idiotypes and Ir gene control of the responseto IgG2aof the b. allotype. J. Exp. Med. 151:13341348 25. Bona,C., and W.E. Patti. 1979.Cellular basis of regulation of expressionof idiotype. I. T-suppressorceils specific for MOPC 460 idiotype regulate the expressionof cells secreting anti-TNPantibodies bearing 460 idiotype. Y. Exp. Med~149:592-600 26. Bottomly, K. 1981. Activation of the idiotypic network: environmentaland regulatory influences. In Immunoglobulin Idiotypes,ed. C. Janeway,E. E. Sercarz, and H. Wigzell, p. 517-532New York: Academic 27. Bottomly,K., and D. E. Mosier.1981. Antigenspecific helper T cells required for dominantidiotyp¢ expression ate not H-2restricted. J. Exp. Med.154: 411-421 28. Britz, J. S., P. W.Askenase, W.Ptak, R. M.Steinman,and R. K. Gershon.1982. Specialized antigen-presenting cells. Splenicdendritic cells andperitoneal. exudatecells inducedby mycobaeteria activate effeetor T cells that are resistant to suppression.J. Exp. Med.155: 1344-1356 29. Buehmilller,T., and G. Corradin.1982. Lymphocyte specificity to protein antigens. V. Conformationaldependenceof activation of cytochromeC-specific T cells. Eur. J. Immunol.12:412-416 30. Burakoff, S. J., R. Finberg, L. Glimcher, F. Lemonnier,B. Benacerraf, and H. Cantor.1978. Thebiologic significance of alloreaetivity. TheontogenyofT-cellsets specificfor alloantigens moditiedself antigens. J. Exp. Med.or148:1414-1422 31. Cantor, H. and R. K. Gershon. 1979. RegulatoryT cell circuitry. Fed. Proc. 38:2058-2062 32. Cazenave,P.-A., J. M. Cavaillon, and C. Bona.1977. Idiotypie determinants on rabbit B- and T-derived lymphocytes, lmmunoLRev. 34:34-49 33. Cazenave,P.-A. 1978.L’idiotypie eomparee des anticorps qui, damle serum d’un lapin immunise enntre la serumalbuminehumaine, sont diriges contre des regions differentes de cet antigen. FEBSLett. 31:348-354 34. Chesnut, R. W., R. O. Endres, and H. M. Grey. 1980. Antigenrecognition by

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Gershon, andH. Cantor. 1978.lmT cells andBcells: reen~tionof crossreactivity betweennative anddenatured munoregulatory circuits among T-cell formsof globular antigens. Clit~ Imo sets. I.T-helper cells induce other T-cell munoLImmunopathol.15:397-408 sets to exert feedback inhibition../. Exp. Med. 147:1106--1115 35. Chesunt,R. W., and H. M.Grey. 1981. Studies on the capacity of B cells to 46.Eardley, D.D.,F.W.Shen, H.Cantor serveas antigen.presenting cells..l.. ImandR.K.Gershon. 1979. Genetic conmuno~126:1075-1079 trolofimmunoregulatory circuits. 36. Ciavarra, R., and J. Forman.1982HGenes linked to theIglocus govern communication between regulatory T2L-restrictedrecognitionof viral antigemin the H-2d haplotype,anti-vesicucell sets. :..Exp. Med. 150:44-51 lar stomatitisviruscytotoxicT cells are 47.Eichmaun, K. 1978.Expression and restricted solely by H-2L.J. Exp. Med. function ofidiotypes onlymphocytes. 156:778-790 .dd~,. Immunol. 26:196 37. Claflin, J. L., and J. M.Davie.1974. 48. Eichmann,K., A. Coutinho, and F. Clonal nature of the immune response Melchers.1977.Absolutefrequenciesof to phosphorylcholine.IV. Idioitypic lipopolysaccharide-reactive B cell prouniformityof bindingsite associatedanducing ASAidiotype in unprimed, tigeulc determinantsamongmouseantistreptococcal A carbohydrate-primed, phosphorylcholineantibodies. J. Exp. anti-A5Aidiotype-seusitized and antiA5Aidietype suppressedA/J mice. ~.. Med. 140:673-86 38. Cohn,M., anti R. E. Epstein. 1978.TExp. Med. 146:1436-1449 cell inhibition of humoralresponsive- 49. Endres, R. O., and H. M. Grey. 1980. ness. Theoryon the role of restrictive Antigenrecognitionby T ceils. I. Suprecognitionin immune regulation. CeIL pressorT cells fail to recognizecrossreactivity betweennative anddenatured Immuno~39:125-153 39. Corradin,G. and S. Card.1983.In Proovalbumin. Y. Irnmuno~ 125:1515tein Conformation as an Immunological 1520 Signal, ed. by F. Celada,¥. $chumaker 50. Erb, P., M. Feldmann,and N. Hogg. 1975. The role of macrophagesin the and E. Sercarz. LondonPlenum: In press generationof T helperceils. III. Inttuence of macrophage-derived factors in 40. Corradin,G., and J. M.Chiller. 1979. Lymphocyte specificity to protein antihelper cell induction.Eur. Y. Immunol. geus.II. FinespecificityofT-cellactiva5:759-766 51. Erb, P., M. Feldmann,and N. Hogg. tion with cytochromec and derived peptides as antigenic probes. J. Exp. 1976. Roleof macrophages in the generMed. 149:436-447 ation of T-helpercells. IV. Nature of 41. Corradin,G., and I. M.Chiller. 19gl. geneticallyrelated factors derivedfrom Lymphocyte specificity to protein antimacrophages incubatedwith soluble angens. III. Capacity of low responder tigens. Fur. J, ImmunoL 6:365-372 miceto beef cytochrome c to respondto 52. Eshhar, Z., R. N. Apte, I. I~wy, Y. aJ. peptide fragment of the moleeule.Fur. Ben-Nerah,D. Givol, and E. Mozes. lmmunoL 11:115-9 1980. T-cell hybridomasbearing heavy 42. Cosenza,H., M. H. Julius, and A. A. chain variable region determinantsproAugustin. 1977. Idiotypes asvariable dttcing (T,G)-A-D-specifichelper facregionmarkers: analogies between tor. Nature286:270--272 receptors onphosphorylcholine-specific 53. Falkoff, R., and J. Kettman. 1972. T andB lymphocytes. ImraunoL Re~,. Differential stimulation of precursor 34:3-33 cells and carrier-specific thymus43.Cunningham, A.J.andE.E.Sercarz. derivedcell activity in the in vimre1971.Theasynchronous development sponse erythrocytes in nfice. to I. heterologous Immuno£108:54-58 ofimmunological memory inbelper (T) and precursor (B)cell lines. Fur. ,i.. Im- 54. Feldmann, M., and S. Kontiainen. munol. 1:413-418 1976.Suppressor cell inductionin vitro. 44.Dzierzak, E.A.andC.A.Janeway, Jr. II. Cellular requirementsof suppressor 1981. Expression ofanidiotype (Id-460) cell induction. Eur. J. Immuno~ 6:302during invivo anti-dinitrophenyl anti305 body responses III. Detection ofId-460 55. Gearhart, P. J., N. D. Johnson, R. innormal serum that does notbind diniDouglas,and L. Hood.1981. IgGantitrophenyl. : Exp.Med.154:1442-1454 bodies to phosphorylcholineexhibit 45.Eardley, D.D.,J.Hugenberger, L. morediversity than their IgMcounterMcVay-Boudrean, F. W.Shen,R. K. parts. Nature 291:29-34

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andsuppressorT cell factor. A miaimal 174. Unanue,E. R. 1978. Theregulation of modelof functional antigen receptor of lymphocyte function by the macroT ceils. Mol. lmmunoL17:867-875 phage. Immunol.Rev. 40:227-255 164. Takaoki,M., M. S. Sy, B. Whitaker,J. 175. Urbain, J., C. Wuiimart, and P.-A. Nepom,R. Finberg, R. N. Germaln,A. Cazenave.1981. Idiotypic regulation in Nisonoif, B. Benacerraf, and M. I. immunenetworks. Contemp.Top. MoGreene. 1982. Biologicactivity of an lec. ImmunoL8:113-48 idiotype-beadngsuppressorT cell fac- 176. yon Boehmer,H., W.Haas, and N. K. tor producedby a long-termT cell hyJeme. 1978. Majorhistocompatibility bridoma. J. ImmunoL128:49-53 complex-linkedimmune-responsiveness 165.Taniguchi,M., andJ. F. A. P. Miller. is acquired b.y lymphoctyesof low1977.Enrichment of specific suppressor responder n~ce differentiating in T cells andcharacterizationof their surthymusof high responder mice. Proc. face markers. Z Exp. Med. 146:1450Nat£ Acad. ScL USA75:2439-2442 1454 177. Walker,E., N. C. Warner,R. Chesnut, 166. Thomas,D. W., M. D. Hoffman,and G. J. Kappler,and P. Marrack.1982. AntiD. Wilner.1982. T lymphocyterecognigen-specific,I region-restrictedinteraction of pcptideantigens:evidencefavortions in vitro betwceen tumorceil lines ing the formationof ncoantigenicdeterand T cell hybridomas. Z ImmunoL minants. J. Exp. Med.156:289-293 128:2164-2169 167. Thomas,D. W., K.-H. Hsieh, J. L. 178. Weyand,C., J. Goronzy, and G. J. Schanstcr, M.S, Mudd,and G, D. WilHammerling. 1981. Recognition of ncr. 1980. Nature ofT lymphocyterecpolymorphic H-2 domains by T lymognitionof macrophage-associated antiphocytes.I. Functionalrole of different gcns. V.Contributionof individualpepH-2domainsfor the generation of altide residues of humanfibrinopeptideB loreactive eytotoxie T lymphoeytesand to T lymphocyteresponses. J. Exp. determinationof precursorfrequencies. Med. 152:620--632 J. Exp. Med.54:1717-1731 168. Thomas, D. W., K.-H. Hsieh, J. L. 179. Weyand,C., G. J. Hammeding, and J. Schauster,andG. D. Wilncr.1981.Fine Goronzy.1981. Recognitionof H-2dospecificity of genetic regulation of mainsby cytotoxic T lymphocytes.Naguinea pig T lymphocytcresponses to ture 292:627-629 angiotensinII and related peptides. J. 180. Wilder, M., C. Demeur,G. Dewasme, Exp. Mea~153:583-594 and J. Urbain. 1980. Immunoregulatory 169. Thomas, W. R., F. I. Smith, I. D. role of maternalidiotypes: Ontogeny of Walkerand I. F. A. P. Miller. 1981. immunenetworks. J. Exp, Met~ 152: Contactsensitivity to azobenzenarson1024-1035 ate andits inhibitionafter interactionof sensitizedcells withantigen-conjugated 181. Woodland,R., and H. Cantor. 1978. Idiotypespecific T helper cells are recells. J.. Exp. Med.153:1124-1137 quired to induce idiotyp¢ positive B 170. Tokuhisa,T., and M.Taniguchi.1982. memory cells 8:600-606 to secrete antibody.Eur. Twodistinct allotypic determinantson J. Immuno£ the antigen specific suppressorandenreachancingT cell factors that are encoded 182. Wilson, D. B. 1974. Immunologic tivity to majorhistocompatibilityalby genes linked to the immunoglobulln loantigens: HARC, effector cells and heavy chain locus. J. Exp. Med. the problemof memory.In Progressin 155:126-139 Immunology, Vol. 2, ed. L Brentand J. 171. Tokuhisa,T., Y. Komatsu,Y. Uchida, Holborow,p. 145-156. Amsterdam:Eland M. Taniguchi. 1982. Monoclonal sevier alloantibodiesspecific for the constant 183. Wysoeki,L. J., and V. L. Sato. 1981. region of T cell antigen receptors. J. Thestrain A anti-p-azophenylarsonate Exp. Med. 156:888-897 majorcross-reactiveidiotype familyin172. Tse, H. Y., J. J. Mond,andW.E. Paul. eludes memberswith no reactivity to1981.T lymj[~_hocyte-dcpcndcnt B-lymwardp-azophenylarsonate. Eur. Z Imphocyteprottt~rative responseto antiCn.I. Geneticrestriction of the stimumunoL11:832-839 tion of B lymphocyte proliferation. J. 184. Yamanehi,K., N. Chao, D. B. Murphy, Exp. Med. 153:871-82 and R. K. Gershon. 1981. Molecular 173. Turkin, D., and E. E. Sercarz. 1977. compositioa of an antigenspecific, Ly-1 Keyantigenic determinantsin the regusuppressor inducer factor. Onemolelation of the immuneresponse. Proc. culebindsantigenandis I-J-, anotheris Nat. Sci. USA74:3984-3987 I-J +, doesnot bind antigen,andimparts

~a

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an IgI-I-variabl¢regionlinkedrestricdifferentiation of"H-2self-recognition" tion. d. ExtT. Med.155:655-665 by T cells: evidencefor dual recognition? Z Exp. MecL147:882-896 185. Yowell,R. L., B. A. Araneo,A. Miller, and E. E. Serearz. 1979.Amputation of 188. Zinkemagel,R. M., and P. C. Doherty. a suppressor determinant on lysozyme 1975. H-2 compatibility requirement reveals underlyingT cell reactivity to for T-cell mediatedlysis of target cells other determinants. Nature279:70-71 infected with lymphocytic chori186. Zinkemagel,R. M., and P. C. Doherty. omeningitis virus: different eytotoxicT1979.MHC-restdeted cytotoxic T cells: cell specificities are associated with structures coded from H-2Kor H-2D. Studies on the biological role of polyZ Exp. Med. 141:1427-1436 morphicmajortransplantation antigens determiningT-cell restriction-specifi189. Zinkernagel,R. M., and P. C. Doherty. city, ftmetion,andresponsiveness.Adv. 1977. Majortransplantation antigens, ImmunoL27:51-177 virus and specilicity of surveillance T cells. The"altered self" hypothesis. 187. Zinkernagel,R. M., G. N. Callahan,A. Althage,S. Cooper,P. A. Klein, and J. Comtemp.Top. Immunbiol. 7:179 Klein. 1978. On the thymus in the

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IMMUNOGLOBULIN GENES

Annu. Rev. Immunol. 1983.1:499-528. Downloaded from arjournals.annualreviews.org by HINARI on 08/27/07. For personal use only.

Tasuku Honjo Department of Genetics,OsakaUniversityMedicalSchool,Kita-kuOsaka, 530 Japan

INTRODUCTION Theimmunoglobulin gene systemis comprisedof three separate gene loci of L~, Lx, andH(light andheavy)chaingenes,each containingvariable (V) and constant (C) genes. TheLK(157), Lx (36), and H (35) chain genes located on mousechromosomes6, 16, and 12, respectively [on human chromosomes 2 (95), 22 (104), and 14 (48, 84), respectively)]. Molecularcloning and structural characterization of the immunoglobulin gene haveshownthat DNA rearrangementplays essential roles in the somaticamplificationof the immunoglobulin diversity that manifestsin two aspects: the ability to bind an enormous numberof antigens, andthe ability to trigger a variety of immunologic reactions. Suchstudies madelongdebating immunologistsmoreor less unanimouson howthe immunoglobulin diversity is generated. Structural analysesof the immunoglobulin gene havealso given newinsights into the evolutionaryprocessthat contributes to the creation of the antibodydiversity. It is nowclear that organization and structure of the immunoglobulin gene per se incorporate the moleculardrive that increases the immunoglobulin diversity through the somatic and evolutionary processes. The basic principles that underlie the genetic mechanism to create the antibody diversity are summarizedas flexible, random,somatic DNArearrangementsaccompaniedby deletion and evolutionary mechanisms dependingon the frequent recombinationand rapid drift. Theantibodies thus created contain not only those that are specific to current antigens, but also those that appearuseless yet mightbe reactive to antigens to be encounteredin future. Thus, the immunoglobulin gene system has Prometheanforesight (116), preparing for unknownantigens by the flexible and randomDNA rearrangement. This system has strong advantages over the fixed rigid geneticsystemthat determinesall the specifities andphysiologicalfunctions a priori in the genome, becausethe latter makesit difficult for the organism to adapt to the drastic changeof the environment. 499 0732-0582/83/0410-0499502.00

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In this review I focus on the organizational and structural features of the cloned immunoglobulingenes and on the implications derived from these studies. Transcription of the immunoglobulingene is not discussed in this review. The encyclopedic citation of references is not the object of this reviewin view of the limited space for such a broad subject. Unlessspecified, described information was derived from studies on mousegenes. Several aspects of this gene were reviewed previously (33, 40, 52, 64, 97).

VARIABLE REGION GENES Structure and Organization THE VARIABLE

REGION

IS

ENCODED BY TWO OR THREE

SEPARATE

DNASEGMENTS The V region was defined originally by the comparison of amino acid sequences of immunoglobulins. The length of the L-chain V region is rather constant (usually aminoterminal 107 residues) whereasthat of the H-chain V region is variable, ranging from 107 to 132 residues (74). Cloning and characterization of the Vx2region gene first demonstratedthat the Vx region is encoded by two separate DNAsegments, i.e. the Vx and Jx segments, as shownin Figure 1 (1 l, 160). The Vxsegment has two exons separated by a 93-bp intron; the 5’ exon codes for a major portion of the leader sequence (residues -19 to -5), which is eventually cleaved off, and the other exon codes for the remaining portion of the leader sequence and the major portion of the V region (residues -4 to 97). The rest of the region (residues 98-110) is encoded by the Jx segment, which is located

Vx2 II

dx2

C7t2

d~4

Cx4

~

Vta V~z ..... V~n

O~

---II-//-tl-//----#l-",;’ I IIII

C~

-"

Figure 1 Organization of immunoglobulin L-chain genes. Each exon is shown by a wide rectangle.

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close to the Cx gene. As described bdow, the expressed V gene is created by the fusion of the germline V and J segments. To distinguish the germline and the complete rearranged forms of the V gene, I refer to the germline form as the V segment throughout this review. The V~ region is also encoded by the V~ and JK segments with lengths similar to the Vx and Jx segments, respectively (102, 137). By contrast, the Vn region is encoded by the three separate DNAsegments, namely, the VH, D, and JH segments (38). Consequently, the complete active VHgene is generated by two somatic recombinations, VH-Dand D-Jn joinings. The D segment encodes primarily the CDR3and varies considerably in sequence as well as in length (88, 89). STRUCTUREAND ORGANIZATIONOF THE V SEGMENTThere are only two V~ segments (Vxl and Vx2) in the mousegenome, whereas a large number of V), segments are expected to be in the human genome. The V~l segment is genetically linked to the C~3and C~,l genes, and the V~2 segmentis linked to the C),2 and Cx4 genes in the mousegenome(14, 105). The VT, segments have not been physically linked to any of the C~genes. The germline V~ segment has the structure essentially similar to the Vx segment. Multiple V~ segments are cloned from mouseand man (9, 113, 142). One of them is a pseudogenecontaining substitutions, deletions, and insertions (9). About 31- to 43-kb DNAfragments carrying two V~ segments were cloned in a cosmid vector, but the distance between two adjacent VKsegments is not known precisely (21). Two human genomic fragments carrying two V~ segmentswith 12.5- to 15-kb spacers were cloned (126). Zeelon et al (78) measuredthe proportion of a distinct V~sequencein the total V~ sequences expressed in mousespleen and estimated the numberof the V~group as 280, assumingthat all the groups are equally expressed. The total numberof the V~ segment is simply calculated to be about 2000 on the assumption that each group has seven members. Cory et al (30) measured numbers of restriction fragments hybridized with 10 probes and concluded that the total numberof the V~segments is between 90 and 320. Bentley &Rabbitts (9) estimated that there are only 15-20 V~segments the humangenome on the basis of the number of hybridized bands with V~probes of three different subgroups. In view of the presence of pseudogenes and the absence of dear knowledge about the extent of cross-hybridization, these values can not be taken seriously. In addition, the ambiguityabout the definition of the V group has to be taken into account. Althoughthe subgroup was originally defined by the aminoacid sequence similarity of the V region framework,analysis at the DNAlevel is consistent with the view that each V subgroup is deter-

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minedby a single germline V segment. The definition of.the group was somewhat arbitrary. For example,VKregions varying at three or moreof the amino-terminal 23 aminoacids wereclassified into different VKgroups. Thedefinition of the group at the DNA level is also arbitrary. Several investigators considerthat all the V segmentscross-hybridizingto a probe belong to the same group. However,cross-hybridization dependson the conditions of hybridization and washing,especially temperatureand salt concentration.Evolutionallyclosely related V segmentsshoudforma group of Vsegments.In somecases, however,there wouldbe no discrete boundary betweendifferent groups because extensive recombinatodalhomogenization of Vsegmentshas taken place, as is discussedlater. TheVnsegmentis also split into twoexonsby an intron at the identical position to the VLsegment.The lengths of two exons are also similar to those of the VLsegments.Several Vii segmentsare physically linked (31, 76, 80). The distance betweentwo adjacent Vnsegmentsis 12-15 kb. The averageVII-VI-Idistanceappearsto be longer(at least 25 kb) thanthe above value, becauseL. Hoodand his associates (personal communication) found 21 VH segmentsin cloned germline DNA fragments that total 492 kb. The total numberof the Vii segmentsis not knowneither. Probablyit wouldfall somewhere between100 and 1000. Kemp et al (81) estimatedthat each group contains 15 members by Southernblot hybridization using three probes of different groups. Assuming that the total numberof VrI groups is about10, they calculatedthat the total number of the Vii segmentsis 160. Three VHsegmentswere mappedin the order Vn76(inulin binding), (phosphorylcholinebinding), and Vm/A8 segmentsby deletion in myeloma cells expressingdifferent Vnsegments(81). Themembersof the sameVii grouptend to cluster (31, 76, 80, 131). However,Hoodand his associates (personal communication) were unable to link the four Vnmembersof the VT~5group even by cloning the germlinc DNA segmentsof 500 kb. It is possible that somemembers of the groupweretranslocated elsewherein the Vncluster. It is importantto stress that members of the sameVngroupdo not always encodethe sameantigen specificity. For example,only one of the four members of the Vx~5groupis used for anti-phosphorylcholinc antibody, the secondis usedfor anti-hemagglutininof influenza virus, the third is used for unknownspecificity, and the fourth is a pseudogene(31; L. Hood, personal communication). STRUCTURE AND ORGANIZATION OF THE J SEGMENT The JK segment is located 1.2-1.3 kb 5’ to each C~gene of mouse(Figure 1). From

the genetic linkage betweenthe Vxand Cxgenes, the Vxl segmentis able to associate with either JK3 or Jkl segment,whereasthe V~2segmentis to

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IMMUNOGLOBULIN GENES

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be joined with either the Jk2 or the Jh4 segment. However,the VX2 was shownto be associated with J)d and Cx3in a hybridoma,suggesting that the Vx2Jx2 Cx2Jx4 Cx4duster is 5’ to the Vx~Jx3 CX3 JM C~,l duster (43). TheJx4 segmentis probably inactive becauseof a 2-bp deletion in the splicing signal. Anotherpseudo-Jsegmentwasidentified betweenthe Jh3 segmentand the Cx3gene (14, 105). There are four JK segments and a pseudo-JK segment in the mouse genome.TheJr segmentsare clustered 2.6 kb 5’ to the CKgene(102, 137). Thefour J~ segmentsare presumedto be capableof joining with any of the V~segmentsfrom amino acid sequence analyses. The humanJ~ cluster contains active five J segmentsabout 2.5 kb 5’ to the C~gene (61). The absenceof the mouse Jr3 equivalentsequencewasexplainedby a duplication event in the humanJ~ duster by unequalcrossing over. In the Hchaingenefamilythere is only oneset of the Jn cluster for eight or nine Clt genes. Thereare four JH andone pseudo-JI.I segmentsin mouse and six Jn and three pseudo-Jnsegmentsin man(130, 139). Althoughthe length of the mouse JL segmentis constant (13 residues), the Jn segments of mouseand manhave varied lengths ranging from 16 to 21 residues. AND ORGANIZATION OF THE D SEGMENT The D segmentis characterizedbyits variability in sequenceas well as in length (88, 89). Theshortest germlineDsegmentidentified so far encodessix amino acid residues, and the longest one encodes17 residues in mouse.At least someDsegmentsare arranged in a relatively small region of the genome. Clusters of 11 and 4 Dsegmentswerefound in mouseand man,respectively (88, 150). Butnot all the aminoacid sequencesof the Hchainsare explained by knownD segments. Worsethan that, the four humanD segments reported are not found in knowncomplementarity-determiningregion (CDR)3sequences. It is possible that moreunknown germline D segments are present in the mouseand humangenomes.Kurosawa& Tonegawa(88) proposedthe D-Djoining as a mechanism to create unexplainedexpressed D region sequences.However,there is not a single case in whichthe D-D joining can explain the knownD region without the point mutation, insertion, or deletion. Thelocation of the D segmentrelative to the Cnand Vnis not known except for one Dsegmentlocated immediately5’ to the JIt segment(138). Evolutionaryconsideration led to a proposal that the D segmentdusters maybe dispersed amongVr~ dusters (65). STRUCTURE

V-(D)-J Recombination Structural comparisonof the germlineand expressedV genes clearly demonstrated that a completeV geneis created by recombinationaljoining of V, (D), and J segments.This recombinationalreaction has three important

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characteristics. First, the combination between different DNAsegments seems to be randombecause the same Vr~segmentsare able to associate with all the Jn segments in the anti-phosphorylcholine antibody H-chain of mouse(53). Secondly, the recombination reaction has flexibility at the joining nucleotide in spite of the strong requirement for the accurate inframe joining. Thirdly, the recombination is accompaniedby deletion. RECOMBINATIONAL MECHANISM Nucleotide sequence determination of the germline V and J segments and their flanking regions has revealed that conserved inverted repeat sequences are located immediately3’ and 5’ to the V and J segments, respectively (38, 102, 137, 139), The D segment is flanked by the similar sequences at both sides (89, 138). The conserved sequence is comprised of a nonomerand a heptamer separated by 12+1- or 23+l-bp essentially random nucleotides. Sequences of the nonomers and heptamers are not identical but are conserved enough to form stable complementary base pairs between the combining pair of segments, as shown in Figure 2. The nonomerand heptamer sequences are, therefore, considered to be at least a part of the recognition signal for putative V-(D)-J recombinase(s). The recognition sequence of the VKand JK segments have 23+l-bp and 12+l-bp spacers, respectively. By contrast, the nonomerand heptamer of the Vn and Jn segments are separated by 23+-l-bp spacers. The D segment has a 12+ 1-bp spacer on each side. Thus, the paired recognition signals have different lengths of the spacer sequences, which is called 12/23-bp spacer rule as originally proposed by Early et al (38). Since one turn of the DNA helix requires about 10.4 bp (168), the conserved spacing of 12 or 23 corresponds to either one or two turns of the helix. The two blocks of conserved nucleotides (nonomer and heptamer) are thus located on the 12

22

w,

A

B

C

D

C

D

12 A

B

22 A

B

C

D

A

B

C

O

Figure 2 Putative recombinational signal for V-(D)-J recombination. Solid rectangles show nonomer and heptamer recognition signals. The pair A and B form complementarybase pairs with C and D. Consensus sequences are A, CACAGTG; B, ACAAAAACC;C, GGTTTTTOT; D, CACTGTG.Numbers indicate average spacer nucleotides.

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sameside of the helix as if they formeda continuousstretch of 16 conserved nucleotides(38, 139). The intervening DNA segmentbetween the V and J segmentswas shown to be deleted in myelomas (27, 137, 145) in the samemanneras in the chain C gene rearrangement(66). Thedeletion is consistent with a stemloop structural modelbasedon the inverted repeat structure (102, 137). alternative modelto explain deletion is a sister chromatidexchangemodel (165) as originally proposedfor the H-chainC-genedeletion (115). modelwas proposedto account for the fact that DNA segments5’ to the JK segmentwere retained in somemyelomas,both of whoseJK segments were rearranged (156, 165). Most(76%)of the region upstreamof J~ retained in the populationof the C~-expressinglymphocytes,eventhough 68%of the J~ loci are rearranged. An inversion model was recently proposedto explain the unusual presenceof the upstreamregion of the J segment(see below). TheV-(D)-Jjoining, in general,is expectedto be preciseat the nucleotide level. Otherwise,the recombinationwouldoften result in the frame-shift mutation. However,the aminoacids at position 96 of somemyeloma kappa chains differ from that encodedin the correspondinggermline DNA segment(102, 137). The V-J joining seemsto have flexibility in the exact nucleotideposition of the recombination as long as it is in phasewith the codingsequence.Thefour possiblealternative sites of the in-framerecombination are shownin Table1. This flexibility providesfurther diversity at junctions of germline DNAsegments, which are located in the CDR. In case of VHgenesthere are manyinsertions or deletions at junctions with Dsegments.Theseinserted portions, termedN regions, wereproposed to be ascribed to the activity of terminal nucleotide transferase during YH’D-JH recombination(1). ABERRANT REARRANGEMENTS The flexibility at the V-J joining costs the generationof missensegenesto the organism.Theaberrant V-J joining wasreported in the kappaas well as lambdalocus (5, 10, 15, 26, 69, 90, 103,167).Eitherdeletionor insertion of a fewbasesresultedin out-of-~phase J sequences.In manyr-producingmyelomas two alleles of kappagenesare rearranged, one of whichis usually aberrantly rearranged. A cell line of MPC11 myelomaproduces an unusual fragment of the kappa chain in addition to a normal kappa chain. Aminoacid sequence determinationson an in vitro synthesized fragmentprecursor showedthat it consisted of a hydrophobicleader sequencejoined directly to the CK region and that the sequencecorrespondingto the Vregion, wascompletely lacking (19, 135). The gene encodingthis fragmentwasanalyzed, which

Annual Reviews 506

HONJO aTable 1 Flexibility

at the recombination site

Germline genes

Codon generated ¯ - - AGT-TCT-CCT-CCCACAGTG ......

V~41

Annu. Rev. Immunol. 1983.1:499-528. Downloaded from arjournals.annualreviews.org by HINARI on 08/27/07. For personal use only.

J

1

l I I[ CACTGTGG-TGG-ACG ........

......

Vt¢41

¯ ".AGT-TCT--CCT-CCCACAGTG ......

J2

....

I Ill CAGTGTGTG-TAC-ACG.........

TGG Trp CGG Arg CCG Pro CCC Pro TAC Tyr CAC His CCC Pro

Trc CTC Leu

J4

¯ .. o - c -cc -cccc oro...... I[il ...... CACTG.GA-TTC-ACG .........

V~41

¯ ¯ ¯ AGT-TCT--CCT-CCCACAGTG ......

CTC Leu

Vt¢41

Illl ......

CCC Pro

ccc ~ro

CACTGTGG-CTC-ACG.........

aTaken from Max et al (102). Codons are shown by hyphens. Recombination signal underlined; vertical lines indicate possible recombinationsites.

showed that it was generated by aberrant recombination of a V~ segment with the middle of the intervening sequence between the J~ segments and the Cr gene (140, 143). The resulting deletion of the Jr segment, including the splicing signals, allowed an abnormal RNAsplicing to create mRNA containing the leader and CKregion sequences. This abnormal rearrangement seems to be due to homologousrecombination between the putative recombinational signal 3’ to the V segment and the homologoussequence in the intervening sequence (IVS) between the Jr segment and the C~gene. Alt & Baltimore (1) found that the inverted Di-Jm and its 3’ flanking sequence is linked to the 5’ side of the JH3 segment in a spontaneously rearranged clone of Abelson routine leukemia virus-transformed cells. In addition, the inverted segment extends through the inverted Jn2 nonomer recognition sequence and ends abruptly at the terminus of the inverted Jn2 heptamer recognition sequence. The inverted Jm sequence is replaced by the noninverted 5’ flanking sequence of a D segmentat this point (Figure 3). Such an unusual structure has to be explained by intrachromosomal recombination because inverted joinings between sister chromatids would have resulted in one dicentric chromosomeand another lacking a centromere. The authors favor the looping-out model for deletion, and argue that the retention of the reciprocal product described above (the region upstream of J) can be explained by inversion. Lewis et al (92) provided further evidence

Annual Reviews IMMUNOGLOBULINGENES 0 2

D,

J~

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I

02 5*flonldng "

J2

Ja

Ji DI J3 I

507

J4

J4

¯

Figure 3 Inverted joining of D and J segments. Top and bottom lines indicate germline and rearranged DNAs,respectively. ~ andS, 5’ and 3’ recognition nanomer, respectively. ( and ~, 5’ and 3’ recognition heptamer, respectively. Arrows indicate direction of DNAin germline configuration. Rearranged from Alt & Baltimore (1).

to support the inversion model using a spontaneously rearranging Abelson virus-transformed cell line, which seemsto be useful in analyzing differentiation steps. SomeT lymphomas,T hybridomas, and T-cell lines were shownto have rearrangement at the Jx segment (28, 50). Most of them seem to be D-Jn rearrangement ($9), indicating that D-Jx recombination initiates at about the same time as the differentiation of T and B lymphocytes.

Somatic Mutation Studies on the murine Lxl chains produced by myelomacells provided the first evidence for somatic point mutation of the V-region gene. Aminoacid sequences of 19 Vxl chains were determined and were divided into seven groups that differed from a common sequence (V~0) by one to three residues (24, 169). Subsequent DNAsequence determination of a rearranged Vxl gene from H2020 myelomaand the germline Vxl gene demonstrated that the two sequencesdiffer at two positions in CDRsand at one in the flanking region (11). More extensive studies on V~,, V~, and VI~ genes, however, indicate that the substitution is not restricted to either the CDRor the coding region and seems to spread into IVS and flanking regions (16, 31, 54, 69, 76, 83, 123, 139, 146). But no mutations were found in C genes linked to V genes. The C genes of both ~l and ),2a chains of the $43 hybridomas are germline DNAsequences, although both V genes contain mutations (16, 141). These results are unable to settle the long argument of whether there exists a special enzymicmechanismthat preferentially introduces mutations to the V gene or whetherthe selection at the cellular level is responsible. However, there seems to be some mechanism that accumulates mutations only around the V-coding sequence and not in the C gene. The Vxl gene of MOPC315 myeloma also shows somatic mutations, although it is an aberrantly rearranged gene (69). Similarly, both of the rearranged V~genes,

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whether expressed or unexpressed, showed similar levels of somatic mutation (123). There are several expressed V genes that have no somatic mutations (144). It is not clear whether inactive germline V segments in antibody-producing cell have substitutions or not, although everybody assumes that DNArearrangement is obligatory for the introduction of somatic mutation into the V segment. Many VL and Vn genes in IgM-producing lymphocytes are germline sequence, whereas most of VLand VI~ genes in IgG- or IgA-producing lymphocytes have substitutions (16, 53). These authors claim that class switching maytrigger somatic mutation. Since the B lymphocyte is stimulated and divides extensively during or after class switching, the chance of somatic mutation is expected to be higher for non-IgM-producing lymphocytes. It is not knownwhether or not class switching is coupled with somatic mutation. Genetic Basis for Variable-Region Diversity After many years of controversy, DNAcloning succeeded in convincing immunologists unanimously on how V-region diversity is generated in a broad view. Nature seems to be generous to everybody who has proposed any mechanismin such a way that everybodyis right in one way or the other (132). Four types of mechanismsgenerate the V-region diversity. First of all, the numberof the germ-line V segment is reasonably great, probably somewhere between 100 and 1000. This is due to the evolutionary process. Secondly, the recombinational joining of the V, (D), and J segments plays a very important role in amplifying the V-region diversity. The combination of each segment is assumed to be random. Thirdly, the flexibility at the recombination sites augmentsdiversity significantly. Finally, the somatic mutation also contributes to increase the V-regiondiversity. It is diiticult, however, to assess the extent of contribution of each mechanism. The minimal possible diversity due to the three mechanismsother than somatic mutation can be calculated as follows: Vr diversity of mouse= number of V~ (100) X numberof JK (4) × flexibility of joining (2) ---- 800; diversity of mouse = number of VH(200) X number of D (20) X number of Jn (4) X flexibility of joining (2 X 2) = 64,000. Provided any V~and n can associate to form a different antigen binding activity, the possible diversity would be 5.1 X l07 (= 800 X 64,000). The basic principle for all the genetic mechanismsto amplify the antibodydiversity is the randomexpression of all the possible genetic information by the enormous flexibility inherent in the immunoglobulin gene system. Obviously, the selection of the randomly expressed information at the cellular level is a key to constructing the useful defense systemfor the

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organism [clonal selection theory (18)]. The number and sequence of segmentsare out of control at the somatic level but certainly are controlled by the balance between evolutionary selection and changes (drift, gene conversion, unequal crossing over, etc).

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Allelic Exclusion In general, only one species of the immunoglobulin is synthesized and secreted by a lymphocyte. The failure of expression of the immunoglobulin genes from more than one chromosomeis knownas allelic exclusion, the mechanismof which has been the subject of somedebate (2, 40, 124). Either a r or k light chain is produced, but not both, in a single lymphocyte (isotype exclusion). It is clear that the exclusion operates at the functional protein but not at transcription of the immunoglobulin genes, as MOPC104E produces both r and k chain mRNA’s,of which only ~ chain mRNA is translated into a stable protein (2, 75, 121). Since manylymphocytes, hybridomas, and myelomascontain at least one unrearranged Jr allele (72, 124), there must be some mechanismthat terminates further V~-J~ rearrangement on the other chromosome.In case the JK segments of both chromosomesare rearranged, one of themis usually defective (10, 15, 26). Theseobservations are consistent with a stochastic model: that the V-(D)-J recombination, which has a certain chance of failure, continues until the complete V genes are formed. The formation of the complete V gene, in turn, wouldshut off the activity of a putative recombinase. In the case of myelomaS107, however, both of the VKalleles are rearranged and transcribed, and two different kappa chains are produced (90). Only one of them is able to form a complex with the alpha chain and is secreted into the medium.The result is also explained by the stochastic model, assumingthe shut-off signal is not the appearance of the L chain in the cytoplasm but the appearance of a complete immunoglobulin on the membraneor in the cytoplasm. The H-chain genes have a higher proportion of nonproductive rearrangements in B lymphocytes as well as in hybddomasand myelomas(25, 26, 70, 114). It is not knownwhether the lower chance of successful joining of the VI~ locus is simply due to one additional recombinational event (V-D and D-J), or to the difference in the recombinatodal machineryas compared with the VL gene. Recently, Wahl& Steinberg (166) found an H-chain-binding protein that binds to free immunoglobulinH chains in pre-B cells, and proposed that the displacement of the H-chain’ binding protein by the L chain terminates the enzymesystem that catalyzes DNArearrangement at the L-chain loci. To explain exclusion of the H-chain locus they also proposed that the H

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chains are so toxic that those cells producingHchains fromboth alleles of the H-chainlocus are killed. Order of the V Gene Rearrangement During Differentiation Duringontogeny,the VHlocus is the first to be rearranged,resulting in the expression of cytoplasmic~ chains in the pre-B cell. Subsequently,the VLlOCUSundergoesthe rearrangement, and IgMmolecules are formedto serve as surface receptors. TheV~geneseemsto be rearrangedbefore the Vxgene, as the J~ geneswereeither ddetedor rearrangedin all 10 of human leukemiacell lines producing h chains (60). By contrast, the Jx genes remainedunrearrangedin eight humanB-cell leukemialines producing r chains. In mice, the hierarchyis less evident, as a nonproductive Vxrearrangementin a r-producing cell line was seen (26). The rearrangement order of the V~and Yx genes maybe regulated developmentally (60). Alternatively, the efficiency of recombination maysimplyfavor the V~gene locus. Evolution

of V-Region Genes

ORGANIZATION The $2

mieroglobulin L chain of the histoeompatibility antigen was shownto have homologousstructure with immunoglobulinL chains and was considered to be evolved from a common ancestor. The /32 microglobulingenewasrecently clonedand wasshownto be interrupted by IVSsat the aminoacid positions +3 and -t-95 (122). Thefirst intron the/32 microglobulingene corresponds to that betweenthe exons of the signal peptide and the V segment. Thelocation of the second IVSin the /32 microglobulingene is similar to the junction betweena VI. and a J~. segmentor betweena Vn and a D segment. The results not only suggest that the prototypeof the immunoglobulin geneis a split genelike V-J, but they also implythat the third exonof the/32 microglobulingenemaybe the evolutionary ancestor to both D and J segments.The V segmentmayhave evolved from the first and second exons. If so, one can assumethat a prototypeV-Jgenerepeatedduplicationeither as a set or as a separateexon. PrototypeJr~ segmentslocated within the Vr~cluster mighthave diverged into D segments, whereas prototype J segmentsclosest to the Ct, gene changedinto the present Jn segments. Fromthis evolutionary consideration, the homologousV segmentsare expected to cluster, as shownin several systems (31, 76, 81, 131). UnknownD segments maybe found dispersedin the Vri cluster. numbers of the V, D, and J segmentsand their variability contribute to the V-region diversity enormously.Thesefactors dependentirely on the evolutionary EVOLUTIONARY ORIGIN OF V-REGION DIVERSITY The

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process. Whatis the mechanismthat allows the accumulationof a large numberof variables and yet maintains the basic frameworkstructure amongthe hundredsof related genes9. This problemhas been discussed theoretically as concertedevolution(51, 154). Ohta(117) showedthat the rate of the aminoacid substitution in CDR is as high as that of the fibrinopeptide,the mostrapidly divergingprotein so far known.Thesubstitution rate in the other portion of the Vregion is one third of that in CDR,in agreementwith the DNA sequence analysis of closely related VHsegments(55). This result suggeststhat the variability in the Vsegmentis mostlydue to the accumulationof randomand neutral base changes. In other words, the V segmentis under weakest selection pressureat the proteinlevel, as the nucleotidedivergencerate at the synonymousposition of the V segmentis the sameas manyother genes(107). The homologyin the frameworkregion is maintained by domain(or segment) transfer betweendifferent loci by either geneconversionor doubleunequal crossing over, as indicated by computersimulationstudies ’ ’ (13, 153). Assuming that the V segmentdiverges by the balance of drift andrecombinational homogenization, Ohta(117) has evaluateddivergencerates of the V segmentusing a populationgenetics approach,and her calculated values fit well with observedvalues. Ohta(118) indicated that a high degree polymorphism and non-allelic diversity in the major histocompatibility complexcan be explained by a similar domaintransfer mechanism. The comparisonof the nucleotide sequencesof the closely related VH segmentsindicates that the rate of the replacementsubstitutions in CDR1 and CDR2are greater than in frameworkregions, indicating that the diversity of the hypervariableregionis generated,at least in part, through an evolutionaryprocess (55). TheVsegmentseemsto movequickly during evolution since restriction fragmentshybridizingto a VHor VKprobeare very different amonglaboratory mousestrains and wild mice (30, 77). Wu& Kabat (171) found that CDR2of a humanV segment contains nucleotides identical to a humanD segment.They proposedthat a minigene-like D segmentmaybe involved in the generation of CDRdiversity by a gene conversion mechanism.The minigene theory was originally proposedas a somatic mechanism to explain V-regiondiversity (73), and it predicted the presence of the Dsegment.Seidmanet al (142) proposed that recombinationand mismatchrepair mightbe a likely mechanismto explain extensive variability in CDR.Theyhaveshownthere is extensive homologyin the flanking regions of related V segments,whichwouldbe consistent with frequent recombinationamongV segments(23). In summary, two factors are involvedin the evolutionarydrive to amplify the V-segmentdiversity: (a) the frequent recombination(gene conversion and/or doubleunequalcrossing over) facilitated by the organizationof the

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V-segment family;and (b) very lowselection pressure at the protein level. Thesetwo factors workin opposite directions and reach the equilibrium. V-REGION GENESIN OTHER SPECIESThe rabbit Vn sequence of a cDNAclone was determined and was shownto contain cDNAsequence homologousto a humanD1 in CDR2 (12). The implication wouldbe that the similar Dsegmentis presentin the rabbit genome andis involvedin gene conversion. Rat J~ segmentswere cloned, and their structural analyses showedthat six JK segmentsseemedto be evolvedby recent two-sequential crossing-overevents as comparedwith the mouseJK cluster (20, 147). CONSTANT-REGION Structure

GENES

and Organization

ORGANIZATION OF CONSTANT-REGION GENESOnly one copy of the C~gene is represented in the mouseand humangenomes.TheC~ genes of mouseand manare located 2.1-2.3 kb 3v to the J segment(61, 102, 137). The distance between the V~segments and C~gene is unknown.The Cx locus is muchmorecomplexthan the C~ locus because there are four Cx genes in the mousegenome.Thepair of the Cx3and Cx~genes and that of the Cx2and Cx4genesare separately linked (14, 105). EachCxgenehas its ownJx segment1.2-1.3 kb upstream. TheCx4gene seemsto be a pseudogene becausethe Jh4 has 2-bp deletion in the heptamerof the recombinational signal. MultipleCa genesare also found in the humangenome(59). Six humanCxgenes were physically linked within a 30-kb region. Each Cxgeneis supposedto haveits ownJx segment.At least five other clones hybridizing with the Cxprobe were isolated, although none of themare linked to the majorCxcluster. Oneof themwasidentified as a processed gene not located on chromosome 22 (63). ThemouseCngene locus consists of eight genes (/x, 8, y3, yl, y2b, y2a, e, and a) that cluster in a 200-kbregion as shownin Figure 4 (149). Theorder of the mouseCagenes5’-Ct~-C~-C~,3-Cyl-C~,2b-C~,2a-C~-Cg-3’ is consistent with the order proposedby the deletion profile of the Cngene myelomas producingdifferent classes of the immunogloblin (66). Unlikethe CLgene, the CHgenes share one set of JH segments(148), whichprovides a genetic basis for H-chainclass switching, as discussedbelow.Themouse Cn gene locus does not contain any well-conservedpseudogene. ThehumanCa genefamily consists of at least nine genes, (/.t, 8, yl, 72, 73, 74, e, al, and or2). Thegeneral organizationof the humanCngene duster is similar to mousein that the C8geneis located several kilobases 3’ to the Ct, gene and that at least someC~, genes are linked (45, 87,

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0

50

I00

150 kiloba~es

513

~00

Figure 4 CH gene organization.

127, 159). However,the Ce-C~region is duplicated in the humangenome (100, 111). As it is not knownwhether or not all the Cy genes form one cluster, we should consider the possibility that the C~,-C,-C~region might be duplicated as a set. In contrast to the mouse CHgene duster, the human CHgene family contains at least three pseudogenes, one Cy (159) and two Ce (7, 49, 100, 112, 164). The S region of the r pseudogene i s r eplaced by c ompletely different reiterated sequences,althoughthe defect in the codingregion is not known.The C~2pseudogenehas deleted the first two exons and the Ce3 is a processed gene. SEQUENCES OF CONSTANT-REGION GENES All the mouse constant-region genes except for the Cx3 and Cx4 genes were sequenced. The C domainof the L chain is encoded by a single exon (4, 11, 15, 101, 170). As shownin Figure 5, the CH genes are composedof three (~, ~) or four (yl, "g2a, y2b, y3, ~, and bt) exons, each encoding a functional and structural unit of the H chain, namelya domainor a hinge region (22, 68, 71, 78, 120, 161, 162, 172, 173; F. Blattner, unpublisheddata). an exception, the hinge region of the C~chain is coded for by the Cmexon (162). Nucleotide sequences of humanCK(62) and Cx (59, 63) genes determined. The presence of the membrane-bound form of the immunoglobulin was suggested by analyses of the surface immunoglobulin of the B lymphocyte (79, 119, 151). These results indicate that the membrane-bound form of the immunoglobulinis larger than the secreted form. Exons encoding the hydrophobic transmembrane segment and the short intracytoplasmic segment were found about 2 kb 3’ to the exons encoding the last secreted domains of most mouse CHgenes: C~, (39, 134), Cy (133, 163, 174), C, (71), and C~ (22). All the membrane domains are encoded by two exons. The hydrophobic segment of the 26 amino acid residues is highly conserved amongall the H chains, suggesting that the membrane NUCLEOTIDE

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291

3~)6 39~ 321

291

306 A8107330 112 321 103

281

542

IZI 321 ~

LSItb I~1

L38kb 131

800

501

81

81

324 81 324 ?8 350 107 1~1~12~8181

Figure 5 Structures of CH genes. Oblique-lined, solid, dotted, and lined rectangles indicate domainexons, membraneexons, hinge exons, and 3’ untranslated regions, respectively. Numbers indicate nucleotides.

form of the immunoglobulinis anchored by a commonmembraneprotein (163, 174). Theintracytoplasmicsegmentsfall into two groups: one group 0,1, T2a, 72b, ~,3, and ¢) has a long segmentof 27 residues; the other (/~ and8) has a short two-residuesegment.If this portion has anything do with transmission of the triggering signal of the antigen-antibodyinteraction, the quality of the signal could be different betweentwo groups. Someof the humanCa genes werecompletelysequenced:~1 (46), ~2 (45), 74 (44), and¢ (49, 100, 164). S-S Recombination DELETION MODEL During differentiation of a single B lymphocyte, a given VHgeneis first expressedin combinationwith the C~geneof the same allelic chromosome, and later in the lineage of the B lymphocyte,the same VHgeneis expressedin combinationwith a different Ca gene. This phenomenonis called H-chainclass switch. Hybridization kinetic analyses in whichcDNA is used have shownthat specific Ca genes are deleted in mousemyelomas,dependingon the Cn genesexpressed(66). Theorder of the Cagenes, 5’-VHgenefamily-spacerCla-Cy3-Cyl-Cy2b-Cy2a-Ca-3’ wasconsistent with the assumptionthat the DNA segment between a Va and the Ca gene to be expressed is deleted, bringing these genesclose to each other. Deletionof the intervening DNA

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segment during H-chain class switch was confirmed by Southern blot hybridization analyses of myelomaand hybridoma DNAs,in which cloned immunoglobulingenes were used (25, 29, 70, 125, 175, 176). Such studies also support the proposed order of Co genes. Comparisonof germline and rearranged H-chain genes led to the proposal that a complete H-chain gene is formed by at least two types of recombinationalevents, as shownin Figure 6 (32, 75, 94, 158). The first type of recombinationtakes place between a given V~I segment, a D segment, and a Jn segment, completing a V-region sequence. After such recombination, referred to as V-D-Jrecombination, the V-region sequence is expressed as a # chain because the Cv is located closest to the JH segment. The second type of recombination is called S-S recombination, which takes place between two S regions located at the 5’ side of each CHgene. The initial S:S recombination always involves the Sv region as one of the pair, and the S region of another class is involved as the other partner, resulting in the switch of the expressed CHgene from C~, to another Ca gene without alteration of the V region sequence. The subsequent switch, such as one from Cy to C, or Co, will require another S-S recombination. Both types of rearrangements are accompanied by deletion of the intervening DNA segment from the chromosome. This model postulates several important structural features in the H-chain gene organization: (a) the order of genes 5’-C#-Cs-Cy3-CTI-CTzb-Cyza-Ce-Ca-3’; (b) the presence of only one set of the JH region segment at the 5’ side of the C~, gene; and (c) the presence of the S region before each Cr~ gene. Studies on the organization of the C~I gene cluster provided direct evidence for the predictions (a) and (b), as described above.

V.

D d.

~ ~ ~3 "~ T2b ~f2o

:-’-"-"-"-"-"---" -m -u -n -m S~.

Sr~ S~ S~zb S~rz,,

.u chcz,’n synthesis V D d

E ~X -m -N S, So~

~ I

~ S- S recombination

synmes/s

S.~ s~a~ ¯ Figure6 Twotypes of DNA rearrangements in H-chaingenelocus. TakenfromHonjoet ~ (6s).

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STRUCTURE OFS REGIONS The S region was originally defined as functionally responsiblefor the class switch(S-S) recombination (75). Nucleotide sequence determination of the region surrounding the S-S recombinationsite showedthat the structural basis of the S region is the tandemrepetition of related nucleotidesequences,Thenucleotidesequence of the St‘ region was shownto be represented by [(GAGCT)nGGGGT]m, wheren varies between1 and 7 (most frequently 3) and m is approximately 150 (109). Theother S regions havealso tandemrepetition of unit sequences that are different from each other but that havein common with the St‘ region the short sequences GAGCT and GGGGT (37, 77, 110). Tandem repetitive sequencesof the S regionare often deletedpartially in different strains of mice(65, 67, 96, 149)and duringcloning(109). TheS region found 1-4 kb upstreamof each codingregion except for the C8gene. The length of S region, whichvaries from about 10 kb (SrI) to about 1 (S,), rather than the sequencehomology with the St, region, is related the amountof the immunoglobulin class in mousesera (110). TheS regions are generally conserved between humanand mouse(112, 129, 158). The St‘, So and S~regions, especially, are highly homologous betweenthe two species. MECHANISM OF S-S RECOMBINATION There are two different interpretations of the S-regionstructure. Onegroup(33, 34) considersthat the S-regionsequences of different CHgenes,say St‘ andS,,, are so different that the homologousrecombination is an unlikely mechanismfor the class switch. Instead, they proposethat class-specific "switching"proteins recognize the St, and Sn regions and bind to one another, thus juxtaposingthe St‘ and S,, regions, and thereby allowinga recombinationalevent to join them.The other (77, 109) puts moreweighton the short common sequences shared amongall the S regions and considers that the recognition of the homologoussequences is the basic mechanismresponsible for the class switch, althoughthey do not excludethe presenceof specific recombinases for class switching. The nucleotide sequences surrounding the S-S recombinationsites of hybridomasand myelomashave revealed the presence of TGGG or TGAG adjacent to the recombinationsite (110). Anotherrelatively conservedsequence, YAGGTTG, was found in the vicinity of the recombination site and wasdispersed in S regions (98). The S-regionsequencecontains many Chi sequences,promotersof the generalizedrecombinationin h phage(82), but the functional significance of such a sequencein the immunoglobulin geneis yet to be seen. Recentstudies with the clonedSt‘ andSa regionshave shownthat preferential recombinationcan take place betweenthe St‘ and S,, regions in Escherichiacoli extracts (T. Kataokaet al., submittedfor publication). Somecultured cell lines switch classes in the presence or

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absence of mutagens(42, 128, 155). The mechanismseems to involve recombination between homologouschromosomes,at least in one case (136). Twoalternative mechanisms could be proposedto explain the deletion of the interveningDNA segmentassociated with the class switch. Thefirst modelpostulates that the DNA segmentis looped out from a chromosome (looping-outmodel).In this case, the S-Srecombination takes place within a single chromosome, whichcan occur at any stage of the cell cycle. The resultant daughtercells are identical, unless the looping-outoccursbetween the S andMphasesof the cell cycle. Theother modelexplains the deletion of the DNAsegment by an unequal crossing-over event between sister chromatids [sister chromatid exchange model (115)]. This mechanism brings about asymmetricsegregation, giving rise to onedaughtercell with the deletion of Ca genesandto anotherwith the duplication. Mostcompelling evidencefor the sister chromatidexchangemodelis the presence of a short S~ sequence between the S~ and Sy~ sequences upstream from the expressed y~ gene in MC101 myeloma(67, 115). Since the S region is located in the IVS, the S-S recombinationdoes not haveto be highly specific to the nucleotidesthat are joined together. Instead, the S region-mediated recombination is expectedto be efficient becausethe class switch takes place within a short period of time after encounteringan antigen. Therefore,not a single exampleof the aberrant S-S recombination results in cripplingthe functionof the gene. However, a numberof abortive S-S recombinations that take place on the unexpressed chromosome are found in myelomasand hybridomas(29, 125, 176). The CH-genedeletion on the unexpressedchromosome appears to be a secondaryreaction because an examplein whichonly an expressed chromosome underwentS-S recombination was clearly demonstrated in BKC~:15 myelomaderived from a BALB/c-C57BL F1 animal (175). The myelomaproduces the y2b chain of BALB/callotype. Using restriction site polymorphismbetween two parental chromosomes, we have shownthat the expressed BALB/c-derived chromosome has deleted the C~ and C~ genes, whereas the CHgenes of the unexpressed C57BL-derivedchromosome are retained. IgG2b-producingMPC11 myelomacarries two rearranged Cy2bgenes, one of which is the expressed gene. The unexpressed chromosomeof MPC11 myelomaunderwent an abortive S-S recombination between the Sy3 andSy2bregions, resulting in the deletionof the Cy3andCr~genes(91). IgA-producingS107 myelomaalso has two rearranged Ca genes, one of whichcorrespondsto the ct chain producedby the S107cell, whereasthe other is an aberrant recombinantin whicha fragmentof DNA,not obviously related to the immunoglobulin Ca gene, has been joined to the S~ ABERRANT RECOMBINATION IN OR AROUND S REGIONS

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region (85). A similar DNA fragment, presumablyunrelated to the immunoglobulinCa locus, was found to be linked to the S,~ regions of an unexpressedCogeneof an IgA-produeingmyeloma J558 (57). Harris et also found that the rearrangementof the DNA segmentoccurs in 15 out of 20 plasmacytomas tested, including 73, 71, y2b, y2a, and ~r producers. Mostof them,however,do not involveS regions. Harris et al (57) suggested that this non-immunoglobulin-associated rearranging DNA maybe related to the frequent translocation of chromosome 15 to chromosome12 in plasmacytomas (86). Reciprocal translocation betweenchromosomes 8 and 14 in the Burkitt lymphoma cell line (Daudi)wasshownto take place near the Vacluster (47). Role of RNASplicing in Class Switching It is knownthat manyB lymphocytescarry morethan one isotype on their surface. Themost frequent double-bearerscarry both/~ and 8 chains with the sameV region on their surface. TheC~and C8genesare not rearranged in /~+8+ lymphomacells, suggesting that the RNA-splicingmechanism rather than the S-S recombinationmaybe responsible for the simultaneous synthesis of the/~ and 8 mRNA’s (93, 108). The completedVr~ gene may be transcribed as a large mRNA precursor containing both C~, and C8 sequences. Subsequentprocessing of the RNAwouldgive rise to mRNA’s codingfor either/~ or ~ chain, both havingthe identical Vasequence.A 8-producinglymphoma has deleted the C~, gene (108), although there is typical S region sequencein the 5’ flanking region of the C8gene(110). Yaoita et al (177) analyzedthe Ca geneorganizationof the/~+e+B cells that weresorted fromspleens of Nippostrogylus-stimulated SJA/9mice and found that none of the Ca genes or their flanking regions had undergone DNA rearrangements.Theresults also suggest that .the simultaneousexpression of the C~and C, genes is mediated by an RNA-splicingmeehanlsm. Yaoita et al proposed that class switching wouldproceed by two differentiationsteps: the first step involvesthe activationof the differential splicing, and the secondinvolves the S-S recombination,as shownin Figure 7. Thesecondstep is a likely place for T cells to affect B-cell switching, probablyby selection. AY2bchain-producingvariant of an Abelsonvirustransformedcell line was also shownto retain the Ct, gene (3). These observationsare consistent with the abovemodel.Noneof the abovestudies, however,haveprovidedthe direct evidencefor the primarytranscript longer than 90 kb, whichis presumablyin scarce quantity.

Evolution of C~Genes IVS-MEDIATED DOMAINTRANSFER Extensive

nucleotide

sequence

data allowed the tracing of the evolutionary history of immunoglobulin Ca genes. Miyata et al (106) comparednucleotide sequences of the

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IMMUNOGLOBULIN GENES

~ pk:~srno

519

cell

Figure 7 Order of DNArearrangements during B-cell differentiation.

mouseC~,l and Cy2b genes and found an intriguing homologyregion containing the Cmdomainand the 5’ half of the IVSbetweenthe Cmdomain and hinge region exons. Since they comparedonly the divergence at the synonymous position, this conservationof the nucleotidesequencesis free fromselective constraint at the protein level. Theyproposedthat this homology region is not due to the selection pressure at the nucleotide sequence,but to the correction mechanism through recombinationsuch as doubleunequalcrossing over. Theyhave predicted fromthe comparisonof the aminoacid sequenc.esof the 72b and 72a chains that a similar mechanismmust haveoperated, betweenthese two genes. Subsequently,the comparison of the nucleotide sequencesof the Cy1, C3,2b, and C~,2agenes revealed, as expected, the different homologyregions dependingon the comparedpair (120, 141, 173). The homologyregions consist of domains and the adjacent IVS. Similarly, the M1exonand the adjacent intron seem to have undergoneIVS-mediateddomaintransfer between the C~,~ and C~,2bgenes (174). There are two possible mechanisms for the recombinational correction: unequalcrossing-overevents, andgeneconversion(6, 41, 152). Unfortunately,it is almostimpossibleto distinguish the two mechanisms experimentally in mammals. Another exampleof dynamicexon shuffling has been obtained from structural analysesof humanC~, genes(159). ThehumanC~,3genehas four hinge exons, one of whichis most homologousto that of the humanC~, pseudogene.Theremaining three hinge exons are most homologousto the C~,~gene. The results suggest that the humanC73gene was created by unequalcrossing over betweenthe C~,~gene and the C~, pseudogene,followedby the successive duplication of the C71-typehinge exon.

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PSEUDOGENE Unlike the mouse CL and CHgenes, the humanC gene families contain several pseudogenes.One C×pseudogene(63) and one C, pseudogene(7, 164) are processed genes that lack entire introns and movedto different chromosomes.The evolutionary origin of such genes is an intriguing problem.In viewof the fact that the introns are removedprecisely at the splicing site, the RNAintermediate seemsto be a likely process. A retro-viral, long-terminal, repeat-like structure wasfoundadjacent to the processedC~pseudogene,suggestingthat retrovirus might have been involved in the creation of the processed gene (164). DIVERGENCEOF EPSILON GENE Comparison of

nucleotide sequences of the humanand mouseC~genes has shownthat the epsilon gene is under the weakestselection pressure at the protein level amongthe immunoglobulin and related genes, although the divergence at the synonymous position is similar (71). Theresults maysuggest that only a small portion of the epsilon chain is important, or that the epsilon gene is dispensable in

accordancewith the fact that, while IgE has only obscure roles in selfdefense,it has an undesirablerole as a mediatorof hypersensitivity.Another explanation proposed is that the humanand murine epsilon genes were derived fromdifferent ancestors duplicated a long time ago (paralogous). A rabbit 9/ chain eDNA clone was isolated from a eDNA library of hyperimmunized rabbit spleen mRNA (58, 99). There are at least two C~, genes in the rabbit genome. A Xenopus C~ chain cDNAdone was isolated from a eDNA library of Xenopusspleen mRNA (17). Theportion of the H-chainsequence contained in the plasmid (22-amino-acidresidues) is 35%homologous mammalian/zand "y sequences. The mRNA hybridized with this plasmid is 2.5 kb, in agreementwith the size of mousebt mRNA. CONSTANT-REGIONGENES OF OTHER SPECIES

ACKNOWLEDGMENTS

I amgrateful to Y. Nishida,T. Kataoka,andA. Shimizufor critical reading of the manuscript. I thank F. Oguni, S. Takeda, and T. Nomafor their assistance in preparingthis manuscript.This investigation wassupported by a special grant-in-aid from the Ministry of Education, Science and Culture of Japan,

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kappaand lambdah.’ght chain messenger RNAsequences m mousemyeloma, MOPC-104E, knownas a lambda chain producer. Biochem.Biophyz gez Commun. 74:796-802 122. Parnes, J. R., Seidman,J. G. 1982. Structure of wild-type and mutant mousefl2-microglobulingenes. Cell 29: 661-69 123. Pech, M., Hoehtl, J., Schnell, H., Zathan, H. G. 1981. Differences between ~ermlineand rearranged immunoglobulin Vxcodingsequencessuggesta localized mutation mechanism. Nature 291:668-70 124. Perry, R., Kelley, D., Coleclough,C., Seidman, J., Leder,P., et al. 1980.Transcription of mouseKchain genes:implicationsSci. for allelic exclusion.Proc.Natl. Acad. USA77:1937-41 125. Rabbitts, J. H., Forster, A., Dunniek, W., Bentley, D. L. 1980. The role of gene delection in the immunoglobulin heavy chain switch. Nature283:351-56 126. Rabbitts, T. H., Bentley, D. L., Milstein, C. P. 1981. Human antibody genes: V gene variability and Cn gene switching strategies, lmmunol.Rev. 59:69-91 127. Rabbitts, T. H., Forster, A., Milstein, C. P. 1981. Humanimmunoglobulin heavychain genes: evolutionary comparisons of C~, C~ and C~genes and associated switch sequences. Nucleic Acids Res. 9:4509-24 128. Radbruck,A., Liesegang,B., Rajewsky, K. 1980.Isolation of variants of mouse m~,elomaX63that express changedimmunoglobulin class. Proc. Natl. Acad. Sci. USA77:2909-13 129. Ravetch,J. V., Kirsch,I. R., Leder,P. 1980. Evolutionary approach to the question of immunoglobulin heavy chain switching: Evidencefromcloned humanand mousegenes. Proc. Natl. Acad. ScL USA77:6734-38 130. Ravetch,J. V., Siebenlist, U., Korsmeyer, S., Waidmann,T., Leder, P. 1982. Structure of the humanimmunoglobulin /~ locus: Characterizationof embryonic and rearranged J and D genes. Cell 27:583-91 131. Reehavi,G., Bienz, B., Ram,D., BenNeriah,Y., Cohen,J. B., et al. 1982. Organizationand evolution of immunoglobulinVn genesubgroups.Proc.Natl. Acad. Sci. USA79:4405-9 132. Robertson, M. 1981. Genes of lymphocytesI: Diversemeansto antibody diversity. Nature290:625-27 133. Rogers,J., Choi,E., Souza,L., Carter, C., Word,C., et al. 1981. Geneseg-

merits encoding transmembranecarboxyl termini of immunoglobulinT chains. Cell 26:19-27 134. Rogers, J., Early, P., Carter, C., Calame,K., Bond,M., et al. 1980. Two mRNAs with different 3) ends encode membrane-bound and secreted forms of immunoglobulin ~u chain. Cell 20:303-12 135. Rose, S. M., Kuehl, W.M., Smith, 13. P. 1977. ClonedMPC11 myelomacells express two kappagenes: a gene for a completelight chain and a gene for a constant region polypeptide. Cell 12:453-62 136. Sablitzky, F., Radbruch,A., Rajewsky, K. 1982, Spontaneousimmunoglobulin class switching in myelomaand hybridomacell lines differs froml~hysiological class switching. Immunol.Rev. 67:59-72 137. Sakano,H., Huppi,K., Heinrich, G., Tonegawa,S. 1979. Sequencesat the somaticrecombinationsites ofimmunoglobulin light-chain genes. Nature 280:288-94 138. Sakano, H., Kurosawa,Y., Weigert, M., Tonegawa,S. 1981, Identification andnueleotide sequenceof a diversity DNAsegment (D) of immunoglobulin heavy-chaingenes. Nature 290:562-65 139. Sakano, H., Maki, R., Kurosawa,Y., Roeder, W., Tonegawa,S. 1980. Two types of somaticrecombinationare necessary for the generation of complete immunoglobulinheavy-chain genes. Nature 286:676-83 140. Schnell, H., Steinmetz,M., Zachan,H. G. 1980. Anunusual translocation of immunoglobulin gene segmentsin variants of the mouse myelomaMPCll. Nature 286:170-73 141. Schreier, P. H., Bothwell, A. L. M., Mueller-Hill, B., Baltimore, D. 1981. Multipledifferences betweenthe nucleic acid sequences of the IgG2# andIgG2ab alleles of the mouse.Proc. Natl. Acad. Sci. USA78:4495-99 142. Seidman,J. G., Leder, A., Nan, M., Norman.B., Leder, P. 1978. Antibody diversity. Science 202:11-17 143. Seidman,J. G., Leder, P. 1980. A mutant immunoglobulinlight chain is formedby aberrant DNA-andRNAsplicing events. Nature 286:779-83 144. Scidman, J.G.,Max,E.E.,Lcdcr, P. 1979.A K-immunoglobulin gencis formed bysite-specific recombination without furthcr somatic mutation. Nature280:370-75 145. Seidman,J. G., Nan, M. M., Norman, B., Kwan,S. P., Seharff, M.andLeder,

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IMMUNOGLOBULIN GENES P. 1980. Immunoglobulin V/J recombination is accompaniedby deletion of joining site and variable region segments. Proc. Natl. Acad. Sc~ USd77: 6022-26 146. Selsing,E., Storb, U. 1981.Somaticmutation of immunoglobulin light-chain variable-regiongenes. Cell 25:47-28 147. Sheppard,J. W., Gutman,G. A. 1982. Rat kappa-chainJ-segment genes: Two recent gene duplications separate rat and mouse. Ce1129:121-27 148. Shimizu, A,, Takahashi, N., Yamawaki-Kataoka, Y., Nishida, Y., Kataoka, T., Honjo,T. 1981. Orderingof mouse immunoglobulin heavy chain genes by molecularcloning. Nature289:49-53 149. Shimizu,A., Takahashi,N., Yaoita, Y., Honjo,T. 1982. Organizationof constant region genefamily of the mouse immunoglobulin heavy chain. Cell 28: 499-506 150. Siebenlist, U., Ravctch,J. V., Korsmcycr, S., Waldmann,T., Ledcr, P. 1981. HumanimmunoglobulinD segments encoded in tandem multigenic fatuities. Nature294:632-35 151. Singer, P. A., Singer, H. H., Williamson, A. R. 1980. Different species of messenger RNAencode receptor and secretory IgMp chains differing at their carboxytermini. Nature285:294300 152. Slighton,J. L., Blechl,A.E., Smithies, O. 1980. Humanfetal oy. and ~T" globin genes: completenucleotidc sequences suggest that DNAcan be exchanged between these duplicated genes. Cell 21:627-38 153. Smith, G. P. 1974. Unequalcrossover andthe evolutionof multigenefamilies. Cold SpringHarborSyrnp. QuantBiol. 38:507-13 154. Smith, G. P., Hood,L., Fitch, W.M. 1971. Antibodydiversity. ~nn. Re~. Biocherr~40:969-1012 155. Stavnezer,J., ~Marcu, K. B., Sirlin, S., Alhadeff, B., Hammering, V. 1982. Rearrangementsand deletions of immunoglobulinheavychain genesin the double-producing B cell lymphomaI29. MoLCell. Biol. 2:1002-13 156. Steinmctz, M., Altenburger, W., Zachau, H. O. 1980. A rearranged DNA sequencepossiblyrelated to the translocat~on ofimmunogiobulingene segments. Nucleic//cids Rex 8:1709-20 157. Swan,D., D’eustachio, P., Leinwand, L., Seidman,J., Keithley, D., Ruddle, F. H. 1979. Chromosomal assignment of the mouset~ light chain genes.Proc. NatL Acad. Scl USA76:2735-39

527

158. Takahashi,N., Kataoka,T., Honjo,T. 1980. Nucleotide sequencesof classswitch recombination region of the mouse immunoglobulin y2b-chain gene. Gene11:117-27 159. Takahashi, N., Ueda, S., Obata, M., Nikaido,T., Nakai,S., Honjo,T. 1982. Structure of humanimmunogiobulin gammagenes: Implications for evolution of a genefamily. Cell 29:671-79 160. Tonegawa,S., Maxam,A. M., Tizard, R., Bernard,O., Gilbert, W.1978. Sequenceof a mousegerm.line genefor a variable region of an immunoglobulin light chain. Proc. Natl.//cad. Sci. USA 75:1485-89 161. Tucker, P. W., Marcu,K., Newel,N., Richards,J., Blattner, F. R. 1979.Sequenceof the clonedgenefor the constant region of murine y~ immunoglobulin heavy chain. Science 206: 1303-6 162.Tucker,P. W.,Slighton,J. L., Blattner, F. R. 1981. MouseIgA heavy chain genesequence:Implicationsfor evolution of immunolobulin hinge exons. Proc. Natl.//cad. Sc~ USA 78:7684--88 A. F., Geronda163. Tyler, B. M., Cowman, kis, S. D., Adams,J. M., Bernard,O. 1982. mRNA for surface immunoglobulinT chainsencodesa highlyconserved transmembranesequenceand a 28-residue intracelular domain.Proc. Natl. Acad. ScL USd79:2008-12 164. Ueda, S., Nakai, S., Nishida, Y., Hisajima,H., Honjo,T. 1982.Longterminal repeat-like elementsflank a humanimmunogiobulinepsilon pseudogene that lacks introns. EMBO Z 1:1539-44 165. VanNess,B. G., Coleclough,C., Perry, R. P., Weigert, M. 1982. DNAbetween variable and joining gene segmentsof immunoglobulin r light chain is frequentlyretainedin ceils that rearrange the K locus. Proc. NatL//cad.Scg US~~ 79:262-66 166. Wabl,M., Steinberg,C., 1982.Atheory of allelic andisotypic exclusionfor immunoglobulin genes. Proc. Natl. Acad. Sct~ US//. 79:6976-78 167. Waltield,A., Selsing,E., Arp,B., Storb, U. 1981. Misalignmentof V and J gene .segmentsresulting in a nonfunctional tmmunoglobulingene. Nucleic Acids Rex 9:1101-9 168. Wang,J. C. 1979. Helical repeat of DNA in solution. Proc.Natl. Acad.Sci. US// 76:200-3 169. Weigert, M., Riblet, R. 1976. Genetic control of antibody variable regions.

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Cold Spring HarborSymp.QuantBiol. 41:837-46 170. Wu,G. E., Govindji, N., Hozumi,N., Murialdo, H. 1982. Nucleotide sequence of a chromosomalrearranged k2 immunoglobulin gene of mouse.Nucleic Acids Rex 19:3831-43 171. Wu,T. T., Kabat,E. A. 1982.Fourteen nucleotides in the secondcomplementarity-deterrnining region of a human heavy-chainvariable region gene are id.en.tieal witha sequencein a human D numgene.Proc. Natl. Acad. ScL USA 79:5031-32 172. Yamawaki-Kataoka, Y., Kataoka, T., Takahashi, N., Obata, M., Honjo, T. 1980. Completenucleotide sequenceof immunoglobulin "~2bchain genecloned from mouse DNA.Nature 283:786-89 173. Yamawaki-Kataoka,Y., Miyata, T., Honjo, T. 1981. Thecompletenucleotide sequenceof mouseimmunoglobulin "~2a gene and evolution of heavy chain genes:further evidencefor interveninlg sequence-mediated domain transl~r. Nucleic AcidzRes. 9:1365-81

174. Yamawaki-Kataoka,Y., Nakai, S., Miyata,T., Honjo,T. 1982.Nucleotide sequences of gene segments encoding membranedomains of immunoglobulin ~/ chains. Proc. NatLAcad.Sci. USA 79:2623-27 175. Yaoita, Y., Honjo,T. 1980.Deletionof immunoglobulin heavy chain genes fromexpressedallelic chromosome. Nature 286:850-53 176. Yaoita, Y., Honjo,T. 1980.Deletionof immuno.globulin heavychain genes aecompames the class switch rearrangement. Biomed.Res. 1:164-75 177. Yaoita, Y., Kumagai,Y., Okumura, K., Honjo, T. 1982. Expression of lymphocyte surface IgE does not require switch recombination. Nature 297: 697-99 178. Zeelon,E. P., Bothwell,A. L. M., Kantot, F., Schechter,I. 1981. Anexperimental approach to enumerate the genes coding for immunoglobulin variable-regions. Nucleic Acids Res. 9: 3809-20

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Ann.Rev.Immunol.1983.1:529-68 Copyright ©1983by AnnualReviews Inc. All rightsreserved

GENES OF THE MAJOR HISTOCOMPATIBILITY COMPLEX OF THE MOUSE Leroy Hood, Michael Steinmetz,

and Bernard Malissen

Divisionof Biology,CaliforniaInstitute of Technology, Pasadena, California91125 INTRODUCTION In the past 2 years our understanding of the major histocompatibility complexhas advanceddramatically becauseof the rapid progress madeby moleculargenetics. Biologists can nowbeginto dissect the molecularnature of one of the mostfundamentalproblemsin eukaryoticbiology--theability of organismsto discriminatebetweenself and nonself. Eventhe mostprimitive of metazoa,the sponges,exhibit cell-surface recognitionsystemscapable of identifying and destroying nonself, presumablyto preserve the integrity of individuals growingin densely populatedenvironments(34). For example,whentwo genetically identical spongesare apposed,the individuals fuse to form a single organism. However,whentwo genetically dissimilarspongesare joined, there is a reactionleadingto tissue destruction at the boundarybetweenthe two individuals (35, 36). Presumably,cellsurface structures recognizenonself and trigger effector mechanisms that lead to the destruction of the foreign tissue. Perhapsthe most striking feature of the moleculesinvolvedin these cell-surface recognitionphenomena is their enormousdiversity or polymorphism. Morethan 900 genetically distinct spongeshavethe capacity to reject oneanother after apposition (35). Self/nonselfrecognitionsystemsin other invertebratesandvertebrates appearto displaysimilar characteristics. Thus,three features are fundamental to self/nonselfrecognitionsystems---cell-surface recognitionstructures, effector mechanisms that lead to the destruction of nonself, and a high degree of polymorphism in the recognition structures. Mammals have self/nonself recognition systems encodedby a chromosomal region termed the major histocompatibility complex(MHC)with pre529 0732-0582/83/0410-0529502.00

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HOODET AL

cisely these features. These systems regulate various aspects of the mammalianimmuneresponse. The same recognition systems were first identified in micebecauseof the existenceof inbred (geneticallyidentical) and congenic(genetically identical but for a single chromosomal region) strains of mice. Bythe grafting of tumors or skin amongsuch mice and followingrejection or acceptanceof the graft, it waspossible to mapthe rejection of nonself to a region on chromosome 17, whichwasthen denoted the MHC (30, 31). Theinitial characterizationof the cell-surface structures responsiblefor graft rejection utilized alloantibodies producedby crossimmunization of micewith spleen cells of micediffering only at the MHC. Later, recombinant congenic mice, which had undergonerecombination within the MHC,were produced. With these recombinantcongenic mice, specific alloantisera to even smaller portions of the MHC could be produced. Thus, inbred, congenic, and recombinantcongenicmice with their attendant potential for r~combinationalanalyses and the generation of highly specific alloantisera permitted immunogeneticists (45, 10Ci), later protein chemists(81, 90, 101), and very recently molecularbiologists characterize the MHC of the mousein considerabledetail. In this review, wediscuss the results obtained primarily from molecularanalyses of the MHC of the mouse. From the data available, the MHC of the mouse appears to be a general modelfor the MHCs of other mammals and perhaps other vertebrates. THE MURINE

MHC

Three Classes of Molecules Alloantisera specifie for gene productsof the routine MHC havepermitted the identification of three classes or familiesof moleculesdenotedI, II, and III (Figure 1). There are two categories of class I genes. Class I genes located in the left-hand side of the MHC or the H-2region encodecellsurface moleculestermed transplantation antigens, denotedK, D, and L, whichmediatethe graft rejection assay initially used to define the MHC (Figure 1). Theclass I geneslocated in the right-handportion of the MHC, denotedthe Qa-2,3and Tla regions, encodecell-surface antigens that are structurally related to the transplantationantigens,but that differ in tissue distribution and presumablyfunction (24, 107) (Figure 1). Class II genes, designatedA~, A~, E~, and E~, encodecell-surface Ia antigens that later werefoundto be identical to the immune response,or Ir genesthat control the magnitudeof the murineimmuneresponsesto different antigens (49). Class III genesencodeseveral components of the activation stages of the complement cascade (1). The availability of recombinantcongenicstrains and specific alloantisera to various geneproductsof the MHC of the mouse

Annual Reviews

MHCGENES 531 has permitted the construction of a detailed genetic map(Figure 1). The MHC of manon chromosome 6 is remarkablysimilar to that of its mouse counterpart, exceptfor a presumedtranslocation in mice, whichled to the separationof the Kfromother class I loci and the existenceof additional class II loci in manwhosehomologueshave not yet been found in mice (Figure 1). Class I and class II moleculesand genes isolated from both murineand humancells are closely related.

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Polymorphism Themost striking feature of the MHC in mouseand manis the extensive genetic polymorphism exhibited by certain class I and class II genes. For example,the Kand D class I genes appear to have 50 or morealldes in both wild and inbred populations of mice (46). Theclass I genes of the Qa-2,3andTla regions are far less polymorphic (24). Certain of the class II genes, Aaand Ea, also appear quite polymorphic, whereasanother, E~, is muchless polymorphic(47). Thefact that somegenesin the class and II families exhibit extensive polymorphism, whereasothers do not, is an interesting feature that mayreflect functionalrequirementsfor diversity

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Annual Reviews

532

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in the polymorphic loci. Individual inbred micewill havedistinct combinations or constellations of these alleles. Eachindividual combinationof alleles is termed a haplotype. For example, the BALB/cmousewhose MHC has been most thoroughlystudied at the molecularlevel is of the d haplotypeand its gene productsare generally denotedby a capital letter d, Ada, w~th a superscript for the haplotypedesignation~e.g. Kd, Da, A~ E , and E_. Theextensive polymorphisms as well as certain other features h~e led t~ the hypothesisthat interesting genecorrection mechanisms are operating within the class I and II genefamilies (see below)(101, 102). Evolution The MHC has been found in all mammals and vertebrates studied to date. In each case with adequateinformation,there appearto be closely linked class I andclass II genes,andin those fewcases wherethe analysis has been carded out, associated class III genes. A controversy has arisen as to whetherthe class III genes should be regardedas an integral part of the MHC complexor, alternatively, as an inadvertent addition comparableto the MHC-linked genes encodingseveral apparently unrelated enzymes(48). Since weknowlittle about the structure, organization,or numbersof class III genesin contemporary or primitive vertebrates, it is dit~cult to argue either sideof this issue. Thelinkageof the class I, class II, andpossiblyclass III genesover the 500 millionyears of vertebrate divergenceposesinteresting questions about the selective constraints that havemaintainedlinkage amongthese genefamilies. Size The MHC spans an enormouschromosomalregion, as indicated by the followingsimplistic calculation. Thehaploid mousegenome includes about 1600cMof DNA by genetic analysis and contains 3 X 109 base pairs by biochemicalanalysis. Therefore, 1 centimorganequals approximately2000kb pairs of DNA.The MHC of the mouseis approximately 1-2 cM in length and, accordingly, should contain about 2000-4000kb pairs of DNA. The cloning of the MHC has involved a variety of approaches. Various eDNAprobes were used to isolate class I and II genes from genomic libraries. Restriction enzymemappingand chromosomal walkinghave defined clusters of linked MHC genes. These genes have been mappedinto regions of the MHC by gene transfer experimentsand restriction enzyme site polymorphisms--as is described subsequently. In the past 2 years, 25-50%of the mouse MHC has been cloned and several new insights concerningl~he structure and organization of these gene families have emerged.

Annual Reviews MHC GENES 533

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CLASS I MOLECULES AND GENES ClassI Polypeptides The most thoroughly characterized class I molecules are the mousetransplantation antigens. These molecules are comprisedof a class I polypeptide that is 45,000 daltons and is an integral membraneprotein noncovalently associated with fl2-microglobulin, a 12,000-dalton polypeptide encoded by a gene located on chromosome2 in the mouse (26, 75, 94) (Figure Aminoadd sequence analyses have demonstrated that the transplantation antigen is divided into five domainsor regions (14, 114). The three external domains, ~1, a2, and a3, are each about 90 residues in length. The transmembraneregion is about 40 residues and the cytoplasmic domainis about 30 residues in length. The ~2 and ~3 domains have a centrally placed disulfide bridge spanning about 60 residues and up to three N-linked glycosyl units bound to attachment points in the el and e2 (Kb, Da) and also in the a3 (Kd, Ld, and Db) domains (68). The transmembrane region contains a hydrophobic and uncharged core of about 25 residues, which presumablytransverses the membrane.Binding studies with peptide fragments from class I molecules have shownthat the fl2-mieroglobulin subunit associates with the a3 domain (128). Prdiminary X-ray analyses of crystals from a proteolytic fragment of a humantransplantation antigen containing the al, ~2, a3, and fl2-microglobulin domainsdemonstrate that this class I molecule has a twofold axis of symmetry(P. Bjorkman,J. Strominger, and D. Wiley, personal communication)and, accordingly, maybe folded so that its four domains are paired in a symmetric manner similar to antibody ANTIBODY

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Figure 2 A schematic representation of the membrane orientations of Thy 1, class I, class II, and antibody molecules. Thy 1 is a T-cell differentiation antigen. Shaded domains indicate sequence homologies that suggest a commonevolutionary ancestry (see text).

Annual Reviews

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domains(Figure 2). Aminoacid sequence analyses suggest that the domain(114) and ~2-microglobulin(88) showhomologyto the constant region domainsof immunoglobulins, thus posingthe provocativepossibility that the genes encodingantibodies and transplantation antigens share a commonancestry. As mentionedabove, the class I genes fall into two categories--those mappinginto the H-2 region and encodingtransplantation antigens and those mappinginto the Qa-2,3 and Tla regions and encodinghomologous antigens that appearto be expressedin a tissue-specific manner(Figure 1). Transplantationantigens are found on virtually all nucleatedcells of the mouse. Somemice, such as the inbred BALB/c,have three or even more distinct transplantation antigens (K, D, L, and perhaps Mor R), whereas other miceappear to express fewer transplantation antigens (21, 33). The cell-surface antigens encodedin the Qa-2,3andTla regions can further be distinguishedfromthe classical transplantation antigens becausethey are significantly less polymorphic (24). Someof the Tla genes encode hematopoieticdifferentiation antigens, whichare found on thymocytesat an early developmental stage as well as on leukemiccells, whereasothers appear to be expressedonly on leukemiccells. TheQaantigens are found on manydifferent types of lymphoidcells. Wedenotethose class I antigens encodedby the Qaand Tla regions as hematopoieticdifferentiation antigens, recognizingthe provisional nature of this designation. Class I cDN/t Clones Twoprocedures were used to isolate mouseand humanclass I eDNA clones. First, eDNA clones wereselected for their ability to bind class I mRNA, which could be identified by in vitro translation and immunoprecipitation of the synthesizedpolypeptides(54, 89). As an alternative approach,short oligonucleotidesweresynthesizedafter reverse translation of the aminoacid sequencesof class I polypeptidesinto DNA languageand wereused as hybridization probes for the identification of class I cDNA clones or as primers to synthesize eDNA’s from unpurified mRNA’s (91, 108). Furthermore, mouseeDNA clones and humangenomicclones also wereisolated by cross-species hybridization with eDNA clones (44, 109). Essentially the sameapproacheswereemployedfor the isolation of mouse and humaneDNA clones for g2-microglobulin(85, 115) and class II polypeptides(3, 32, 51, 60, 64, 113, 118, 124), whichis discussedsubsequently. The class I eDNA clones were characterized by DNA sequenceanalysis to verify their authenticity. Mostof the mouseeDNA clones havenot been correlated with knownclass I moleculesmainlybecauseof the paucity of aminoacid sequencedata currently available. However,the eDNA clones d (56, 126) molecules have been identified. for the Kb (91), b ( 93), and K

Annual Reviews MHC GENES 535 The class I cDNAprobes were then used to screen genomic libraries to obtain clones that could be used to analyze the structure and organization of class I genes. Structure

ORGANIZATION Three mouse class I genes have been completely sequenced, and their structural features are depicted in Figure 3A. d) encode Ld transplantation Two of these genes (27.5 and CH4A-H-2L antigens (23, 76), whereas a third (27.1) has been mappedinto the Qa-2,3 region (111). It is not knownwhether gene 27.1 is expressed. The exonintron organization of these class I genes is remarkablysimilar. Each gene is divided into eight exons, which correlate precisely with the domainsof the class I polypeptide (111). The first exon encodes the leader sequence, whereas the second, third, and fourth exons encode the t~l, a2, and a3 domains. The fifth exon encodes the transmembranedomainand the sixth, seventh, and eighth exons encode the cytoplasmic domain and 3’ untranslated region of these genes (Figure 3.4 .) The exon-intron organization of a human class I gene is similar to the mouse genes, with the only difference being that the humanclass I gene contains two rather than three cytoplasmic exons (67). Additional sequence data on humanclass I genes will be necessary to determine whetheror not this feature is characteristic of all humanclass I genes. The general features of the class I genes resemble those of other eukaryotic genes. The upstream and downstream intron boundaries typically have the classical GT/AGnucleotides, which appear to be important RNA splicing signals (99). The 5’ flanking sequenceof gene 27.1 appears to have sequence elements characteristic of other eukaryotic promoter regions-CAATand TATAboxes at positions 81 and 53 upstream of the AUG initiation codon(111 ). The consensus recognition sequence, whichprecedes the site of polyadenylation, AATAAA, appears twice, adjacent to one another approximately 390 nucleotides downstreamfrom the stop codon in exon 8 of the 27.1 gene (111). According to the nucleotide sequence, the two reported Ld genes are functional in that there are no stop codons at inappropriate positions, the RNAsplicing junctions are all characterized by the appropriate signal nucleotides, and there are no deletions or insertions that changethe reading frames of the exons (23, 76). In contrast, gene 27.1 appears to be a pseudogene by several criteria (111). First, there is a charged aspartic acid codon in the middle of the highly hydrophobic transmembrane exon. Second, there are termination codons near the end of the transmembraneand seventh exons. Third, the upstream RNAsplice signal for the sixth exon does SEQUENCE

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of Class I Genes

Annual Reviews 536

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7 5

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Figure3 (A) A schematicdiagramof the exon-intronorganizationof antibody,class I, class II, and #2-microglobulin genes.L denotesthe exonsencodingthe signal or leader peptides; el, e2, and e3 represent exonsencodingthe external domains;TMdesignates the transmembrane exons; CYTsymbolizesthe exonsencodingthe cytoplasmicdomains;and 3’ UTdenotes the 3’ untranslated region. Darkboxesrepresent coding sequencesand hatchedboxes3’ UT regions. (B) Schematicdiagramof the exon-intronorganizationof two mouseclass I genes. Nonhomologous sequencesbetweenthe L~ and Qa(27.1) genes are indicated by stippling. Exonsare numbered;3’ untranslatedregions are hatched.Arrowsshowthe localization of type I andtype II Alu-like repeat sequences. not agree with the consensus sequence. Thus, it is clear that gene 27.1 cannot encode a full-length class I polypeptide. However, with the exception of the aspartic acid codon in the transmembrane exon, the aberrations in gene 27.1 are all in the 3’ portion downstream from the stop codon at the end of the transmembrane exon. Thus, gene 27.1 may encode a membrane-bound class I polypeptide that is missing the cytoplasmic domain, or alternatively, it may be expressed as a soluble transplantation antigen (39) (see below). PATTERNS OF VARIABILITY FOR CLASS I GENES AND POLYPEPTIDES A detailed comparison of 12 class I amino acid and translated nucleotide sequences has appeared recently (68). The overall homology

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MHC GENES 537 between six polypeptides for which extensive sequence information exists [Kb, Db, L°, Kd, D°, Qa (27.1)] is 76-94%(Table 1). From these comparisons it is obvious that the alleles Kb and Kd or Db and Dd are not more related to each other than to other class I polypeptides. This has been denoted a general lack of "D-ness" and "K-ness" and is discussed below. However,close inspection of the sequences reveals the existence of short allele-speeitie sequences located mostly in exons 4 to 8 (68). Particularly interesting is the close relationship betweenthe Ld band D amino acid sequences (94%), whereas theDb and Dd polypeptides are only 84%homologous.This observation raises the possibility that the Ld and Db genes maybe alleles (68). Alternatively, the gene correction mechanisms that operate to generate polymorphisms (see below) may be capable correcting nonallelic genes extensively so that they resemble each other. The variability in the extracellular part of the analyzed class I molecules appears to be clustered in three regions in the first two external domains (residues 62-83, 95-121, and 135-177)(68). In contrast, the third external domainwhich interacts with fl2-microglobulin is more highly conserved. Exon6, encoding part of the cytoplasmic domain, is completely identical in seven functional class I sequences that can be compared(gene 27.1 has a different exon 6 sequence, but also contains a premature stop codon upstream of exon 6). The significance of this finding is unknown. THE Qa (27.1)

GENE SHOWS STRIKING

HOMOLOGY TO CLASSICAL

TRANSPLANTATION ANTIGENSThe overall homology between gene 27.1, which mapsinto the Qa-2,3 region, and the classical transplantation antigens (K, D, L) is 78-84%(Table 1). Therefore, the Qa-2,3 gene is closely related to the genes encoding transplantation antigens as they are to one another. These homologies suggest that the Qa and transplantation antigens descended from a commonancestor. It will be interesting to find out whether or not the structural homologyis reflected in similar functions as well. Peptide mapanalyses of Qa-2,3 antigens also have revealed strucaTable 1 Percent homology between mouse class I molecules Molecules bH-2K bH-2D dH-2L dH-2K dH-2D

bH-2K

bH-2D

dH-2L

dH-2K

dH-2D

Qa (27.1)

83

83 94

77 76 76

89 84 87 76

79 80 79 78 84

Qa (27.1) aAminoacid sequences compared ranged from 113 to 342 residues. Maloy & Coligan (68).

Table adapted from

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tural homology to transplantation antigens (107), whereasTLpolypeptides appearto be very different (127). Onthe other hand, the similar size and binding to/32-microgiobulin of the TLmolecules, as well as the strong cross-hybridization of these genes to a probe for the exonencodingthe /82-microglobulin-binding domainagain suggest homologyand a shared ancestry (I 12). DNA sequenceanalyses of the Tla genesare nowunderway.

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ALU REPEATS AND CLASS I GENES

The human genome has an element

repeated300,000times that is termedthe Alu repeat becausethe restriction enzymeAlu I cleaves within the repeat and thus permits the visualization of this DNAon Southern blots. The mousegenomehas an homologous elementtermedan Alu-like repeat. The3’ untranslated regions of class I genescan be distinguished by the presence or absenceof Alu-like repeats (11, 109). Alu-likesequenceshavethe general characteristic of transposons with terminal direct repeats encompassinga central sequencecomposed of the Alu-typeand an A-rich sequence(43). Theycan be classified into two groupscalled type I (described above)and type II sequences.TypeII Alu sequencescontain an additional non-Alurepetitive sequence.Alu-like repeat sequencesof type II havebeenfoundat the 3’ endof the 3’ untranslated region of three class I eDNA clones [pH-2II(109), pH-2d-1(56),and 203(93)] and include the AATAAA polyadenylationsignal as a part of the A-rich sequence. One of these eDNA clones (pH-203)has been shown encodethe Db polypeptide(68, 93). ThepH-2IIclone encodesa polypeptide very similar (except for five positions) to the d polypeptide 7( 6, 109) a clone pH-2d-1appears to encodethe Dd polypeptide (10, 68). Atype Alu-like repeat also has been found at the 3’ end of the 3’ untranslated region of the L~ gene (76). Thus, class I genes encodedin the D region appearto contain Alu-likerepeat sequencesin their 3’ untranslatedregions. The eDNA clones encoding the Kb (92) and the d ( 126) polypeptides apparentlylack Alu-like repeat sequences.Their 3’ untranslatedregions are highly homologous(80-90%)to the D region eDNA clones for the first -,-300 bp, whereasthe remainingportion of ~120-160nucleotides encompassingthe Alu-like repeat in the Dgroupof genesis completelydifferent for the Kgenes (93, 126). Indeed, this region of the eDNA clone homologousto the Kd polypeptidehas beenusedas a specific probefor the identification of the Kd gene(126). Gene27.1 also does not contain the Alu-like repeat at its 3’ end(111) but a sequencethat is distantly related to the corresponding portion of the Kgene, possiblyreflecting a closer evolutionary relationship between Qa and K region genes than between K and D region genes. Cross-hybridizationof 5’ flanking sequencesbetweenKand Qa-2,3 genes but not betweenK and D genes of the b haplotype support this notion(27).

Annual Reviews MHC GENES 539

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A comparison of the two genomicsequences of the genes 27.1 and Ld has shownthat the introns and exons have diverged about equally from one another apart from the insertion of a sequence of approximately 1000 nucleotides in the intron between exons 3 and 4 of gene 27.1 (76)(Figure 3B). This insertion is boundedat its 3’ end by a type II Alu-like repeat sequence and contains a type I Alu-like repeat sequence close to its 5’ end (Figure 3B). Thus, transposon-like Alu-like repeat sequences may have mediated this insertion of 1000 nueleotides. ARETHERETWOLa dGENES?The translated sequences of the two L genes correspond to the Ld protein sequence at 77 of 77 positions that can be compared(23, 76) and encode a class I polypeptide that can be detected with anti-L a monoclonalantibodies after gene transfer into mouseL cells (23, 29) (see below). However,these two genes appear to differ from another by 20 to 30 nucleotides. These differences might be explained in different ways. First, someof the differences might be due to DNA sequencing errors. However,the differences appear to be too extensive to be explained exclusively in this manner. Second, since these Ld genes were derived from two distinct populations of BALB/cmice, perhaps some genetic polymorphismhas already occurred since their separation. Finally, there maybe two distinct class I genes that both encode molecules recognized by anti-L d monoclonal antibodies. This latter hypothesis is being tested by chromosomalwalking techniques, which are discussed in a subsequent section. POSSIBLE SECRETORY CLASS I MOLECULES An unusual category of class I mRNA’s with two interesting features has recently been described (16). First, these mRNA’s contain several codons for charged aminoacids in the transmembraneregion, which is usually hydrophobic and uncharged. Second, a termination codon is found at the end of the membraneexon. Gene27.1 has precisely these same characteristics (111) and thus may capable of synthesizing a similar mRNA.These unusual class I mRNA’s are slightly smaller than the mRNA’s encoding full-length class I polypeptides and can be identified by a distinctive probe that again has been derived from the 3’ end of the 3’ untranslated sequence (17). It appears that these RNAmolecules are expressed only in liver cells and not in other mouse tissues that have been tested (17). Although there is to date no formal evidence that these mRNA’s are translated into proteins, it is attractive to speculate that they maydirect the synthesis of soluble class I gene products. ALTERNATIVE PATTERNS OF RNA SPLICING AT THE 3’ END OF THE CLASS I GENES Antibody heavy chain genes can be transcribed and then

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HOODET AL

differentially processedby RNA splicing at their 3~ ends to generate the membraneand secreted forms of immunoglobulins (22) (Figure 3A). eral intriguing observations suggest that class I genes also mayundergo alternative patterns of RNA splicing at their 3’ ends(Figure 4). First, the C-terminal aminoacid sequences of class I polypeptides and translated eDNA clones appear to be heterogeneousin length and they exhibit blocks of aminoacids whichdiffer (111) (Figure 4A). Theseproperties would expectedif different codingregions (exons) could be employedfor the termini of these polypeptides. Second, two mouseclass I cDNA clones (pH-2Iand pH-2II) reveal a notable sequencedifference. The 3’ ends these two cDNA clones are homologousto one another, apart from an insertion of 139 bp in the pH-2IIdone (111) (Figure 4B). This 139 pair sequencecorrespondspreciselyto the seventhintron of the class I gene. Thus,it is attractive to suggest that the seventh intron wasnot removed fromthe mRNA represented by clone pH-2II, whereasit wasfor the pH-2I and Ld-like mRNA’s (Figure 4C). Third, comparison of the cDNAsequenceof pH-202encodingthe Kb moleculewith the sequenceof gene27.1 suggestsa third pattern of RNA splicing for the seventhintron (92). Togive rise to an H-2Kb-likemRNA, exon 7 wouldbe spliced to a sequence 27 nucleotidefurther upstreamof the splicing point used for the generation of pH-2I or La-like mRNA’s (Figure 4C). These alternative patterns RNAsplicing wouldlead to C-terminal sequences differing in size and sequence.This particular pattern of RNA splicing also has beendescribed for the gamma geneof fibrinogen(18). It remainsto be seen whetherthese alternative formsof mRNA’s result fromthe transcription of the sameclass I gene. Nevertheless,the heterogeneitygeneratedby different RNA splicing mechanismsin the cytoplasmic domainsof class I molecules might be importantfor different effector functionsthroughinteractions withdifferent componentsof the cytoskeleton.

Organizationof the Class I GeneFamily A MULTIGENE FAMILY The possible homologiesof the class I and antibodygeneshaveraised several questions about the organizationof class I genes. First, howmanyclass I genesare encodedin individual mice?Second, do class I genes undergosomaticrearrangementsas do their antibody genecounterparts duringthe differentiation of cells that expressclass I molecules?Southernblot analyses of the DNAs fromvarious strains of mice with class I cDNA probes display 12-15 bands (13, 69, 86, 100, 104, 109, 111). Therefore,it is presumedthat there are 12 or moreclass I genesin mice. Althoughmostcells including liver express class I molecules,sperm do not express transplantation antigens. There are no gross DNArearrangements of class I genesin cells actively synthesizingthese molecules,

Annual Reviews MHC GENES 541

b H2-K pH-2l]

G SQ T~DLSL ~$~E

M~G

PO CKV MVH PP H $ O T

L

G S DW G G A MWT

pH-21

Annu. Rev. Immunol. 1983.1:529-568. Downloaded from arjournals.annualreviews.org by HINARI on 08/27/07. For personal use only.

139bpinsertion TERM

pH-2lI TERM

pH-2Z

41 voriotions in272bp 85%homotogous

3 wriofions in 85bp 96%homologous

d L

pH-2]~

b K

Figure 4 Differential RNAsplicing patterns at the 3’ end of class I genes. (,4) Comparison b polypeptide and the polypeptides encoded by the of the C-terminal sequences of the H-2K class I, pH-2II, and pH-2I eDNAclones. The shaded area indicates the sequences encoded b sequence. (B) Comparison by exon 7. A straight line indicates identity to the H-2K of the DNAsequencesat the 3’ end of the class I eDNAclones pH-2II and pH-2I, indicating close homologyexcept for a 139-bp insertion in clone pH-2II. Termination codons are boxed. Vertical bars indicate a nacleotide substitution. Gapswere introduced to achieve maximum homology.(C) Threepossible differential RNA splicing patterns of class I genes consistent with the structures of the Ld polypeptide, the pH-2II-like mRNA’s,andthe K~ polypeptide. Numbered black boxes indicate exons. The 3’ untranslated regions are hatched, andtermination codons are shownas black boxes at the mRNA level. (Adapted from 111.)

since liver and spermDNAshave the samehybridization patterns in Southern blots (109). Furthermore, completeclass I genes, uninterruptedby long stretches of noncodingDNA,are present in spermDNA,as shownby gene cloning and sequencing. Therefore, it is unlikely that majorDNArearrangementsof widely separated exons occur in class I genes as they do in antibodygenes. Multiple class I genes also have been identified in human and pig DNA(10, 83, 105). CLUSTERS OFCLASSI GENES Several laboratories have used the cloning of large DNAfragments (30 to 50 kb in size) in cosmidvectors to study the complexity and linkage relationship of class I genes in mice and man (27, 66, 112). The most extensive study so far has been carried out with

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HOODET AL

BALB/cDNA(112, 125). Some56 cosmidclones were identified in library of BALB/cspermDNAwith class I cDNAclones (112). These clones weremapped with10 infrequentcutting restriction enzymes to permit the identification of cosmidclones with overlappingDNA inserts. On the basis of these analyses, these clones couldbe orderedinto 13 gene clusterscontaining 36distinct class I genesin a total of 840kbpairsof DNA (Figure 5). Comparison of genomicSouthernblots with pooled cosmid DNA analyzedwith class I cDNA clones as hybridization probesshowed that most of the class I genes of the BALB/c mousehadbeencloned. Thelargestof thesegeneclusters, duster1, containssevenclass I genes spreadover 191 kb of DNA(Figure 6) andmapsinto the Qa-2,3 region Cluster

Location Mapping Expression Lenglh{kb)

Organization

o

~’o ,~o ,~o

" kb 200

27.1 L

3

Overlapping clones

~

Qo-2,5 P

L

Tie

191

17

68

2

I03

9

TLo 4

~ ~

,5

Tie

TL

64

9

distaltoD

TL

49

4

63

5

TIo

58

3

Qa-2,3 between D,Qa-2,3

Co

~

7

---re.m----

Qo-2,5

8

~

TIo

47

2

9

~

0a-2,3

38

2

IO

~

Tie

42

I

II

K ~

K

43

2

12

~

distal to D

39

I

13

D ~

O

35

I

840

56

K

O

36 ¢105sI genes Figure 5 A schematic

representation

of the 13 cosmid clusters

and their

locations.

The

locations were determined by genetic mapping through restriction enzyme site polymorphisms and by expression in DNA-mediatedgene transfer experiments (see text). The class I genes are indicated as dark boxes. (Adapted from 111.)

Annual Reviews MHC GENES 543 ~ 5’ 3 C~oI KpnI Sma I Sac~I XhoI XmoTrr

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Scale

27.1

~ ,

I 0

I 25

I 50

I 75

I I00

I 125

I 150

I 175

I 200 kb

Figure 6 Restriction map of the class I cosmid gene cluster 1 isolated from BALB/cDNA. Class I genes are indicated as black boxes. Homologousregions according to the restriction mapare indicated graphically by ovals, rectangles, and wedges(see text). Arrowsindicate the 5’ to 3’ directions of transcription. Sal I, Cla I, etc, are restriction enzymes.The location of gene 27.1 is indicated. (From111.)

(112) (see below). The spacing between these genes ranges between 7 28 kb. Thestructure of cluster 1 is interesting in that the three class I genes to the 3’ side of the cluster as well as their flanking sequencesare indistinguishable from one another by restriction mapanalysis. These genes probably arose by relatively recent duplication events, presumably by homologousunequal crossing-over. The two genes located at the 5’ end of cluster 1 also are related to one another, as revealed by cross-hybridization of their 3’ flanking sequences.Thus, the genesin cluster 1 suggest that class I genes can be ordered into closely related subgroupsof class I genes that are continually undergoing expansion and contraction by homologousunequal crossing-over. In this example,moreclosely related class I genes seem to be located adjacent to one another. Seventeen distinct class I genes located on seven cosmid clusters have been isolated and characterized from B10 mouse DNA(27). One of these gene clusters, cluster 3, containing five class I genes mapsinto the Qa-2,3 region and appears to be the homologto cluster 1 isolated from BALB/c DNA.Further characterization of the class I genes of B10mice is necessary to determine whether B10 mice contain only half as manyclass I genes as BALB/cmice. Such a difference is not apparent from Southern blot analyses (13, 69, 86, 109). If such a difference in class I genes amonginbred mice is real, extensive gene expansion and contraction must occur. Somemousestrains fail to express the L polypeptide at the serological level (e.g. B6, AKR,A.SW).To determine whether or not this failure expression results from deletion of the L gene, a DNAprobe has been isolated from the 5’ flanking sequence of the Ld gene (112). According Southern blot analyses, this sequence is missing in the DNAfrom mice of the b (B6) and the k (AKR)haplotypes, indicating that nonexpression might arise from ddetion of the L gene. Likewise, in mutant BALB/cmice denoted dml and din2, which also fail to express the Ld gene, the same 5’ flanking sequence is ddeted (H. Sun, personal communication). It will

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544

HOODET AL

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interesting to determinethe precise 5’ and3’ endpoints of these deletions by chromosomalwalking experiments, since a hybrid class I molecule comprisingthe N-terminalpart of the Dd moleculeand the C-terminalpart of the Ld moleculeappears to be present in BALB/c dm1 mice(123). These data also suppo~the hypothesisthat class I genesare undergoingcontinual gene expansionand contraction. GENETIC MAPPINGBY RESTRICTION ENZYMESITE POLYMORPHISMS Strategy Themosteffective approachfor mappingthe class I clusters to their respective locations in the MHC has beengenetic mappingby restriction enzymesite polymorphisms(27, 125). This approach employsthree steps. First, single- or low-copyprobes,whichwill hybridizeonly to oneor a few genomicrestriction fragments,are isolated from each of the cosmid clusters. Second,these probes are used to examinethe DNAs of congenic and recombinantcongenicmice for restriction enzymesite polymorphisms. Theoretically, three types of polymorphisms can be observeda changein restriction fragmentsize (mutation),a loss of the restriction fragment(deletion), or an increase in the numberof restriction fragments(duplication). Third, these restriction enzymesite polymorphisms are then correlated with the serological polymorphismsof the various congenic and recombinant congenicmousestrains analyzedto allow the mappingof a particular site to one of the four class I regions--K, D, Qa-2,3and Tla (Figure 7). Molecularmapof class Igenes Single- or low-copyprobes were isolated fromeach of the 13 class I geneclusters fromthe BALB/c mouseand were used to mapthe geneclusters with respect to the genetic mapof the MHC (125)(Figure 8). Fourstriking results emerged.First, all 36 class I genes could be mappedto the MHC.Noclass I genes have been found so far mappingto chromosomes other than 17. In contrast, processed pseudogenes fromother multigenefamilies such as the antibody (6, 37), globin (59), and tubulin (121) genefamilies mapto chromosomes other than those containingtheir functional counterparts.Second,31 of 36 class I genesmap to the Qa-2,3and Tla regions. Thus, these two regions contain morethan 80%of the class I genes. Threegeneclusters containingfive class I genes mapto the K and D regions, whichencodethe classical transplantation antigens. Thesedata are in agreementwith mappingstudies of class I gene clusters carried out for B10mouseDNA (27) and with previous conclusions derived from Southernblot analyses that most of the class I genes are located in the Qa-2,3and Tla regions (69, 86). However,these DNA blot

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Annual Reviews MHC GENES

545

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HOODET AL

analysesalso suggestedthat one or moreclass I geneswerelocated outside of the MHC (27, 86), whereasthe restriction enzymemappingstudies with cloned BALB/c genesfailed to identify any such class. I genes(125). Perhaps morethan 36 class I genes are present in the BALB/c mouseand one or moreof the unclonedgenes falls in this category. Third, restriction enzymesite polymorphisms occur with considerably greater frequencyin the K andDregions than in the Qa-2,3and Tla regions (125). This observation is in completeaccordancewith the extensive serological polymorphism of the K and Dmoleculesas contrasted with the limited serological polymorphismof Qa-2,3 and TL molecules. Hence, the extensive polymorphismsexhibited by the K and D genes also are seen in their flanking sequences, whereasthe morelimited polymorphisms of the Qa-2,3 and Tla genesalso are reflected by a lack of polymorphism in their flanking sequences.Hence,these differences mayreflect the differential operationof mechanisms for generating polymorphisms (see below). Finally, the results obtainedusing 13 single- or low-copyprobesto analyzethe numberof class I bandsin four different inbred strains suggest that modestexpansionand contraction of the class I gene family occurs, presumablyby homologous unequalcrossing-over (125). Theseobservations are in accord with those discussed earlier on the gene homologiesin the cluster 1 of BALB/c DNA andthe loss of the L genein certain strains. An elegant approachhas been used to study the organization of human class I genes (83). Treatmentof humancell lines with gamma rays leads the randomdeletion of various chromosomalsegments throughout the humangenome,including the loss of various portions of the humanMHC or HLAcomplex. The corresponding loss of one or more humanclass I genescan be followedby Sbuthernblot analyseswith class I probes. These studies havepermittedthe correlation of particular bandsdetected on the Southernblot with certain HLAalleles. Thesestudies should reveal the location of the as yet unidentified humanclass I genesequivalentto those of the mouseQa-2,3and Tin regions. Mechanisms for the Generation of Polymorphism COMPLEX ALLOTYPES,

SPECIES-ASSOCIATED

RESIDUES,

NO "K-

NESS"OR"D-NESS" Three observations madewith the initial N-terminal K

I

S

D,L

Oa2,5

Tla

Figure 8 Locations of the 13 claso I gene clusters in the MHC.The order of the clusters in a region is not known.Clusters 5 and 12 mapeither to the Qa-2,3 or Tin regions. Arrowspoint to the locations of the probes used for mapping. Open boxes indicate expressed genes by DNA-mediatedgene transfer, whereas dark boxes represent nonexpressed genes. (From 125.)

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MHC GENES 547 protein sequencedata on transplantation antigens suggestedthat the individual membersof the class I multigenefamily could be rapidly expanded andcontractedor correctedagainst oneanother(101, 102). First, the alleles of the K(and D) locus differ fromone anotherby multipleresidues. These multiplysubstituted alleles arc denotedcomplexallotypes (102), and they represent a contrast to the typical simple allotypes of the giobins, which differ generallyby single aminoacid substitutions. Thus,a special genetic mechanism appears necessaryto accountfor the extensive allelic ditferences. Second,the class I polypeptides of humanand mouseare distinguishedfromoneanotherby species-associatedresidues at various positions (102). Thus, the multiple mouse(and human)class I genesmust evolve parallel by a processtermedcoincidentalevolution(38). Onceagain, coincidental evolution amongmultiple genes requires the operation of mechanismsfor gene correction (38). Finally, the N-terminalregions of the alleles andthe Dalleles exhibitedno "K-ness"or "D-hess";that is, in a pool of sequencesfor KandD alleles, no features permittedthe Kalleles to be distinguished from their D counterparts (101, 102) (see also 81). These observationssuggestthat there is a rapid exchangeof informationbetween Kand Dgenesso that they fail to evolveindependentcharacteristics. The mechanismof gene conversion--the correction of one sequenceagainst a second--is also suggestedby data on the mutanttransplantation antigens. MUTANT TRANSPLANTATION ANTIGENS Mutant transplantation antigens weredetected by carrying out reciprocal skin grafts among siblings

in an inbredstrain of mice(80). Occasionally oneanimalwill reject the graft fromhis siblings. Subsequentcharacterization of these mutantsoften reveals that there is an aminoacid sequencevariation in one transplantation bantigen that leads to the subsequentgraft rejection. Mutantsof the K transplantation antigen appearto be remarkablein several contexts (Figure 9 ~/). First, they are very frequent, occurringat a frequencyof approximately5 XlO-~/cell/generation. Second,there are generally two or more aminoacid substitutions that are relatively closely spacedto one another in each mutantantigen. It appears improbablethat one isolated mutation wouldalwaysbe followedby a second closely linked mutation. Third, the sameindependentlyderivedvariants are seen repeatedly. Finally, virtually all of the Kb mutationsoccur at positions wherethe mutantresidues are observedin other class I molecules.For example,the L° polypeptideappears to contain mostof the substituted aminoacids foundin the Kb series of mutants(23, 87). Presumably one or moreclass I genescontainingthese residues also exist in the b haplotypemouse.Thesedata suggest that the Kb gene can undergoa single geneconversionevent with one of the other class I genes leading to variants containing multipleaminoacid substitutions (23, 56).

Annual Reviews 548

HOOD ET AL

I H-2 Kb[

91 ~

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bml brn5 brn4. bm 5

18~ I

275 I

\~26/ -~,~ ’li I1 ....

I I

brn 6 bin7 bm8 bin9 bm I0 broil bml6

"II

II

145 150 152 155 156 160 165 HIS Ly5 TRP~LU ~LNALk GLY~LUALAGLUARGLEUAR~ALATYRLEU~LUGLYTHR

b K Kbm-I

ARG

ALA~TYR TYR

GLU-

Figure 9 (,4) Schematicrepresentation of aminoacid substitutions in 11 independently isolated Kb mutants.The top illustration showsthe presumptiveexon organization of the Kb mRNA. Substituted residues in the mutantsare indicated by vertical bars. (Adaptedfrom 80.) (B) Sequencecomparisonof the ~, Kbmm,and Ld genes from codon position 145 to 165. Horizontallines indicate identity to the Kb protein or DNA sequences.(Adaptedfrom120.) The class I gene encoding the mutant Kbml transplantation antigen has recently been isolated (97, 119). This mutant class I polypeptide differs positions 152, 155, and 156 from the wild-type Kb antigen. DNAsequence analyses of the Kb, Kbin1, and Ld genes demonstrate that the Kbin1 mutant has seven nucleotide substitutions arising over a stretch of 13 nucleotides, which lead to the three amino acid substitutions (Figure 9B). All seven these substitutions are seen in the Ld bml gene, which is identical to the K gene, over a stretch of 52 nucleotides encompassing this region. This observation raises the possibility that the mutant Kbin1 gene arose by gene conversion against a b haplotype class I gene identical in sequence to the L~ gene in this region. These data also may be explained by a double recombinational event, which appears less likely. Since this mutant sequence is not

Annual Reviews

MHC GENES 549 locatedin the single class I genecloselylinkedto Kb (119), the putativegene conversionof the Kb genepresumablyoccurswith class I geneslocated 0.3 or morecMaway(Figure 1). Thus, gene conversion apparently mayoccur over extensivedistances(41, 42).

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GENE CONVERSION

AND GENE EXPANSION

AND CONTRACTION

CREATE POLYMORPHISMS Gcnc correction mechanisms such as gene expansionand contraction or geneconversionare required to explain complex allotypes, species-associatedresidues, the lack of "D-hesS"and "Khess," and the patterns of substitutions seen in the mutanttransplantation antigens (see 38 for a discussionof these issues). Bothmechanisms appear to play a fundamentalrole in generatingthe complexallotypes of immunoglobulin Ca genes(4, 81a, 96). Theanalyses of class I genenumberswith low- andsingle-copyprobes clearly indicate that duplication and deletion of class I genes occurs, presumablyby homologous unequalcrossing over. Furthermore,the mutantbmlgene appearsto havearisen by a geneconversion event. Since manymutantclass I polypeptideshaveproperties similar to those of bml, it is logical to concludethat they also arose by gene conversion and that this process is common throughout the class I gene family. Geneconversionalso appears to accountfor interesting aminoacid sequencerdationships in humanclass I molecules(65). Apparentlygene conversioncannotoccur amongall class I geneswith the sameefficiency. For example,the Db gene showsa 10-fold lower mutation rate than the Kb gene (45). The important point is that both gene expansionand contraction and genecorrection mechanisms play a fundamentalrole in generating the extensive polymorphism of certain class I genes. Anintriguing question is whetherone or both of these genecorrection mechanisms also give rise to the extensive flanking region polymorphisms of the K and D gene dusters, or alternatively, whetherthese polymorphisms reflect the operation of yet a third unknowngenetic mechanism.Onemust ask why certain regions and subregions [K, D, I-A (see below)] appear to exhibit extensive polymorphisms, whereasothers [Qa, Tla, I-E, S (see below)] not.

Expressionof Class I GenesAfter GeneTransfer APPROACH Theunequivocalidentification of serologically defined class I genes requires gene transfer experimentsin whichthe cloned gene is expressedin a foreign cell so that its gene product can be identified by serologic techniques.ClassI geneshavebeensuccessfullytransferred into mouseL cells lacking the thymidine kinase gene by co-transfer with a herpes virus thymidinekinase gene with the calcium phosphatetransfer technique(5, 23, 29, 63, 74, 105). Underappropriateselective conditions,

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cells expressingthe herpes thymidinekinase genewill grow(1 in 104-105 L cells), and about 90%of the thymidinekinase-positive cells expressthe newlyintroducedclass I geneproducton the cell surface. Mouse L cells are derived from C3Hmice of the k haplotype, whichappear to express only two transplantation antigens, Kk and Dk. Monoclonalantibodies can readily distinguisheachof the BALB/c (d haplotype)serologicallydefinexlclass I products from their C3Hcounterparts. The new(BALB/c)class I molecule can be identified by radioimmunoassay, by the fluorescence-activated cell sorter, by two-dimensionalgel electrophoresis, and by peptide map analysis. Data obtained by these meanscan then be correlated with the results obtained by DNA sequenceanalysis and the mappingof the gene by restriction enzymesite polymorphisms. IDENTIFICATION

OF SEROLOGICALLY

DEFINED

CLASS

I

GENES

Thegenetransfer techniquehas beenusedto identify all of the serologically defined class I genes in the BALB/c mouseexcept for the Qa-1gene (28). Thereis no monoclonalantibodyto the Qa-1antigen, and identification of class I geneproductsin mouseL cells after genetransfer is difficult with heterogeneousalloantisera. Six serologically defined genes have been locatedin their correspondingcosmidclusters andare identified in Figures 5 and 8. TheKd, Dd, Ld, Qa-2,3, and two Tla genes havebeen identified. In each case the mappingby restriction enzymesite polymorphisms correlates with the mapping by serologicalidentification after genetransfer (125). TheKb gene isolated from the C57BL/6mousealso has been identified by gene transfer experiments(74). Theb gene resides i n aclass I gene cluster containingtwoclass I genes,whichis similar in organizationto the BALB/c Kd gene cluster (27, 112, 125). Genomicclones for humanclass I moleculesalso have been transferred into mouseL cells to study expressionof HLA antigens (5, 62, 63). These studies haverevealed that humanclass I genes can be expressedin mouse fibroblasts and that these products associate efficiently with mouse$2microglobulinand in somecases induce conformationalchanges of mouse $2-microgiobulin(62). Four humanclass I genes encodingHLA-A2, HLAA3, HLA-B7,and HLA-CW3 antigens were identified with monoclonal antibodies (5; and F. Lemonnier,personal communication). NOVEL CLASSI GENEPRODUCTS Six of the 36 class I genes isolated from BALB/c DNA appear to encode serologically defined antigens (28). To determinewhetherany of the remainingclass I genes are expressed, a radioimmunoassay wasdevelopedfor cell-surface fl2-microgiobulin (28). MouseL cells express relatively constant amountsof the Kk and Dk molecules on their cell surface. Since eachknownclass I moleculeis associated with ~82-microglobulin,the amountof/~2-microglobulinassociated with

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MHC GENES 551 endogenousclass I gene products on the cell surface does not vary. When foreign class I genes are transferred into mouse L cells, the amountof surface ~2-rnicroglobulin generally increases, because foreign class I gene products also must associate with/32-rnicroglobulin to be expressed on the cell surface, and excess/~2-microglobulin appears to be synthesized. Thus, each of the 36 class I genes was independently transferred into mouseL cells and I0 novel gene products were identified by the/~2-nficroglobulin immunoassay(Figure 8). This assay wouldnot identify class I gene products expressed in low levels or not associated with ~2-microglobulin.All of these genes mapto the Qa-2,3 and Tla regions. Indeed, the two additional class I genes in the K and L gene clusters do not appear to be expressed by this gene transformation assay. One of the transformed L cells expressing a novel gene product was used to immunizeC3Hmice, the strain from which the L cells were derived (28). A specific alloantiserum to the novel gene product was raised. Analysis by two-dimensional gel electrophoresis after immunoprecipitation with this antiserum established that the novel gene product was 45,000 daltons and was associated with ~2-microglobulin. Thus, the novel gene product has the typical features of a class I molecule. Furthermore,this novel gene product appears to be present on spleen cells but not on cells from other tissues in the mouse. Alloantisera to these novel gene products should be useful in defining their tissue distribution, their developmentalregulation, and ultimately their functions.

Functionof Class I Genes Cytotoxic or killer T cells carry out an immunosurveillance to eliminate host cells that have been infected with virus (129). The T-cell receptor must recognize both the foreign viral antigen and a self transplantation antigen. The transplantation antigen is termed a restricting element, which permits the T cell to detect foreign antigens in the context of self. Aew~ordingly, the transplantation antigen plays an important role in permitting the cytotoxic T-cell system to distinguish self from nonself. DNA-mediated gene transfer permits the functional analysis of individual class I genesin two ways.First, the restricting elements (K, D or L) that permit different viruses to eliminated by cytotoxic T cells can be determined. Second, in vitro mutagenesis either by the shuffling of exons betweendifferent class I genes or by mutation of individual nucleotides can be used to perturb T-cell recognition and killing functions. Cellular assays such as the one depicted in Figure 10 have been employed to analyze cytotoxic T cells raised in response to a particular viral infection against mouseL cells expressing various class I genes (74, 82). Cytotoxic T cells raised in a mouseof a particular haplotype can only employrestrict-

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ing elements from the same haplotype. Thus, cytotoxic T cells raised in BALB/c mice against the LCMvirus can kill LCMvirus-infected mouse L cells only if they have been transformed with the appropriate BALB/c transplantation antigen. A summaryof the results of T-cell killing studies from several laboratories is presented in Table 2. Several stalking results emerge. First, a particular virus often uses a particular transplantation antigen as restriction element (e.g. the LCMvirus infection in the BALB/c d mouse employs the L element and the influenza infection in the C57BL/10 mouse employs the Kb element). Second, cytotoxic T cells raised in mutant d haplotype animal lacking the L gene (BALB/cdin2) do not employ the Ld gene as a restricting element (82). Thus, the dm2 mice appear to capable of employing another class I molecule as restriction element to L- cell transformants

~

LCMV i.p.

LCMV ~ 2 days ¯

7 doys spleen

trypsinize wash Slcr- labelling wash

suspension

cytotoxicity assay

Figure 10 A schematic ofthe procedures used in the testing of LCM virus-specific H-2 restriction withL-cell transformants.L cells transformedwiththe Ld geneare infected with LCM virus and then are labeled with 5~Cr.CytotoxicT cells isolated fromLCM virus-infected BALB/c mice are then used to showby 5~Crrelease that the Ld moleculeon the surface of the transformedL cells can serve as a restricting elementfor LCM virus.

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553

Table2 Productsof transfectedH-2class I genesact as Iestriction element for anti-viralcytotoxicTcells Transfected gene bH-2K dH-2L

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d H-2L

Virus Influenza Lymphocytic choriomeningitis virus Vesicular stomatitisvirus

References 74 82 25

achieve T-cell recognition and killing of LCMvirus-infected cells. Therefore, the systemis flexible. Third, cloned T cells behaveessentially the same way as their heterogeneous counterparts. Cloned T cells will be essential reagents in the in vitro mutagenesis experiments described bdow. Since a transplantation antigen is required to evoke a cytotoxic T-cell response against a particular virus, this element maybe mutated to determine which portions of the molecule are important for the recognition and effector functions of T-cell killing. Preliminary exon shuffling experiments between the Ld gene and gene 27.1 have been carried out (I. Stroynowski, personal communication).The ~ element can no l onger serve as a restricting element if both the a 1 and a2 exons are exchanged with homologous 27.1 exons. Thesedata suggest that the T-cell receptor recognizes antigenic determinants on the crl and/or or2 domains. Moreextensive in vitro mutagenesis and exon shuffling experiments for class I genes are being carried out nowin a numberof different laboratories. Structure and Expression of the ~2-Microglobulin Gene The exon-intron organization of the/~2-microglobulin gene, which appears to be a single-copy genein contrast to the class I multigenefamily, has been determined by DNAsequencing (84). This gene consists of four exons, with most of the coding region present in one exon (amino acids 3-95) (Figure 3.4). Fromthe comparison in Figure 3.4, it is obvious that the B2-microglobulin gene has a very similar exon-intron structure to the antibody, class I, and class II genes. This homologyalso is reflected at the aminoacid (88) and DNAsequence level (73, 84) and indicates an evolutionary relationship between these genes (see below). The gene for/~2-microglobulin is located in a region on chromosome2 encoding several non-MHC-linkedtransplantation antigens and several immuneresponse genes (75). Although/~2-microglobulin and class I genes are located on different chromosomes, they are coordinately regulated. Both genes are not expressed in teratocarcinoma stem ceils, which are presumablyequivalent to early embryoniccells, whereasboth are expressed in differentiated cell fines

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derived from the teratocarcinomastem cells (19, 77). Theregulation these genesappearsto be controlled transcriptionally. Analyses of t Haplotype Mice with Class I cDNAProbes Proximalto the MHC on chromosome 17 is a large genetic~region termed the t complex(7, 8, 103). Some25%of wild micecarry a mutationin this region that results in early embryonicdeath whenit is present on both chromosomes. Thesemutations fall into eight eomplementation groups(7, 8). A chromosome 17 that carries one of these mutations has been termed a t chromosome and its DNA is said to be of t haplotype origin. All t haplotypes showseveral unusual properties. First, males transmit the t chromosometo their progeny with a muchhigher frequency than the normalchromosome 17. Second, genetie recombinationbetweenthe t and the normalchromosome 17 is suppressedover a region of 20 cMthat starts fromthe centromereand includes the H-2complex(7, 8, 103). Avariety of biochemical, serological, and genetic data have suggested that the t complexmaybe evolutionarily related to the MHC complex(2). To analyze the t complex,a ehromosomal walk from the H-2 complexto the t complex maybe feasible (110). Asa first approach,class I eDNA clones have been usedto analyzeclass I genesby Southernblot analysesin different t haplotype micethat expressantigenicallydistinct class I molecules(100, 104). Surprisingly, restriction enzymesthat revealed a considerable polymorphismbetweenthe class I genesof different normalinbred strains of mice failed to showa similar kind of polymorphism betweendifferent t haplotypes. This finding has beeninterpreted to indicate a close evolutionary relationship of all t haplotypes.Thesignificanceof these observations,as well as the possibility of an evolutionaryrelationshipbetweenthe t complex and the MHC, remainsfor future studies to elucidate. CLASS II

GENES AND POLYPEPTIDES

Class H Polypeptides The I region of the MHC encodes the elass II molecules and has been divided by recombinationalanalyses into five distinct subregionsdenoted I-A, I-B, I-J, I-E, and I-C (78) (Figure 11). Twoof these subregions, and I-E, contain genes for the class II molecules, whichhave beenwell characterized by serological and biochemicalmethods.The two class II molecules encodedin the I-A and I-E subregions are both heterodimers composed of alpha and beta chains (Figure 11). Thealpha chains range molecularweight from30,000-33,000and the beta chains range in molecular weightfrom27,000-29,000.Thedifference in molecularweightbetween the t~ and ~ chains is primarily dueto differences in glycosylation.Thea

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MHCGENES chain contains two carbohydrate units, whereasonly one is boundto B chains. TheA~, A~, and E~genesare containedwithin the I-A subregion, whereasthe Ea geneis located within the I-E subregion(Figure 11). Athird subregion,I-J, appears to encodepolypeptidesthat are subunits of the suppressorfactors secreted by suppressorT cells. Althoughthe I-J polypeptideshavebeencharacterized in functional assays, their biochemical characterization is only beginning(53, 117, 120). Thecharacterization of suppressorfactors andtheir geneshas beenof vital interest becauseit represents a uniqueopportunity to study one form of the elusive T-cell rer, eptor. TheI-B andI-C subregionshavebeendefinedexclusivelyon the basis of their ability to modulatecertain immune responsesin mice, and someimmunologistsquestion whetherthese subregions exist (48). Thestructures of the class II polypeptideshavebeendeterminedprimarily fromthe analysis of humaneDNA clones (3, 32, 51, 57, 58, 60, 64, 113, 118, 123) and morerecently fromthe analysis of mouseand humangenomic clones (9, 50, 61, 71, 73). Onlyvery limited aminoacid sequenceinformation is availablefor mouseclass II molecules(116), whereasmoreextensive sequenceinformation has been obtained for humanclass II polypeptides (98). Thesedata suggest that the mouseI-A and I-E moleculesare homologousto the humanDCand DRmolecules, respectively (12) (Figure 1). counterpart to the third humanclass II molecule, SB(40), has not been found in the mouse.The three humanclass II moleculeseach have a and /5 chains denoted DCa,DC0, DRy, DR0, SBa, and SBO.Each class II polypeptideis composed of twoexternal domains,each about 90 aminoacid residues in length, ~1 and ~2, or fl 1 and ~2, a transmembrane region of about 30 residues, and a very short cytoplasmicregion of about 10--15 residues (Figure 2). Three of the four external domainshave centrally placed disulfide bridges (~2,/~1, and/~2). Thus,the class I and class moleculesappearto be very similar in overall structure and domainorganization (Figure 2). Theclass II moleculesserve as restricting elementsthat permitregulatory T ceils (helper, suppressor,amplifier)to viewantigenin the contextof self on the surface of other T cells, macrophages, or B cells (49, 79). Class genesappearto control the proliferationof regulatoryT cells as well as the effector reactionsearried out by these T cells, suchas the amplificationof other T-cell subsets and the promotionof B-cell differentiation. I A A~ A~ E~,

B

J

E

J?

E~

C

Figure11Geneticmapof the I region(seetext).

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DNA sequences the E~H - (73), Structure of ofClass GenesEk~(71), and A~(M. Malissen, personal communication) geneshavre beende’~erminedand ~he exon-intronorganization of these genesis illustrated in Figure12. Onceagainthere is a striking correlation betweenthe organizationof exonsandstructural domainsof the class II genes and molecules. Both gene~contain separate exons for the leader peptide and the a 1 and a2 domains.However,the E~gene employs a single exon to encodethe transmembrane and the cytoplasmicdomains, whereasthe A#gene appears to contain an intervening sequencein the cytoplasmicportion separating the exons encodingthe transmembrane and part of the cytoplasmicdomains.The Eogene has a fourth intron located in the 3’ untranslatedregion(71). Thus,the structures of the class II a and B genesappear to differ in several regards. Thegenefor B2-microglobulin showsan exon-intron organization very similar to that of the E~gene (Figure 3.4). For both genes, the exonencodingthe leader peptide is separated by a long intervening sequencefromthe rest of the codingsequence [2.8 kb for fl2-microglobulin(77) and about 2.3 kb for Eo (71)] and intervening DNA sequenceis found in the beginningof the 3’ untranslated region. TheA~genedisplays a structure moresimilar to a class I genein that transmembrane and at least part of the cytoplasmicregions are separated by an intron. Thesedata suggest that the class II ~ genes are more closely related to B2-microglobulin,whereasclass II ~ genes are more closely related to class I genes. ThehumanDRogene has been sequenced and has the sameexon-intron organization as the E~gene(50, 61). Comparison of the aminoacid sequencesencodedby the A~ and Ek genes has revealed an overall homologyof 50%betweenthe two proteins (9). Quite surprisingly, the most conservedregion is the transmembranedomain, whichexhibits 78%homology,perhaps indicating structurally and functionallyimportantinteractions of these parts of the moleculeswith the fl chains or other transmembrane proteins. ~’1

L

,81

,’,2 TM/CY/5’UT 3’UT

,82

TM

Figure 12 Schematicrepresentation of the exon-intronorganizationof the E~ and A~genes. Blackboxesshowexons; 3’ untranslated regions (3’ UT)are hatched; TMdenotes transmembrane; andCYindicates cytoplasmic.Theexonsencodingthe leader peptide and part of the cytoplasmic region and the 3’ untranslated region have not yet been identified by DNA sequencingfor the Aa gene. (Adaptedfrom70; and M. Malissen,personal communication.)

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MHC GENES 557

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Homology of Class L Class IL /~2-Microglobulin,Antibody, and Thy 1 Genes Thereare several similarities between class I, class II, 82-microglobulin, and antibodygenes(Figures2 and3A).First, all genesexhibit a precise correlation betweenexonsand the protein domainsthey encode.Second,the sizes of the external domainsandthe central placementandsize of the disulfide bridges are similar. Third, RNA splicing alwaysoccurs betweenthe first and second baseof the junctional codonsaccording to the GT/AG rule for both genefamilies. In other genesandgenefamilies, RNA splicing also can occurafter the secondand third base of the junctional codons(99). Fourth, X-ray data (P. Bjorkman,J. Strominger,and D. Wiley, personal communication) suggestthat the class I and antibodymoleculeshavea twofoldaxis of symmetry and, accordingly,probablyexhibit paired domains(Figure 2). Fifth, the DNA sequencesof the class I a3 exons, the class II 02 and 82 exons, and 82-microglobulinare homologous to the exon sequencesencoding antibodyconstantregions(9, 11, 50, 57, 58, 71, 73, 84, 109)(Figure A lowlevel of homology also has beenobservedbetweenthe class I a 1 and the class II 81 domains(58). Sixth, the class I al and~2 andthe class a 1 exonsdo not showsignificant sequencehomology to one another nor to the antibodygenes. However,their sizes are similar to those of the other class I, class II, and antibody exons. Furthermore,the class I a2 exon encodesa centrally placeddisulfide bridge. Theseobservationssuggestthat the completeclass I, class II, andantibodygenesshareda common ancestor andmarkedchangesoccurredafter divergenceof the genesto fulfill different functions. Finally, homology to the T-cell differentiation antigen, Thy 1, also is observedat the protein level (122). Becauseof their sequenceand structural relationships,it is attractive to postulatethat all of these genes ~lass I, class II, 82-microglobulin, immunoglobulin,and Thy 1--must have descendedfrom a common ancestor and are therefore membersof a supergenefamily. Twoqualifications mustbe raised. First, it is impossible to distinguish betweendivergent and convergentevolution. Accordingly, the membrane-proximal domainsof these molecules mayhave arisen from distinct genes that convergedtowardone another becauseof common functional constraints. If this hypothesisis correct, onewouldhaveto arguethat convergentevolution also generated moleculeswith external domainsof similar size and with similar disulfide bridge placement.This possibility appearsunlikely, but cannotbe formallyexcluded.Second,perhapsonly the membraneproximal domains evolved from a commonancestor, whereas the remainingportions of these genescould haveindependentorigins. Once again, the sharedsize andcentrally placeddisulfide bridgesof the external domainsmakethis an unattractive but formally possible hypothesis.

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Linkage Relationship of Class H Genes A continuous stretch of about 230 kb of DNA has been isolated from the I region by first screening a BALB/c spermcosmidlibrary with a human DR~eDNAprobe and then by using single-copy probes for chromosomal walking(110). Theclonedregion appearsto contain genesencodingall four serologieally defined class II polypeptides.Theclass II genes havebeen identified through the use of synthetic DNA probes whosesequenceswere generatedfrom protein data, mouseB cell-specific eDNA probes, and humaneDNA probes that cross-hybridized to the mouseclass II genes. These data are depicted in Figure 13. This 230-kbregion of DNA includes the right-hand boundaryof the I region, because the structural gene for the complementcomponentC4 mappinginto the S region of the MHC could be identified (M. Steinmetz, L. Fors, A. 0rn, unpublishedresults). TheC4genelies about 90 kb distal to the E~geneand wasidentified with a synthetic oligonueleotide probe specific for the N-terminal portion of the C4 a subunit. Theleft-hand boundaryof the I region cannot be defined at present becausethe cosmid clones do not extend into the K region. Five class II genes, extending over a stretch of DNA encompassing. approximately90 kb, have beenidentified. TheE~(73), A,, (20), and (M. Malissen,personal communication) genes havebeenidentified by direct DNA sequenceanalysis, the EOgene has beenidentified by hybridization with a specific oligonucleotide probe (110), and the EO2gene has been identified by cross-hybridization to a humanDCachain eDNA clone and the mouseEo gene (110). Identification of the Et~ gene waseonfumed the use of restriction enzyme site polymorphisms to localize a serologically defined EOrecombinantto the middle of the EOgene (110). Whetherthe E02gene is functional or represents a pseudogeneis unknown. The A0and Eo geneshavethe same5’ to 3’ orientation, whereasthe E,, geneshowsan opposite orientation. The orientation of the A~gene has not yet been determined. Thelocation of the A~genebetweenthe AOandEOgenesis different from the gene order of A~-Ao-E0proposed from peptide mapanalyses of the A~polypeptide (95). Strain A.TL,whichis an intra-H-2 recombinantdedyed from a cross between B10.S (A~), and A.AL(A~) was found bear an A,, chain whosepeptide fragments appeared to be a composite of As and Ak peptides. If this interpretation of the data is correct, the Aa ggne wott~d mapproximalto the Aagene. However,other investigators could not find a difference betweenthe A~polypeptides isolated from A.TLand B10.A(1R)mice (15, 72). Until the recombination point in the A.TLmice has been mappedat the DNA level this will remain con-

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troversial. However,the gene cloning data unequivocally suggest that the class II gene Order in BALB/cmice is 5’-A~-Aa-E~-Eo-3’. The region corresponding to that containing the five class II genes in BALB/cDNA(d haplotype) also has been isolated from the DNAof AKR mice (k ha..plotype) by screening an AKR cosmid library (M. Steinmetz, Fors, A. Orn, unpublished results). Twoimportant points arise from comparisonOf the Iregions from the k and d haplotypes (Figure 14). First, the same five class II genes are present in the k haplotype DNA.Moreover, their organization is virtually identical in both haplotypes without any major insertions, ddetions, or rearrangements. Second, a striking boundary of polymorphismproximal to the E~ gene is observed whenthe restriction maps of d and k haplotype DNAare compared. Although the two DNAs are almost identical to one another distal to this boundary, they show extensive restriction enzymesite polymorphismproximal to this point. Interestingly, this polymorphismbreakpoint correlates with a hot spot of recombination in the I region (see below). This difference in restriction enzyme site polymorphisms has been noted previously by Southern blot analyses of various mouse strains (110) and correlates nicely with the serological polymorphismsof the corresponding genes (47). Only two alleles each are knownfor the Eo, C4, and Sip genes, whereas the Aa and E# genes appear to be as polymorphic as the class I genes K and D. The important point is that the sequence conservation or restriction enzymesite polymorphismsare not confined to coding sequences but cover large regions of flanking DNA.This observation obviously has important implications for evolutionary mechanismsthat attempt to explain the generation of the extensive polymorphismof certain MHC alleles (see earlier discussion). Southern blot analyses have been carded out on mouseDNAwith t~ and fl probes under conditions that favor extensive cross-hybridization in an V//A HIGHLY BALB/c

0

~ CONSERVED

~?._ t?~

.._~ t? ~ ?~?_.,o_ ~_ ~ E/~ E#2 Ee

A#

A a

=~ ~=t?~

AKR

POLYMORPHIC

:~ 50

t~t?t~

:~__~?~I~_~ _~ ~ ~t?t~ I00 ~5Okb

~

S~lI

~

CIoI

~ Kpnl

Figure 14 A polymorphie breakpoint i~ the I region. Cloned regions of the I region BALB/e(d haplotype) and AKR(k haplotype) mice are compared over a stretch of of DNA.The region to the left ofthe breakpoint exhibits extensive restriction enzymesit~

lx~lyraorphisms, whereasthe regionto Nefight doesnot. Thepolymorphism breakl~int locateddoseto the recombinational hotspot at the 3’ endof the E~gene.(From M.Steinmetz L. ForsandA.~rn, unpublished results.)

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MHC GENES 561 attempt to define the total numberof a and fl genes (110). Thesedata suggest that there are two a genes and four to six ~ genes in the mouse genome.Thus,the total numberof class II genesthat can cross-react with the available class II DNA probes will be less than 10, althoughthe existence of more distantly related class II genes in the mousecannot be excluded.This striking paucityof class II genesstands in sharpcontrast to the exquisite specificity of immune reponsivenessregulatedby the class II genes. Thesedata suggest that the specificity of immune responsivenessis likely to be governedby the repertoire of T-cell receptors. Correlation of the Molecular with the Genetic Mapof the I Region Tensingle-copyDNA probeswere isolated fromvarious portions of the 230 kb of clonedDNA in the I region (110) (see Figure 13). Theseprobes used to search for restriction enzymesite polymorphisms in various inbred, eongenic, and recombinantcongenicstrains of mice. Thesepolymorphisms werethen correlated with the serological polymorphisms, whichdefine the various subregionsof the I region. In this mannerthe molecularmapof the I region (Figure 13) could be correlated with the genetic mapproduced recombinationalanalysis of serological polymorphisms (Figure 11). These analysesmapped eachof the four serologicallydefinedclass II genesto the appropriatesubregions(Figure 15). Thus,the A0, the A~, and at least the 5’ half of the E0genes are all encodedin the I-A subregion, whereasthe E~gene is encodedin the I-E subregion. The genetic and molecularmaps are consistent in the locations of these genes. Aninconsistencyexists betweenthe genetic and molecularmapsfor the I-B and I-J subregions. The analysis of nine recombinantcongenicmice defining the right-handand left-hand boundariesof the I-A and I-E subregions, respectively, haveled to the conclusionthat these twosubregionsare separated by no morethan 3.4 kb of DNA (110) (Figure 15). To complicate mattersfurther, the Yhalf of the Eogeneresides in at least 2.4 kb of this 3.4-kb region. Thus, about 1 kb of DNAremains for the I-B and I-J subregions. Indeed, it is possible that the I-A and I-E subre~ionsjoin directly to oneanother. Twoobservationsexcludethe possibility that the BALB/c mousehas deleted or rearranged the I-B and I-J subregions in comparisonto other mice. First, extensiveSouthernblotting data indicate that the organization of BALB/cDNA around the 3.4-kb region is no different fromthat foundin three other mousestrains (b, k, s haplotypes), whichhavebeenused to define the I-B and I-J subregions (110). Second, the corresponding regions in the cloned k and d haplotype DNAsare co-linear (M.Steinmetz,L. Fors, A. t)rn, unpublishedresults) (Figure

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O --O

Z _o~ =~

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MHC GENES 563

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These observations pose a paradox for explaining the location of the genes of the I-B and I-J subregions. Others have suggested that the I-B subregion can be explained by gene complementation between the I-A and I-E subregions (48). Our data supported the notion that the I-B subregion does not exist. However,it is moredifficult to dismiss the I-J subregion because I-J polypeptides have recently been identified by monoclonal antibodies (T. Tada, personal communication).

The Paradox of the 1-J Subregion Howcan the expression of the I-J polypeptides be encodedby 3.4 kb or less of DNA? There are two possible categories of explanations (110). First, the J gene or genes may be encoded outside that segment of DNAresiding between the I-A and I-E subregions. For example, the interpretation of recombinantevents in the recombinant congenic mice could be incorrect in that these mice mayhave undergone multiple recombinational rather than the usually assumedsingle recombinational events within the MHC.Alternatively, this 3.4-kb segment of DNAmaycontain a regulatory element that controls the expression of a battery of polymorphicJ genes located elsewhere in the mousegenome. A second category of hypotheses suggests that at least part of the J polypeptidesare encodedin this 3.4-kb region. The I-J gene might be identical with the Ea gene and appropriate post-translational modification(e.g. glycosylation, ere) could generate the serological determinants of the J polypeptide. Alternatively, the J polypeptides maybe encoded by someEa exons, whichare then linked to distinct I-J exons by alternative patterns of RNA splicing muchas alternative splicing exists for ~membrane and /x~retea mRNA’s of antibody heavy chain genes. Finally, the J gene may be transcribed off the DNAstrand opposite that which encodes the E a gene. The possibility that the J gene is encodedentirely in the remaining 1 kb of DNAin this region appears remote since most eukaryotic genes require far more DNA. Evidence has already been obtained that argues against the second category of explanations (52). RNAisolated from a numberof suppressor T-cell lines does not show hybridization to DNAsequences covering this 3.4-kb region. These RNAsalso fail to hybridize with extensive regions of DNA upstream and downstreamfrom the 3.4-kb region. These data suggest that the structural gene for I-J is not located betweenthe I-A and I-E subregions. The location of the J gene is a question of considerable importance because this polypeptide is expressed alone or in conjunction with another polypeptide in two distinct kinds of T-suppressor factors. These T-suppressor factors have been viewed by manyas the easiest way to approach the structure and molecular biology of the T-cell receptor. Hence, isolation of the I-J gene could be of considerable significance as an approach to the cloning of the T-cell receptor.

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Nonlinearity of Recombination Nine recombinanthaplotypes for the I-A and I-E subregions have been examinedby mappingof restriction enzymesite polymorphisms and in all strains the point of crossing-overfalls withina region of 8 kb of DNA (110) (Figure 15). Indeed, these recombinationsmayall have occurred at precisely the samepoint. Theseobservationssuggestthat recombination in the eukaryotiegenome is not randomlyscattered throughoutbut, at least in this case, is highly localized to a discrete region. Theoccurrenceof hot spots of recombinationhas obvious implications for any attempts to translate centimorgansof DNA from the genetic or recombinationalmapsinto kilobases of DNA from molecular maps. Such a conversion must assumethat recombinationis randomlyscattered throughout the mousegenome.Since the Aa, E~, and C4genes havebeen linked by molecularcloning, recombination frequenciesfor this region can be comparedto physical distances: As and Ea are 0.1 cMapart by recombinationalmappingand are separated by 85 kb of DNA.The E~ and C4 genes separated by 0.11 cMare 90 kb fromone another. Earlier calculations based on the assumptionof random recombinationsuggested that 1 eMshould equal 2000kb. Thusthe actual distances are shorter by about a factor of twoto three than the theoretical distances. It will be interesting to examineother chromosomal regions of the eukaryotic genomewheremultiple recombinations have been mapped to determinewhetherthe occurrenceof recombinational hot spots is specific to the I region or whetherthis is a muchmore general phenomenon.

Expression of Class H Genes Fourinbred strains of mice(b, f, q, s haplotypes)do not express the I-E moleculeon the cell surface. Twoof these (b and s) do not express the E~polypeptidebut have cytoplasmicEOchains, and two (f and q) neither expressE,, nor E~. BySouthernblot analyseswithspecific probes,it appears that the failure to expressthese genesis not dueto a completedeletion of these genesin the respective haplotypes(11). Moredetailed analysesof the structure and transcription of the Ea genein these inbred strains has revealed that at least two different mechanismsare responsible (70). transcription of the E~geneis observedin miceof the b and s haplotypes, presumablybecausea small deletion occurredin the promotorregion of the gene. Obviouslydefective RNAtranscripts of various sizes are found in mice of the f and q haplotypes. Noone has succeededunequivocallyin expressingclass II genesin mouse L ceils after DNA-mediated gene transfer. This mightbe due to the presence of a weakpromotorregulating class II genetranscription, inappropriate splicing, or instability of mRNA’s. Furthermore,the expressionof a

Annual Reviews MHC GENES 565 third chain, called the invadant (Ii) chain, might be necessary for the transport of class II a and fl chains to the cell surface (55). The powerful approachesof molecular biology will certainly lead to class II gene expression within the relatively near future. The long-termgoal is to transfer class II genes into biologically rdevant cells such as macrophages,B cells, and T cells so that their functions can be assessed and perturbed by in vitro mutagenesis.

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THE FUTURE Molecular genetics has given us stalking insights into the structures and organization of genes encoded by the MHC.The tools of molecular biology, when employedin conjunction with the well-developed assays of cellular immunology,provide a unique opportunity to dissect the functions of these multigene families involved in self-nonsdf recognition phenomena.It also will be interesting to determine whetheror not additional multigenefamilies fall into the supergenefamily, whichincludes the antibody, class I, and class II genes. Future experiments will showthe extent to which strategies of regulation and information expansion are shared by these homologousgene families. ACKNOWLEDGMENTS Wethank R. Flavell, B. Mach, H. McDevitt, and S. Nathenson for sending preprints of their workprior to publication, B. Larsh for her careful preparation of the manuscript, and E. Kraig and J. Parnes for thoughtful comments. M.S. was supported by a Senior Lievre Fellowship from the California Division of the American Cancer Society and B.M. was supported by a CNRSand DGRSTFellowship from the French Government. The work of L.H. and M.S. was supported by grants from the National Institutes of Health. LiteratureCited 1. Alper,C. A.1980TheRoleof the Major Histocompatibility Complexin lm. munology, ed. M.E. Doff,pp. 173-220. NewYork: Garland STPM 2. Artzt, K., Bennett,D. 1975. Nature 256:545-47 A.J., Roux-Dos3. Auffray,C., Korman, seto, M.,Bono,R., Strominger, J. L. 1982.Proc. NatL.4cad. Sc~USA79: 6337-41 4. Baltimore,D. 1981.Cell 24:592-94 5. Barbosa,J. A., Kamarck, M.E., Biro, P. A., Weissman, S. M.,Ruddle,F. H.

1982. Proc. NatLAcad.ScL USA79: 6327-31 6. Battey,J., Max,E. E., McBride, O.W., Swan,D., P. 1982.Proc.Natl. Acaa~ ScLLeder, USA79:5956-60 7. Bennett,D.1975.Cell 6:441-54 8. Bennett, D. 1980. HarveyLectures 74:1-21 9. Benoist,C. O., Mathis,D.J., Kanter, M.R., Williams, V.E., McDevitt, H.O. 1983. Proc. Natl. Acad.Sc£ USA.In press 10.Biro,P. A., Pereira, D., Sood,A.K., DeMartinville, B., Francke,N., Weiss-

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man,S. M. 1981. ImmunoglobulinIdiotypes, ICN-UCLASymposium on Molecular and Cell Biology, ed. C. Janeway, E. E. Sercarz, H. Wigzell, 20:315-26. NewYork: Academic 11. BrSl~Sg~re, F., Abastado, J. P., Kvist,$., Rask,L., Lalanne,J. L., et al. 1981. Nature 292:78-81 12. Bono,M.R., Stominger,J. L. 1982.Nature 299:836-38 13. Cami,B., Br~g~g~re,F., Abastado,J. P., Kourilsky, P. 1981. Nature 291: 673-75 14. Coligan,J. E., Kindt,T. J., Uehara,H., Martinko, J., Nathenson,S. G. 1981. Nature 291:35-39 15. Cook,R. G., Capra,J. D., Bednarczyk, J. L., U’hr,J. W.,Vitetta,E. S. 1979.J. lmmunol. 123:2799-803 16. Cosman,D., Khoury,G., Jay, G. 1982. Nature 295:73-76 17. Cosman,D., Kress, M., Khoury, G., Jay, G. 1982.Proc. Natl. dcad.Sc~USA 79:4947-51 18. Crabtree, G. R., Kant,J. A. 1982. Cell 31:159-66 19. Croc¢, C. M., Linnenbach, A., Huebner,K., Parnes, J. R., Margulies, D.H., et al. 1981.Proc.NatLAcad. US.478:5754-58 20. Davis, M. M., Nielsen, E., Cohen,D., Steinmetz, M., Hood, L. 1983. Proc. NatLAcad. ScL USA.In press 21. D6mant,P., Ivb~nyi, D., OudshoornSnoek, M., Calafat, J., Roos, M. H. 1981. Immunol.Rev. 60:5-22 22. Early, P., Rogers, J., Davis, M., Calame,K., Bond,M., et al. 1980. Cell 20:313-19 23. Evans, G. A., Margulies, D. H., Camerini-Otero, R. D., Ozato,K., Seidman,J. G. 1982. Proc. Natl. .4ca~ ScL USA79:1994-98 24. Flaherty, L. 1980. TheRole of the Major Histocompatibility Complexin Immunology,ed. M. E. Doff, pp. 33-57. NewYork: Garland STPM 25. Forman,L, Goodenow, R. S., Hood,L., Ciavarra, R. 1983. J. Exp. MecLIn press 26. Goding, J. W. 1981. J. ImmunoL 126:1644-46 27. Golden,L., Mellor,A. L., Weiss,E. H., Bullman,H., Bud,H., et aL Submitted for publication 28. Goodenow, R. $., McMillan,M., Nicolson, M., Sher, B. T., Ealde, K., et al. 1982. Nature300:231-37 29. Goodenow, R. S., McMillan,M., ~rn, A., Nicolson,M., Davidson,N., et al. 1982. Science 215:677-79

30. Gorer,P. A. 1938.J. Pathol.Bacteriol. 47:231-52 31. Gorer, P. A., Lyman,S., Snell, G. D. 1948. Proc. R. Soc. London Set. B 135:499-505 32. Gustafsson, K., Bill, P., Larhammar, D., Wiman,K., Claesson, L., et al. 1982. Scand. J. Immunol.16:303-8 33. Hansen,T. H., Ozato, K., Mellno, M. R., Coligan,J. E., Kindt,T. J., et al. 1981. Z ImmunoL126:1713-16 34. Hildemarm,W.H., Clark, F. A., Raison, R. L. 1981. ComprehensiveImmunogenetics~NewYork: Elsevier 35. Hildemarm,W. H., Johnston, I. S., Jokiel, P. L. 1979. Science 204:420-22 W,H., Jokiel, P. L., Bigger, 36. Hildemann, C. H., Johnston,I. S. 1980. Transplantation 30:297-301 37. Hollis, G. F., Hieter,P. A., McBride, O. W., Swan,D., Leder, P. 1982. Nature 297:83-84 38. Hood,L., Campbell,J. H,, Elgin, S. C. R. 1975..4nr~ Rev. Genet. 9:305-53 39. Hood, L., Steinmetz, M., Goodenow, R. 1982. Cell 28:685-87 40. Hurley, C. K., Shaw,S., Nadler, L., Schlossman,S., Capra, J. D. 1982. J. Exp. Med. 156:1557-62 41. Hurst, D. D., Fogel, S., Mortimer,R. K. 1972. Proc. Natl. Acad. Sc~ USA 69:101-5 42. Jackson,J. A., Fink,G. R. 1981.Nature 292:306-11 43. .4nn~ Jelinek, W.Biochem. R., Schnfid, C. W.1982. Rev. 51:813-44 44. Jordan, B. R., Br6g6g~re,F., Koudlsky, P. 1981. Nature290:521-23 45. Klein, J. 1975. Biology of the Mouse Histocompatibility Complex.Berlin: Springer 46. Klein, J. 1979. Science 203:516-21 47. Klein, J., Figueroa,F. 1981.Immunol. Rev. 60:23-57 48. Klein, J., Figueroa, F., Nagy,Z. A. 1983. Anr~ Rev. Immunol.1:119-42 49. Klein,J., Juretic, A., Baxevanis, C. N., Nagy,Z. A. 1981. Nature291:455--60 50. Korman,A. J., Auffray, C., Schambook,A., Strominger,J. L. 1982.Proc. Nat£ Acad. Sc£ USA79:6013-17 51. Korman,A. J., Knudsen,P. J., Kaufmann, J. F.,Acad. Strominger, L. 1982. Proc. Natl. Sc~ USAJ. 79:1844--48 52. Kronenberg, M., Steinmetz, M., Kobori, J., Kraig, E., Kapp,J., et al. Manuscriptin preparation 53. Krupen,K., Araneo,B. A., Brink, L., Kapp,J. A., Stein, S., et al. 1982.Pro¢. Natl. Aca~ Scl USA79:1254-58 54. Kvist, S., Br6g6g&e, F., Rask, L, Cami,

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74. Mellor, A. L., Golden,L., Weiss, E., Bullmann, H., Hurst, J., et al. 1982.Nature 298:529-34 75. Micha¢lson,J. 1981. Immunogenetics 13:167-71 76. Moore,K. W., Sher, B. T., Sun, Y. H., Eakle, K., Hood, L. 1982. Science 215:679-82 77. Morello,D., Daniel, F., Baldacci, P., Cayre,Y., Gachelin,G., Kourilsky,P. 1982. Nature 296:260-62 78. Murphy,D. B. 1980. The Role of the MajorHistocompatibility Complexin Immunology,ed. M.E. Doff, pp. 1-32. NewYork: Garland STPM 79. Nagy,Z., Baxevanis,C. N., Ishii, N., Klein, J. 1981. ImmunoL Rev. 60:59-83 80. Nairn, R., Yamaga,K., Nathenson,S. G. 1980. ,4n~ Rev. Genet. 14:241-77 81. Nathenson, S. G., Uehara, H., Ewenstein, B. M., Kindt,T. J., Coligan,J. E. 1981. ,4nr~ Rev. Biochem.50:1025-52 81a. Oilo, R., Rougeon,F. 1983. Cell. In press 82. Orn, A,, Goodenow, R. S., Hood,L., Brayton, P., Woodward, J. G., Frelinget, J. A. 1982. Nature297:415-17 83. Orr, H. T., Bach,F. H., Ploegh, H. L, Strominger, J. L., Kavathas, P., DeMars, R. 1982. Nature London 296:454-56 84. Parnes,J. R., Seidman,J. G. 1982.Cell 29:661-69 85. Parnes,J. R., Vdan,B., Felsenfeld,A., 65. Lop6zde Castro, J. A., Strominger,J. Ramanathan,L., Ferrini, U., ct al. L., Strong, D. M.,Sc£ Orr, H.79:3813-17 T. 1982. 1981. Proc. Natl. ,4cad. ScL US,478: Proc. Natl. ,4cad. USA 2253-57 66. Malissen, M., Damotte,M., Birnbaum, 86. Pease, L. R., Nathenson,S. G., LoinD., Trucy,J., Jordan,B. R. 1982.Gene. wand, L. A. 1982. Nature 298:382-85 20:485-9 87. Pease, L. R., Schulze,D. H., Pfaifen67. Malisscn,M., Malissen,B., Jordan, B. bach, M., Nathenson, S.InG. 1983. Proc. G. NatL ,~cad. Sc[ USA Press R. 1982. Prot~ NatL ,~cad. ScL 79:893-97 88. Peterson, P. A., Cunningham, B. A., 68. Maloy,W.L., Coligan,J. E. 1982.ImBerggard, Edelman, G. M. 1972. Proc. Natl.I.,~4cad. Sc£ USA 69:1697munogenetics16:11-22 69. Margulies,D. H., Evans,G. A., Flah1701 erty, L., Seidman,J. G. 1982. Nature 89. Plocgh,H. L., Orr, H. T., Strominger, 295:168-70 J. L. 1980.Proc. Natl. ~4cad.ScLUS,4 70. Mathis,D. J., Benoist,C. O., Williams, 77:6081-85 V. E., Kanter, M., McDevitt, H. O. 90. Ploegh,H. L., Orr, H. T., Strominger, 1983. Proc. Natl. ,4cad. ScL USA.In J. L. 1981.Cell 24:287-99 press 91. Reyes,A. A., Johnson,M.J., Sch~51d, 71. Mathis,D.J., Benoist,C. O., Williams, M., Ito, H., Ike, Y., et al. 1981. ImV. E., Kanter, M., McDevitt, H. O. munogenetics14:383-92 Submittedfor publication 92. Reyes,A. A., SchiSld,M., Itakura, K., 72. McMillan,M., Frelinger, J. A., Jones, Wallace,R. B. 1982.Proc. Natl. .4ca~ P. P., Murphy,D. B., McDevitt,H. O., ScL USA79:3270-74 Hood, L. 1981. Z Exp. Med. 153: 93. Reyes, A. A., Sch~ld, M., Wallace,R. 936-50 B. 1982. Immunogenetics16:1-9 73. McNicholas,J., Steinmetz, M., Hun- 94. Robinson,P. J., Lundin,L., Sege, K., kapiller, T., Jones, P., Hood,L. 1982. Graf, L., Wigzell,H., Peterson, P. A. Science. 218:1229-32 1981. Immunogenetics14:449-52

B., Garoff, H., et al. 1981.Proc. NatL ,4cad. ScL USA78:2772-76 K., Claesson,L., Pe55. Kvist, S., Wiman, terson, P. A., Dobberstein,B. 1982.Cell 29:61-69 56. Lalanne,J. L., Br6g6g~re, F., Delarbre, C., Abastado,J. P., Gachelin,G., Kourilsky, P. 1982. Nucleic Acids Res~ 10:1039-49 57. Larhammar, D., Gustafsson, K., Claesson, L., Bill, P., Wiman, K., ¢t al. 1982. Cell 40:153-61 58. Larhammar,D., Schenning, L., Gustafsson, K., Wiman, K., Claesson,L., et al. 1982. Proc. NatLAcad.Sci USA79: 3687-91 59. Ledcr, A., Swan, D., Ruddle, F., D’Eustachio,P., Leder,P. 1981. Nature 293:196-200 60. Lee,J. S., Trowsdale,J., Bodmer, W.F. 1982. Proc. Natl. ,~ca~ Sc~ USA79: 545-49 61. Lee,J. S., Trowsdale, J., Travers,P. J., Carey,J., Grosveld,F., et al. 1982.Nature 299:750-52 62. Lemonnier,F. A., Le Bouteiller, P., Malissen,B., Golstein, P., Malissen, M., et al. 1983. Z Immunol.In press 63. Lemonnier,F. A., Malissen, M., Golstein, P., LeBouteiller, P., Rebai,N., et al. 1982. Immunogenetics 16:355-61 64. Long,E. O., Wake,C. T., Strubin, M., Gross,Natl. N., Acc, olla,Sc£ K. US.4. S., et 79:7465-9 al. 1982. Proc. ~lcad.

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Proc. Natl. Acad. ScL USA79:5966-70 95. Rose,S. M., Cullen, S. E. 1981.J. Im114. Strominger,J. L., Orr, H. T., Parham, munol. 127:1472-77 96. Sehreier, P. H., Bothwell, k. L. M., P., Ploegh, H. L., Mann,D. L., et al. Mueller-Hill, B., Baltimore, D. 1981. 1980. Scand. J.. Immunol.11:573-92 Proc. Natl. Acad. ScL USA78:4995-99 115. Suggs,S. V., Wallace,R. B., Hirose,T., 97. Schulze,D. H., Pease, L. R., Wallace, Kawashima, H., ScL Itakura, K. 1981. Proc. Natl. E. Acad. USA78:6613-17 R. B., Nathenson,S. G., Seier, S. S., Reyes,A. A. Submittedfor publication 116. Sung,E., Hunkapiller,M. W., Hood,L. 98. Shackelford, D. A., Kaufman,J. F., E., Jones, P. P. Manuscriptin preparaKorman, A.Rev. J., Strominger, tion Immunol. 6:133-87 J. L. 1982. 117. Taniguchi, M., Tokuhisa, T., Kanno, 99. Sharp, P. A. 1981. Cell 23:643-46 M., Yaoita, Y., Shimizu,A., Hoajo,T. 100. Shin, H. S., Stavnezer,J., Artzt, K., 1982. Nature 298:172-74 Bennett, D. 1982. Cell 29:969-76 118. Wake,C. T., Long,E. O., Strubin, M., 101.Acad. Silver,SeL J., Hood, L. E. 1976. Proc. Natl. USA73:599-603 Gross, N., Accolla, R., Carrel, S., Mach,B. 1982. Proc. Natl. Acad~Sci. Silver, J., Hood, L. 1976. Contemporary 102. USA. 79:6979-83 Topics in MolecularImmunology,ed. 119. Weiss, E. H., M¢llor, A., Golden,L., H. N. Eisen, R. A. Reisfeld, 5:35-68. Fahrner, K., Simpson,E., ¢t aL SubmitNewYork: Plenum ted for publication 103. Silver, L. 1981. Cell 27:239-40 120. Wieder,K. J., Axaneo,B. A., Kapp,J. 104. Silver, L. M. 1982.Cell 29:961-68 A., Webb,D. R. 1982.Proc. Natl. Acad. 105. Singer, D. S., Camerini-Otero,R. D., Sc~ USA79:3599-3603 Satz, M.L., Osborne,B., Sachs, D., C. D., Crowther,C. E., Cripe, T. Rudikoff,S. 1982.Proc.Natl. Aca~ScL 121. Wilde, P., Gwo-ShuLee, M., Cowan,N. J. USA79:1403-7 1982. Nature 297:83-84 106. Snell, G.D., Dausset,J., Nathenson, S. 1976. Histocompatibility. NewYork: 122. Williams, A. F., Gagnon,J. 1982. Science 216:696-703 Academic 107. Soloski,M.J., U-hr,J. W.,Flaherty,L., 123. Wilson,P. H., Nairn,R., Nathenson,S. O., Sears, D. W.1982. Immunogenetics Vitetta, E. S. 1981.~. Exp. Mea~153: 15:225-37 1080-93 108. Sood,A. K., Pereira, D., Weissman, S. 124. Wiman,K., Larhammar,D., Claesson, L., Gustafsson,K., Schenning L., et al. M. 1981. Proc. Natl. Aca~ScL USA 1982. Proc. NatL Acae~ Sc~ USA 78:616-20 79:1703-7 109. Steinmetz,M., Frelinger, J. G., Fisher, 125. Winoto, A., Steinmetz, M., Hood,L. D., Hunkapiller,T., Pereira, D., et al. 1983. Proc. Natl. Acad. Sc£ USA.In 1981. Cell 24:125-34 press 110. Steinmetz,M., Minard,K., Horvath,S., 126. Xin, J. H., Kvist, S., Dobberstein,B. McNicholas,J., Frelinger, J., et al. 1982. EMBO J. 1:467-71 1982. Nature300:35-42 127. Yokoyama, K., Stockert, E., Old, L. J., 111. Steinmetz, M., Moore,K. W., Frelin. get, J. G., Shcr,B. T., Shen,F.-W.,et al. Nathenson, S. G. 1981. Proc. Natl. Acad. ScL USA78:7078-82 198LCell 25:683-92 K., Nathenson,S. G. 1983. 112. Steinmetz,M., Winoto,A., Minard,K., 128. Yokoyama, J. Immunol.In press Hood,L. 1982. Cell 28:489-98 113. Stetler, D., Das, H., Nunbcrg,J. H., 129. Zinkernagel, R. M., Doherty, P. C. Saiki, R., Shcng-Dong, R., et al. 1982. 1980. Adv. Immunol.27:51-177

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AnmRev. ImmunoL1983 1:569-607 Copyright©1983b.v AnnualReviewsIn~ All rights reserved

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GENETICS, EXPRESSION, AND FUNCTION OF IDIOTYPES Klaus Rajewsky and Toshitada Takemori Institute for Genetics,Universityof Cologne, Cologne, Federal Republicof Germany

1. INTRODUCTION In this article we speak about the networkof idiotypes in the immune system. Westart by defining the vocabularyweare goingto use. Wethen discussthe structural basis of idiotypicdeterminants (i.e. antigenicdeterminants on the antibodyvariable region; also called idiotopes) and of their complementary anti-idiotypic antibodybindingsites (anti-idiotopes). Idiotopes and anti-idiotopes are encodedby antibody V, D, and J genes. The idiotypic repertoire thus reflects the repertoire of these genesas they are expressed, selected, and somatically modifiedin the immune system. The idiotypicrepertoire, its geneticbasis andits selectionin ontogeny,is a main themeof the present view.This relates directly to the functional idiotypic network:Whichrules do idiotypie interactions in the immune systemfollow andwhatis their physiologicalrole? Wewill see that idiotypic interactions seemto be involvedin the growthcontrol of lymphocyte clones with defined receptorspecificity andmaythus be essential for the generationof a diverse systemof interacting ceils. Theyalso stabilize the expressionof a given receptorrepertoire in active immune responses.Idiotypie interactions occur within the compartment of B lymphocytes,but they also involveT cells. We discuss the crucial issue of whetheror not recognitionof B-cell idiotypes is oneof the principles by whichT cells are selected in the immune system. Thisarticle cannotdeal with all facets of idiotypic research. Werestrict ourselves largely to the discussionof experimentalworkin the mouseand, in particular, wedo not discussin anydetail the role of idiotypic interactions in regulatory T-cell circuits controlling immune responses. Detailed modelsof immunologiccontrol through cell interactions have emerged fromrecent workin this fidd. Werefer the disappointedreader to a few 569 0732-0582/83/0410-0569502.00

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reviews on this and other matters that are not discussed here (9, 22, 57, 73a, 78, 95, 184, 198, 204).

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2. VOCABULARYAND STRUCTURAL PRINCIPLES OF IDIOTYPIC INTERACTIONS BETWEENANTIBODIES Anidiotype (129, 155) is a set of idiotypic determinants or idiotopes expressed on the V region of a particular antibody or the V regions of a set of related antibodies. Idiotypes and idiotopes are defined serologically, i.e. by anti-idiotypic and anti-idiotope antibodies, respectively. Anti-idiotypic antibodies recognize, in general, a set of idiotopes and are therefore polyclonal. Anti-idiotope antibodies recognize individual idiotopes and are therefore, in general, monoclonalreagents. The idiotype of a given antibody distinguishes the latter from most other antibodies, but, as is discussed in more detail below, idiotypic crossreactions are frequently observed. In Figure 1 we depict schematically the interaction of two complementary antibodies, antibody 1 (abl) and antibody 2 (ab2). Wemayconsider ab2 as an anti-idiotope, raised against abl and binding with its hapten binding site (paratope) to an idiotope of ab 1. However,this distinction into anti-idiotope and idiotope-beadng antibody is merely operational: we might as well have immunized an animal with ab2 and obtained, as an antiidiotope, abl. In this situation the anti-idiotope wouldbind with a structure outside of the hapten binding cleft (paratope) to the idiotope-bearing ab2, and the idiotope recognized wouldbe the paratope of ab2 itself. The scheme in Figure 1 indicates that idiotypic interactions between antibodies occur through complementarystructures in antibody V regions and must involve aminoacid residues on protrusions and clefts of the interacting V regions. Becauseof the selectivity of idiotypic recognition, the interacting complementary structures must be largely determined by the hypervariable regions

pten1

~’~1~hapten2

Figure1 Schematic drawingof twointeracting antibodies.Complementary structures are meantto be in the Vregions.

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IDIOTYPES

571

of the V domains, an expectation verified by a large body of experimental work(see Section 3). It is satisfying to see that in the available threedimensional models of antibody V domains, obtained by X-ray diffraction techniques and indirect methods, the hypervadable regions are indeed partly arranged as pockets and partly exposed on the surface of the V domain(reviewed in 75, 159). If this were not the case, idiotypic interactions of the exquisite specificity observed wouldbe impossible.1 The structural requirements of idiotypic interactions as depicted in Figure 1 are no morethan a restatement of the "sticky end" modelof antibody V domains developed by Hoffmann (88). The matter has recently been discussed in detail by Jerne et al (101), and wewouldlike to point out, along with the latter discussion, a fewspecific features of idiotypic interactions as they can be deduced from the schemein Figure 1. If we had raised ab2 as anti-idiotope against abl, 2 we would find that the anti-idiotope would not competewith hapten (hapten 1) for binding to its target (abl). In classical nomenclature we would call the idiotope recognized by the anti-idiotope non-binding-site related. If, on the other hand, ab 1 were the aati-idiotope it wouldcompetewith the hapten (hapten 2) for binding to its target (ab2); here the idiotope recognized wouldbe binding-site-related and, in fact, could be the binding site itself. In the original formulation of the network hypothesis (97), the structure on abl recognizing ab2 is the internal image of hapten 2. In line with these considerations, binding-site-related (28) and nonbinding-site-related idiotopes (114) have been described in a variety experimental systems. It is clear, however,that a hapten-inhibitable antiidiotope must not necessarily be a true internal image of the corresponding hapten, but may recognize an idiotope that is associated with the hapten binding site for structural and/or genetic reasons. Genetic associations even of nonbinding-site-related idiotopes with certain binding specificities have indeed been observed and are discussed below (Section 4.3). On the other hand, there is also goodevidence for the existence of true internal images of ligands from the word outside the immunesystem within the immune systemitself, at the level of antibodyidiotopes. Stalking examplesof this are anti-idiotypic antibodies against various hormone-specific antibodies. SubtThearrangement of the hypervadable loops in antibodyVdomainshas presumably been selectedin evolutionnot onlyto permitefficient idiotypieinteractionsof antibodies.The interactionof antibodieswithexternalantigenssuchas proteinsmayoftennot be restricted to a cleft in the Vregion,as suggested for smallhaptensbythe availablethree-dimensional models,but mayalso involveresiduesat the Vregionsurfaceas in the interactionof the V regionof proteinKolwithits ownFcpiece(90, 138). 2Inthe nomenclature of Jerneet al (101),ab2in Figure1 would be an anti-idiotope (against abl) of the ~ type(ab2a),whereas abl wouldbe anti-idiotope(against ab2) of the~ ty (ab2fl).

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sets of such anti-idiotypic antibodieshavebeenshownto be able to mediate hormone-specific effects (183, 193). Likewise,a selected anti-idiotypic antiserum against antibodies with specificity for tobaccomosaicvirus (TMV) coat protein wasable to induce anti-TMV antibodies in animalsof several species (132). Thereis also evidencefor internal imagesof constant region allotypes at the level of antibodyidiotypes in the rabbit (101, 178). As shownbelow, the idiotypic repertoire in any given animalis genetically restricted so that not any possible three-dimensionaldeterminantis expressedat the idiotopelevel (see Section4.3). Internal imagesmaytherefore exist for certain antigenic determinantsbut not for others. Let us reiterate that idiotope-bearingand anti-idiotopeantibody(idiotype and anti-idiotype) are operational terms by whichwelabel the partners in an idiotypic interaction (Figure 1) on the basis of an ad hoe criterion (mostlyconsideringas "anti-idiotope" the antibodyraised by immunization with the "idiotope-bearing"antibody). Whenone considers the functional role of idiotypic interactions in the immune system,oneshouldbear in mind that the anti-idiotope maycontrol the expressionof the idiotope-bearing antibodyas well as vice versa. As is shownbelow, this fundamentalpoint has not beensufficiently taken into considerationin the interpretation of regulatory experiments. 3. STRUCTURAL

LOCALIZATION

OF IDIOTOPES

Canwestructurally localize idiotopes within theV region? Thisquestion isofimportance notonly fortheunderstanding ofidiotypic interactions (see above), buta/soforthegeneticist whowants touseidiotopes asmarkers ofthegenes thatencode antibody V regions (Section 4.3). Intheabsence ofdirect information based uponX-ray analysis, oneisleftwithattempts tolocalize idiotopcs onsubunits oftheantibody molecule andtocorrelate amino acidsequences withidiotopc expression. Asonemight expect, a complex picture emerges fromthiskindofanalysis. Chain reassociation experiments haveshown thatidiotypic determinants usually require both heavy (H)andlight (L)chains forfull expression, although incertain cases some reactivity ofisolated chains with theanti-idiotypic antibody isfound (e.g. 19a,30,33a, 34,74,79,90a,134, 180a, 190a, 213;seealso 57,89). Anti-idiotypic reagents specific fordeterminants oneither L orH chain can bcselectively induced byimmunizing withhybrid antibody molecules in whicheither theH or theL chains arcderived frompooled "normal" immunogiobulin (I90,220). Suchrcagcnts arcparticularly useful forstudyingthegenetic basis ofV gcncexpression. Inspection ofamino acidsequences inidiotypically related mycloma andhybddoma antibodies support thenotion thatidiotopes arcphcnotypic markers of complementarity-

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IDIOTYPES 573 determining regions (CDR)(33, 34). In the case of a(1-3)-dextran (4-hydroxy-3-nitrophenyl)acetyl (NP)-binding k-chain-bearing murine antibodies, attempts have beenmadeto correlate aminoacid sequencesof the V, D, and J segmentsof the H chain with the expression of certain idiotypic determinants.[k-Chainbearingmurineantibodiesare particularly useful for this purposesince ), chains showlittle variability in the mouse (207).] In the case of ¢(1-3)-dextran antibodies, wherethe aminoacid sequencesof a large numberof closely related antibodies havebeendetermined(181), it appearedthat the expressionof a certain idiotypic determinant wasdependenton two positions in the D segment,and that of another was dependent on two residues in the second CDR(CDR2in V~(38). However,as the authors noted, the situation is muchmore complexthan wouldappearat first glance. Table1 showsthe reaction of three monoclonal anti-idiotope antibodieswith a series of monoclonal anti-dextranantibodies of definedprimarystructure. Inspectionof the data reveals that the interaction of idiotype and anti-idiotope (whichin all cases also dependson the presenceof the ~ light chain) canin no casebe attributed to a single stretch of aminoacids, and sequencesin V, D, and J seemto be involvedin various combinations.Table1 also contains sequencesand idiotypic characteristics of a series of closely related anti-NPantibodies together with a non-NPbindingsomaticvariant (the sourcesof these antibodies are given in Table 1). Althoughthe D region is again clearly of importancefor idiotope expression, the Vri region is also involved. (Comparethe expression of idiotope Ac146in antibodies B1-8, B1-8.V3,B1-48, and B1-8.V1;and of idiotope Ac38in antibodies B1-8, B1-48, G100.24,and B1-8.V3.For the contributionOf VHto idiotope Ac38,see also Section4.3.) Thus,the situation is again morecomplexthan originally anticipated (172). That amino acid residuesin both DandVHcan contributeto the constructionof a single idiotopeis also stronglysuggestedby the workof Potter andhis collaborators on the idiotypic propertiesof a series of interrelated galactan-binding antibodies (160). Oneof the idiotopes they analyzedappearedto be influenced by amino acid residues in D and CDR2,and three-dimensional modelsgenerated by a computerprogramindicate that the residues in questionare exposedon the surface of the V domain,in reasonabledistance from each other. Other idiotypic determinants in the samesystem also appear to involve the participation of morethan a single CDR. Despitethe complexstructure of idiotopes, very smallchangesin primary sequencecan cause drastic changesin idiotypic specificity. Anexampleof this is antibodyB1-8.V3in Tablel, wherea single aminoacid exchangein Dleads to the loss of oneof the idiotopes, the modificationof the second, and, in fact, to the loss of six other idiotopes of the parent molecule.The variant antibody fully retains NP-bindingspecificity (A. Radbruch,S. Zaiss, K. Rajewsky,K. Bcyreuther, to be published).

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Table 1 Idiotopes and primary structure regions

of c~(1-3)-dextran

bV region segments

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a Antibody J558 Hdex9 Hdex36 Hdex2 Hdex25 Hdexl2 Hdex24 MIOC104E

B1-8 B1-48 G100.16 G100.24 B1-8.V3 B1-8.V1

V H 1 3 1 1 1 1 1 1

186-2 186-2 186-2 186-2 186-2 186-2/102

cIdiotopes

D

JH

RY RY RY NY SY GN SS YD

1 1 2 1 1 4 2 1

YD. YYGSS -L LG Y.. - .... --TH ..... R-....

and NP-binding antibody

2 1 4 2 2 2

EB3-72

EB3-16

AB3

+++ ++ ++ ++ ++ ++ ++ -

+++ + + -

+++ ++ +++ ++ -

Ac38

Ac146

NP-bdg

+ + + +

+ dND eCR -

+ + + + + -

acffl-3)-dextran (upper panel) and NP-binding(lower panel) myelomaand hybridomaproteins. bAminoacid sequences of heavy-chain variable regions in abbreviated form (the L chains are germline-encoded ~.1 chains in all cases). TheHsegment si i dentical i n most of t he dextran-binding (VHI) and the NP-binding molecules (V186-2). VH3differs from VH1in 4 positions in CDR2. VH186-2/102 differs from VH186-2in 10 positions, most of them in CDR2.The sequences of the Dsegmentare given in the one-letter code. The JHsegmentis also indicated. The sequencedata on dextran binding moleculesare taken from Schilling et al and Clevinger et al (38,181). Thesequences of the NP-binding molecules are from Bothwell et al (23) (B1-8); A. Bothwell, M. Paskind, Baltimore, personal communication(B1-48); H. Sakano, K. Karjalainen, personal communication (G100.16and G100.24); S. Zaiss, K. Beyreuther, personal communication(B1-8.V3); and Dildrop et al (49) (B1--8.V1). CEB3-72,EB3-16,and AB3are idiotopes of J558. The typing results are from Clevinger (37). Ac38and Ac146are idiotopes of B1-8. NP-bdg, NP-binding. The typing of B1-8.V3was done by A. Radbruch. dNot determined. eAffinity of anti-idiotope 10× lower than on B1-8.

Thedatain Table1 also showthat the absenceof a binding-site-related idiotope[Ac146(174)] is not necessarilyaccompanied by a changein hapten bindingspecificity. Clearly,therefore,the corresponding anti-idiotope andthe hapteninteract with the target antibodyin different ways.The anti-idiotypemaybind to a larger surfacearea than the haptenor to an idiotopethat is, for structuraland/orgeneticreasons,in its close vicinity. Anidiotope of the latter kind could, at the level of the total antibody repertoire,be mostlyassociatedwithantibodiesof the samehaptenspecificity--as indeedhas beenobservedin the case of idiotope Ac146--although the corresponding anti-idiotopedoesnot carrythe "internalimage"of the hapten.

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To concludethis section, idiotypic determinantsappear to be complex structures to whichoften both Hand L chains and, within one chain, more than a single CDRmaycontribute. Theymaybe distant from, or closely associatedwith, a hapten-binding site of the antibody,but evenan idiotope invariablyassociatedwith antibodiesof a givenhaptenspecificity andrecognized by a hapten-inhibitable anti-idiotope does not necessarily coincide with the haptenbinding site. 4.

THE IDIOTYPIC

REPERTOIRE

4.1 What are the Questions? Idiotypesare serological markersof antibodyVregions, andthus in principle allowthe analysis of the expressionandselection of antibodyVregions in the immune system. In viewof the diversity of V regions, any analysis of the repertoire remains a priori fragmentary. However,a numberof questionscan be askedthat allowstraightforwardanswers.(a) Is the idiotypie repertoire generatedat the B-cell level restricted andcan wecorrelate it with the genetic informationavailable in the antibodystructural genes? (b) AreB cells selected accordingto their receptoridiotype whenthey leave the bone marrowand enter the peripheral immunesystem?(c) Howare cells selected in antigen-driven immune responsesand, specifically, are idiotypicinteractionsinvolvedin this selection?(d) If idiotypieinteractions are involvedin B-cell selection at any stage, whatis its mechanism? Thesequestions lead to the field of regulatory T cells, whichare known to be able to specifically recognizeandexpressidiotypic markers.Wedis- " cuss later the problemsof T-cell idiotypes and their regulatory function. 4.2 Analyzing the Idiotypic Repertoire of B-Cell Receptors Theclassical wayof studyingidiotype expressionin the immune systemis the idiotypic analysis of antibodies producedin antigen-driven immune responses.This approachis limitedin severalways.First, it is restricted to antibodies of a given antigen-bindingspecificity, and thus does not allow one to study the representation of a given idiotypic markerin the total population of antibodies. This limitation can be overcomeby inducing idiotypic responsesby immunization with anti-idiotypic antibodies, an approach pioneered by the groups of Urbain (205) and Cazenave(35). analysis of suchresponsesis complicatedby the fact that an anti-idiotypic antibody will induce the production of two types of complementary antibodies, namely(a) antibodies carrying the idiotype defined by the antiidiotype used for immunization,and (b) anti-anti-idiotypes. Theusual serological assays used for idiotype titration do not distinguish between these twotypes of molecules.This has led to confusionin the literature for

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the following reason: If any complementary antibody produced in response to immunizationwith anti-idiotypc is considered to express the corresponding idiotypc, then the idiotypc will be found expressed in any immune system, since each system is sufficiently diverse to allow the induction of complementaryantibodies. Several ways arc open to distinguish between idiotype and anti-anti-idiotypc produced in response to anti-idiotype. One is to identify idiotypc by a variety of anti-idiotypic reagents in addition to the one used for immunization (18, 217). Another one is to identify the idiotype by an unusual structural property such as, for example, expression of k chains in the case of the mouse. Since in mice k chains are expressed in less than 5% of total immunoglobulin, anti-anti-idiotypes would bc expected to express r light chains. The latter approachhas been successfully used (170, 171, 199), and its validity has been recently verified in an independent (third) way that is generally applicable (19), namely, by showing that the idiotypic molecules were indeed encoded by V genes similar to those encoding the original idiotypic antibody (M. Siekevitz, R. Dildrop, K. Bcyrcuthcr, K. Rajcwsky, to be published). Methods of this kind are essential for the identification of idiotypic molecules producedin response to anti-idiotope. One should bear in mind, however, that they all give a minimumestimate of the idiotypic response and might classify an unknown fraction of idiotypic moleculesas anti-anti-idiotopes. The mainproblem of analyzing idiotypic repertoires a.t the level of induced serum antibodies is that the cells producing these antibodies have already been subject to complexregulation, be it before or after immunization. Onewouldtherefore like to see howthe idiotypic patterns determined at the level of serum antibodies compare to the frequencies of B cells expressing the corresponding idiotypic determinants at the various stages of B-cell ontogeny. Several methodsare available for this purpose. One is the limiting dilution assay for mitoge~-reactive B cells (4, 5), in whichthe frequency of mitogen-reactive B cells expressing a #oven antibody V region can be determined. The assay is a priori limited to mitogen-reactive cells --~ the case of bacterial lipopolysacchadde (LPS), roughly every third cell in the spleen of a young mouse(4). Since LPS-reactive cells can generated from bone marrowor fetal liver pre-B cells in vitro (66, 141), frequency determinations of, for example, idiotype-expressing cells in the population of newly generated B lymphocytes are possible (147). During ontogeny, certain fractions of the newly generated (LPS-reactive) B cells becomeresponsive to T-cell help (77, 120, 189) and (or?) enter the pool long-lived, recirculating B cells (64, 191, 192). As T-cell lines delivering help to B cells in vitro in a polyclonal or antigen-specific fashion have recently becomeavailable (6, 105, 117, 162), frequency determinations B cells responsive to T-cell help in an in vitro systemappear feasible, but

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they havenot yet been carried out. However,the morecomplicatedtechnique developedby Klinmanand his colleagues (119), in whichlimiting numbersof B cells are injected into irradiated hosts andthe T-cell dependent responsesof single cell clonesin splenicfragmentsculturedin vitro are recorded,is thoughtto allow frequencydeterminationsin just that B-cell population.Thereis thus a variety of methodsavailable for the determinations of V-regionrepertoires in B cells, eachmethoddeterminingthe repertoire expressedin cells at a particular stage of differentiation.

4.3 The GeneticBasis of the Idiotypic Repertoire Classicalworkon the expressionof idiotypesat the level of inducedantibodies (reviewedin 57) has revealed two types of idiotypic markers,private (minor) idiotypes, expressedirregularly in someindividuals of a given species or strain, and recurrent (major, cross-reactive) idiotypes, which regularly appear in certain immune responsesin the members of a certain speciesor strain or evenseveral species(102b,200a).In the case of strainspecific recurrentidiotypes,geneticanalysis has shown that idiotypeexpression is almost invariably controlled by antibodystructural genesonly, in most eases by the Vn(reviewed in 208), in someinstances by both the VI~and the VLlocus (51, 91, 107, 149). The interpretation of these resuits wasthat idiotypes are markersof antibodyVgenes, that strain-specific idiotypes are due to V-genepolymorphism, and that the predominant control by the VHlocus reflects moreextensive polymorphism of VHthan V~. genes. (Wenowknowthat D and J region polymorphism must also be taken into account.) Byusing recombinantmousestrains it waspossible to construct roughgenetic mapsin whichregions controlling various idiotypic markers were separated from each other by recombination breakpoints. Thesesubjects have been extensively reviewed(56, 137, 208). The modem recombinantDNA techniques have madeit possible to study the genetic basis.ofidiotypeexpressionat the molecularlevel. Suchstudies havelargely confirmedthe classical workand, in addition, dramatically improvedour understandingof the problem.The molecularanalysis of several immune responses dominatedby recurrent idiotypes showedthat each of these responsesis basedon the expressionof a single or very few Vnanda single or very few VLgenesin the germline. This is the case in the responseof miceto arsonate [dominatedby the Arsidiotype (148)], a(1-3)-dextran J558 and MOPC104E idiotype (17, 182)], phosphorylcholine(PC) [domimated by the T15 idiotype (41, 133)], and NP[dominated by the b idiotype (137, 93)]. In these four systems,the antibodyresponseis known to consist of oneor a fewantibodyfamilies, each of whichis characterized by the expression of a single Vngene in combinationwith several D and JI~ segmentsand of one or a few V~and Ji~ genes(23, 24, 33a, 38, 48, 72,

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78, 173, 174, 181, 187) [for the molecularbiology of antibodystructural genes, see Honjo(88a) in this volume].Themembers of each familyare thus closely related to eachother, but the families are verylarge, as in addition to combinatorialdiversity, somaticmutationsin the rearranged variable region genes occur, sometimesat high frequency(11, 23, 24, 48, 72, 161, 181,187, 207; for further families ofidiotypicallyrelated antibodiessee 78). Thus,at least in responsesto certain epitopes, the Y-regionrepertoire is controlled and limited by a few germlinegenes, despite extensive somatic mutation--amodernversion of the "germlinetheory" of antibodydiversity. Asimple interpretation of the phenomenon of recurrent and private idiotypes in antigen-inducedimmuneresponses emerges:recurrent idiotypes are markers of those germline ¥ genes from whichthe antibodies in the correspondingimmune responseare derived and of part of their somatically mutatedprogeny.Private idiotypes are characteristic for somaticmutants. Wehavetested this propositionwhichhas beenwidelyentertained [78] with a pair of monoclonalanti-NP antibodies derived from C57BL/6mice. AntibodyB 1-8 expresses a germline-encodedV region; antibody $43 uses the sameV-regiongenesbut carries somaticpoint mutationsboth in VII and YL(23, 24,173). Non-cross-reactingidiotopes on antibody B1-8and $43, respectively, were defined by monoclonalanti-idiotope antibodies. As expected, the B 1-8 idiotopes are regularly expressedin anti-NPresponsesof C57BL/6mice (111, 174, 175). In contrast, the $43 idiotopes are only occasionallydetectable, and eventhen at very low levels (G. Wildner,T. Takemori,K. Rajewsky,to be published). Theresult is consistent with the viewthat recurrent idiotopes are markersof germlineV(and possibly D) genesand that idiotopes specific for somaticvariants are irregularly expressedand thus private. This does not, of course, necessarily implythat all private idiotypic markersmust be the result of somaticmutation, nor that all germlineV genesare regularly expressedin the immune system;we return to these pointslater. Thefindingof extensivesomaticmutationin B cells engagedin a specific immune responsealso providesa potential explanationfor a finding that has puzzledimmunologists since its original description (156), namely,that idiotypic cross-reactivityof antibodiesdirected at different determinantsof the immunizing antigen (64a, 87b, 102a, 108, 125, 143, 170, 199, 214a,217). Onemightspeculate that in a given B-cell clone, activated in responseto oneof the antigenic sites of the immunizing antigen, somaticvariants arise, someof whichexpress specificity to another determinant on the same antigen. Suchvariants wouldthen be further stimulated by the immunogen and maybe selected for higher affinity by subsequentsomaticmutation.The mutantsmaystill share idiotypic specificity with the antibodyexpressedby the original ("wild type") clone, and the idiotypie networkmightthus also

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IDIOTYPES 579 contribute to their selection (97). This picture has recently emergedfrom the elegant experimentsby W.Gerhardand his colleagues in whichmonoclonal antibodieswith specificity for influenzahemagglutinin wereisolated from individual immunizedmice and were comparedin terms of primary structure andepitope andidiotypic specificity. Theevidencesuggestsstepwisemutationin single B-cell clones, resulting in the productionof antibodies with different, fine specificities (194; W.Gerhard,M.Weigert,personal communication). These considerations add an important aspect to the problemof the generationof the antibodyrepertoire. If somaticB-cell mutantswith altered bindingspecificity constitute a substantial part of the repertoire of functional B cells they might play an important role in responsesfor which suitable V, D, and J genes have not been selected in the germline, e.g. responses against variants of common pathogens. Wecan only say at this point that somaticmutationis not extensive enoughto obscurethe clear pattern of germlinedeterminationin responseslike those to NP,Ars, and PC. Geneticdeterminationandrestriction of the idiotope repertoire can also be demonstratedwhenidiotope expressionis studied in the total antibody populationrather than in populations defined by a given antigen-binding specificity. In the initial experimentsof this type, rabbits wereimmunized with anti-idiotypic antibodies (ab2 in the designationof the authors) produce antibodies complementaryto ab2 (ab3). The population of ab3 containedlittle or no antibodieswith specificity for the antigen, whichwas recognized by the idiotypic antibody (abl) against whichab2 has been raised; however,uponimmunizationwith that antigen, the ab2-sensitized animalsproducedantigen-specificantibodies(ab 1’) that wereidiotypieally indistinguishable from abl (35, 205). Theseexperimentswere later reproducedin other systems(18, 20, 131, 132, 170, 171, 199, 217). It wasalso shownthat in certain experimentalsystemsthe response to ab2 wasdominatedby ab 1, as definedby its antigen-binding specificity (178). Thus,a way appearedopento determinethe idiotypic repertoire in the functionally active B-cell population.Suchexperimentsrequired, however,the differentiation in the ab3populationof idiotype-bearingantibodiesfromanti-antiidiotypes. When this wasdone(as outlinedin Section4.2), twoclear results emerged.In general, the populationcontains a large component of idiotypebearing antibodies that do not share antigen bindingspecificity with the idiotype-bearingantibodiesusedfor the inductionof ab2 (18, 170, 171,199, 217). Togetherwith the workon idiotype sharing betweenantibodies of differentfine speeiticity, the structural data discussedin Section3, andother work(52a, 60), this clearly demonstratesthat the distributions of idiotopes and antigen-bindingspecificities in antibody-Vregions are overlappingbut

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not identical. This important point is exemplified by the data in Figure 2, where the expression of two idiotopes of the germline-eneoded anti-NP antibody B 1-8 in the total antibody population is analyzed, upon immunization with the corresponding cross-linked anti-idiotopes. Clearly, one of the two idiotopes (Ac38) is mainly expressed in non-NPbinding antibodies, whereas another (Ac146) is largely restricted to antibodies carrying binding sites. The appearance ofidiotope Ac38in non-NPbinding antibodies is not due only to somatic mutation or to different waysof V-D-Jjoining. The structural analysis of a somatic variant of antibody B1-8, in whichpart of the VrI region is encodedby a different germ line Vri gene (30, 49) and of anti-idiotypically induced Ac38+ monoclonalantibodies (R. Dildrop, M. Siekevitz, K. Rajewsky,K. Beyreuther, to be published), demonstrates that a series of closely related Vri genes, only one of which is involved in encoding NP-specitic antibodies, encodes Vn regions of Ac38+ molecules. This is direct evidence for idiotype sharing between products of V genes in the germline expressedin antibodies differing in antigen-bindingspecificity. A second result can also be read from the data in Figure 2, namely, that also in this approachidiotope expressionturns out to be strictly genetically ~l-bearing

antibodies(~ug/ml)

1.0 10 100

1.0 10 100

1.0 10 100

÷ A. Ac38

÷ B. Ac146

÷ C. A6-24

1.0 10

C57BL/6 BALB/c CB.20 D. NPb

Figure2 Analysisof idiotopeexpressionby immunization witha cross-linkedmonoelonal anti-idiotopeantibody.Thisantibody(Ae38)detectsan idiotope(Ae38)on the germlineencodedNP-binding antibodyB1-8.Thelatter antibodyalso carries idiotopesAc146and A6-24,definedbymonoclonal anti-idiotopes.Alltheseidiotopesare recurrentlyexpressed in anti-NPresponsesof micecarryingthe Ighb haplotype.Idiotope-bearingandNP-binding molecules in the sera of miceimmunized withcross-linkedantibodyAc38weredetectedon plates coatedwitheitheranti-idiotope(left threepanels)or NP-bovine serumalbumin (right panel),witha radioactive monoclonal anti-k1 antibody [the anti-idiotopes carry~¢chains,the idiotope-bearing molecules carryk chains(174)].Absorption oninsolubilizedantibodyAc38 removed> 98%of all idiotypie and NP-binding molecules,absorptionon NF-Sepharose + and 17%Ac38 + moleculesin strain C57BL/6 removed83%Ac146 (Ighb/b). Strain CB.20 b carries the Igh locusonthe BALB/c background. BALB/c is Igh~/°. Thedata are takenfrom Takemori et al (199),wherefurtherdetails canbe found.

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IDIOTYPF-,8 5 81 controlled, again by genesin the Igh linkage group. Thus,Ighb/b but not Igha/a mice express the Ac146and A6-24idiotopes, and although both + antibodies, it is only in Ighb/b micethat strains of micecan produceAc38 this idiotope is associated in part with NPbinding specificity. Since in + NP-bindingantibodies of the IgG1class all (Ighb X Igha)Fl mice, Ac38 C~ 95%)carry the b allotype (199), it wouldappear that indeed the D, and JH genes in the Igha haplotype are unable to encode an anti-NP antibody with the Ac38idiotope. TheF1 experiment,also carried out in anotherexperimentalsystem(53), argues against the possibility that lack of idiotope expressionis due to anti-idiotypic (network)control. Astrong argument against this possibility has also beenmadeby Siekevitzet al (186) for the Ars idiotype, whosestrain-specific expressioncould be correlated with the presence or absenceof the correspondingstructural Vri gene. Strains lackingthis genedo not expressthe Arsidiotype, althoughthey are undercertain conditionsable to produceidiotypically cross-reactiveantibodies (136)~presumably products of related, but non-identical, V genes. Asimilar situation appearsto emergein the case of the M460 idiotype(131). Asis discussedin Section6.1, lack of idiotypeexpressionhas beenascribed to anti-idiotypic control in certain other systems. To summarizethis section, recurrent idiotypes have turned out to be encodedby single, or small groupsof, germlineVaand VLgenes and some of their somaticallymutatedprogeny.Theidiotypic repertoire is thus in part controlled andrestricted by the Vgenesin the germline,anddifferent strains in a speciesmaydiffer in their repertoirebecauseof Vgenepolymorphism. The systemgenerates somatic mutants, and since this process appears to be based on randommutation, idiotypes characteristic for such mutantsare probablyusually nonrecurrent,i.e. private. Thetotal repertoire of idiotopes in the immune systemis the sumof the repertoire of recurrent andof private idiotypes. Wewantto stress again specifically that although certain immune responseslike those to PC, NP,Ars, ere, are dominatedby recurrent idiotypes (and somaticmutationmayin these cases mainlyserve to improvethe specificity of the response), other responsesand cellular interactions maydependentirely on somatic mutants whosegeneration mighttherefore be of vital importancefor the system. Finally, the data discussedin this section showthat idiotopes can be sharedbetweenantibodies of different antigen-binding specificity andvice versa, andit providesa clue of howidiotypicallyrelated antibodieswithdifferent epitopespecificity might arise in immune responses. 4. 4 ldiotypic Repertoire at the Level of B-Cell Precursors Animportant approachto studying the sdeetion of Vregions in the immunesystemis to analyze the V-regionrepertoire expressedin newlygener-

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ated B cells, whichappear constantly in large numbersin the bonemarrow (154), i.e. the "available" repertoire, and to compareit to the V-region repertoire expressedat later stages of B-cell ontogenyand in immune responses-the "functional" repertoire. Unfortunately,studies of this type are still in their infancy.Theearliest analysesof frequenciesof idiotypically defined B cells showedthat T-dependent(188) and T-independent(TI-2) (42) T15positive precursorcells occurin spleencells at a frequencyof 1-2 X10-5. Theseresults werelater confirmedby Fung& K/Shier(71 a). Clearly, B cells in the spleenmayalreadybe selected fromthe "available"repertoire. This notion is compatible with subsequent results of Kllnmanand his colleagues (189), whichshowthat T15-positive, T-dependentprecursor cells couldbe detectedin the spleenonly whenthe micewereat least 7 days old. In fact, at the level of B-cellprecursorsdefinedby their antigenbinding specificity, the late appearanceof certain specificities has beenobserved repeatedly(reviewedin 120a).In line with this result, Fung&K/Shler(69) recently found only T-independent(TI-1) T15+ responses in B cells from very younganimals; T-dependentresponseswere observedonly later. Frequencies of T15+ precursors in newlyarising B cells have not yet been determined.In other systems, frequencies of LPS-reactive, idiotypically defined B precursor cells were measured.Eichmannet al (60) determined + LPS-reactivesplenic B cells to be 4 X 10-4. [ASA the frequencyof ASA is a recurrent idiotype in the responseof A/J miceto groupAstreptococcal carbohydrate(54).] In a recent stqdy, Nishikawaet al (147) measured frequencyof LPS-reactivesplenic B cells expressingidiotopes of the germ line-encodedantibodyB 1-8 (23, 24, 173; see also Table1 andFigure2). One of the idiotopes (Ac38,see Section4.3) known to be presenton the products of several VHgenes in combinationwith the Vxl domainoccurred at a frequencyof"d0-a; another one, restricted to antibodies with an NPbinding site (Ac146,Section 4.3), wasfoundat a frequencyof -5. Identical frequencies wereobservedin LPS-reactivecells from spleens of newborn and adult animals and in cells generatedfrom bonemarrowpre-Bcells in vitro, in agreementwith similar data of Jui et al (106a). Overall, the frequenciesmeasuredfor recurrent idiotypes or idiotopes in the various assaysystemsrangefrom10-3 - 10-5 (see also 10). Thereis the indication that in newlyarising B cells, the frequency dependson the numberof genes involved in the expression of a given idiotypic marker (147). However, to date, there is not a single analysis availablein whichfor a given idiotypic markerfrequencydeterminationshavebeencarried out at the various stages of B-cell ontogeny.LPS-reactivecells mayrepresent immatureB cells (70, 139), and it is thus not surprising to find similar precursor frequencies in such cells, be they isolated fromthe spleen or generatedin vitro frompre-Bcells. It will be essential for an understanding

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IDIOTYPES 583 of B-cell selection in generalandthe idiotypic networkin particular to see howthese frequenciescompareto those in B-cell populationsat later stages of ontogeny,e.g. B edls responsiveto specific T-cell help and/orlong-lived, recirculating B cells (64, 69, 71, 191, 192). Below(Section5.3) wegive examplethat strikingly showsthat idiotypic frequencies in LPS-reactive spleencells may,in certain instances,not at all reflect whatis expressedin the immuneresponse generated by the sameanimal’s B-cell population. Otherinformationthat is lacking are the frequenciesof precursor cells expressing,at the various stages of ontogeny,idiotypic markerscharacteristic for somaticmutantsand, in the samecontext, of cells expressingantiidiotypesagainst recurrentidiotypicmarkers.Thelatter point has to be seen in connectionwith the so far unresolvedquestion of whetheror not the germlinedirectly encodesa networkof idiotypes (see Section4.3). In addition, the frequencyof anti-idiotypesmaybe of importancefor the activation of idiotype-bearingcells in the system.

4.5 ImmunoglobulinIdiotypes on T Cells After the initial findingsthat anti-idiotypieantisera couldspecificallyinduce idiotype-bearing T helper (TH)cells (62) and that alloantibodies sharedidiotypes with alloreactive T cells (14), a bulk of experimentalevidence has further demonstratedthe expressionof recurrent antibody idiotypesin functional T cells andantigen-binding material(factors) of T-cell origin. Earlyliterature on this subject has beenreviewed(15, 135, 169), and wequote here only morerecent workon idiotype or idiotope expressionin TH (59, 104, 144, 194a) and T suppressor(Ts) (81, 132a, 185, 193a, cells, in T cells mediatingdelayed-typehypersensitivity(DTH)(7, 96, 196, 201, 218), andcytotoxicreactions(68) in antigen-binding materialof T-cell origin (46, 47, 127) including suppressor(73, 86, 150) and helper factors (145). However,with respect to the molecularnature of the T-cell receptor for antigen, idiotypic cross-reactivity of this kind doesnot havemuchsignificance, in particular if one remembers the discussion of "internal images" and the idiotypic cross-reactions betweenanti-hormoneantibodies and hormonereceptors (Section 2). It was only after the demonstrationof the ~ontrol of both T- andB-cell idiotypesby the Igh locus that the hypothesis could be advancedthat antibodyV genes[and specifically VHgenes, since neither VI. determinantsnor control of idiotype expression by VLgenes couldbe identified in T cells (46, 57, 196a)]participate in encodingT-cell receptors for antigen (15, 169). Despitethe availability of specific antiidiotypic reagents andof clonedT-cell lines, little progresshas beenmade over the last years with respect to the elucidationof the structure of T-cell receptors. In addition, although recent immunogenetic data indicate that

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genes also linked to the Igh complexmayencodeconstant regions of T-cell receptors (158, 202), the available evidencefrommolecularbiology argues against the notion that the sameVII genesthat encodeantibodyV regions are expressedin T cells: regular Vngenerearrangement has so far not been observedin suchcells (128, 130). Wedo not wishto go into this complicated matter further in this context, in particular since the molecularbasis of T-cell recognitionof antigen has not, to date, been resolved. However,wewant to maketwo points, which lead directly into the topic of the next sections, namely,that of idiotypic regulation. First, since T cells expressidiotypes [and, consequently,also anti-idiotypicspecificities (3, 27, 36, 61, 76, 86, 116, 157,163, 197, 276)], they are by definition partners in the idiotypic network.Second,if T-cell idiotypes are not encodedby antibody V genes, wehave to explain how idiotypic polymorphism developsin parallel in the B- and T-cell compartment. If wedo not want to invoke a complicatedmechanism of adaptation andrectification of separatesets of Vgenesin evolution,wehaveto consider the possibility that T cells are selected in the immune systemin ontogeny such that their receptor idiotypically resemblesthat of B-cell receptors. Sucha mechanism wouldimplya functional idiotypic networkin the most fundamentalsense. It wouldbe expectedto lead to a situation wherethe idiotypic (and anti-idiotypic) repertoires expressedin the twosets of cells wouldbe similar, but not quite identical, in keepingwith substantial experimental evidence (13, 15, 76, 102, 126, 146, 163). Wecomeback to this matter in Section6. 4. 6 Co-Existence of Lymphocytes with Complementary Receptors." The ldiotypic Network Lymphoeytes expressing complementaryreceptors co-exist in the immune system.At the level of B cells, anti-idiotypic antibodiescan be inducedin animals knownto possess B-cell precursors expressing the corresponding idiotype (reviewedin 57). In addition, animalsoften produce,in the course of an immune response,anti-idiotypic antibodiesagainst idiotypic determinants of their ownantibodies, althoughmostlyin low concentration(12, 39, 67, 77a, 94, 110, 121, 140, 177, 197a,200). At the level oft cells, idiotypebearingand anti-idiotypic T helper and suppressorcells can be inducedin animalsthat expresssimilar idiotypes andanti-idiotypesat the B-cell level (see Sections5 and6). Thereis thus no questionthat self-recognitionat the level of receptor idiotopes is possible within the immune system,quite as Ramseier and Lindenmann (171a) pointed out 10 years ago. Whatare the physiological consequencesof this situation? Wecan look at the problemfrom the standpoint of classical immunology and ask how the systemis able to establish self-tolerance since it allowsthe expression

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IDIOTYPES 585 of idiotypesin immune responses,undisturbedby anti-idiotypic cells. Aswe shall see, regulatory mechanisms havebeenidentified that potentially resolve this difficulty. Fromthe standpointof Jerne’s hypothesis(97, 98), the co-existenceof idiotypesandanti-idiotypesis an essential feature of the immune system. Cells with complementary binding sites interact with each other and an equilibriumof idiotypes and anti-idiotypes is established. Immune responsesare seen as disturbancesof this equilibriumand attempts of the systemto re-equilibrate. In addition, idiotypic interactions mightbe essential for the generationof the functionalreceptorrepertoire, in the sense of both promotingsomaticdiversification and restricting diversity. Quantitative modelsof idiotypic networkshavebeendeveloped(2, 87, 88, 176), and the essential points of three of themhavebeen summarized by Jerne (99). In the sections belowwediscuss someof the experimentalevidence that supports the notion of a functional idiotypic network. 5. IDIOTYPIC REGULATION OF THE RECEPTOR REPERTOIRE BY ANTIBODIES 5.1 Effect of .4nti-Idiotype on Idiotype Expression In this andthe next section weconsideronly experimentsthat approachthe physiologicalsituation in that antibodiesare applied in native formandin low doses. Underthese conditions, anti-idiotypic antibodies are extraordinarily potent regulators of idiotype expression,in the sense of enhancementor suppression. In general, thoughnot without exception (18, 61, 164a, 165, 203), anti-idiotype, whengiven in native form, did not directly inducesynthesis of idiotypic molecules.Rather,its effect appearedto be a long-lastingmodificationof the functionalidiotypicrepertoire. Theclassical workin this area, largely basedon xenogeneicand allogeneic conventional anti-idiotypic antibodies, has beenextensivelyreviewed(22, 57). Weconcentrate the present discussion on morerecent workand on the mechanisms throughwhichanti-idiotypes exert their regulatory function. Themonoclonalantibody technique (122) has madeit possible to study antibody-mediated idiotypic regulation at the level of individual idiotopes and with isogeneic monoclonalidiotype-bearingand anti-idiotope antibodies. Experimentsof this type, carded out in our laboratory, showedthat anti-idiotopes of mouseorigin injected into the mousewereas ettieient in terms of idiotypic regulation as werexenogeneic(guineapig) anti-idiotypic antibodies injected into the mouse(54, 55, 62): 10-100ng of anti-idiotope enhanced,whereas10/~g suppressedthe expressionof the target idiotope in immune responses induced4-8 weekslater (111, 112, 175). Theinteresting observationthat the class of a xenogeneicanti-idiotype determinesits regulatory function in the mouse[guinea pig IgG1 enhanced

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whereasIgG2suppressed idiotype expression (54, 55, 62)] could not reproducedwith the monoclonalmudneanti-idiotopes. In recent experiments(C. Miiller, K. Rajewsky,to be published), two families of such antibodies were constructed and employed.The membersof each family carry the sameV region on different heavy-chainconstant regions [spontaneousclass switchvariants (167)]. It wasshownthat anti-idiotopes of the IgG1, IgG2a, and IgG2bclass all enhancedidiotope expression at nanogram doses and were suppressive at microgramdoses. In the isologous situation, the regulatoryfunction of anti-idiotopeantibodiesof these three classes is thus determinedonly by the antibody dose. Onewonders,however, whetherthe nature of the target idiotope mayalso play a role, since in earlier experimentswehad failed to induceenhancement with a particular anti-idiotope andsuspectedclass-specific regulation(175). Theexperimentsdescribed abovewererestricted to the analysis of idiotype expressionat the antibodylevd. Anti-idiotypes havealso been successfullyusedto induceidiotypiceffeetor cells in cellular reactions, suchas T cells mediatingDTH (7, 96, 196, 201). Theclass of the anti-idiotope may play a role in these cases. Astriking effect of anti-idiotypic antibodiesis also seen whensuchantibodiesare either injected into newborn animals(1, 8, 71, 87a, 109,115, 116, 124, 182, 209) or enter into the newborn’scirculation fromthe mother, either through the placenta or the milk (210). As a consequence,longlasting suppressionof idiotype expressionis generallyobserved,althougha case of enhancement has also beenreported (87a, see also Section6.1). Very smalldosesof anti-idiotypeare requiredfor suppression(1, 8, 71, 109, 115, 116, 182, 209). In our ownexperimentswehavefound that 1/.tg of monoclonal anti-idiotope injected into the newbornwassufficient for essentially complete idiotope suppression over several months (T. Takemori, K. Rajewsky,submittedfor publication).

5.2 Effect of Idiotype on Idiotype Expression Experimentshave also been done in whichthe effect of injection of an antibody with a given idiotype on the expression of that sameidiotype (instead of complementary binding sites, as in the experimentsdescribed above)wasassessed. All experimentsin whichsoluble "idiotypic" antibody wasapplied had in principle the sameresult. Urbainand co-workersimmunized a pregnant female rabbit with anti-idiotype to producecomplementary antibodies. In the offspring of the immunizedanimal, the correspondingidiotype was recurrently expressed in the immuneresponse, in contrast to the controls (214). Similarly, Rubinsteinet al (179) injected small doseof an anti-levan antibodywhoseidiotype is only rarely expressed in the anti-levan response into newbornmice. Whenthe animalshad grown

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IDIOTYPES 587 up, the sameidiotype wasdominantin their anti-levan antibodies (179). Enhancement of idiotype expression was also seen whenmonoclonalantibodiescarrying a recurrent idiotype wereinjected into adult mice. In one study, the induction of idiotype-expressingplaque-formingcells wasreported, without deliberate addition of antigen to the system(92; however see also 85a). In an experimentdone in our laboratory it wasshownthat injection of 10 #g of antibody B1-8led to enhancedexpression of B1-8 idiotopes in an anti-NP response induced6-8 weekslater. Ananalogous result wasobtainedby Ortiz-Ortiz et al (153) in the Ars system,but much higher dosesof idiotype wereused in this case. Formally,these results can be describedas idiotypic memory:the immune systemsees the structure of an antibody variable region and reproducesit later on in an immuneresponse. Idiotypic antibodyhas also beeninjected into animalsafter it hadbeen chemicallycoupledto syngeneicthymocytesor spleen cells (50, 195). contrast to the aboveexperiments,this resulted in suppressionof idiotype productioneither at the level of antibodies or of cells mediatingDTH. 5.3 Mechanisms of ldiotypic Regulation by Antibodies Since enhancement ofidiotype expressionby idiotype occursundera variety of experimentalconditions, morethan a single mechanism might mediate the effect, suchas inductionof idiotype-specitie T-cell help (179) and/or enhancinganti-idiotypic antibody(112) or elimination of suppression anti-idiotype (153). Thesuppressioninducedby cell-boundidiotype is due to the inductionof anti-idiotypic suppressorT-cell circuits (50, 195). Howdoesanti-idiotype exert its effect on idiotype expression?Enhancing doses sensitize idiotype-expressing Tr~cells (16, 62, 103), T edls mediating DTH(7, 196, 201), and B cells whosefrequencyis found enhancedin the population of LPS-reactive spleen cells (60; T. Takemori,K. Rajewsky, unpublishedresults); it is not clear, however,whetheror not the antiidiotypic antibodysensitizes B cells directly. Alternatively,anti-idiotypic Ta ceils (see section 6.1), whichmightalso be inducedby anti-idiotypie antibody,possiblyvia idiotypicTri cells, couldbe responsiblefor this effect (84). Amplification of idiotype-bearingB cells couldalso be dueto interaction with idiotypic Ta ceils via bridginganti-idiotypic antibody(61). It difficult to distinguishexperimentally betweenthe latter twopossibilities. However, the role of T cells in anti-idiotype-indueed B-cell sensitization is indirectly supportedby the finding that T-cell populationssensitized by anti-idiotype preferentially collaboratewith idiotype-bearingB cells (84). In the case of idiotype suppressionby anti-idiotypic antibody, wefind again that both B and T calls are involvedin this phenomenon. Suppressor T cells that specifically preventedidiotypeexpressionin the B-cell response

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were first demonstratedin the ASAidiotypic system(55) and subsequently identified in a variety of cases of adult and neonatal suppressionof both B-cell and DTH responses(71, 113, 116, 157, 218). In someinstances, the suppressorcells wereshownto expressanti-idiotypic specificity by virtue of their ability to bind idiotype (116, 157). However, the situation is more complicatedin that the T-suppressorpathwayincludes interacting T-cell subsets, such that, for example,an idiotypie suppressorinducercell could activate an anti-idiotypic suppressoreffector, thus mimicking the effect of the latter (150, 197, 211,219).Anidiotype-expressingcell wouldof course appear as the natural target of anti-idiotypic antibodies. Wedo not go further into these complicatedmatters (whichinclude the problemof the target of suppressoreffeetor cells) in the contextof the presentdiscussion. This particular subject has recently beenreviewed,and werefer the reader to this literature (9, 73a, 78, 95, 184,198). As in the case of enhancement,idiotype suppressioncan also be demonstrated within the B-cell population itself. In adult mice injected with microgram amountsof anti-idiotype, evidencefor transient nonresponsiveness of idiotypicB cells wasobtained(85) despite the fact that the frequencies of idiotypic LPS-reactiveB-cell precursorsin such animalsare usually only slightly reduced(60; T. Takemori,K. Rajewsky,submittedfor publication). This phenomenon mayrepresent a classical ease of B-cell tolerance or "anergy"[see Nossal(149a)in this volume].Alongthe lines of the work of Nossaland his colleagues,Nishikawa et al (147)haverecently shownthat anti-idiotypic antibody in microgramconcentrationspreventedthe maturation of pre-Bcells into idiotype-expressingLPS-reactiveB cells in vitro. This result suggests that if a given antibody is producedin the immune systemin such concentrations, the generation in the bone marrowof functional B cells expressingcomplementary (anti-idiotypic) receptors may shut off. Theworkof Iverson(92a) and of Rowleyand his colleagues(179a) together with the inability of Sakato&Eisen (180) to generate anti-Tl5 antibody in mice in which microgramamountsof T15+ molecules were circulating (133)supportthis notion(see also section 5.4). Long-lasting unresponsivenessin the B-cell compartmentwasobserved in systems of neonatal suppression. Whennewbornmice were suppressed + precursorB cells could not for the T15idiotype by anti-T 15 antibody,T15 be detected in the micefor several months(1, 40, 70). Thepossibility that this effect wasmediatedby T cells has beenshownto be unlikely in other cases where suppression of the MOPC104E and T15 idiotypes was achievedby injecting anti-idiotype into newborn(T-cell deficient) BALB/ cnU/numice(115, 209). Onthe other hand, there is also someevidencefor the existenceof idiotypespecific suppressorTcells in animalsinjected with anti-idiotypie antibodyat birth (71, 116). Theeffect of neonatallyapplied

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IDIOTYPES

589

anti-idiotype on both T and B cells is exemplified by an experiment we depict in Figure 3. Micewere injected at birth with 100/~gof anti-idiotope antibody Ac38, recognizing an idiotope recurrently expressed in the primary anti-NP response (see Section 4.3). At 13 weeksof age, splenic T and B cells were isolated from the animals and were injected, together with T and B cells from normal controls, in various combinations into irradiated syngeneie hosts. The hosts were then immunized with NP coupled to chicken gammaglobulin, and the expression of the target idiotope in the humoral anti-NP response was recorded (T. Takemod, K. Rajewsky, submitted for publication). As a result, the B cells from the suppressedanimals mounted a normal anti-NP response but were unable to express idiotope Ac38 even though they were stimulated in the presence of normal T calls. This is particularly remarkable, since in limiting dilution analysis of LPSreactive precursor cells, the frequencyof Ae38+ precursors was identical to a

100 ¯

8 ¯ ¯

¯

Bcell" cell

N N

¯

N N

N S

¯

N S S S

¯

S

N

S S

S N

Figure3 Effectof neonatalinjectionof a monodonal anti-idiotopeantibodyonT andB cells in the adult mouse.Antibody Ac38(100/zg),whichdetectsa recurrentidiotope(Ac38) the anti-NPresponse(174;legendto Figure2), wasinjected into newborn C57BL/6 mice. At week13, T andB cells fromthe spleensof the injected animals(S) andfromcontrol spleens(N)weretransferedin variouscombinations (bottomof Figure)into irradiatedhosts (10Tcells of eachtypeper host). Thehosts wereimmunized withNPchickengamma globulin andthe sera weretitrated 12dayslater for total IgG1anti-NPantibody(whichwasthe same in all groups;data notshown) andfor anti-NPantibodies expressing the Ae38idiotopeeither togetherwitha secondidiotope(Ac146, left panel)or the Ac38idiotopealone(fight panel). Antibody titers in individualsera are indicated.Fordetails of titration methods seeKelsoe et al (112).Dataare fromT. Takemori, K. Rajewsky, submittedfor publication.

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that of control animals(data not shown).Thus,as discussedin Section4.4, the repertoire of LPS-reactiveprecursors mightnot be the sameas that expressedin the mature, functionally active B-cell population. Alternatively, the idiotypic B-cell responsecouldbe suppressedby anti-idiotypic B cells whosefrequencyin the suppressedanimalsremainsto be determined. Mostinteresting in this context are recent data of Okumura et al (151), who ascribea critical role in idiotypic Controlto a B-cellpopulationexpressing the Lyt-1 surface antigen. Comingbackto the experimentin Figure3, the T cells of the suppressedanimalswerealso affected in that they did not allow the expressionof the target idiotope Ac38in a normalB-cell population, althoughthey helpedthe B cells to mounta normalanti-NPresponse. Mixingexperimentshaveso far failed to establish whetherthis deficiency is dueto lack of idiotype-specifichelp, active suppression,or both. Interestingly, although nonresponsiveness in the B-cell compartment comprisesall antibodies expressingidiotype Ac38,the T cells seemto see only a subset of these antibodies, namelythose co-expressinga secondidiotope (Ac 146). This not only controls for the purity of the B-cell populationin the experiment,it also suggeststhat the idiotypic determinantsseen by T andB cells maynot be the same. Wecomeback to this point in Section 6.2. Toconcludethis section, smalldoses of antibodies, appliedto the animal in native form, profoundlyaffect the equilibrium of the corresponding idiotypes and anti-idiotypes in both the T- and the B-cell compartments of the immunesystem. 5.4 Interpretation of the Regulatory Effects of ldiotypic and Anti-ldiotypic Antibodies Theinterpretation of the results described abovehas been madedifficult because of a problem of nomenclature, namely, the terms idiotype and anti-idiotype. Certainly, any idiotype arising in the immune systemhas to deal with the problemof anti-idiotypes; however,whenweinject an "antiidiotype"into the mouse,it is incorrect to interpret this experimentsolely in the sense of control of the corresponding"idiotype," as has usually been done. Asdiscussed in detail in section 2, idiotype and anti-idiotype are operational terms for functionally equivalentcomplementary bindingsites, and anyidiotype is equally well an anti-idiotype and vice versa. Whenweinterpret the data on this basis, a surprisingly simple pattern emerges.At low doses (nanogramrange), antibodies enhancethe expression of complementary binding sites in the system. This enhancement is not at the level of effector functions, suchas high-rate antibodyproduction,but presumablyit involves mainlythe recruitmentof T and B cells expressing complementary bindingsites. It is attractive to think that the enhancement phenomenon reflects a fundamentaldriving force in the immune system in the senseof the networkhypothesis.Lowlevel antibodyproduction(natural

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IDIOTYPES 591 antibody) could accompany and promoteinteractions betweendiverse sets of cells with complementary receptors (61). Oncethe concentrationof certain antibodybindingsites is abovea certain threshold (microgramrange), for examplewhenthe system engagesin active immune response,a different set of mechanisms is set in motion.Now the systemis designedto lock itself into the expressionof a givenantibody repertoire. The"suppression"experimentsdiscussed abovereveal someof these mechanisms: microgram quantities of antibodies ("anti-idiotypes" in this case) shut off, in the bonemarrow,the productionof cells with complementary("idiotypic") receptors and suppressor T cells suppressing the activation of suchcells in the peripheryare induced.Thesystemstabilizes the expressionof those bindingsites (in this case the "anti-idiotype") whichit has committed itself. This is strikingly demonstrated by the finding that idiotype can facilitate the expressionof the sameidiotype["idiotypic memory,"Section 5.2; the problemof stabilizing immuneresponses has beendiscussedfrommanydifferent angles (e.g. 82, 83, 88)]. Theentire set of data discussed above can thus be interpreted in a coherentwaythat makessense in terms of the physiologyof the systemand ascribes the idiotypic networka physiologicalregulatoryrole. Afinal remarkabout idiotypic control of antigen-specific antibodyresponses: As discussedin Section 4.3, such responsesconsist of antibody families in whichsets of idiotopes are expressedon the members in various combinations.Ananti-idiotypic responseagainst the morecommon of these idiotopes would have as its commondenominator, because of its heterogeneity,the bindingsites for these idiotopes. Becauseof this asymmetry it couldunidirectionallyregulate the expressionof the idiotypic antibodyfamily, with the implication, however,that regulation could extendto members of the familythat do not possess the antigen-bindingspecificity of the initial immune response. Theabl-ab2-ab3 experimentsof Urbain, Cazenaveand others (204; section 4.3) can be consideredto exemplifythis and can be interpreted in this waywithoutcomplicatedgenetic assumptions (168). Idiotypie control wouldbe strictly specific for the antigen-induced responseif the activatedcells wereparticularlysusceptibleto the regulatory signals (36), anda priori so in caseswherethe anti-idiotypic responsewould see the antigen-binding site as the common denominatorin the antigeninducedantibodypopulation, and thus consist largdy of "internal images" 000, 101). 5.5 Antibody Idiotypes and Growth Receptom on Pre-B and B Cells A potentially importantissue has not beenmentionedso far, namely,that of idiotypic relationshipof antibodiesandgrowthreceptorsexpressedin the B-cell lineage. Coutinhoandhis collaborators (44) havefoundthat certain

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anti-idiotypic antibodies against recurrent idiotypes can bind to a large fraction of B andpre-Bcells andcan drive these cells into proliferation. A similar phenomenon was subsequentlydescribed for antibody M460(164), whoseidiotype is recurrently expressedin BALB/c anti-dinitrophenyl responses(21, 51, 52, 131). Theantibodiesare thoughtto interact with a set of growthreceptors expressedin different combinations on different subsets of pre-B and B cells, and a theory was developed according to which antibodyidiotypes and B-cell growthreceptors havebeenselected in evolution for efficient interaction, providinga mechanism for self-stimulation within the B-cell system (43). This mechanism wouldbe operating already at the pre-Bcell level, that is before the cells expresssurface Ig [it is interesting in this connection,however,that pre-B cells maycarry small amountsof free/~ chains on the surface (158a, 206)], and maternalantibodies mightthus influence B-cell development in the organismfight fromthe beginning(10). Thestudies in this field are still in their infancyin terms molecularanalysis. However,together with other evidence (163a, 165a, 183, 193), they point in a direction that mayturn out to be of physiological importancein a general way,namely,the possible involvementof antibody idiotypesin interactionswith surfacereceptorsother than the antigenreceptors of the immunesystem. 6.

T CELLS AND IDIOTYPE

REPERTOIRE

6. ] Silent Clones and Idiotype Dominance Thediscussionin the previoussections has shownthat antibodyidiotypes, be they expressedon circulating or cell-boundantibodies, can specifically induceT-cell activities. It is thus not surprising that in antigen-driven immune responses[in whichin addition to antigen-specificantibodies spontaneous auto-anti-idiotypes often appear and have beenascribed a regulatory role (39, 67, 77a, 110, 121, 153, 197a, 200; see also Section 4.6)] idiotype-beadngand anti-idiotypic Tn and Ts cells have beenobservedin a variety of experimentalsystems, including antibodyand DTH responses. Theinduction of such cells is thus not restricted to manipulationswith anti-idiotypic antibodies. Basedon sophisticated workwith techniquesof cellular immunology, idiotypic interactions of regulatory T cells havebeen incorporatedinto schemesof T cell circuits regulating immune responses. Thediscussion of these matters is beyondthe scopeof the present review andwerefer the reader to the literature (9, 73a, 78, 95, 184, 198). However,since the immunesystem continuously lives with antibody idiotypes, starting in ontogenywith immunoglobulins of maternal origin, idiotype-dependentmodulationof T-cell activities must be expected to occurthroughoutlife. Thisleads us directly to the issue of silent clonesand

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IDIOTYPES 593 ofidiotype dominancein the B-cell compartment. There is evidence,mainly fromthe workof Urbain, Cazenaveand Bonaand their colleagues, that the bone marrowproducesclones of cells not expressedas recurrent or dominant idiotypes in immune responses,either becauseof suppressionby T cells or becauseof the lack of T-cell hdp (21, 106, 179, 204). Alongthese lines, several groupshavepresented data suggestingthe dependenceof dominant expressionof recurrentidiotypeson idiotype-specificTHcells (3, 25, 36, 85, 216).It seemsclear that classical, cartier (i.e. antigen)-specifichelpercells are sufficient to drive B cells expressingantibodiesof the IgMandIgGclass (and also a "dominant"idiotype) into proliferation and antibody production in vitro (31), but this doesnot excludethe possibilitythat anti-idiotypie TI~ cells often participate in the preselection and/or expansionof certain B-call clones that dominatecertain immune responses. Preferential expansion by anti-idiotypic helpers couldbe responsiblefor the dominance of an idiotype in the anti-lysozymeresponse, being established only late in the response(142). Clearly, such evidencehas to be weighedagainst the classical conceptof selection in the immune responseof ceils expressingantibodies with high affmity for antigen (63). Such selection .(in which idiotype-specific T cells could also be involved)mayfinally explain many eases of idiotype dominance,in particular since the correspondingantibody Vregions (often specific for bacterial antigens) mighthave beenalready selected in evolution(29, 65, 215). Theessential role of antigen in driving immune responsesis evident fromthe fact that the expressionof idiotypes in antigen-inducedresponses is usually, although not always (156), restricted to antigen-bindingmoleculesin the serum(e.g. 54, 148, 170, 217). 6.2 Antibody Idiotypes and the T-Cell Repertoire Theconcept that the existence of silent clones and idiotype dominance reflects idiotype-specitic T-cell control represents a complicationfor the discussionof the geneticdetermination of the idiotypic repertoirein B cells (Section 4.3). Thus,if a givenidiotype is not expressedin the B-cell compartment,wemightconsiderthis to be unrelatedto the absenceor presence of certain antibodystructural genesanddue to suppressionby T cells. Let us try to clarify the situation. Almostwithoutexception,idiotypeexpression is exclusivelyunderthe control of the Igh locus (Section 4.3). Therefore, if a given idiotype is expressedin a givenstrain, but not in another,then the idiotype-negativestrain either lacks the structural informationfor that idiotype (as exemplifiedin several cases, see Section4.3) or a regulatory elementrequired for its expression. That elementmustalso be located in the Igh locus. Clear candidatesfor such hypothetical regulatory elements are genesencodinganti-idiotypes. If suchgeneswereexpressedin T cells,

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these cells would,in principle, be able to interact with idiotype-bearingB cells, either in the senseof suppression(generatingsilent clones)or enhancement(generatingidiotypic dominance). However, this type of strain-specific indirect control of idiotype expressionvia regulatory T cells wouM again be due to polymorphism of genesin the lgh locus. If, indeed, the repertoire of antibody V-regionstructural genes were sharedbetweenT- andB-cell receptors, the extensiveidiotypic interactions betweenthe two types of lymphoeyteswouldnot be surprising. In particular, idiotypic polymorphism wouldbe expectedto be expressedequally at the level of T and B cells, as is indeedfound(Section 4.5). However, as discussedabove,evidencefrommolecularbiology arguesagainst the notion that the V, D, and J genesexpressedin antibodies are also expressedin T cells. Furthermore, there is evidencethat idiotypic control of B-cell activities by regulatoryTcells andwithinregulatoryT-cell circuits is restricted by the Igh locus,suggestinga selectionof T cells for the recognitionof B-cell idiotypes expressedin the samesystem(see 9, 26, 106, 198, 219). These considerationslead to the far-reachinghypothesisthat the T-cell repertoire mightbe selected in ontogenynot only for the recognitionof self-histocompatibility (MHC) antigens(118, 222), but also for (self) B-cell idiotypes. its extremeform, this hypothesisexplains idiotype sharing betweenT and B cells by idiotypicinteractions betweenthese cells leadingto the selection of a T-cell receptor repertoire complementary to that of B-cell receptors. Since the latter contains complementary receptors in itself, and since, in addition, anti-idiotypic T cells selected for B-cell idiotypes mayin turn expandidiotypic T cells, a situation wouldresult in whichnot only efficient idiotypic T-Bcell interactions wouldbe guaranteed,but also the receptor repertoire of T cells wouldbe idiotypically (and anti-idiotypically)similar, althoughnot identical, to that of the B cells. Theexperimentalresults in Figure3 can easily be interpreted in this sense. Thereis suggestiveevidencealso in other systemsthat T and B cells see similar, but not identical, idiotopes on antibodies (76, 80, 102, 163). One mightspeculate that "regulatory" idiotopes in the sense of Paul & Bona (159a)mightbe those that T ceils are able to see. Apriori, a B call appears to be an ideal target to be seen by T cells: It expressesclass I andclass II MHC antigens on its surface, as well as a unique structure, the immunoglobulin idiotype. Furthermore,a close structural similarity of immunoglobulin domainsand the domainsof class I and class II antigens has recently emerged(130a, 152, 221). This has led to the speculation that domainsas well as MHC antigens mightrepresent restriction elementsfor T cells (25, 26, 95, 106), and that Vregions could havebeenselected evolutionfor structural similarity with MHC antigens (45). Thelatter could relate to the findingthat helper andsuppressorT cells see different idiotypic

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IDIOTYPES 595 determinantsin certain systems(80, 184), and to the issue of silent clones and idiotype dominance(Section 6.1). An answerto the question as to what extent the T-cell repertoire is determinedby idiotypic interactions with B ceils will require the analysis of T-cells that haveditfercntiated in the absenceof B-ceil idiotypes.Experimentsof Bottomlyand her co-workerssuggest that T cells from Ig-suppressedmicestill contain THcells specific for conventionalantigens (25); but of course the suppressedmiceare not free of the antibody idiotypes present on the suppressingantibody. Bottomlyand Mosier(27) have also suggested that anti-idiotypic THcells are absent from mice lacking the correspondingidiotype in the B cell compartment. This result is controversial at present (32, 166). Further experimentsalong these lines appear urgently needed.Theissue is whetheror not idiotypic interactions are the natural wayfor T cells to communicate with each other and with B cells. 7.

CONCLUSIONS

Westarted our discussionwith the principles of idiotypic interactions and the structural andgenetic basis of idiotypic dctcrminants.Thereis clear evidencethat the germlinecontrols and limits the availableidiotypic repertoire in newlyarising B cells. Thecells in the immune system arc continuouslyexposedto idiotypic interactions. A pre-B cell arising in the bone marrowmayalready be positively selected by antibodyidiotypcs throughinteraction with growth receptorson its surface. When the cell expressesits Ig receptor, it becomes subject to idiotypic control of several types. Thereis cvidcnccthat cells arising in the bone marrowand expressing receptors complementaryto idiotypes present in the circulation in microgramamountsare turned off. Onthe other hand, nanogram doses of idiotypes in circulation lead to the expansionof cells with complementary receptors. Idiotypes maythus play a crucial role in establishing the functional repertoire in the system,by recruiting B cells that are otherwiseshort-lived into the pool of recirculating, long-livedcells and/orthat of B cells responsiveto specific T-cell help (these two compartments could bc overlappingor identical). In the idiotypicselectionof B cells, regulatoryT cells can be specifically involved,they themselvesbeing targets of regulation by antibodyidiotypes. Theinterplay of T and B cells via idiotypic interactions will lead to the selection of sets of complementary cells whosereceptor repertoire might well differ fromthat of the newlygeneratedcells. Idiotypc sharing between T andB cells andthe control of idiotypcs by the Igh locus couldthus either indicate the involvementof Vngenesin the receptors of both cell types or the adaptationof the T-cell repertoire to B-cell idiotypesin ontogcnyand/

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or evolution, similar to its adaptation to the (structurally related) MH antigens. There is experimental evidence that the receptor repertoire of T cells is idiotypically related but not identical to that of antibodies, and hints that T cells might adapt to the recognition of antibody idiotypes in ontogeny. If it will turn out that idiotypically selected cells represent an importan part of the functional repertoire, then immuneresponses might indeed, quite in the sense of the original network hypothesis (97), depend on the interference of antigen with interactions of complementarycells and reflect release from suppression. Experimentaldata pointing in this direction have recently been presented (58, 123). Oncethe system expresses a given repertoire of binding sites in an immuneresponse, the expression of these binding sites is stabilized via idiotypic control ("idiotypic memory"). A functional network of idiotypes maynot only exist at the level of T-B (or T-T) cell interactions but also in the B-cell compartmentitself. Here could either have been selected in evolution or reflect an interplay of germ line encoded with somatically mutated V regions (168). The question whether the germline encodes an idiotypic network of antibody V regions can now be directly approached by the tools of molecular biology. ACKNOWLEDGMENTS

Weare grateful to K. Beyreuther, M. Cramer, A. Radbruch, M. Siekevitz, F. Smith, and H. Tesch for valuable criticism and advice, and to E. Sieg: round, C. Tomas, and W. Muntjewerf for typing the manuscript. Wealso thank K. Karjalainen for samples of monoclonal antibodies, and him and H. Sakano, A. Bothwell, M. Paskind, D. Baltimore, S. Zaiss, and K. Beyreutber for letting us have unpublished sequence information. Wewish to acknowledgespecifically the contributions of M. Reth to our work and thinking. The work from our laboratory was supported by the Deutsche Forschungsgemeinschaft through SFB 74. LiteratureCited 1. Accolla, R. S., Gearhart, P. J., Sigal,N. H., Cancro,M. P., Klinman,N. R. 1977.Idiotype-spccificneonatalsuppressionof phosphorylcholine-responsire B cells. Eur.Z ImmunoL 7:876-81 2. Adam,G., Weiler, E. 1976. Lymphocytepopulationdynamicsduring ontogeneti¢ generation of diversity.In TheGeneration of Antibody Diversity:A NewLook,ed. A. J. Cunningham, pp. 1-20. NewYork: Academic 3. Adorini,L., Harvey,M.,Sercarz,E. E. 1979.Thefine specificityof regulatory T cells. IV. Idiotypiccomplementarity

andantigen-bridging interactionsin the anti-lysozyme response.Eur. J. Im. munol.9:906--9

Anderson, J., Coutinho,A., Melcher F. 1977.Frequencies of mitogen-rea tire Bcells in the mouse. I. Distributi in different lymphoidorgans from differentinbredstrainsof miceat differ ent ages. Z Exp.Med.145:1511-19 Anderson, J., Coutinho,A., Meleher F. 1977.Frequencies of mitogen-rea tire B cells in the mouse.II. Freque cies of B cells producingantibodi~ whichlysesheepor horseerythrocyte

Annual Reviews IDIOTYPES and trinitrophenylated or nitroiodophenylated sheep erythrocytes. Z Exp. MetL145:1520-30 6. Anderson,J., Schreier, M. H., Mdchera, F. 1980. T-cell-dependentB-cell stimulationis H-2restricted and antin dependent onlyat the restinl~ B-cell vel. Pro~ NatL Acad. Sc£ USA77: 1612-16 7. Arnold,B,, Wallich,R., H’fimmeding, G. J. 1982. Elicitation of delayed-type hypersemdtivity to phO,l?orylcholine by monoclonal anti-idiotyplc antibodiesin an allogenicenvironment. ,/.. Exp.MetL 156:670-74 8. Augustin, A., Cosenza, H. 1976. Expression of newidiotypes following neonatal idiotypic suppression of a dominantclone. Eur. Y. ImmunoL6: 497-501 9. Benacerraf,B., 1980.Geneticcontrol of the specificity of T lymphocytesand their regulatoryproducts.In Progressin immunology:Immunology80, ed. M. Fougereau,J. Dansset, 4:419-31. New York: Academic 10. Bernab~,It. A., Coutinho,A., Cazenave, P.-A., Forai, L. 1981.Suppression of a "recurrent"idiotyperesults in profoundalterations of the wholeB-ceil compartment. Pro~ NatL Aca~L US.478:6416-20 11. Bernard, O., Hozumi,N., Tonegawa, $. 1978. Sequences of mouse immunoglobulin fight chain genes before and after somaticchanges.Cell 15:1133--44 12. Binion,S. B., Rodkey,L. $. 1982.Naturally inducedauto-anti.idiotypic anti. bodies. Induction’byidentical idiotopes in somemembersof an outbred rabbit family. Y.. Exp. MetL156:860-72 13. Binz, H., Ffischknecht, H., Shen, F. W., Wigzell,H. 1979. Idiotypic determimmtson T-cell subpopolations. Exp. Me&149:910-22 14. Binz, H., Wigzell,H. 1975.Sharedidiotypic determinants on B and T lymphocytesreactive against the sameantigenic determinants. I. Demonstration of shnilar or identical idiotypeson IgG molecules and T-cell receptors with specificityfor the samealloantigens.,L. Exp. Med. 142:197-211 15. Binz, H., Wigzdl,H. 1977. Antigenbinding idiotypic T-lymphocyte receptors. Contemp. Top. Immunobio~7: 113-77 16. Black,S. J., ITdmmerling, G. J., Berek, C., Rajewsky,K., Eichmann,K. 1976. Idiotypic analysis of lymphocytes in vitro. I. Specificityandheterogeneity of B and T lymphocytesreactive with anti-

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idiotypic antibody../. Exp. Med.143: 846-6O 17. Blomberg,B., Geckeler, W.R., Weigeft, M.1972. Geneticsof the antibody response to dextran in mice. Science 177:178-80 18. Bluestone,J. A., Epstein,S. L., Ozato, K., Sharrow,S. O., Sachs, D. H. 1981. Anti-idiotypes to monoclonalanti-H-2 ~ antibodies. II. Expressionof anti-H-2 idiotypesonantibodiesinducedby antiidiotyp¢ or H-2k antigen. Z Exp. Med. 154:1305-18 19. Bluestone,J. A., Krutzsch,H. C., Auchiucloss, Jr., H., Cazenave,P.-A., Kindt, T. L, Sachs, D. H. 1982. Antiidiotypes against anti-H-2 monoclonal antibodies: Structural analysis of the moleculesinducedby in v~voanti-idiotype treatment. Proc. Nat~Acad~Sc~ USA24:7847-51 19a0 Bluestone, J. A., Mctzger, Knode,M. C., Ozato, K., Sachs, D. H. 1982. Anti-idiotypesto monoclonal anti-H-2antibodies.I. Contribution of isolated heavyandlight chainsto idiotype expression. Mo~ImmunoL19:515--24 20. Bona,A., Heber-Katz,E., Paul, W.E. 1981.Idiotype-anti-idiotyperegulation. I. Immunizationwith a levan-binding mydoma protein leads to the appearance of auto.anti-(anti-idiotype) antibodies and to the ~tivation of silent clones. Z Exp. Med.153:951-67 21. Bona, C., Paul, W.E. 1979. Cellular basis of regulationof expressionof idiotype.I. T-suppressor cells specific for MOPC 460 idiotype regulate the expressionof cells secreting anti-TNPanfibodies bearing 460 idiotype, d. Exp. Med. 149:592-600 22. Bona,C., Casenave,P.-A. 1980. LympyhOCytic Regulationby AntibodiegNew ork: Wiley 23. Bothwell,A. L. M., Paskind, M., Reth, M., Inmnishi-Kari, T., Rajewsky,K., Baltimore, D. 1981. Heavychain vari~ able region eontfibution to the N1 family of antibodies: Somaticmutation evident in a T2avariable region. Cell 24:625-37 24. Bothwell,A. L. M., Paskind,M., Reth, M., Imanishi-Kari, T., Rajewsky,K., Baltimore,D. 1982. Somaticvariants of murineimmunogiobulin h light chains. Nature 298:380-82 25. Bottomly,K., Janeway,C. A. Jr., Mathieson, B. J., Mosier,D. E. 1980.Absenceof an antigen-specifichelperT cell requiredfor the expressionof the T 15 idiotype in micetreated with anti-p antibody. Eur. J. ImmunoL10:159-63

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26. Bottomly,K., Maurer,P. H. 1980. Antigen-specifichelperT cells requiredfor dominant production of an idiotype (ThId) are not under immuneresponse (It) gene control. J. Exp. Med~152: 1571-82 27. Bottomly,K., Mosier,D. E. 1979.Mice whoseB cells cannot producethe T 15 idiotype also lack an antigen-specific hdper T cell required for T 15 expression. J. Exp. Med.150:1399-409 28. Brient, B. W.,Nisonoff,A. 1970.Quautitative investigationsof idiotypicantibodies. IV. Inhibition by specific haptens of the reactionof anti-haptenantibodywithits anti-idiotypicantibody.J. Exp. Med. 132:951-62 29. Briles, D. E., Forman,C., Hudak,S., Claliin, J. L. 1982. Anti-phosphorylcholine antibodiesof the T 15 idiotype are optimallyprotective against Steeptococcus pneumoniae. J. Exp. Med. 156:1177-85 30. Briiggemann, M., Radbruch, A., Rajewsky, K. 1982. Immunoglobulin V regionvariants in hybridoma cells. I. Isolation of a variantwithaltered idiotypic and antigen-bindingspecificity. EMBO J. 1:629-34 31. Cammisuli,S., Schreier, M. H. 1981. Individual clones of carder-specific T cells help idiotypicallyandisotypically heterogeneousanti-hapten B-cell responses. Immunology43:581-89 32. Cancro,M. P., Klinman,N. R. 1980. B ceil repertoire diversity in athymic mice..i.. Exp. Meg151:761-66 33. C.apra,J. D., Kehoe,J. M.1975.Hypervariableregions,idiotypy,andthe antibody-combiningsite. Adv. ImmunoL 20:1-40 33a. Capra,J. D., Slaughter,C., Milner,E. C. B., Estess, P., Tucker,P. W.1982. The cross-reactive of A-strain ndce. Immunologyidiotype Today 3:332-39 34. Car~n, D., Weigert, M. 1973. Immunochemical analysis of cross-reacting idiotypes of mousemyelomaproteins withanti-dextranactivity andnorreal anti-dextran antibody.Proc. NatL Acad. ScL USA70:235-39 35. Cazenave,P.-A. 1977. Idiotypic-antiidiotypic regulationof antibodysynthesis in rabbits. Pro~NatLdcad. ScLUSA 74:5122-25 36. Corny,J., Caulfield,M.J. 1981.Stimulation of specificantibody-forming cells in antigen-primed nude mice by the adoptivetransfer of syngeneic anti-idiotypic T cells. J. ImmunoZ 126:2262-66 37. Clevinger,B. 1982.In Idiotypes~"Antigens on the Inside, ed. I. Westen-

38.

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Selmurr, pp. Roche Clevinger,B., Davie,J. M.1~ of eross-reaetis pie determinan to a-(l-~3) 151:1059-70 Cosenza,H. 19 otype reactive phosphorylcho 6:114-16 Cosenza,H., J A. A. 1977.Idi markers: Anal on phosphoryl lymphocytes.~ Cosenza,H., I~ inhibitionof pl phoryleholine1 body. Science Cosenza,H., ~ 1975. Antilx~ horyleholinei equencyof ] clone size and5~ J. Immuno£

~r

43. Coufiaho,A. 1 cfiminationan, tion of the a~ Immunol. (PaJ 44. Coutinho, A., | 1980. The pob munoglobulin nants on the n their precurso~ munopathoL3 45. Coutinho,A., 1 Ivars, F. 1983. Tautologic?It Geneticsof the andE. MiSller, press 46. trainer, M.,I~ I., Imanishi-K Givol, D., R@ hapten-bindin~ lymphocytes. munoglobulin hydroxy-3-nitr specificrecept~ B and T lymph 9:332-38 47. Cramer, M., R 1,981. T Cell ~ Immunoglobut pression,ed. J E., Wigzell, H Academic 48. Crews, S., (~ Calame,K., H H genesegmen

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IDIOTYPES sponse to phospholTlcholine:Somatic mutationis correlatedwith the class of the antibody. Cell 25:59-66 49. Dildrop, R., Brii~gemann, M., Radbruch, A., RajewsKy,K., Beyreuther, K. 1982. Immunoglobulin V region variants in hybridoma cells. IL Recombination between V genes. EMBO d. 1:635-40 50. Dohi, Y., Nisonoff, A. 1979. Suppression of idiotype and generationof suppressor T cells withidiotype-conjugated thymocytes, d. Exp. Meal150:909-18 51. Dzierzak,E. A., Janeway,C. A. Jr., Rosenstein, R. W., Gottlieb, P. D. 1980. Expressionof an idiotype (Id-460) during in vivoanti-dinitrophenylantibody responses. I. Mapping of genes for Id460expressionto the variableregionof immunoglobulin heavy-chainlocus and to the variable region of immtmoglobufin k-light-chain locus, d. Exp. Med. 152:720-29 52. Dzierzak, E. A., Rosenstein, R. W., Janeway,C. A. Jr., 1981.Expressionof an idiotype(Id-460)duringin vivoantidinitrophenyl antibody responses. II. Transient idiotypic dominance..LExp. Meal 154:1432-41 52~ Dzierzak, E. A., Janeway,C. A. Jr., 1981.Expressionof an idiotype(Id-460) duringin vivo anfi-dinitrophenylantibodyresponses.III. Detectionof Id-460 in normalserumthat does not binddinitrophenyl, d. Exp. Meal154:1442-54 53. Eichmaun,K. 1973. Idiotype expression andthe inheritance of mouseantibodyclones. £ Exp. Med. 137:603-21 54. Eichmmm, K. 1974. Idiotype suppression. I. Influenceof the doseandof the effectorfunctionsof anti-idiotypicantibody on the production of an idiotype. Eur..L ImmunoL 4:296-302 55. Eichmaun,K. 1975. Idiotype suppression. IL Amplification of a suppressorT cell withanti-idiotypicactivity. Eur.~. ImmunoL5:511-17 56. Eichmann,K. 1975. Geneticcontrol of antibodyspecificity in the mouse.Immunogentics2:491-506 57. Eichnmnn, K. 1978. Expression and function of idiotypes on lymphocytes. Adv. ImmunoI26:195-254 K. 1982. Idiotypes.Antigens 58. Eichmmm, on the Inside, ed. I. Westen-Schnurr, pp. 209-21. Basel: Editiones Roche 59. Eichmann,K., Ben-Neriah,Y., Hetzelberger, D., Polke,C., Givol,D., Lonai, P. 1980. Correlated expression of Vn frameworkand Vn idiotypic determinantson T helper cells and onfunctionally undefinedT cells binding groupA

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streptococcal carbohydrate.Eur. L Immuno£10:105-12 60. Eiehmaun,K., Coutinho,A., Melehers, F. 1977. Absolute frequencies of Lipopolysaecharide-reaetive B cells producing A5Aidiotype in unprimed, streptococcal A earbohydrate-primed, anti-A5Aidiotype-sensitized and antiA5Aidiotype-suppressed A/J mice. Exp, Med. 146:1436-49 61. Eiehmaun,K., Falk, I., Rajewsky,K. 1978.Recognitionof idiotypes in lymphocyte interaetions. II. AntigenindependentcooperationbetweenT and B lymphocytes that possesssimilar and complementary idiotypes. Eur. ~. lmmuno~8:853-57 62. Eiehmann,K., Rajewsky,K. 1975. Induction of T and B cell immunityby anti-idiotypic antibody. Eur. ~. ImmunoZ5:661-66 63. Eisen, H. N., Siskind, G.W.1964.Variations in affinities of antibodiesduring the immune response. Biochemistry3: 996-1008 64. Elson, C. J., Jablonska,K. F., Taylor, R. B. 1976.Functional half-life of virgin and primed B lymphocytes.Eur. £ Immunol. 6:634-38 64a. Enghofer, E. M., Glandemans,C. P. L, Bosma, M. L 1979. Immunogiobufins withdifferentspeeificitieshavesimilar idiotypes. MoLImmunoL16: 1103-10 65. Etlinger, H. M., Julius, M.H., Heusser, Chr. H. 1982. Mechanismof clonal dominancein the murine anti-phosphorylchofineresponse.I. Relationbetweenantibody avidity128:1685-91 and elonal dominance. J. Immunol. 66. Fairchild, S. S., Cohen,J. J. 1978.B lymphocyteprecursors. I. Induction of Lipopolysaccharideresponsivenessand surface immunoglobulin expression in vitro, d. ImmunoL121:1227-31 67. Fernandez,C., M/511er,G. 1979. Antigen-inducedstrain-specific autoantiidiotypie antibodies modulatethe immuneresponse to dextran B512.Proc. NatLAcad.Sci. US.476:594zl---47 68. Frischknecht,H., Binz, H., Wigzell,H. 1978.Inductionof specific transplantation immune reactions using anti-idiotypic antibodies. £ Exp. Med. 147: 500-14 69. Fung,J., KShler,H. 1980. Late clonal selection andexpansionof the TEPC-15 germ-line specificity, d.. Exp. Meal 152:1262-73 70. Fung, J., KShler, H. 1980. Mechanism of neonatal idiotype suppression. I.

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merit at birth with idiotypes is assoasymmetryin idiotypic interactions. ciated with the expansionof idiotypeAnn. ImmunoLIn press 169. Rajewsky,K., Eichmaun,K. 1977. Anspecific helper T cells. J. Exp. Med. tigen receptors of T helper cells. Con156:506-21 179a.Rowley,D. A., Griflith, P., Lorbach,I. temp. Top. ImmunobioL7:69-112 170. Rajewsky,K., Reth, M., Takemori,T., 1981. Regulation by complementary Kelsoe,G. 1981.A glimpseinto the inidiotypes. Ig protects the cloneproducnet life of the immune system. In The ing it. J. Exp. Med.153:1377-90 Immune System,ed. C. M.Steinberg, I. 180. Sakato,N., Eisen, H. N. 1975. AntibodLefkovitz,2:1-11. Basel: Karger ies to idiotypes of isologous immtmo171. Rajewsky,K., Takemori,%, geth, M. globulins. J. Exp. Med~141:1411-26 1981.Analysisand regulation ofVgene 180a.Schitf, C., Boyer,C., Milili, M., Fougeexpression by monoclonalantibodies. reau, M. 1981. Structural basis for MOPC 173 idiotypic determinants disIn Monoclonal.~ntibody and T Cell Hybridoma:Perspectiveand Technical tinctively recognizedin syngeneicand vances,ed. G.J. I-I’~immerling, U.Hiimallogenc~cimmunization:contribution merling, J. F. Kearney,pp. 399--409. of DH,JH, and JK regions to an idiNewYork: Elsevier otyperecognizedby allogeneicantisera. ~4ng Immunol. Ins~ Pasteur 132C: 171a. Ramseier, H., Lindenmann,J. 1972. Aliotypic antibodies. Tra~plant. Rev. 113-29 10:57-96 181. Schilling,3., Clevinger, B., Davie,J. M., 172. Reth, M., Bothwell, A. L. M., RaHood,L. 1980. Aminoacid sequenceof homogeneous antibodies to dextran and jewsky,K. 1981. Structural properties DNA rearrangementsin heavy chain Vof the haptenbinding site and of idib otopes in the NP antibody family. In region gene segments. Nature 283: ImmunoglobulinIdiotypes: ICN-UCLd 35-40 Symposia on Molecular and Cellular 182. Schuler, W., Weiler,E., Weiler, I. J. Biology,ed. C. JanewayJr., E. E. Ser1981. Biological and serological comcarz, H. Wigzell,20:169-78.NewYork: parison of syngeneicandallogeneicanAcademic ti-idiotypic antibodies. MolecularImmunology18:1095-105 173. Reth, M., I-~knmerllng, G. J., Rajewsky,K. 1978.Analysisof the reper- 183. Scge, K., Pcterson, P. A. 1978. Useof toire of anti-NPantibodies in C57BL/6 anti-idiotypicantibodiesas cell-surface miceby cell fusion. I. Characterization receptor probes. Proc. NatLAcad.ScL of antibodyfamilies in the primaryand USA75:2443-47 hyperimmuneresponse. Eur. J. Im- 184. Sercarz, E. E., Metzger, D. W.1980. Epitope-specificand idiotype specific munoL8:393-400 cellular interactions in a modelprotein 174. Reth, M., Imanishi-Kari,T., Rajewsky, K. 1979. Analysisof the repertoire of antigen system. Springer Semin. Immunopatho£3:145-70 anti -(4 -hy.droxy-3ultrophenyl)acetyl (NP) antibodies in C57BL/6mice 185. Sherr, D. H., Doff, M.E. 1982.Haptenceil fusion.II. Characterization of idispecificT ceil responsesto 4-hydroxy-3otopes by monoclonalanti-idiotope anmtrophenylacetyl. XIII. Characterizatibodies. Eur. Y.. Immuno£ 9:1004-13 tion of a third T cell L~pulatiunin175. Reth, M., Kelsoe, G., Rajewsky,K. volvedin suppression of in vitro PFC 1981.Idiotypic regulation by isologous responses. J. ImmunoL128:1261-66 monoclonal anti-idiotope antibodies. 186. Siekevitz, M., Gefter, M. L. Brodeur, Nature 290:257-59 P., Riblet, R., Marshak-Rothstein,A. 176. Richter, R. H. 1975. A networktheory 1982.Thegeneticbasis of antibodyproof the immunesystem. Eur. Y.. Imduction: The dominantanti-arsonate muno~5:350-54 idiotype response of the strain mice. 177. Rodkey,L. S. 1974.Studies ofidiotypic Eur. J. Immuno~12:1023-32 antibodies. Productionand characteri- 187. Siekevitz, M., Huang,S. Y., Gefter, M. zationof autoanti-idiotypicantisera. J. L. 1983. Thegenetic basis of antibody Exp. Med~139:712-20 production: Onevariable region heavy 178. Roland,J., Cazenave,P.-A. 1981. Rabchain geneencodesall moleculesbearbits immunized against b6 allotype exing the dominantanti-arsonate idiotyp¢ press s’mdlaranti-b6 idiotopes. Eur.J. in the strain A mouse.Eur. I. ImmunImmunoL11:469-74 ol. In press 179. Rubinstein,L. J., Yeh,M., Bona,C. A. 188. Sigal, N. H., Garhart, P.J., Klinman, 1982. Idiotype-anti-idiotype network. N. R. 1975. The freque~myof phosII. Activationof silent clonesby treatphorylcholine-specific Bcells in conven-

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189. Sigal, N. H., Picard, A. R., Metcalf,E. S., Gearhart, P. J., Klinman,N. R. 1977.Expressionof phosphorylchollnespecific B cells duringmurinedevelopment. J. Exp. Med.146:933-48 190. Sogn,J. A., Yarmush,M.L, Kindt, T. J. 1976.Anidiotypic markerfor the VL region of an homogeneousantibody. .,Inn. ImmunoL(Paris) 127C:397-408 190a.Somm6, G., Semi,J. R., Leclercxl, L., Moreau,J.-L., Mazi6, J.-C., Momier, D., Fougereau, M., Th~ze, J. 1982. Contribution of the H- and L-chains andof the bindingsite to the idiotypic specificities of mouseanti-GATantibodies. MoLImmunoL19:1011-19 191. Sprent, J. 1973. Circulating T and B lymphocytes of the mouse.II. Life span. Cell. lmmunoL7:40-59 192. Strober,S. 1972.Initiation of antibody responsesby different classes of lymphocytes. V. Fundamentalchanges in the physiological characteristicsof virgin thymus-independent ("B") lymphocytes and "B" memory cells..L Exp. Mea~136:851-71 193. Strosberg,D. A., Couraud,P.-O., Shreiher, A. 1981.Immunological studies of hormone receptors: A two-way approach. Immunol. Today2:75-9 193a. Sugimura,K., Kishimoto,T., Maeda, K., Yamamura,Y. 1981. Demonstration of T15idiotype-positive effector and suppressorT cells for phosphorylcholine-specificdelayed-typehypersensitivity response in CBA/N or (CBA/N X BALB/c)Ft male mice. Fur. d. ImmunoL11:455-61 194. Staudt, L. 1982. In Idiotypes-dntigens on the Inside, ed. I Westen-Schnurr, pp. 96-111. Basel: Editiones Roche 194a. Suzan, M., Valstedt, F., Boned,A., Rubin, B. 1982. Genetic chasing of T helper cell idiotype andallotypegenes. Immunogenetics16:229-41 195. Sy, M.-S., Bach, B. A., Brown,A., Nisonoff, A., Benacerraf, B., Greene, M. I. 1979. Antigen- and receptordriven regulatory mechanisms.IL Inductionof suppressorT cells with idiotype-coupled syngeneicspleencells, d. Exp. Med. 150:1229-40 196. Sy, M.-S.,Brown,A. R., Benacerraf,B., Greene,M.I. 1980. Antigen-andreceptor-driven regulatory mechanisms. III. Induction of delayed-type hypersensitivity to azobenzenearsonate with anti-cross-reactiveidiotypicantibodies,d. Exp. Med. 151:896-909 196a. Sy, M.-S., Brown,A., Bach, B. A., Benacerraf,B., Gottlleb, P. D., et al. 1981. Geneticand serological analysis

of the expressionof crossreactiveidiotypic determinants on anti-p-azobenzenarsonate antibodies and p-azobenzenarsonate-specificsuppressorT cell factors. Proe. NatL Acad. ScL USA78: 1143-47 197. Sy, M.-S., Dietz, M. H., Germain,R. N., Benacerraf,B., Greene,M.I. 1980. Antigen- and receptor-driven regulatoT mechanisms.IV. Idiotype-bearing I-J suppressor T cell factors induce second-ordersuppressor T cells which express anti-idiotypicreceptors,d.. Exp. Med. 151:1183-95 197a. Szewczuk,M. R., Campbell, R. J. 1981.Lackof age-associatedauto-antiidiotypic antibodyregulation in mucosM-associated lymphnodes. Eur. J. Immunol. 11:650-56 198. Tada, T., Okumura,K., Hayakawa,K., Suzuki, G., Abe,R., Kumagai,Y. 1981. Immunologicalcircuitry governed by MHCand VH gene products. In Immunoglobulin Idio~ypes: ICN-UCLM Symflosia on Molecular and Cellular Biology, ed. C. JanewayJr., E. E. Serearz, H. Wigzell,20:563-72.NewYork: Academic 199. Takemori,T., Tesch,H., Reth, M., Rajewsky, K. 1982. The immuneresponse against anti-idiotype antibodies.I. Induction of idiotope-bearingantibodies andanalysis of the idiotoperepertoire. Eur. Z ImmunoL12:1040-46 200. Tasiaux, N., Leuwenkroon, R., Bruyns, C., Urbain,ft. 1978.Possibleoccurence and meaningof lymphocytes bearing auto anti-idiotypic receptorsduringthe immune response. Eur. Z ImmunoL 8:464-68 200a. Th~ze,J., Morean,J.-L. 1978.Genetic control of the immune response to the GATterpolymer. ,~nn. ImmunoL (Paris) 129C:721-26 201. Thomas,W.R., Morahan,G., Walker, I. D., Miller,J. F. A. P. 1981.Induction of delayed-typehypersensitivityto azobenzenearsonateby a monoclonalantiidiotype antibody, d. Exp, Med. 153: 743-47 202. Tokuhisa,T., Komatsu,Y., Uchida,Y., Tani~. ehl, M. 1982. Monoclonalalloantlbodiesspecific for the constantre#on of156:888-97 T cell antigenreceptors. ,L ~xp. Med. 203. Trenkner, E., Riblet, R. 1975. Induction of antiphosphorylcholineantibody formationby anti-idiotypie antibodies. d. .Exp. Meal142:1121-32 204. Urbam,J., Wuilmart, C., Cazenave, P.-A. 1981.Idiotypic regulation in immunenetworks. In ContemporaryTop-

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munesystem. Common idiotypic speicsinMolecular Immunology, ed.F. P. Inwan,W.J. Mandy,8:113-48. New citicites betweenidiotypesandantibodYork:Plenum ies raised againstanti-idiotypicantibod205.Urbain, J.,Wilder, M.,Franssen, J.D., ies in rabbits. Z Exp. Med.150:184 Collignon, C.1977. Idiotypic regulation 215. Williams,K, R., Claflin, J. L. 1982. oftheimmune system bytheinduction Clonotypesof anti-phosphocholineanofantibodies against anti-idiotypic antitibodies inducedwith Proteusmorganii bodies. Proc. Natl. Acad. Sc~ USA (Potter). II. Heterogeneity,class and 74:5126-30 idiotypie analysesof the repertoires in 206. Yowler, L. B., Preod’homme,J. L., BALB/c and A/I-Id mice. J. Immunol. Scligmarm, M., Gathings,W.E., Grist, 128:600-7 W.M., et al. 1981. Diversity of im- 216. Woodland,R., Cantor, H. 1978. Idimunoglobulinexpression in leukaemie otype-speeific T helper cells are recells resemblingB-lymphocyte precurquired to reduce idiotype-positive B sors. Nature290:339-41 memory cells to secrete antibody.Eur. Y. lmmunol. 8:600-6 207. Weigert,M,, Riblet, R. 1976, Genetic control of antibody variable regions, 217. Wysocki,L. J., Sato, V. L. 1981. The ColdSpringHarborSyrup. Quant.Biol. strain A anti-p-azophenylarsonatcma41:837-46 jor crossreactive idiotypic family in208. Weil~ert dudes memberswith no reactivity to, M., Riblet, R. 1978. Thegewardp-azophenylarsonatc.Fur. J. Imnetic coatrol of aatibodyvariable regions in the mouse.SpringerSemi~Immunol. 11:832-39 munopathol.1:133-69 218. Yamumoto,H., Nonaka, M., Katz, D. 209. Weiler,E. 1981.In ldiotypes-Antigens H. 1979.Suppressionof hapten-speeitie on the lnside, ed. I. Westen-Sclmurr, delayedtype hypersensitivityresponses in miceby idiotype-specitic suppressor p.p.. 142-49.Basel: EditionesRoche 210.weiler, I. J., Weiler,E., Sprenger,R., T cells after administration of anti-idioCosenza,H. 1977. Idiotype suppression typic antibodies. Z Exp. Med. by maternalinfluence. Eur. Z lmmunol. 150:818-29 7:591-97 219. Yamauchi,K., Chao, N., Murphy,D. 211. Weinberger, J. Z., Get’main, R. N., B., Gershon, R. K. 1982. Molocular Benacerraf,B., Doff, M.E. 1980. Haptcomposition of an antigen-specificLy-1 en-speeifie T cell responses to 4T suppressorinducer factor. Onemolehydroxy-3-nitrophenylacetyl. V. Role culebindsantigenandis I-J-, anotheris of idiotypesin the suppressorpathway. I-J+, does not bindantigen, andimparts an Igh-variable region-linkedrestricZ Exp. Med. 152:161-69 212. Weinuerger,J. Z., Germain,R. N., Ju, tion. J. Exp. Med.155:655-65 $.-T., Greene,M.I., Benacerraf, B., 220. Yarmush,M., Sogn,J. A., Mudgett,M., Doff, M.E. 1979.Hapten-speeifie T cell Kindt, T. J. 1977. The inheritance of responses to 4-hydroxy-3-nitrophenyl antibodyV regionsin the rabbit: Linkacetyl. II. Demonstration of idiotypie .age of an H-chain-specificidiotype to determinantson suppressor T cells. Z unmunoglobulin allotypes. Z Exp. Med. Exp. Meal. 150:761-76 145:916-30 213. Wells, J. V., Fudenberg,H. H., Givol, 221. Yang, C., Kratzin, H., GiSt2, H., D, 1973.Localizationof idiotypicantiThiunes,F. P., Kruse,T., et al. 1982. genie determinantsin the F, region of PrimLrstruktur mensehlicher Hismurine myeloma 315. tokompatibilit~tsantigene der KlasseII Proc. Natl. Acad.protein Sc£ USAMOPC 70:1585-87 2. Mitteilung: Aminos~uresequenz der 214. Wilder, M., Demeur,C., Dewasme, G., N-terminalen179Reste der ~-Kettedes Urbain, J. 1980. Immunoregulatory HLA-Dw2/DR2-Alloantigeus.Hopperole of maternalidiotypes. Ontogeny of Seyler’s Z. Physiol. Cher~363:671-76 immunenetworks. :. Exp. Meal. 152: 222. Zinkernagel, R. M., Doherty, P. C. 1024-35 1974.Restrictionof in vitro T-cell me214a. Wilder, M., Fraussen, J.-D., Colligdiated eytoxieity in lymphocyticehorinon, C., leo, O., M~’iam6,B,, et al. omeningitis within a syngeneic orse1979. Idiotypic regulation of the iramiallogcneic system. Nature 248:701-2

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EPITOPE-SPECIFIC REGULATION Leonore .4. Herzenberg, Takeshi Tokuhisa, and Kyoko Hayakawa Genetics Department, Stanford California 94305

University

School of Medicine, Stanford,

INTRODUCTION Longbefore lymphocyteshad been identified with certainty as the precursors of antibody forming ceils, immunologists and immunogeneticists were well aware that animals immunizedwith the complex antigens like bovine serum albumin individually produce antibodies to different subsets of the epitopes (determinants) on the antigen. Over the years, manyof the cells and cell interactions that regulate antibody production have been defined in great detail; however,the processes that control the characteristic individuality of antibody responses still remain shrouded in mystery. The epitope-specific regulatory system described here offers a workableexplanation of howthis variation is generated and maintained. Furthermore, as we shall show, the joint operation of the carrier-specific induction mechanismand the epitope-specific effector mechanismthat defines the characteristics of the regulated response provides an explanation for manyof the odd observations encountered in studies of immunologicmemoryand carrier-specific regulation. Current concepts of howcarrier-specific regulation influences antibody responses to the epitopes (immunogenicstructures) on carrier molecules date from the early 1970s, whenMitchison (I) and Rajewskyet al (2) first demonstrated that carrier-primed T cells help hapten-primed B cells give rise to adoptive secondary antibody responses. These studies, which showed that "... the antigen [hapten-carrier conjugate] is recognized by two receptors, one directed to the hapten and the other to a determinanton the carrier protein" (1), in essence formulated the contemporary definition of a dependentantigen, i.e. a macromoleculewith at least one "carrier determi609 0732-0582/83/0410-0609502.00

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nant" recognized and used by T cells to regulate antibody responses to the various epitopes on the antigen. The hapten-carrier bridge mechanismsuggested for carrier-specific helper activity in these early studies is as viable today as it was whenfirst stated (and still remains unproven); however,the simplistic two-cell model (helper T and memoryB) introduced initially has undergone considerable expansion. Twofunctionally distinct carrier-specific regulatory T cells have been added, one which suppresses antibody responses (3-8) and another which contrasuppresses such responses (9). Furthermore, several carrierspecific cells have been identified as part of developmentalcascades leading to the emergenceof the functional cell types. Finally, on a theoretical level, a series of carrier-specific regulatory circuits have been proposed locating the various functional cells in a self-limiting ("feed back") type systemthat controls antibody production by controlling the supply of carrier-specific help (10). This construct is consistent with most of the available data; however, it fails to explain a numberof relevant observations (several of whichpreMate its inception). In particular, becauseit is predicated on the idea that carrierspecific regulatory interactions do not selectively influence antibody production to individual epitopes on an antigen, it tends to trivialize findings that suggest links between cartier-specific and epitope-specifie or other regulatory mechanisms.In essence, it has led to consistent disregard of evidence suggesting that immunizingcarrier-primed animals with a "new" hapten coupled to the priming carrier results in the induction of specific suppression for antibody responses to the hapten (see below). Mitehison (1) and Rajewskyet al (2) overtly chose to ignore this peculiar response failure to simplify consideration of the mechanismsinvolved in carrier-specific help. Thus, although they carefully cite evidence from several studies (including their own) showingthat carder-primed animals often fail to producedetectable levels of antibodies to haptens introduced subsequently on the priming carrier, they put more trust in opposing evidence showingthat significant anti-hapten antibody production can be stimulated by this "carrier/hapten-carrier" immunizationprotocol. Rajewskyet al (2) makethis point quite directly by stating: "Wesuggest that, in general, an animal pretreated with free carrier and receiving a secondary injection of this carrier eomplexedwith a hapten will be found to produce a better anti-hapten response than without the pre-treatment, if the experimental design is aimed at detecting this effect." This "leap in faith" (although largely incorrect) was probably crucial the orderly progress of early studies exploring the mechanismsregulating antibody production. The idea that carrier-priming wouldaugment (rather than suppress) subsequent antibody responses to a hapten presented on the

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EPITOPE-SPECIFICREGULATION 611 primingcartier cleared awayconfusion and allowed rational planning of adoptiveand in vitro experimentsthat followedfromthe newlypublished carrier-specific help studies. However,although this idea wasadvanced originally as a prediction and had beenshownto be invalid undercertain circumstances,it rather rapidly assumed the mantleof truth. Consequently, several years later, we(and mostof our colleagues)werequite surprised the suppressedrather than augmentedanti-hapten antibody responsesthat weobtained followingcarrier/hapten-earrier immunization(11). Theseunexpectedfindings (e.g. see Figure1) engendereda rapid series of experimentsexploring the mechanism of the suppressionand a somewhat moreleisurely search throughthe literature lookingfor precedentsfor our observations. Thus,by the time wediscoveredthat previousinvestigators had ascribed this kind of responsefailure to impairedanti-hapten memory development (14) or to the presenceof anti-carrier antibodies(12, 13), hadalready ruled out these possibilities andrealized that weweredealing with a previously unrecognized"epitope-specific" regulatory systemthat selectively controls antibodyproductionto individualepitopes accordingto the dictates of carrier-specific (and other) regulatoryT cells presentin the immunologicenvironmentwhensuch epitopes are first introduced (11, 15-22). In pursuingour studies of this system,wehavebeenconcernedprimarily with finding out howit works; however,we have also put considerable thought and someexperimentaleffort into determininghowit could have escapednotice for so manyyears. Not surprisingly, the answersto these questions often merge.For example,in attempting to establish adoptive assays for the inducerandetfector cells responsiblefor the suppression,we foundthat quite strong in situ suppressionis diti~cult to transfer to irradiated recipients, particularly whenmeasuredas the ability to suppress a responsemountedby a co-transferredcell population(16). This characteristic tendencyfor help to predominate over suppressionin irradiated recipients put several investigatorsoff the track, includingSarvaset al (14) who in 1974reasonedcorrectly that cartier-specific suppressorT cells induce suppressionfor anti-hapten responsesin carrier/hapten-carrier immunized virgin B ....... > EarlyMemory ....... > Mature Memory .......... > IgGAFC

:

DEVELOPMENT

: : EXPRESSION

Figure 1 Regulation of IgG (memory) responses. Mechanismsthat regulate memoryB-cell developmentregulate the potential for IgG antibody production (22). Mechanismsthat regulate memoryB-cell expression control which and howmanyof the memoryB cells present in a given animal actually give rise to AFC(17).

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animals(see below),but discardedthe idea becauseit "requires postulating that the effect of these suppressorcells is abolishedin cell transfer experiments.’’ Thegeneral tendencyto measureanti-hapten antibody productionexclusively whilestudyingthe mechanisms involvedin carrier-specific regulation also appears to haveplayed a majorrole in "hiding" the epitope-specitic system. Ishizaka & Okudaira(23), for example,demonstratedthat antihapten responses were suppressed while anti-carrier antibody responses proceededto secondarylevels in carder-primedanimalsstimulated with a haptenon the primingcarrier. Discussingthis work(in 1973),these investigators referred to work by Tada & Okumura(24-26) demonstrating the existence of suppressorT cells and suggested"... that immunizationwith 1 to 10 #g of ovalbuminmayresult in the formationof so called [carrierspecific] suppressorT cells that mightsuppresspreferentially the primary anti-DNPresponse" (23). Immuno-historywouldprobablybe quite different had Tada(or others interested in carder-specific suppressorT cells) taken this suggestionseriously enoughto test anti-carrier antibody responsesin suppressionassays routinely. All in all, the attractivesimplicityof the idea that carrier-specificsuppressor T ceils regulate antibodyproductionby depleting carrier-specific help appears to be sutticient to explain the willingness of manylaboratories (including our own)to ignore subtle inconsistencies and leave a fewmosscoveredstones unturned.Perhapsthis has beenmainlyfor the good,since the mechanisms involved in epitope-specific regulation are considerably morecomplexandwould,in fact, havebeendifficult to explorewithoutthe levd of technologyand theoretical understandingreachedwith the last few years. In any event, we nowappear to have reached the time whenreevaluationof past evidenceis essential to present progress. Thesections that follow describe various aspects of our studies of the epitope-specific regulatory system. Webegin with an overviewsection (immediatelybelow)that broadly outlines the systemas a whole, documenting statements with references rather than with evidence. Theremainingseetions, in contrast, discussaspects of the systemin moredetail andinclude muchof the evidence uponwhichour conclusions are based. The workwediscuss here has mainly been conductedin our laboratory at Stanford; however,studies with carder-specific suppressorT cells and soluble factors were conductedin Dr. MasaruTaniguchi’s laboratory in Chiba, Japan. Almostwithout exception, the evidence we report derives from experimentsthat repeat earlier workbut include key controls aimed at distinguishingcarrier-specific fromepitope-specificregulatoryeffects. Thus, weindependentlymeasureboth the anti-hapten and anti-carder antibodyresponsesfollowingvarious antigenic stimulations, andweconfirmthe

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EPITOPE-SPECIFICREGULATION 613 induction of epitope-speeific suppressionfor a given anti-hapten antibody response by a final immunizationwith the hapten conjugated to an unrelated carder molecule.As weshall show,the application of these rather straightforwardtechniquesprovidessurprising insights into the older data andreveals the outlines of a highly flexible central regulatorysystemresponsiblefor controlling all aspectsof antibodyresponses.

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EPITOPE-SPECIFIC

REGULATION:

AN OVERVIEW

Theepitope-specific systemapparently provides a common channelthrough whichcarrier-specific, isotype-specific, allotype-speeific, and 1-regiondefined mechanisms exert control over antibody responses(11, 15-21). plays a key role in regulating IgGantibodyresponsesto haptensand native epitopes on commonly used carrier moleculessuch as KLH (keyhole limpet hemocyanin)and CGG(chicken gammaglobulin) (11, 15-18). Furthermore,it is active in regulating IgGresponsesto epitopes on the synthetic ter-polymerTGAL, even in genetic "non-responders"to this antigen (20). Carder-specificinteractions induce it to suppressantibodyproduction(to individual epitopes on the carrier); however,onceinducedto suppress given anti-epitope response, it will suppressthat responseevenwhenthe epitope is presented in immunogenic form on a different carder molecule (II, 15-17). Theeffector mechanism in this systemcontrols memory B-cell expression (as opposedto development;see Figure 1) and appears to be the ultimate arbitor of whichandhowmanysuch B cells will be permittedto differentiate to IgGantibody-formingcells (AFC)in response to a given antigenic stimulus.It is epitopespecific in that it independently regulatesthe amount and affinity of the antibodyresponseproducedto each of the epitopes on a complex antigen(11, 15-17);andit is Igh restricted in that it selectively controls the IgChisotype andallotype expressionin such responses(17-19). Theseproperties suggestthat the overall systemis composed of individually specific elements,each chargedwith the regulation of a subset of B cells committedto produceunique or closely related IgGmolecules(17). Theflexibility of this (compound) regulatorysystemandthe nature of its effects on in situ antibodyresponsesare extraordinary. It is capable of suppressingvirtually the entire primaryand secondaryantibody response to a given epitope. Therefore, it can completelyconceal the presence of normalanti-epitope memory B-cell populations clearly demonstrablein adoptive transfer assays (11, 15, 16). Alternatively, it can support the expression of a subset of memory B ceils and suppress the expression of others, thereby defining the uniquespectrumof an antibodyresponse producedby a given animal(17-19).

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Carrier-specific and other regulatory mechanisms operative in the immunologicalenvironmentwhenan epitope is first introduced determine whichcomponentsof the antibody response will be suppressed and which will be supported(11, 15-17). Prior immunization history, genetic predisposition towardresponsiveness(20) and perinatal immunologic "conditioning" (18) all contribute to this determination.Thus,the epitope-specific systemconstitutes an ideal candidate for a central integrative mechanism capableof resolving conflicting signals fromvarious peripheral regulatory systemsand translating a coherent decision to the antibody-producing apparatus. In keepingwith this role, this systemoffers a uniqueregulatorycapability that providesboth the stability requiredto maintaina responsepattern once inducedand the flexibility to modifythat responsepattern whenstimulatory conditionschangedramatically.In essence, the individual epitope-specific, Igh-restricted elementsin the systemappearto behavesimilarly to bistable electronicbinary("flip-flop") circuits that can be switchedinitially into "on"or "off" positionby a smallelectrical force andthen require a substantially larger force to switchthemto the oppositeposition. Thatis, initial immunization conditions that induce epitope-specific elementsto suppress a givenIgGantibodyresponsewill usuallyfail to do so oncethe systemhas beeninducedto supportthat response; and, similarly, conditionsthat inducethe systemto support a response will be far less effective oncethe system has been induced to suppress the response. Nevertheless, either suppression or support can be reversed by sul~cient stimulation in the oppositedirection (17). The evidenceuponwhichthese conclusions (and hypotheses) are based is presentedin detail in several publicationsthat haveappearedwithin the last few months(16-19). Therefore, in the discussion whichfollows (and in Tables 1 and 2), we summarizethese findings and concentrate more intensively on several recent studies defining the T cells that mediateepitope-specific suppression and demonstrating someof the more complex aspects that exist within the system. CARRIER/HAPTEN-CARRIER IMMUNIZATION INDUCES EPITOPE-SPECIFIC SUPPRESSION FOR IgG ANTI-HAPTEN RESPONSES Theepitope-specific regulatory systemcan be specifically inducedto suppress primaryand secondaryIgG antibody responses to the dinitrophenyl hapten (DNP)without interfering with antibody responses to epitopes the carrier moleculeon whichthe DNPis presented(see Table3). Furthermore,onceso induced,it specifically suppressesantibodyresponsesto DNP presented on unrelated carrier molecules. Themagnitudeof a suppressed

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primary anti-DNP response is usually about 30%of the normal primary response; however,the affinity of a suppressedresponse is about 10-fold lower than normal. Suppressedsecondaryanti-DNPresponses are typically less than10%of normalandhaveaverageaffinities that are at least 100-fold below normal(11, 16). For most of our studies, we induce this suppression by immunizing animals sequentially with a carder molecule such as KLH (keyhole limpet hemocyanin) and then DNPconjugated to" the carrier (i.e. with the Table 1 Induction of epitope-specific tions

suppression occurs under a wide variety of condia Result

Variable tested Epitope

DNP, TNP, NIP

Suppression induced for both epitopes by carrier/hapten-carrier; suppression inducible for KLHepitopes by other protocols

Carrier

KLH, CGG, OVA, TGAL

All prime for suppression induction; 100 ~.g on alum sufficient; somegenetic restrictions (see table 2)

Age

KLHat 8 weeks to >6 months

Suppression equally strong at all ages

Timing

1 to 13 weeks between KLH and DNP-KLH

Suppression equally strong for all intervals between carrier and hapten-carrier

KLH/DNP-KLH then DNP-KLH or DNP~-CGG up to 1 yr later

Suppression equally strong for all intervals between first and second hapten-carrier immunizations

BALB/C, BAB/14, SJL, SJA, C3H, C3H.SW, A/J, (SJL × BALB/C), C57BL/10, C57BL/6

Suppression inducible in all strains

Strains

aSummarized from References 1 through 7. Table 2 Epitope-specific suppression in genetically controlled regulatory conditions Condition I-region controlled responses Impaired response to KLHcarrier (non-MHC) Allotype suppression for Igh-lb (IgG2a) allotype production in (SJL × BALB/C)F1

Immunization

Result

TGAL/TNP-TGAL in C3H(H-2k) and C3H.SW(H-2b) KLHpriming in A/J or C57BL/10

Suppression induction occurs in responder and non-responder strains (20)

DNP-KLHat 8 weeks of age (prior to midlife remission from allotype suppression)

Poor suppression induction by KLH/ DNP-KLH;normal induction by CGG/ DNP-CGG (21) Igh-lb responses to DNPand KLH specifically suppressed during remission (18)

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Table 3 Anti-hapten antibody production is specifically hapten-carrier immunizedmice aImmunizations Second DNP

K -K -K --

-D-K D-K D-C D-C D-K D-K

-----D-K D-K

K ---

D-K D-K D--C

D-C D--C D-C

K

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In situ IgG2a b antibody responses

First DNP

Carrier

suppressed in carrier/

Anti-DNP c~tg/ml (Ka) < 3 (< 0.3) 5 (<0.3) 35 (5) 20 (2) 13 (1) 9 (0.5) 120 (300) 6 (<0.3) 60 (100) 85 (400)

Anti-KLH units

Anti-CGG units

20 170 15 --370 130 ----

---21 11 --8 9 100

a K = KLH(keyhole limpet hemocyanin); C = CGG(chicken gammaglobulin); DNP= 2,4-dinitrophenyl hapten; D-K -- DNP-KLH;D-C = DNP-CGG.(BALB/c SJL)FI mice injected i.p. with 100/~g of the indicated antigen on alum at approximately six week intervals. bSerumantibody levels measuredby RIA two weeks after last indicated immunization. Anti-carrier antibody expressed as percentage of antibody in a "standard" secondary response serum pool. CKaM-I X 106 measured by RIA (22).

KLH/DNP-KLH immunizationsequence). Several weeks later we again immunizewith DNP,this time either on KLH or on an unrelatedcarrier moleculesuch as CGG (chickengamma globulin). Weinject 100 #g of each antigenon alum,usually at 4-weekintervals, andcompare the IgGantihaptenandanti-carderresponsesobtained2 weeksafter eachimmunization to the responsesraisedin (age, sex, andstrain matched) controlgroupsthat are not primedinitially with the carrier protein andthus are immunized only withthe appropriatehapten-carrier conjugates. ThiscarrierFaapten-carrier immunization protocolinducesmarked suppressionfor IgGanti-haptenantibodyproduction(as indicatedabove)but doesnot interfere withanti-carderantibodyresponsesor withthe devdopmentof normalanti-haptenmemory B-cell populations.Thatis, splenic B cells from either KLH/DNP-KLH immunized animals or DNP-KLH primedanimalsgive rise to equivalentanti-haptenmemory responsesin adoptiverecipients supplemented with an appropriatesourceof carrierspecific help. Furthermore,IgG anti-DNPresponses in KLH/DNP-KLH immunized animalsare suppressedto belowprimarylevel while anti-KLH responsesare equivalentto the (secondary)anti-KLH responsesobtained fromcontrol animalsimmunized a similar number of times withthe carrier protein, i.e. twice with KLHor DNP-KLH (11-16),

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EPITOPE-SPECIFICREGULATION 617 Comparison of responses in KLH/DNP-KLH/DNP-CGG immunized animals and DNP-KLH/DNP-CGG immunizedcontrols demonstrates the specificity of the suppressionfor anti-DNPresponsesevenmoredramatically. Controlanimalsproducetypical high magnitude,high affinity in situ secondary IgG anti-DNPresponses. The experimental group produces IgG anti-DNPresponsesthat are still belowprimarylevels. Nevertheless, all animals (experimentaland control) produceequivalent primaryIgG antibodyresponses to the CGGepitopes on the second hapten-carrier conjugate. Thus, carder-primedanimals fail to produceantibodies to a "new" hapten presented subsequently on the priming carder and animals immunized in this waydevelopa persistent suppressionspecific for responsesto the hapten, evenwhenpresented next on a different carder molecule(11,

15, 10. Althoughthe failure of anti-hapten responsesin carder/hapten-carder immunizedanimals appears novel from a contemporaryperspective, this phenomenonwas well knownin an earlier era (ca 1970), having been described in the landmarkpapers demonstratingadoptive carder-specific help interactions (1, 2). It waslater attributed to interferencewith memory B-cell development (12) and then largely forgotten as attention shifted using adoptive andin vitro assays for characterizing the carder-specific (and other) mechanisms regulating antibody responses. Weviewthe loss this key "immunologic fact" as understandable(17) but nonethelessregrettable, since someserious misconceptions(discussedbelow)could havebeen avoidedif it hadnot fallen fromsight.

CARRIER-SPECIFICSUPPRESSORT CELLS INDUCE EPITOPEoSPECIFICSUPPRESSION Carrier-specific suppressorT cells (CTs)that arise shortly after priming with a carder molecule(4-6) are responsible for inducing the epitopespecific system to suppress IgG anti-hapten responses to "new"epitopes presented on the carrier molecule(15, 16). Thesewell-known regulatory cells werecommonly believed to regulate antibodyproductionby interfering with carder-specifichelp; however,by repeatingthe original CTstransfer experiments (4, 5) with additionalcontrolsthat define the specificity the mechanism mediatingthe suppressionin CTsrecipients, wehaveshown that KLH-specificCTsregulate responses by inducing typical epitopespecific suppressionfor anti-DNPresponseswhenthe recipients are immunized with DNP-KLH (15, 16). Thus, whether KLH-pdmed animals are immunized directly with DNP-KLH (KLH/DNP-KLH immunization sequence) or whetherT cells from KLH-primed animals are challenged with

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DNP-KLH in (non-irradiated) recipients, anti-DNPresponses are persistently suppressedwhereasanti-carrier responsesproceednormally. The demonstrationof CTsin KLH-primed animals generated someconfusion initially becausesplenic T cells fromsuch animalsprovidean excellent source of carrier-specific help (CTh)rather than suppression (irradiated) adoptivetransfer recipients. This differenceappearedto be due to the use of aqueousKLHfor generating CTsand alum-precipitated KLH for generatingCTh;however,our results indicate that primingeither with aqueousor alum-precipitated antigen induces both CThand CTsin the immunized animals,that both kinds of regulatory T cells are active in such animals, and that each can be demonstratedindependentlyin the in vitro or adoptiveassaysdevelopedto reveal its activity. The aqueousKLHpriming protocols usually used to generate CTs did prove to be somewhatmoreeffective in primingfor in situ suppression induction than the alum-KLH priming protocols commonly used to generate KLH-specitichelper T cells; however,as a rule, wehave used alumKLHpalming in the carder/hapten-earder sequence and in adoptive studies with KLH-primed T cells, and have generated strong epitopespecific suppression.In fact, as weshall showbelow, roughlyequivalent suppression is induced (by DNP-KLH) in animals primed with KLH alum, in Freund’s adjuvant or in aqueousform. In essence,these studies collectivelydemonstrate that the epitope-speeifie systemconstitutes the major, if not the only, etfector mechanism through whichCTscontrol antibody production. Thus, they define a newrole for CTs(as inducers of epitope-specific suppression) and cast these cells "conditioners" of the immunologic environmentthat, whenpresent, alter howthe epitope-speeifiesystemrespondsto (carder-borne)epitopes that has not "seen" before. Coupledwith the bistable properties of the epitopespecific system, this construct explains howpalmingwith an antigenic (carrier) moleculecan simultaneouslyprepare the animalto producetypical secondaryantibodyresponsesto epitopes encounteredinitially on the priming antigen and yet to specifically suppressantibody productionto "new" epitopes encountered subsequently on the sameantigenic molecule. We return to this point in a later section exploringthe consequences of bistable regulation. The revised view of howCTs regulate antibody production discussed aboveis consistent with recent evidencefromstudies with T-cell lines and hybrids demonstratingtwo carrier-specific inductive pathwaysthat terminate in epitope-specificcells (7, 8), onethat suppressesantibodyproduction and the other that augmentsantibody production. Relationships (if any) betweenthese cells and the epitope-speeifie effeetor mechanism described here haveyet to be established; however,the separatecarrier-specific path-

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EPITOPE-SPECIFICREGULATION 619 waysthey define supportthe inductiverole our evidenceassignsto carrierspecific regulatorycells in in situ antibodyresponses.

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EPITOPE-SPECIFIC REGULATION IS IGH-RESTRICTED The induction and maintenanceof suppression (in carder/hapten-carder immunized animals)varies in efficiency for individual isotype anti-hapten responses (17-19, 21). IgMresponses showno evidence of suppression. IgG2a, IgG2b, and IgG3 responses are easily suppressed whereas IgG1 responsesare morerefractory to suppressionin that they tend to be suppressed in fewer animals under suboptimalsuppression-induction conditions and to escape from suppressionmorefrequently than the other IgG isotypes after a given numberof restimulationswith the hapten. This characteristic isotype hierarchy prevails in animalsin whichthe induction of epitope suppressionis either genetically impairedor experimentallyminimizedby immunizing initially with lowdosesof the carrier protein. In fact, wheneversuppression is weakinitially or begins to waneafter repeated antigenic stimulation, IgG1antibodyresponsesare alwaysthe first to appear (17, 21). IgG2a, IgG2b,and IgG3responses showabout the samesusceptibility to suppression; however,whensuppressionis weakor waning,these isotypes often "escape"individually or in randompairs (very occasionallyin animalsthat remainsuppressedfor IgGresponses). This selective expression demonstratesthe independentcontrol exerted by the eptitope-specific systemwith respect to isotype representationin antibodyresponses.Studies with allotype-suppressedmicesimilarly showthat the epitope-specitic systemcan specifically suppressIgh-lb (IgG2aallotype) responsesto individual epitopes in an allotype heterozygotewithoutinterfering with production of the (allelically determined)Igh-la responsesto the sameepitopes (18). Thus,the individual elementsthat mediateepitope-specific regulation appear to be restricted to controlling the productionof antibodies with the sameor closely related combining-sitestructures and a single heavychain constant-regionstructure (allotype/isotype). Theoreticalconsiderationssuggestthat this Igh constant-regionrestriction maybe basedexclusivelyon the recognitionof allotypic (rather than isotypic) structures. Thatis, since isotypic structures are sharedbetween allotypically different heavychains, isotype-restricted regulation cannot explain the selective regulation of Igh-lb allotype antibodies. Allotyperestricted regulation, in contrast, can clearly accountfor selective isotype regulation since nearly all Igh allotypic structures are uniqueto (and thus can identify) the heavychain isotype on whichthey are found (27). Thus,

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it is likely that the selectiveregulationof bothisotypeandallotyperepresentation in individual anti-epitope responsesderives froma requirementfor recognitionof polymorphic (allotypic) regions of Igh heavychain constant regions.

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EPITOPE-SPECIFICREGULATION IS BISTABLE Bistable systems, by definition, have two alternative steady states with mutuallyexclusive functions. Whenconfronted initially with a stimulus favoringone state or the other, these systemsmoverapidly to the favored state. Stabilization mechanisms then maintainthe initially inducedstate, so that a substantially strongersignal is required to moveto the other steady state than wouldhavebeenrequired to establish that state initially. Thus, bistable systemstend to remainas initially inducedbut nonethelessremain capableof shifting to the alternate state if stimulatoryconditionsso dictate (17, 28). The characteristics of the regulation providedby the epitope-specific system meet these criteria (17). As we have indicated, carrier/haptencarder immunizationinduces suppressionfor IgG responsesto the hapten. Onceinduced, this suppression tends to be maintained(especially for IgG2a, IgG2b,and IgG3responses). Repeatedstimulation with the hapten (on any carder), however, eventually induces IgG anti-hapten antibody production (morequickly for IgG2than for the "moresuppressible" isotypes). Antibodyproduction, onceinitiated, also tends to be maintained. Carder/hapten-carrierprotocolsthat inducestrong suppressionfor anti-hapten antibodyresponsesin virgin animalsare substantially less effective in animals producingan ongoingprimaryIgGanti-hapten response (e.g. due to stimulation with the haptenon an unrelated carder prior to completionof the carrier/hapten-carder sequence). Underthese conditions, detectable suppressionis inducedin about half the animaland, wheninduced, mainly affects the moresuppressible isotypes. Thus, the initiation of antibody productionimpairsthe subsequentinduction of suppression,andthe initial induction of suppressiontends to preventsubsequentinitiation of antibody production(17). This reciprocal relationship defines a bistable regulatorymechanism that fixes long-termantibodyresponsepatterns accordingto the conditions under whichit first "sees" individual epitopes. Thus,it providesa vehicle through whicheven quite transient conditions in the initial regulatory environmentcan strongly influence the characteristics of subsequentantiepitope responses.

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EPITOPE-SPECIFICREGULATION 621

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CONSEQUENCES OF BISTABLE REGULATION Although memoryB-cell induction and developmentare required for ananmestic(memory) responses, the epitope-specific systemand the mechanismsthat induce it to suppressor support memory B-cell expressionplay a key role in determiningwhichand howmanyof these B cells will be expressedwhenan animalre.encountersa givenepitope. In fact, as a general rule, the characteristics of in situ memory responsesprovidea muchbetter measureof the status of the epitope-speeitie system than of the memory B-ceil populationsthat havebeengeneratedby a particular primingprotocol. Ourevidenceon this point suggests that muchof the informationfrom studies usingin situ secondaryresponsesto evaluate the effects of priming conditions on the development of memory B cells requires re-evaluation to distinguish conditions that truly influence B-cell development from those that influencethe in situ expression(epitope-specifieregulation) of memory B-cell responses. Carrier/hapten-carrier immunization,for example,induces normal anti-hapten memorypopulations that can be revealed in adoptive recipients (11, 15, 16); however,these memory cells remainentirely cryptic in situ under the conditions usually used to test for the presence of immunologic memory,e.g. repeated boosting with 1/zg aqueous antigen (unpublishedobservations). Stimulatoryconditions that overcome the initially inducedepitope-speciticsuppressionreveal the presenceof the cryptic memory populations, but this generally requires several immunizations with primingdoses of the antigen (e.g. 100/~gof alum-precipitated hapten-carrier conjugatewere used for each immunizationin the suppression-reversal experimentsdiscussedabove). Adoptivestudies in whichtransferred spleen cells (co-resident B plus T) are used to evaluate memorydevelopmentsuffer from muchthe same problem,since suppressionis maintainedwhenspleen cells fromsuppressed animalsare transferred to adoptiverecipients (16, 21). In essence, wehave foundthat the only reliable wayto reveal anti-hapten memory that is not expressedin situ is to transfer T-cell depletedsplenic (B cell) populations into recipients supplemented with carrier-primed T cells (preferably from animalsprimedwith alum-precipitatedcarrier protein plus B. pertussis). Theseconsiderations lead us to question the idea that variation in the memoryB-cell populations generated in individual animals immunized with a typical protein antigen accounts for the well-knowntendencyfor such animalsto produceantibodies to different subsets of the epitopes on the immunizingantigen (17). B-cell "clonal dominance"mechanismsmay contribute to this individualizationof antibodyresponses;however,considerable variability will also be introduc~lby stochastic processesinherentin

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the operation of a bistable regulatory system in whichthe major force inducingthe systemto suppressantibody production(CTs) maturesto full function several days after the initial immunization with a carrier protein andits associatedepitopes(17, 19). Thatis, since a bistable mechanism tendsto maintainitself in its initially inducedconfiguration,anti-epitope responsesestablishedprior to the emergence of a functional CTspopulation will tend to continue despite the presenceof these cells; however,anti-epitope responsesthat could not (or did not) becomestabilized rapidly enoughwill tend to be suppressedonce CTsbecomeactive. Therefore, the probability that a given epitope on a primingantigen will inducestable antibodyproduction(or stable suppression) in a particular animalwill be a functionof the rate at whichthe epitope induces support for antibody production in comparisonwith the rate at whichCTs mature (17). In practice, this "horserace"can be expectedto result in the induction of suppressionfor responsesto essentially randomsubsets of primingantigenepitopes in individual animals.Someepitopes, however,will tend to be morelike the DNPhapten, whichinduces support so rapidly that stable antibodyproductionis established well before CTsmaturein virtually all immunizedanimals (perhaps because such epitopes bind to a very wide variety of antibodycombiningsites and consequentlycan stimulate a large number of B cells). Otherepitopes will be notablyless successfulin establishing antibody production. Thus(in accord with common serologic experience), the frequencyof responsesto individual epitopes obtained in group of immunized animalswill be relatively reproduciblewhile the combination of epitopes detected by the antibodies producedby individual animals will vary from animal to animal.

TESTING THE HYPOTHESIS If CTsinducesuppressionfor responsesto carrier-borne epitopes that have not as yet inducedstable support for antibody production, then antibody responsesto the all of the epitopes on a primingantigen (even DNP)should fail if the level of antigen-specificCTsactivity is sufficient to initiate the induction of epitope-specific suppressionimmediatelyafter priming. This condition is rarely (if ever) metnaturally since the developmental cascade that results in the appearanceof functionalCTsonly beginsafter the first (priming) encounter with an antigen; however, it can be approximated experimentallyby priminganimalswith a hapten-carrier conjugateshortly after they havebeeninjected with appropriatelyspecific CTs-secretcdfactors (CTsF)that mediatethe induction of epitope-specific suppression. Underthese conditions (if our concept of bistable immunoregulation is

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EPITOPE-SPECIFIC REGULATION

623

correct), antibody responses to the hapten and to the native epitopes on the carder should all be suppressed. Data in Table 4 show that, as predicted, anti-DNP and anti-KLHantibody responses are suppressed in animals that received KLH-specific CTsF shortly before being primed with DNP-KLH.Furthermore, anti-DNP responses predictably remain suppressed in these animals when the hapten is presented subsequently on an unrelated carder molecule (DNP-CGG), whereas antibody responses to the (CGG)epitopes on the second carrier molecule proceed normally. Thus, this immunizationprotocol results in the induction of typical epitope-specific suppression for antibody responses to DNPand (by inference) to all other epitopes presented on the priming carrier. These findings (T. Tokuhisa, M. Tagawa, M. Taniguchi, manuscript in preparation) directly demonstratethat all epitopes on hapten-carrier conjugate are treated equivalently to the hapten in the carder/hapten-carrier immunizationsequence whenCTs activity is artificially introduced prior to priming with a hapten-carder conjugate. Nevertheless, under normal circumstances, the emergence of active CTs shortly after priming does not interfere with antibody production to (at least someof) the epitopes on the priming antigen. This apparent paradox confirms the existence of a bistable mechanismthat allows priming antigen epitopes to induce specific protec-

Table 4 Carrier-specific a pression

suppressor T-cell factor (CTsF) induces epitope-specific

sup-

dIgG2a antibody responses BALB/c bKLH-TsF

cImmunizations

-+ -+

D-K D-K D-K D-K

-+ -+

D--C D-C D-K D-K

D--C D--C

Strain BALB/c BALB/c BALB/c BALB/c BALB/c BALB/c C57BL/6 C57BL/6

DNP ~g/ml)

KLH (units)

CGG (units)

73 18 102 19 22 30 71 102

24 3 -------

--7 6 -----

aT. Tokuhisa, M. Tagawa, M. Taniguchi, manuscript in preparation. bKLH-specific suppressor factor (CTsF) prepared from BALB/cthymocytes as previously described (5); yield from 108 thymocytes injected per animal 24 hours prior to first immunization. CTsFprepared from BALB/c(H-2d) is not active in C57BL/6(H-2b) c 100 ~g each antigen on alum at two week intervals. dMeasured by RIA 2 weeks after last indicated immunization; anti-carrier responses expressed as percentages of "standard" adoptive secondary responses to the indicated antigen.

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tion (support) for antibody production before CTs gain sufficient strength to induce specific suppression for unprotected responses.

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EXCEPTIONSTO THE RULES During the course of studies characterizing the epitope-specific system and the conditions that induce it to suppress or support antibody production, we tested the effects of a wide variety of protocol modifications (different antigens, doses, timing, adjuvants, mousestrains, ere). In nearly all cases, the results we obtained (summarizedin Tables 1 and 2) were consistent with the properties of the bistable regulatory system defined by our original carrier/hapten-carrier immunizationstudies; however, we noted two striking exceptions, one concerning adjuvant effects on the carrier-specific suppression induction mechanismand the other (somewhat more surprising) concerning the maintenance of suppression for anti-DNP responses in sequential immunizations with DNPon different kinds of carriers. The adjuvant studies show that although KLH/DNT-KLH immunization resultsin theinduction of typicalepitope-specific suppression when animals are primed with aqueous KLH, KLHon alum or KLHon alum plus complete Freunds adjuvant (CFA), priming with KLHon alum plus Bordatella pertussis completely prevents the subsequent induction of suppression for anti-DNP responses (see Table 5). Coupling the B. pertusais Table 5 Carrier immunization with Bordatella pertussis prevents subsequent suppression induction

aImmunizations

with KLH(K) or DNP-KLH(D-K)

First

Second

--

---

K alum + PV K alum K alum K CFA K aqueous K aqueous -K alum -K alum K alum

-K alum --K aqueous ----K alum

a 100/~g each antigen at 4 week intervals; assay 2 weeks after last immunization.

Third

Anti-DNP in serum ~g/ml) IgG2a

IgG 1

alum alum alum alum alum CFA CFA

64 60 < 13 <6 < 15 <6 <7 70 <9

145 125 125 15 63 55 <28 200 128

D-K alum + PV D-K alum + PV D-K alum + PV

90 33 < 13

225 225 45

D-K alum D-K alum D-K D-K D-K D-K D-K D-K D-K

antibody responses measured by radioimmune

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625

with the DNP-KLH immunization in the sequence, in contrast, does not appear to interfere substantially with suppression induction since anti-DNP responses are clearly lower than in non-preimmunizedcontrols that received DNP-KLH plus B. pertussis. The interference with suppression induction does not appear to be due to the increased amounts of anti-KLH antibody produced by the KLH/ pertussis immunized animals since KLH/CFAimmunization stimulates roughly the same amountof antibody but does not interfere with suppression induction (see Table 6). These findings suggest that KLH/pertussis induces a carrier-specific call populationthat prevents epitope-specific suppression induction when animals are subsequently immunizedwith DNPKLH.Since this population appears to be functionally similar to the carrier-specific "contra-suppressor" population described by Gershonand colleagues (9), we wonderwhether carrier immunization with B. pertussis might not be an excellent way to stimulate contra-suppressor ceils and whether, in fact, such cells might not be the effector mechanismthrough which B. pertussis acts as an adjuvant to augment antibody responses. Results from current carrier/hapten-carrier immunization studies with DNPcoupled to sheep erythrocytes (D-SRBC)introduce another, more disquieting, exception to the otherwise consistent behavior of the epitopespecific system (19). SRBC/D-SRBC immunization induces what appears to be typical epitope-specific suppression in that the IgG anti-DNPresponse is substantially smaller and has a lower affinity than control responses (in D-SRBCimmunizedanimals), whereas the IgG anti-SRBC response climbs to secondary levels comparable to those in SRBC/SRBC-immunized controis. Similarly, IgG anti-DNPresponses remain suppressed after a second immunization with D-SRBC,and anti-SRBC responses proceed normally. However, when SRBC/D-SRBC-immunizedanimals are immunized with DNP-KLH,their IgG anti-DNP responses are barely suppressed! Table 6 Priming with KLHin complete Freund’s adjuvant or plus Bordatella pertussis yields similar responses bIgG anti-KLH response

aImmunizations First

Second

Status

IgG2a

IgG 1

KLH on alum

DNP-KLH on alum

primary secondary

8 100

6 106

KLH plus CFA

DNP-KLH on alum

KLHon alum plus B. pertussis

DNP-KLH on alum

primary secondary primary secondary

48 87 15 99

36 140 18 236

aSee legend to Table 5. bpercentage of a "standard" adoptive secondary anti-KLH response.

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Severalother carders (includingficoll and certain synthetic aminoacid copolymers) yield similar results visa vis their failure to inducesuppression that extends to responses to DNPon KLHor CGG(unpublished observations). Furthermore,althoughwehavenot extensively cross-checkedthese carders, the suppression they induce does not appear to extend to DNP presented on any of the others. Thus, weare faced with a heterogeneous groupof carders (cells, carbohydrates,artificial proteins) that apparently induce a suppressionspecific for anti-DNPresponses(since anti-carder antibodyresponsesproceednormally)and yet do not inducesuppressionfor such responsesper se (since presentation of DNPon other carders induces IgG anti-DNPantibody production). This puzzlingset of findings wouldbe explainedif the suppressioninducedin eachcase affected productionof a different subset of the combining sites in the anti-DNPrepertoire. This wouldmeanthat DNPpresented on eachof these carders evokesa substantially different antibodyresponseand a correspondingly uniqueset of regulatorycells specific for that response. This idea is consistentwith theoretical considerationsconcerningthe structure of combining sites likely to bind DNPin different structural environments (29-31; A. Edmondsen, personal communication);however,whether it will provecorrect remainsto be seen.

EFFECTOR CELLS IN THE EPITOPE-SPECIFIC SYSTEM Early attempts to characterize the cell(s) responsiblefor epitope-specific suppressionwerestymiedby difficulties in reliably measuringsuppressive activity in adoptiverecipients. Recentstudies (T. Tokuhisa,M.Tagawa,M. Taniguchi, manuscriptin preparation) havehad moresuccess using an in vitro assay in which cells from CGG/DNP-CGG immunizedanimals suppress anti-DNPantibody production by spleen cells from DNP-KLH or DNP-OVA (ovalbumin)-primedmice. These rather elegant studies demonstrate that the epitope-specific suppressionis mediatedby Thy-1positive cells that havea specific receptor for DNPand (as Table 7 shows)can removedon DNP-BSA coated plates. At present, data are consistent with one or moreDNP-specific suppressor T cells beingrequired for suppressionin this assay andwith the suppressor cell(s) servingas direct mediatorsof suppressionor as inducersof cells that mediatesuppression(since the respondingspleen cells providea sourceof T cells that potentially can be inducedto suppressantibodyproduction). However,in either event, these studies clearly define a newcategory of regulatorycells by demonstrating the existenceof epitope-specificsuppressor T cells (ETs) that control antibody production independentlyof the carder on whichthe epitope is presented.

Annual Reviews EPITOPE-SPECIFICREGULATION 627 Table 7 Epitope-specific suppressor T cells (detected in vitro) bind specifaically to DNPocoatedplates DNP-KLH primed b spleen 3

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3 3 3 3 3 3 4 5

CGG/DNP-CGGimmunized spleen c b Cell population Cells added -T cells (anti-MIg depleted) T ceils (anti-MIg depleted) T cells bound to DNP-BSA T cells boundto BSA Unseparated spleen ---

IgG1 anti-DNP (ng/ml)

None 1 2 0.4 0.4

238

1 2 None None

88 100 450 625

25 13 50 288

aT. Tokuhisa, M. Tagawa,M. Taniguchi, manuscript in preparation. bCells cultured (X 105). cSpleen cells that failed to bind to anti-MIg coated plates (anti-Mlg depleted fraction) were applied to DNP-BSAor BSA coated plates and incubated 30 minutes at 37°C. Plates were then washed, chilled and the boundcells were eluted by gentle pipetting. About1 percent of the cells in the original spleen-cell suspension were recovered from the DNP-BSA coated plate.

A MODELFOR A BISTABLE REGULATORYMECHANISM Several years ago, we introduced a set of hypothetical immunoregulatory circuits whoseoperationprovides bistable, Igh-restricted, epitope-specitic regulation for antibody responses (28). This modelproposed a system central (Core) "circuits," each composedof two helper and two suppressor T cells and each providing the basic "on-oil’ regulation for productionof antibodies carrying a particular antibody-combining site coupledto a particular Ig heavychain (isotype/allotype). By allowing each suppressorcell in the circuit to attack oneof the helperceils andbe helped(to differentiate andexpand)by the other (see Figure1), we arrived at a set of cell interactions that approximatesthe behaviorof an electronic binary ("flip-flop") circuit in that the circuit tends to stabilize in a help or suppressionmode but nevertheless can shift to the opposite configuration in response to a dramaticshift in stimulatory conditions. The decision as to whethera given Corecircuit in the modelpermits or suppressesproductionof the antibodyit regulatesis partially internal to the circuit but also dependson the relative strengths of positive and negative signals transmittedto the Corecircuit froma series of functionallydistinct auxiliary regulatorycircuits that directly sense the antigenic environment. Thus, this modelplaces Ig-specific regulation to the B cell being regulated andestablishes a systemwherebya variety of positive andnegative auxiliary

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HERZENBERGET AL CIRCUIT

TS O,~,~Th id÷ id-

@~ B

CRC [

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ThO id-

+ id

Figure 2 Modelfor a bistable regulatory circuit. In this theoretical cell-interaction circuit (28), a B cell carrying Idd- VHIg surface receptors is helped by a T cell, Thl, that has complementary(id-) receptors. Thl (analogous to an idiotype-specific helper T cell) is pleted by a suppressor T cell, Tsl, (analogous to an idiotype suppressor T cell) which carries id+ receptors similar to the B cell and therefore tends to bind the same haptenic determinant as the B cell. Tsl is helped by Th2, and Th2is depleted by Ts2 (which is helped by Thl). Th2 and Ts2 are distinguished from Thl and Tsl by non-VHrelated surface determinants. The configuration of this circuit is such that it will tend to stabilize either with Thl and Ts2 dominantor Th2and Tsl dominant, since either of these pairs will decrease the activity of the other. Thusthis circuit will tend to maintainitself either in a help or suppressionconfiguration dependingon howit is induced initially by conditions of antigenic stimulation (for further explanation, see 28).

stimulatory signals are integrated (by the Core circuits) to determine whether one or more of the possible antibody populations shall be represented in a response. In framing the model, we deliberately avoided considering auxiliary circuits containing CTs since we could see no straightforward way of rationalizing the then-current view (that CTs regulate antibody production by depleting carder-specific help) with the central regulatory system we were proposing. Working now, we would draw a CTs-containing circuit that induces the Core circuits to suppress antibody production (to newepitopes on the carder). Similarly, concepts of contrasuppression (9) were nascent (or unknown) when this model was drafted but would now be included auxilary circuits that favor help rather than suppression. Thus, although the model as published "shows its age," the basic principles it embodies are more viable than ever. Wewould be pleased to claim that our studies on the epitope-specific system were directly instigated by this "theoretical exercise;" however, serendipity had moreto do with the initiation of these studies than rationality. That is, having forgotten the earlier work (1, 2) showingthat in situ anti-haptcn responses fail following immunizationwith (what we nowcall) the "carricr/haptcn-carricr" sequence, we set up an allotypc suppression experiment in which we hoped to augment anti-hapten antibody production by pre-immunizing with carder (32). The minimal anti-hapten and normal anti-carder responses we obtained in control animals intrigued us sufli-

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EPITOPE-SPECIFICREGULATION 629 ciently to trigger a further seres of experiments.Thus,weinadvertantly reopenedan old question and, armedwith moderumethodsfor measuring memory B-cell developmentand expression, woundup somethree months later with evidence(11) outlining a previouslycryptic (epitope-specific) regulatorysystemwhoseproperties, wefound, werelargely predicted by the theoretical regulatory circuits wehadproposedearlier. Thecoincidence of these properties adds credibility to the proposed model(28) and opensthe wayfor a direct test of someof its morespecific predictions(e.g. if the modelis correct, epitope-specificregulationas described here should be basedon co-ordinatedidiotype-specific regulatory interactions that can be dissected by introducingconditions that perturb idiotype representationin anti-epitope antibodyresponses). However, aside fromits value as a guide to future experimentation,this model(correct or incorrect) serves a clear and current purpose.Thatis, it demonstratesthat a workableset of cell interactionscan be devisedto accountfor the bistable regulation of antibodyresponsesdemonstratedin the epitope-specific system. Thusit de-mystifiesthis novelregulatorycapability andbrings it into the realm of possibility for a systemthat has evolvedcomplexcellular mechanisms to protect the animalagainst invasion by deleterious agents.

GENERALITY OF EPITOPE-SPECIFIC

REGULATION

In essence, the evidencewehavepresentedcasts the epitope-specificsystem as an integrative central mechanism responsible for shapingantibody responses according to the dictates of the regulatory environmentwhenan epitope is first introduced. Thestatus of the carrier-specific regulatory system, as wehaveshown,plays a keyrole defining the properties of this environment (11, 15, 16). Thestatus of allotype-specificandI-regiondefined regulatory mechanisms contribute significantly, apparently by preventing rapid initiation of antibodyproductionand henceallowingCTsactivity to predominate(18, 20). Thus,throughthe agencyof this central system, the activities of a variety of independentlystudiedregulatoryinteractions come together as co-ordinatedinfluences on the antibodyresponsesproducedby a given animal(17). Perusalof the literature suggeststhat an analogousepitope-specificsystem centrally regulates cellular immuneresponses. For example,recent studies demonstratethat the induction of allergic encephalomyelitis(AE) by an encephalitogenic peptide-carder conjugate can be inhibited (suppressed) by prior immunization with the carrier protein, i.e. by carrier/hapten-carrier immunization(33). Similarly, the mechanisms regulating drayed-typehypersensitivity(34) showa specificity for epitopes not unlike

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the mechanisms described here. Therefore, it is reasonable to suspect that each of the major types of immune responses are controlled by "Core" systems that duplicate the properties of the epitope-specific system controlling antibody responses. Taking this supposition one step further, the existence of such Core systems would constitute an overall mechanism through which cellular and humoral responses could be co-ordinated, perhaps by direct communication or perhaps by differential responsiveness to common"auxiliary" systems. The complexity inherent in such a mechanism is staggering; however, given the extraordinary versatility of the immunesystem as it operates even in its earliest evolved form, we (as investigators) will indeed be lucky if immune response regulation proves to be based on such a simplistic view. ACKNOWLEDGMENTS We thank David Parks, who played a major role in developing the concepts of bistable regulatory circuits discussed here, and Leonard A. Herzenberg, who has continuously provided invaluable scientific criticism, advice, sup-

port, andeditorial help for these studies. Thisworkwassupported in part by NIHgrants HD01287, AI-08917, and CA-04681. Literature Cited 1. Mitchison, N. A. 1971. The carder effect in the secondaryresponseto hapten-protein conjugates. I. Measurement of the effect andobjectionsto the local environmenthypothesis. Eur. J. IrnmunoL1:10-17 2. Rajewsky,K., V. Sehirrmacher,S. Nase andN. K. Jerne. 1969. Therequirement of morethan one antigenic determinant for immunogenieity. J. Exp. Med.129: 1131-1143 3. Gershon,R. K. 1974. T Cell Control of Antibody Production. In "Contemporary Topicsin Irnrnunobiology", Vol. 3., ed. M. D. Cooperand N. L. Warner,pp 1-35. NewYork: Plenum 4. Tada, T., K. Okumuraand M. Taniguchi. 1972. Regulation of Homocytotrophic AntibodyFormationin the Rat: VII. Carrier Functions in the Antihapten HomocytotrophicAntibodyResponse. J. lmmunol.108:1535 5. Tada, T. and K. Okumura.1979. The role of antigen-specificT cell factors in the immuneresponse. Adv. lmmunol. 28:1-87

6. Sercarz, E. E., R. L. Yowell,D.Turkin, A. Miller, B. A. Araneoand L. Adorini. 1978. Different FunctionalSpecificity Repertoires for Suppressorand Helper T Cells. ImmunologicalRev. 39:109136 7. Tada, T. and K. Hayakawa.1980. Antigen-specificHelperandSuppressorFactors. In "Immunology 80; Progress in ImmunologyIV". ed. M. Fougereau and J. Dausset, NewYork: Academic. 389 pp. 8. Taniguchi, M., T. Saito and T. Tada. 1979.Antigen-specificSuppressiveFactor Producedby a Transplantable I-J bearing T cell Hybddoma.Nature 278:555 9. Yamauchi, K., D. R. Green, D. D. Eardley, D. B. Murphyand R. K. t3ershon. 1981. IramunoregulatoryCircuits that ModulateResponsivenessto Suppressor Cell Signals. Failure of B10 Miceto Respondto SuppressorFactors Can be Overcomeby Quenching the ContrasuppressorCircuit. J. Exp. MecL 153:1547-1561

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REGULATION

631

10. Green,D. R., R. K. Gershon,and D. D. 20. Herzenberg,L. A., T. Tokuhisaand K. Eardley. 1981. Functional deletion of Hayakawa.1982. Lack of immuneredifferent Ly-1T cell inducersubset acsponse(IR) genecontrol for the inductivities by Ly-2 suppressor T lymtion of epitope-specificsuppressionby phocytes. Proc. Nat. Acad. Sci. USA the TGALantigen. Nature. 295:32978:3819-23 331 11. Herzenberg,L. A., T. Tokuhisaand L. 21. Herzenberg, L. A. 1981. NewperspecA. Herzenberg.1980. Carrier-priming tires on the regulationof memory B cell leads to hapten-specific suppression. developmentand expression. Thesis, Nature 285:664-666 Doctorated’etat et sciences, University 12. Katz, D. H., W.E. Paul, E. A. Goidl, de la Sorbonne Paris VI, France. andB. Benacerraf.1970. Carrier func247pp. tion in anti-hapten immune responses: 22. Herzenberg, L. A., S. J. Black, T. I. Enhancement of primary and secondTokuhisaand L. A. Herzenberg.1980. ary anti-hapten antibody responsesby Memory B cells at successivestages of carrier preimmunization.J. Exp. Med. differentiation: Affinity maturationand 132:261-282 the role of IgDreceptors. J. Exp. Med. 13. Paul, W.E., D. H. Katz, E. A. Goidl 141:1071-1087 and B. Benacerraf.1970. Carrier func23. Ishazaka, K. and H. Okudaira. 1973. tion in anti-hapten immune responses. Reaginie antibody formation in the II. Specificpropertiesof carrier cells camouse.II. Enhancementand supprespable of enhancinganti-haVen antision of anti-hapten antibodyformation bodyresponses. £ Exp. Med.132:283in primingwith cartier. J. lmmunologl, 304 110:1067-1076 14. Sarvas, H., O. Makela,P. Toivanenand 24. Tada, T., M. Taniguchi and K. A. Toivanen. 1974. Effect of carrier Okumura.1971. Regulation of homopreimmunization on the anti-hapten recytotropic antibody formation in the sponse in chicken. Scand. J. Immunol. rat. II. Effectof X-irradiation. J. Im3:455-60 munology106:1012 15. Herzenberg, L. A. and T. Tokuhisa. 25. Okumura,K. and T. Tada. 1971. J. Im1981. Carrier Specific SuppressionOpmunology. Regulation of homocytoerates Throughthe Hapten-Speeifie tropic antibody formationin the rat. System.In: lmmunoglobulinldiotypes III. Effect of thymectomy and splenecand Their Expression, ICN-UCLA Symtomy. 106:1682 posia on Molecular and Cellular 26. Tada, T., K. Okumuraand M. TaniguBiology, VolumeXX,ed. Charles Janechi. 1972. Regulation of homocytoway,Eli E. Serearz, HansWigzell and tropic antibody formationin the rat. C. Fred Fox. pp. 695-707NewYork: VII. Carrier funtions in the anti-hapten Academic homoeytotropicantibody response. J. 16. Herzenberg,L. A., T. Tokuhisa.1982. Immunology108:1535-41 Epitope-specificregulation:I. Carrier27. Herzenberg, L. A. and L. A. Herzenspecific induction of suppressionfor berg. 1978. MouseImmunoglobulin AIIgGanti-hapten antibodyresponses. J. lotypes: Descriptionand Special MethExp. Med. 155:1730 odology. In "Handbookof Experimen17. Herzenberg,L. A., T. Tokuhisa,D. R. tal Immunology"ed. D. M. Weir, ed. Parks andL. A. Herzenberg.1982. Epipp. 12.1-12.23. Oxford:BlackwellSci. tope-specific Regulation:II. Bistable 3rd ed. Control of Individual IgGIsotype Re28. Herzenberg, L. A., S. J. BlackandL. A. sponses. £ Exp. Med.155:1741 Herzenberg.1980. Regulatorycircuits 18. Herzenberg,L. A., T. Tokuhisa,andL. A. Herzenberg.1982. Epitope-specifie and antibody responses. Fur d. lmmunol. 10:1-11 Regulation:III. Inductionof allotyperestricted suppressionfor IgGantibody 29. Singer,S. J. 1964.Onthe heterogeneity responses to individual epitopes on of anti-hapten antibodies, lmmunochem. 1:15-20 complexantigens. Eur. d. lmmunoL 30. Haber,E., F. F. Richards,J. Spragg,K. 12:814-8 19. Herzenberg,L. A., K. Hayakawa, R. R. F. Austen,M.Vallottonand L. B. Page. Hardy,T. Tokuhisa,V. T. Oi andL. A. 1967. Modifications in the heteroHerzenberg.1982. Molecular,cellular geneityof the antibodyresponse.In Anand systemicmechanisms for regulating tibodies, ColdSpring HarborSymposia IgCH expression. ImmunoL Rev. on Quantitative Biology,Vol. 32. pp. 67:5-31 233-310

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31. Levine,B. B. 1965. Studies on delayed hypersensitivity. I. Inferences on the comparativebindingaffinities of antibodies mediatingdelayed and immediate hypersensitivity reactions in the guinea pig. J. Exp. Med. 12h873-888 32. Herzenberg,L. A. 1983. Allotype Suppression and Epitope-specific Regulation. Immunol.Today32:In press 33. Rausch,H. C., I. N. Montgomery and R. H. Swanborg. 1981.Inhibition of Ex-

fimental Allergic Enccphalomyditis ~y Carrier AdministeredPrior to Chal-

lenge with EncephalitogenicPeptideCarrier Conjugate. Eur. J. ImmunoL 11:335-338 34. Bullock, W. W., D. H. Katz and B. Benacerraf. 1975. Induction of T-Lym~ahOCyte Responses to aJ.Small r Weight Antigen. Exp.MolecuMed. 142:275-287

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Annual Reviews www.annualreviews.org/aronline

Ant~ Rev. Immuno£1983. 1:633-55 Copyright©1983by AnnualReviewsInc. All rights reserved

T-LYMPHOCYTE CLONES

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C. Garrison Fathmanand John G. Frelinger Division of Immunology, Departmentof Medicine, Stanford University, Stanford, California 94305

INTRODUCTIONAND HISTORICAL OVERVIEW Several discoveries and technological developmentshave revolutionized the field of cellular immunology during the past decade. Of primary importance was the development by Kohler & Milstein of hybddomacell lines which immortalized production of monoclonal antibodies (1,2). Another important advancewas the utilization of molecular genetics to study regulatory and effector products of cellular immunology.A third important development has been the adaptation of culture conditions to allow the growth and maintenance of T-lymphocyte clones in long-term culture (3). This rapid advance is all the more remarkable when one considers that the field of cellular immunologyhas developed so very recently. In fact, less than 25 years ago the function of lymphocytes was still unknown.A 1958 review by Trowell (4) on the lymphocyte included the following: "Thesmall lymphocyte seemsa poorsort of cell characterizedby mostlynegative attributes:smallin size withespeciallylittle cytoplasm, unableto multiply,dyingonthe least provocation, survivingin vitro for onlya fewdays,living in vivoperhapsa few weeks...Although lymphocytes havebeenstudiedfor overa hundred years,not a single functioncanyet be ascribedto themwithanyconfidence,andtheir role in the body remainsbothan enigmaanda challenge.Broadlyspeaking,tworival and mutually exclusiveviewshavebeenentertained.Theoneis that lymphocytes are primitivestem cells capableof differentiatinginto all the othertypesof bloodcells, andalso into reticuloendothelial cells andflbroblasts.Theotheris that theyare endcells dyingafter a fewhourswithouthavingperformed any known function." Perhaps none of the advances has been as potentially far reaching as the recent adaptation of tissue culture techniques that allow cloning and functional immortalization of T lymphocytes. Because this was primarily a technical advance, the areas in which T-cell clones have played a critical role have been diverse. In part, this was a reflection of the methodsused to generate T-lymphocyteclones, each of which has allowed different T-cell functional properties to be retained. In this review, we will present a brief 633 0732-0582/83/0410-0633 $02.00

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overviewof howT-cell cloneswerederived,describe the as yet unsuccessful attemptsto isolate T-cell receptors, and surveythe advancesmadein quite varied areas of cellular immunology that weremadepossible by the use of T-cell clones.

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METHODS OF IMMORTALIZING

T CELLS

There are basically three waysto developT-lymphocyteclones. Onemay obtain clonal derivatives of T cells by the fusion of a T-cell lymphoma and a normalT lymphocyteso as to establish an immortalizedcell line that maintainsfunctions of interest. Or one maychoose one of two other approachesthat allow the extendedgrowthof T lymphocytesin the absence of somaticcell fusion. T cells can be selected and growncontinuouslyin mediacontaining large amountsof T-cell growth factor(s) (TCGF), designatedInterleukin 2 (IL-2). Thefinal, and perhapsthe mostlaborious techniqueinvolves maintainingT cells in mediacontaining little or no exogenousIL-2, and requires repeated antigenic stimulation and serial reculture. This methodrequires the presenceof antigen-presentingcells for helperT-cell clones,or the presenceof appropriatetarget cells for cytolytic T-cell clones. Hybridomas Oneof the first successfulfusionsbetweenfunctional T cells anda thymoma line wasreported by Tanaguchi& Miller (5). Takingadvantageof the fact that suppressorcells bind antigen andexpressI-J determinants,these investigators utilized two selective procedureswhichincreasedthe probability of their isolating a suppressorT-cell hybridoma.Antigen-binding T cells were enrichedfromthe spleens of micethat had beenpreviouslyrenderedtolerant to humangammaglobulin(HGG)by absorption to and elution from HGG-coated plastic dishes. Theseantigen-bindingT cells werefused to a T-cell tumorline. Usingthe fluorescence-activatedcell sorter, Tanaguchi& Miller selected and ultimatdy propagatedthose hybrid cells whichwere stained with antibodiesdirected against productsof the I-J subregionof the murine major histocompatibility complex(MHC).There have been many subsequentreports of the establishmentof functional T-cell hybridomas by the fusion of T lymphoma cell lines and primedlymphocytes(6-10). Despite the relative ease with whichsuppressor T-cell hybridomaswere developed, it wasinitially difficult to isolate other typesof functionalT-cell hybridomas followingfusion. In retrospect, this mayhavebeen due to the lack of an appropriatemethodof assay. Thus, althoughhelper T ceils have antigen specificity, such cells "see" antigen in the context of an "MHCrestricted" recognition. Antigen-specific hdper T cells do not bind free

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T-LYMPHOCYTECLONES

635

antigen, and thus cannot be selected simply by antigen-binding criteria. This problem was circumvented in a very elegant series of experiments by Marrack & Kappler and their coworkers (11, 12). Although helper T cells not bind free antigen or MHC products, helper T cells do bind to antigenpulsed presenting cells of the appropriate haplotype. The laboriousness of studying antigen-binding-cell:antigen-presenting-cell interactions following physical association by enumeration precluded this as a conventional assay technique. However, Marrack & Kappler noted that hybridomas formed between a helper T-cell line and a drug-marked thymomasecrete lymphokines in response to challenge with the appropriate combination of antigerl and MHC.These investigators were able to isolate helper T-cell hybridomas using the production of TCGF-IL-2by the hybridomas as an assay method. Nabholzand his collaborators have utilized somatic cell hybridization to develop cytotoxie T-lymphocyte (CTL) hybrids derived between CTLlines and drug-marked thymomas(13, 14). These workers are developing techniques for analysis of CTLfunction by somatic cell genetics and are studying the molecular basis of the most distinctive traits of the CTLphenotype: specificity, cytolytic competence, and TCGFdependence. Thus, utilizing the technique of somatic cell hybridization, it has been possible to generate stable hybridomalines of suppressor, helper and cytolytic cell lineages. Thesehybrid cells are relatively easy to growand maintain, and they can be obtained in large numbers. The major disadvantages of this technique for the generation of T-cell clones are the unknown contributions of the fusion partner and, at least in somecases, the instability of the differentiated function of the T-cell hybrids.

IL-2 Dependent Lines The second approach which has allowed the isolation and propagation of T-cell clones is the extensive use of TCGF.As early as 1965, it was demonstrated that mediumobtained from lymphocytes that had been stimulated in mixed lymphocyte culture caused unrelated third party lymphocytes to proliferate (15, 16). It wasnot until muchlater that the significance of these observations was appreciated by cellular immunologists.In fact, credit for the concept of TCGFis often attributed to Gallo and his colleagues, who were studying the effects of growth-promotingsubstances on humangranulopoiesis. Their studies showed that conditioned mediumfrom phytohemagglutinin(PHA)-stimulated human lymphocytes had not only conventional colony-stimulating factor activity, but also caused extended growth of lymphoblasts (17). Initially, it was assumed that these were lymphoblasts which had been transformed as a result of infection with Epstein-Barr virus. Later, when Gallo and his associates were again at-

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tempting to bring about long-term growth of humangranulopoietic cells, they examined lymphoblasts which grew out of such cultures and found, to their surprise, that these lymphoblasts were mature T lymphocytes. They subsequently characterized the active agent in such conditioned medium, the lymphokineIL-2, and namedit "T-cell growth factor" (17, 18). With the discovery of TCGF,the stage was set for cloning T lymphocytes. Within the following year, long-term lines of mousecytotoxic T cells were being routinely maintained in the presence of TCGFs(19), and soon a variety cells were cloned (20-24). The source of the TCGFused in these cultures (IL-2) is usually conditioned media obtained following the stimulation lymphooytes with mitogen, although the "induced" supernates of certain T-cell lymphomalines can also be used. There is a variety of products in such conditioned media in addition to TCGF. One of the remarkable findings which has recently cometo light concerns the karyotypic evolution of cytolytic T-cell lines maintained by TCGFs. Johnsonet al (35) have karyotyped a variety of murine CTLlines, and have shown that there is a remarkably high frequency of chromosomal rearrangementin manyof the lines. In fact, none of the clones that they studied were karyotypically normal. Their studies suggested that rearrangements of chromosomeswere more frequent than numerical aberrations. This is in contrast to previous reports on the spontaneous evolution of other murine cell cultures (26). Thus, it must be borne in mind that T cells which are maintained in long-term tissue culture in the presence of TCGFswill undoubtedly develop karyotypic abnormalities. One must be aware of this possibility whenusing IL-2 dependent T-cell lines in designing experiments which depend upon a normal karyotype. Serial .4ntigenic Stimulation The third methodology which has allowed the growth and maintenance of T-cell clones dependsupon repeated antigenic stimulation of cloned populations of antigen-specific T cells. This methodwas an outgrowth of in vitro studies of mixed lymphocyte reactions in which lymphocytes were repeatedly challenged with stimulator cells (27, 28). T-cell lines (29-31), subsequently clones which were alloreactive (32, 33), cytolytic (33-36) nominalantigen-reactive (37, 38), were isolated using antigenic restimulation and TCGFs.This method has the advantage that the selection and growthconditions continuously require the retention of antigenic specificity of the T cells. The major disadvantages of this technique are that: (a) maintainingthese lines is relatively laborious; Co) it is difficult to obtain large numbersof cells; and (c) there is obligate contamination of T-cell clones with the antigen-presenting cells. However,utilizing serial antigenic challenge as the methodof selection and propagation, T-cell clones have

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been developedwith functions whichare perhaps morecharacteristic of normalcells.

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THE HELPER T-CELL A CONUNDRUM

RECEPTOR FOR ANTIGEN:

ThehdperT-cell receptor is virtually uncharacterizedbiochemically,as can be seen by the list of its propertiesin Figure1. Includedin this Figurefor comparisonare the properties of immunoglobulins, whichare the antigenspecific receptorsof B cells, andthe propertiesof solublefactors whichare secreted fromsuppressorT cells (these entities are discussedin other reviewsin this volume).Ascan be seen fromthis list, very little is currently knownabout the properties of antigen-specificreceptors on helper T-cells. It has not beenpossibleto demonstrate that helper T cells bind free soluble antigen. Rather, helper T cells seemto recognizeantigen only as it is displayedon the surface of antigen-presentingcells in associationwith an MHC-encoded cell surface protein, the Ia molecule(39). Suchassociative recognition has been termed "MHC-restdctedrecognition," and it is a feature not only of helper T cells whichexhibit Ia restriction, but is also a hallmarkof effector T cells. EffectorTcells, suchas CTLs,recognizetarget antigens only in association with the K/D-like(class I) moleculesof the MHC(40-43). Onebasic tenet of the clonal selection theory is that on each antigenspecific cell there are receptorswith specificity for antigen.Although receptors havenot yet beenisolated fromhelper T cells, there is evidencethat an individual helper T lymphocyte and its clonal progenyrecognizeantigen specificity. Themostthoroughlystudiedantigen-specificrecognitionsystem Properties of T-CellReceptors for Antigen Property

T H N

T s Y?

Bcell(Ig)

Clonally Restricted (Idiotype) Y Allelicexclusion ?

Y Y

Y?

Y ? y?

Y

Y?

N?

Associated withSoluble Factor Y? Functional Domains

Y?

Y

Soluble Agbinding

Ig-likeIdiotype MHC Restriction

Y

Y

FigureI A comparison of the propertiesof the receptorson helperT cells, suppressorT cells, and Bcells. Y = Yes, N ---- No, ? = Unknown, (2 followingY or N indicates uncertainity or conflictingreports). (From2.)

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is the immunoglobulinreceptor on B cells. Oneof the major questions concerningT-cell receptors for antigen concerns the homology that such receptors mightshare with immunoglobulin molecules. Ahallmarkof antigen-specificreceptorson B cells is the geneticmechanism of allelic exclusion, whichallows only one immunoglobulin genotypeto be expressed as the cell surface receptor. Allelic exclusionis a sine qua non for surface immunoglobulin, and maybe a necessity for clonally-restricted receptors for antigenon the surface of T cells. Anotherquestionstill not completely answeredis whetherthere are immunoglobulin-likeidiotypes present on T-cell receptorsfor antigen. Finally, are suchT-cell receptors expressedas solublefactors whichare secreted or shedfromhelper T cells, as they seem to be from suppressorT lymphocytes?Are there functional domainswithin each molecule analogous to the functional domainsof immunoglobulin molecules?These questions have by no meansbeen addressed adequately, yet should nowbe approachablethrough the utilization of helper T-cell clones. The ease with whichB-cell receptors for antigen were identified once clonedlines wereavailable raised false expectationsconcerningthe relative ease with whichT-cell receptors for antigen mightbe characterized once cloned T-cell lines becameavailable. As mentionedpreviously, the major problemwith understandingthe nature of helper T-cell receptors for antigen is that helper T cells do not recognizefree soluble antigen, but yet recognizeantigen in associationwith Ia molecules.This associativerecognition is shownin Figure 2, wherethe three methodsby whichsuch a ternary complexcan be formedhave been diagrammaticallyillustrated. There are three potential methodsin whicha ternary complexcomposedof the "Tcell receptor," the antigen, and the MHC-encoded Ia molecule can combine. Our ignorance concerningthe mannerin whichthese three elements interrelate is practically complete.TheIa moleculemayin somewayspecitieally combinewith antigen to forma binary complexwhichis then recognized by the T-cell receptor. Alternatively, it is possible that the T-cell receptorhas someaffinity for either the free Ia molecules or for free antigen, and that one of these binary complexesis sufficiently stable to allow the third member of the ternary complex to bind, resulting in T-cell activation. Until weunderstandeither the basis for the associationof Ia moleculesand antigen or the nature of the antigen-specificreceptor on helper T cells, we will not be able to understandwhatunderlies the recognition and subsequentactivation of antigen-specifichelper T cells. lmmunoglobulin Homology Oneof the mostlogical approachesto studyingT-cell receptors wasto study immunoglobulin expression in cloned T cells. Theoretically, the use of

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1IA

+ AG

~ IAoAG

+

~

Figure 2 Possible ways that the recognition of Ia and antigen by the T-cell receptor(s) may take place. IA = Ia or class II molecules, AG= antigen, T = T cell. (suggested by J. Kappler, personal communication.)

B-cell V-region genes in T cells wouldeliminate the need for two entirely separate antigen-binding systems. Additionally, it has been shownthat T cells which respond to a variety of antigens, including carbohydrates (44), haptens (45), MHC alloantigens (46, 47), proteins (48), and synthetic peptides (49, 50), express idiotypic determinants in common with antibodies to the same antigens. Perhaps the most elegant studies have been those by Eichmanand his collaborators, whohave shownthat anti-idiotypic sera in somemanneraffect T-cell functions (51). The advent of cloning strategies and technologies has allowed the isolation of relatively large numbersof homogeneous cell populations for studies on the elusive T-cell receptor for antigen. These cells should provide the starting material for the isolation of T-cell receptors, muchas myelomas provided material for the isolation of immunoglobulins. Unfortunately, studies by molecular geneticists have demonstrated that the rearrangements of the constant regions of light-chain (52-56) or of heavy-chain(52-55, immunoglobulingenes are not obligatory in either functional cloned helper or cloned killer T-cell lines. Additionally, there was no transcription of light-chain constant region sequences in any homogeneous T-cell line tested (52). Further, Kronenberg (52) found no evidence for transcription Jn, Cbt or Ca sequencesin cloned T-cell lines, although C/~ transcripts have been found in some T-cell lymphomas(58), and in thymocyt~s (59) hybridomas (54). These data taken as a body are rather convincing, and suggest that constant regions ofimmunoglobulins are not utilized by T cells. If the explanation for shared idiotype is a shared structural gene, then the expression of immunoglobulinidiotypes in T lymphocytes must be due to the expression of either all or part of a VHgene. Studies are currently underway in several laboratories in an attempt to demonstrate VHgene expression in T cells utilizing homogeneousT-cell lines which have an idiotype (idiotope) in commonwith immunoglobulins. The general ap-

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proachis to utilize a Vri gene probederived froma hybridomacell line whichexpresses a characterized idiotype. This Vri gene probe wouldthen be used to assay transcription and DNA rearrangementsin cloned T-cell lines whichexpressthe sameidiotype. Thus,it shouldbe possiblein the near future to answerthe question of whetheror not T-cell lines utilize any immunoglobulingene segmentsas a portion of the T-cell receptor for antigen. Additionalquestions to be addressedconcernthe potential for allelie exclusion of T-cell receptors on homogeneous T-cell lines derived from heterozygotemice.Studies to addressthis questionare currentlyin progress in our laboratory, as well as elsewhere.Basically,these studies makeuse of monoclonalantibodies reactive with murineT-cell surface products which are controlled by genes residing on the murinetwelfth chromosome (Igl linked) (60-62). Suchantibodies are producedby purposeful immunization betweenimmunoglobulin congenicstrains of mice using an inoculumcontaining T cells, for instance BALB/c anti-CB.20con Ablasts. Subsequently, monoclonal antibodieswhichhavespecific reactivity for T cells are selected. Evidenceto date wouldsuggest that these antibodies recognize a product which,for simplicity, has beennamedCt (62). It shouldbe possible, utilizing antigen-specificT-call clones frommiceheterozygous for the Ig 1 locus (carrying both the Iga andIgb allotype), to addressthe questionof whether a, or not FI T-call clones can be divided into clones whichexpress only Ct versus T-cell clones whichexpress only Ctb products. Evidencefor allelic exclusion of a gene product expressed on cloned T ceils wouldindeed be intriguing. Despite the fact that T-cell clones have beenused in attempts to characterizethe T-cell receptor for antigen, these attemptshavenot yet beensuccessful.

Dualversus Single Receptor T-cell cloningoffers a methodof studyingwhetherthere are single or dual receptors on T calls to account for MHC restriction. Twogroups have addressedthis topic, using T-cell hybridomas,andhavecometo eontticting results, thoughthe general approachutilized by each wasvery similar (11, 12, 63). In both cases, a T-cell hybridoma specific for a given antigen and a Ia molecule(restriction site) werefusedto anotherT cell witha different restriction specificity. Theresultant hybrids werescreenedto see if any wouldrespondto the first antigen whenpresented by cells expressingthe second restriction dement(Ia molecule). Suchrecognition wouldimply that the receptors involvedin bindingof antigen, and the restriction element,weredistinct, and thus favor a dual receptor modelfor T cells. The opposite result wouldbe most compatiblewith a single receptor model. Kappler and Marrackconstructed a series of T-cell hybridomasby the

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fusion of two already existing hybddomas each restricted to a different Ia moleculeand havinga different antigenic specificity. Of 27 resultant hybddomastested, 16 irthedted the antigen and MHC specificities of both T-cell parents. Noneof the 16 hybridstested could recognizea mixturethat hadparental-antigen-recognition andMHC-restriction specificities (11, 12). Thus, the prediction that antigen and MHC could be recognized independently by twototally different T-cell receptorsdid not appearto be correct. However,data recently reported by Lonaiet al (63), whoused the production of H-2restricted helperT-cell factors as an assaysystem,are contradictory. In fact, their results support the dual receptor model.Hybridomas were derived by the fusion of T cells which recognized chicken gamma globulin and were H-2b restricted to BW-5147,an H-2k thymomaline. Lonaiet al reasonedthat althoughBW-5147 has no knownantigen specificity, since it is an H-2k thymoma, its restriction specificityshouldbe for self MHC--that is, H-2k. Lonai and his collaborators isolated hybddomas from a fusion of BW-5147 and chicken gamma-globulin-specificH-2b-restdcted T cells whichproducedhelper factors specific for chickengamma globulin, but wereH-2k restricted. This scramblingof antigen specificity and MHC restriction suggestedthat there weretwoindependentreceptors (63). These contradictory data havenot yet been resolved, although the solution may lie with the different assaymethodsutilized. If there are two independent receptors, they must be closely associated and recognize a complexof antigen and MHC products, becauseeven Lonai et al (64) did not find the binding of soluble antigen in the absence of MHC products. Therefore,wemustleave questions on the nature of, and the numberof, T-cell receptor(s) for antigen unanswered at the present time. It is to hopedthat the methodologies outlined abovewill soonallow resolution to this perplexingproblem. SUCCESSFUL RESEARCH

AREAS OF T-CELL

CLONE

Rather than dwell on other questions whichhave been inadequately addressedutilizing T-cell clones, it is pertinent to discuss research topics whichhave beensuccessfully studied by such techniques. There has been substantial progressin understandingseveral critical areas of cellular immunology using T-cell clones. For example,the dual specificity of T cells for nominalantigen as well as alloantigen has been conclusivdydemonstrated using T-cell clones. T-cell clones havebeen used to examinethe repertoire of T cells. Antibodiesto T-cell cloneshaveallowedthe biochemical andfunctionaldefinition of cell surfacemarkerson T cells. Studieswith T-cell clones haveled to the observationthat a single helper T-lymphocyte

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clone can provide help for at least two independentpathwaysof B-cell differentiation. Further, T-cell andantigen-presenting cell interactions have beenexaminedusing T-cell clones to showclearly that B cells can present antigen. It has beenshownthat T-cell clonescan recognize,in a functional manner,hybridor combinatorialIa molecules.Finally, the in vivo activity of T-cell clones has beenexamined with the aimof studyingboth the biology of T cells andthe potential usesof T-cell clonesas therapeuticagents.These areas will be discussedbelow. Alloreactivity Theuse of clonedT calls has allowedinvestigators to addressinteresting questionsaboutthe alloreactivity of antigen-specificT-cell clones. Oneof the mostperplexingquestions concernedthe extraordinarily high incidence of T cells whichrecognize and respond to antigens of the MHC of other membersof the same species. Due to this high incidence, it had been suggestedthat the cell populationwhichrespondedto antigen mustoverlap to a large degreewith the T-cell populatignrespondingto alloantigens(65, 66). T-cell cloning has allowedthe direct and unequivocaldemonstration that a single T cell has specificity for both an alloantigenandanotherforeign antigen in association with a self MHC geneproduct. Thefirst such demonstration was by yon Boehmerand his colleagues (22). They isolated eytotoxieT-cell clonewith specificity for the maleantigen(H-Y)in associab MHC tion with self H-2D products. At least one clone which was H-Y b additionally killed allogeneictargets which specific andrestricted by H-2D d, wereH-2D irrespective of the sex of the mousefromwhichthe target ceils were derived. Kanagawa et al (67) haveconfirmedand extendedthis observation by finding that approximately 20%of the clones specific for H-Yand b exhibit this samecrossreactivity with H-2D d. In a similar vein, H-2D Braciale and coworkershaveisolated influenza-specificH-2-restrictedcytolytic T-cell clones (35), one of whichalso wasalloreactive. This clone d, yet could also lyse recognizedthe immunizing virus associated with H-2K normal (uninfected) H-2Kk-expressingtargets (68). Additional and vincing data fromthe laboratory of Schwartzand his colleagueshaveshown that antigen-specificproliferating T-cell clonesalso possessalloreactivity (69, 70). Theyisolated a clone derived from B10.Amicewhichrecognized DNP-OVA in association with B10.Aantigen-presenting cells, but was equally well able to recognizeallogeneic B10.Sspleen cells in the absence of DNP-OVA. Further, these antigen-reactive clones could be passagedfor 68 dayson alloreactive stimulators and still retain their original antigen specificity (70). Thehigh frequencyof finding T-cell clones with such dual specificity demonstrated not only by Schwartzandhis collaboratorsbut also by others (71; M. Kimoto, unpublished data) has proven that a large

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percentageof T cells performboth functions. Althoughwecan state with certainty that a single T cell can be both antigenreactive andalloreactive, it has not yet been possible to determineunequivocallywhethersuch alloreactive andantigen-reactiveT-cell clones use twoindependentreceptors, or use one receptor unit with crossreactivity. T-Cell Repertoire Therepertoire of T-cell receptors has beenmostextensivelyanalyzedat the clonal level usingCTLclones derivedby limited dilution cloning.Obtaining a representativesampleof all the possiblereactivities of Tcells is critical to an analysis of repertoire. Hence,the use of short-termclonedlines may be preferable to using clones derived from long-term bulk cultures. The latter cell lines mayhave undergonemoreselection, so that potentially reactive clones have been eliminated. Shermanhas analyzedalloantigenspecific CTLclones using a panel of target cells fromKb mutantmice(72). Themicehad previously beenshownto possess distinct and defined amino acid substitutes in the Kb molecule(73). Thereactivity patterns on this paneldistinguish betweenCTLclones that use different receptors for antib response, Sherman gen. Using clones derived from a B10.D2anti H-2K found47 distinct reactivity patterns (72). Thus,there mustbe at least different types of clonally distributed cytolytic T-cell receptorsusedby the B10.D2mouseto recognizethe B6Kb molecule.Usingthese data in analyzing the frequencyof Kb-restdctedCTLprecursors, the minimum size of the CTLrepertoire for a given antigenic determinantcan be calculated to be about 20,000SlYdCificities.Becausethis is a minimum estimate, the repertoire of T-cell receptors maybe extensive. Thenatm’eof the determinantsseen by T-cell receptors is uncertain. It has beenreported that helper T cells respondto both native and denatured antigen equally well (74), whereassuppressorcells and antibodies react predominantlywith the native formof the moleculeoriginally used as the immunogen (74, 75). HelperT-cell clones can respondequally well, on molarbasis, to spermwhalemyoglobinor to cyanogenbromidepeptides of this molecule(76). This findingis consistent with the generalizationraised above. However, other studies suggestthat manymurinealloreactive helper T-cell clones directed against I-A moleculesrecognizethe combinatorial associationof the a- and//-chainIa polypeptides(32, 77, 78). Althoughnot madein the identical strain combination,monoclonalantibodies raised followingalloimmunizationagainst these Ia moleculesseemto recognize the ~ or the ~ chainindependently,in the sense that mostmonoclonal antiIa antibodies do not see combinatorial determinants whichrequire the specific pairing of a given a and ~ chain. Using a pand of monodonal antibodiesreactive with the Iak molecule,derivedby Pierres et al (79),

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were able to precipitate hybrid Ia molecules from cells of a (b X k)F~ heterozygous mouse(J. Frelinger, unpublished data). This confirms the lack of preferential recognition by anti Ia antibodies of the "parental" Ia conformation (Figure 3). Theseresults suggest that recognition of Ia molecules T ceils is more dependent upon tertiary conformation than is the recognition of Ia molecules by antibodies. However,this is not true in every case, since monoclonal antibodies can react with combinatorial Ia determinants Annu. Rev. Immunol. 1983.1:633-655. Downloaded from arjournals.annualreviews.org by HINARI on 08/27/07. For personal use only.

(80). In a series of experiments utilizing B6-dedved CTLclones induced in response to a point mutation in the H-2Kb molecule, it was found that a single done could frequently recognize two or more mutants. These mutants differed from each other with respect to the position of their amino acid substitution (73). These data suggested that CTLclones respond conformational changes in H-2 molecules, and that the same determinant recognized by a CTLclone can be generated by amino acid substitutions at separate locations within the molecule. An analogous panel of monoclonal antibodies in a parent anti-mutant combination has not yet been successfully generated. Interestingly, even MHC restriction, once thought to be the exclusive province of T cells, has recently been shownto be a property of certain antibodies. Selected monoclonal antibodies raised against influenza-infected cells wouldouly bind to infected cells and not to free virus (82). It is clear from these data that T ceils can recognize subtle conformational changes of MHCmolecules, perhaps better than antibodies do. Helper T cells maybe less sensitive to the denaturation of nominal antigen than are antibodies. However, these apparent preferences might also be related to the exact experimental design and previous selection in the system(e.g., mutants are selected by skin graft rejection, whichrequires a vigorous T-cell response). HYBRID I-A ANTIGENS Configuration {-A k |_Ab

Tranacomolementing "hybrid" I-A molecules

~

A~ chain A~achain

OOOO00 O000OOOOe

A~ chain A~ chain

¯ ¯ ~, ~, ~, ¯

A~ chain A~ chain

~ OO000000~

A~ chain A~ chain

(AaA~)

C (A~ A~)

Figure3 Schematic representation of the hybridI-Aantigensof an (H-2~’ X H-2k) F,mouse. Configurations AandB indicatethe parentalI-Amolecules. Configurations CandDindicate the unique"hybrid"I-Amoleculesformedby the combinatorial associationof the a- and fl-chainpolypeptides (reprintedbypermission of the editorsof Immunological Reviews).

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Antibodies

to Murine T-Cell

645

Clones

The combination of monoclonal antibodies and T-cell clones affords a powerful tool for studying cell interaction in the immunesystem at a biochemical level. The two general approaches for examining this interaction have been either to look for monoclonalantibodies which bind to the immunizingclone, or alternatively to screen for an effect of the monoclonal antibodies on a functional activity of the T-cell clone. As a general method, this will give a battery of reagents whichcan be used to study the structure and function of the cell surface molecules on T cells. However,in a more directed sense, it was hoped that some monoclonal antibodies would be clonally specific, and thus might be reactive with the T-cell receptor. Several investigators have made antibodies directed at CTLclones. In screening monoclonalantibodies by the inhibition of cytotoxicity, a repeated finding has been that anti-Lyt-2 antibodies inhibit the cytotoxic function of manyclones (83-86). However, the clones were heterogenous in their ability to be blocked by anti-Lyt-2 antibodies (84-86). Variant clones lacking Lyt-2 had markedly reduced levels of eytotoxic activity, although this diminished functional activity could be overcome by the addition of lectins (87). However,Lyt-2 maynot be a universal requirement for cytolytic cells, since an H-2-unrestdcted CTLclone does not require Lyt-2 for its lyric function (88). It was thus proposed that Lyt-2, while perhaps not obligatory in the cytolytic process, might be involved in the stabilization of effector-cell: target-cell interaction. Veryrecently, in a similar screen for inhibition of cytotoxicity, a monoclonal antibody~GK1.5 --was found which may be directed to the routine analog of the human OKT4antigen (D. Dialynas, personal communication).It is likely that this approach will generate a series of monoclonalantibodies which may ultimatdy define all the componentsinvolved in the process of T-cell-mediated reactivity. This approach can be generalized to include the definition of molecules involved in any cell:cell interaction in which homogenouscells take part and for which functional assay techniques are available. Antibodies to T-cell clones whichreact in a clonally specific mannerare rare. Wehave, however, succeeded in making antibodies to alloreactive T-cell clones whichstimulate the immunizingclones specifically (89). Since the antibodies Can distinguish between or amongclones derived from the same strain of mouse, we have called these antisera "anti-idiotypic." Our initial antibodies were madeby injecting a strain A alloreactive clone which responds to B6 into a (B6 X A) 1 mouse. T his F 1 a nti-parent r esponse should generate antibodies which are highly restricted. By a variety of immunization schemes, including a totally syngeneic immunization protocol, we have been able to makesimilar antibodies to antigen-reactive T-cell clones (J. Frelinger, unpublished data). Thus, the ability to make such

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antisera is not an aberrantfindingresulting fromthe alloreactive immunization scheme.Although these antisera are functionallyclonally specific, they bind to inappropriate clones, probablybecausethere are other antibodies in the antisera. To date, wehavenot generatedmonoclonal antibodies with exactly the samefunctional activities. A monoclonalantibody--384.5-whichboth bindsand specifically blocksa cytolytic clone has recently been describedby others (86). However,these investigators wereunable to immunoprecipitateproteins with this antibody.Theysuggestedthat the difficulty resulted fromthe low level of expressionof the moleculeon the T-cell clone. Byflow cytofluorometry,it wasestimated that monodonal antibody 384.5 recognizedonly 104 moleculesper cell. Thus, extremelysensitive assay techniques will need to be developedto analyze moleculesof such limited expression.Althoughthe approachesutilizing clonally-specitieantibodies have not yet producedany solid data addressing the biochemical nature of the T-cell receptor, the general methodremainsa promisingone.

HelperActivity of T-Cell Clones Asingle hdperT-cell clone can provide hdp for two independentpathways of B-cell maturationinto antibody secreting cells (90). Asanoet al had originally shownthat a KLH-specificT-cell clone could provide help to hapten (TNP)-primed B calls, as measuredin a plaque assay. Further, the source of the TNP-primed B cells could be varied, using either CBA/CaHN (Lyb-5+ and Lyb-5- B cells) or CBA/N (Lyb-5- B cells) mice as donors. The results of these experimentscan be summarizedas follows. At low concentrationsof antigen, the T-cell clone cooperatedwith hapten-primed B cells from either CBA/CaHN or CBA/N mice to generate predominantly an IgGresponse.Suchhdpwashapten-earrierlinked, and the T-cell: B-cell interaction was MHC restricted. At high concentrations of antigen, the T-cell clone could provide help to CBA/CaI-IN B cells, but not to the CBA/N B cells. TheT-cell help generatedunder these conditions is "nonspecific." Althoughthe interaction of the helper T-cell done with the antigen-presentingcell wasstill MHC restricted, the T-cell:B-cell interaction wasnot genetically restricted. This "high-doseantigen" interaction resulted in a predominantlyIgMresponse, and in the presence of other antigens the help wasnot antigen specific. Thus, the sameT-cell done, under conditions of different concentrations of antigen, could help two distinct B-cell subsets produceantibody. Since the "nonspecific" help seen with high antigen concentrations seemedto be mediatedby soluble factors, wewereinterested in developing a systemin whichwecould assess the helper activity of the T-cell done while simultaneouslyassayinglymphokine productionand T-call proliferation. Usingan in vitro antibody formationsystem, wefoundthat the pro-

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duction of antibodies could be monitoredfrom supernates by a sensitive enzyme-linkedimmunoassay technique. Beeausethe amountsrequired for assaywereso small, the sameculture supernatescouldbe tested for a variety of lymphokines.Ourpreliminaryanalysis, in collaborationwith E. Vitteta and coworkers, indicated that the production of lymphokines,like the proliferation of T-cell clones, wasrelated to the antigen concentration. Further, in the samesystemwecould clearly separate proliferation of the T-cell clonefromits helper activity. At lowerconcentrationsof antigen, the clonedid not proliferate, yet it couldprovidesuti~cienthelp to allowB-cell differentiation and productionof IgG(M. Shigeta, unpublisheddata). Using a cloned helper line specific for influenza virus, Lambandcoworkers (91, 92) showedthat the mostet~cient helper activity of the clone was an antigen concentration approximately10-fold lower than that required for maximalproliferation. Thesedata also support the notion that T-cell proliferation and T-cell help are independent. The Nature of T-Cell:Antigen-Presenting-Cell Interaction Workwith T-cell clones has provideddefinitive insight into antigen-presenting cell function. Thegeneral questionof whatcircumstancesare necessary and sufficient to present antigen to T cells has been surroundedby controversy.For example,can B eells present antigen to T cells? In part, the question arose becauseof the difficulty of isolating homogeneous cell populations.In particular, the possibility of contaminant-antigen-presenting cells being mixedin with either the T- or B-cell populations always existed. Withthe advent of T-call clones and T-cell hybridomas,and the availability of B-cell tumorlines, investigators wereable to addressthis question moreexactly. McKean et al showedthat Ia+ B-cell tumor lines couldpresent antigenandinduceproliferation in an antigen-specificT-cell clone (93). Similarly, Glimcheret al showedthat + tumors could present antigen to long-termT-cell lines (94). Kappleret al showedthat B-cell hybddomas could present antigen to T-cell hybddomas (95). Finally, humanT-cell clones specific for tetanus toxoid wereinducedto proliferate using Epstein-Barr virus B lymphoblastoidcells as a source of antigenpresentingceils for the tetanus toxoid (96). Thesedata conclusivelydemonstrate that someIa+ cells of the B-cell lineagecan effectivdypresentantigen to T cells. This type of analysis, using clonedpopulationsof interacting cells, is still in its infancy,but it will eventuallyprovidedear answersto manypuzzles of cell interaction in the immune system. Definition and Functional Expression of Hybrid la Antigens Thedescriptionof hybridIa antigensas funetional entities wasmadepossible using T-cell clones. Initially, using alloreactive clones, investigators

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were able to demonstratethe existence of uniqueneoantigenswhichwere present only on FI cells (hybrid antigens)and werenot present on the cells of either parent (32, 77, 78, 97). ThesehybridIa antigens wereformed the combinatorialassociation of a and/3 chains (Figure 2). Later, these hybrid Ia molecules were shownto allow effectively the recognition of nominalantigen by antigen-reactiveT-cell clones (37, 69, 76, 77, 98). These results were complemented by the biochemicaldemonstrationof hybrid Ia molecules(99, 100). Byusing anti-IA monoclonalantibodies, both in immunoprecipitation assaysandin T-cell proliferation-blockingassays, it was possible to determinewhichof the FII-A complexesa T-call clone recognized (101). Further, on a given molecularcomplexthe existence of multiple functional sites for "restriction" of antigen presentation could be demonstrated.Ona panel of T-cell clones restricted to the AkA~ complex, three different blocking patterns witha panel ofanti-I-A monoclonal antibodies wcrcsccn(102). Thcsc results indicated thatthcrc arcmultiple functional sites ona given Iamolecule. Additionally, using a I-Amutant mousc---bm12--it waspossible todemonstrate multiple functional sites on a given IAcomplex 003). Thecrucial observation wastheidentification of clones which wouldrespond to B6 andthemutant bin12, whereas other clones would respond onlytocclls fromB6andnotbml2. Since thebml2 mutationis a/~-chain mutation,this suggestedthat there are two distinct sites on the AoAacomplexdeterminedby the/~ chain. Theseobservations demonstratethat there arc two epigenctic mechanisms for increasing antigcn presentation. First, the numberof restriction sites is expandedby the creation of hybrid antigens formedby the combinatorialassociation of a and/~chains. Second,on a given Ia molecule,there are multiplefunctional sites or "domains." These mechanismsgreatly expandthe ways through’ whicha limited numberof Ia polypeptidescan present antigen to T cells. T-cell clones canrespond toquantitative aswellasqualitative changes inlaexpression. Thelevel ofexpression oflamolecules ina variety of strains expressing theAeE~complex wasmeasured byquantitative fluorescence-activated cytofluoromctry andby immunoprecipitation andtwodimensional gelanalysis. There wasshownto bcadirect correlation between thclevel ofIaexpression andtheability ofcells topresent antigen toT-cell clones (I04, 105). A similar conclusion wasreached fromstudies using T-cell clones ina proliferation assay which madeuseofF1 orhomozygousantigen-presenting cells tovarygenetically theamount oflaonthe surface oftheantigen-presenting cells. Theamount oflaonthesurface again correlated with theability topresent antigen (106).

ClonedT Cells Usedfor Reconstitution In Vivo The examinationof the waysin whichT-cell clones function in vivo is importantin understanding howT cells work,as analysis of cellular interac-

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tions in vitro mayor maynot allow extrapolation regarding their performance in vivo. In particular, studies on T-cell clones in vivo are an important step in the potential developmentof T-cell clones as a therapeutic tool. CTLclones are effective in reducing the size of seeded tumors when both are injected in the samesite---for example,into the peritoneal cavity --but are relatively ineffective when clones and tumors are administered separately by different routes (107, 108). This has been attributed to two major problems.First, the homingor trafficking patterns of these clones are abnormal; second, the clones are dependent on IL-2 for growth (112, 113). Thus, their relative inefficiency in vivo might be due to an inability to circulate to the site of the tumor, or to their relative short lifetime in vivo. One apparent exception is a clone specific for influenza virus, which protected mice from a respiratory infection with virus (111), perhaps by the abnormalhomingof the cloned T cells to the lungs (109, 110). The problem of IL-2 dependenceof T-cell lines studied in vivo maybe partially overcome by the addition of exogenous IL-2 (112). For example, the intravenous injection of certain Ly2+ T-cell clones which do not require exogenousIL-2 to growin vitro induced the destruction of allogenic tumor cells within the peritoneal cavity of immunosuppressedhistocompatible mice (113). The activity of helper T cells in vivo seems somewhatmore efficient, although this may reflect the assay systems used. Tees & Shreier have shown that nude mice reconstituted with T-cell clones will makeantibodies specific for the antigen the clones recognize (114); no "bystander" effect is seen. Studies in our laboratory (P. Nelson, unpublished data) on the reconstitution lethally irradiated syngeneic recipients using cloned antigen-specific T-cell clones and hapten-primedB cells demonstrate essentially the sameresults. The anti-hapten response requires hapten-carrier linkage, and no bystander effects are observed. The homingpatterns, as in the case of CTLclones, are abnormal (110). However,B cells can cooperate with the T-cell clones produce antibody. This maybe due to the fact that although migration of these cells is abnormal, significant numbersof both T and B cells hometo the spleen, wherethey can interact effectively.

SUMMARY To date, the most successful uses of T-cell clones have been in the demonstration that a single type of cell can perform multiple functions. However, their potential usefulness is enormous,and the study of call interactions using clonal populations has just begun. The development and study of more cloned populations will surely lead to a clearer analysis of c~llular interactions in the immunesystem. The use of T-cell clones and hybridomas to analyze T-cell receptors and/or factors is well under way, and will continue to be an area of intense investigation. Molecular biologists will

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undoubtedly make moreextensiveuseof T-cell clonesin the future,both as a sourceof cloningmaterialandas transfectionrecipients.Themost excitingareafor development, froma medical pointof view,is thepotential for use of these cell lines or their productsin immunotherapy andin providing a mechanism for specifically modulating the immune response.

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ACKNOWLEDOMENTS We thank Frank Fitch for directing us to Trowell’s delightful review. We thank John Kappler for providing us with the outline for Figure 2, and Patsi Nelson, J. A. Frelinger, and Jane Parnes for hdpful criticisms of this manuscript. Special thanks also go to Karen Carpenter for careful and patient secretarial assistance. C. G. Fathman is supported by grant RCDAAI-00485 and J. 13. Frelinger is a Fellow of the Jane Cotfm Childs Foundation. We are also supported by grants AI-18716 and AI-18705. Literature Cited 1. Kohlcr,G., Milstein, C. 1975. Continuousculturesof fusedcells secretingantibodyof predefmedspecificity. Nature 256:495-497 2. Kohler,G., Milstein, C. 1976.Derivation of specific antibody-producing tissue culture and tumorline by fusion. Fur. d. ImrnunoL6:511-519 3. Fathman,C. G., Fitch, F. W., eds. 1982. Isolation, Characterizationand Utilization of T LymphocyteClonez NewYork:Academic 4. Trowell, O. A. 1958. Thelymphocyte. lnt. Rev. Cytol. 7:235-293 5. Taniguchi,M., Miller, J.F.A.P. 1978. Specificsuppressivefactors producedby hybn’domas derived fromthe fusion of enriched suppressor T cells and a T lymphomascell line. J. Exp. Med. 148:373-382 6. Kapp,J. A., Araneo,B. A., Clevinger, B. L. 1980.Suppressionof antibodyand T cell proliferative responses to Lt° glutamicacid~°-L-alanine~°-L-tyrosine by a specific monoclonal T cell factor. J. Exp. Met£ 152:235-240 7. Kontiainen,S., Simpson,E., Bohrer,E. M, Beverley, P. C. L, Herzenberg,L. A., et al. 1978.T-cell lines producing antigen-specificsuppressorfactor. Nature 274:477-480 8. Hewitt, J., Liew,F. Y. 1979. Antigenspecific suppressorfactors producedby T cell hybridonmsfor delayed-type hypersensitivity. Eur. d. Immunol.9: 572-575

9. Taussig, M. J., Holliman, A. 1979. Structureof an antigen-specificsuppressor factor producedby a hybrid T-cell line. Nature277:308-310 10. Taniguchi,M., Saito, T., Tada,T. 1978. Antigen-specific suppressive factors producedby transplantatable I-J bearing T cell hybridoma.Nature278:555558 11. Kappler,J. W.,Skidmore,B., White,J., Marraek,P. 1980. Antigen-inducible, H-2restricted, interleukin-2-producing T cell hybridomas:Lackof independent antigen and H-2recognition. J. Exp. Med. 153:1198-1214 12. Marrack,P., Graham,S., Lcibson, H. J., Roehm,N., Wegmatm, D., Kappler, J. 1982.In Isolation, Characterization andUtilization of T Lymphocyte Clones, ed. C. G. Fathman,F. W.Fitch, pp. 119-126. NewYork: Academic 13. Nabholz, M., Conzelmann,A., Aeuto, O., North, M., Hass, W., et al. 1980. Established murine cytolytic T-cell lines as tools for a somaticcell genetic analysis of T-cell functions. ImrnunoL Rev. 5h125-156 14. Nabholz,M., Cianfriglia, M., Acuto, O., Conzclmann,A., Hass, W., ¢t al. 1980.Cytolyticallyactive routineT-cell hybrids. Nature 287:437-440 15. Gordon,J., MacLean,L. D. 1965. A lymphocyte.stimulating factor producedin vitro. Nature208:795-796 16. Kasakura,S., Lowcnstein,L. 1965. A factor stimulating DNAsynthesis

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stant re#on gene segments. J. Exp. Med. 152:1745-1761 53. Kronenberg,M., Kraig, E., Horvath,S. J., Hood,L E. 1982. ClonedT cells as a tool for moleculargeneticists: approachesto cloninggenes wkiehencode T cell antigen receptors. In Isolation, Characterizationand Utilization of T LymphocyteClones, ed. C. G. Fathman, F. W.Fitch, pp. 467-491. NewYork: Academic 54. Zuniga, M. C., D’Eustaekio,P., Ruddie, N. H. 1983. Immunoglobulin heavy chain gene rearrangement and transcription, In MurineT Cell Hybridsand TLymphoma~In press 55. Cayre,Y., Palladino, M.A., Mareu,K. B., Stavnezer,J. 1981.Expressionof an antigenreceptor on T cells does not require recombination at the immunoglobulin Ja-C~locus. Proc. NatLAcad. Sci. USA78:3814-3818 56. Forster, A., Hobart, M., Hengartner, H. M., Rabbitts, T. H. 1980. An immunoglobulinheavy-chaingene is altered in two T cell clones. Nature 286:897-899 57. Kurosawa,Y., von Boehmer,H., Haas, W., Sakano, H., Traurieker, A., Tonegawa, S. 1981. Identitieation of D segments of immunoglobulin heavy chain genesandtheir rearrangements in T lymphocytes.Nature 290:566--570 58. Kemp,D. J., Harris, A. W., Cory, S., Adams,J. M. 1980. Expressionof the immunoglobulinC~ gene in mouse T and B lymphoid and myeloid cell lines. Proc. Natl. AcacL Sc£ USA 77:28762880 59. Kemp,D. J., Wilson,A., Harris, A. W., Shortman, K. 1980. The immunoglobulin p constant region geneis expressed in mousethymoeytes.Nature 286:168170 60. Owen,F., Spurll, G. W., Panageas,E. 1982. Tthy~, A newthymoeytealloantigen linked to Igh-1. J. Exp. Med. 155:52-56 61. Spurll, G. M., Owen,F. L. 1981. Afamily oft cell alloantigenslinkedto Igh-1. Nature 293:742-744 62. Tokuhisa,T., Komatsu,Y., Uchida,Y., Taniguchi, M. 1982. Monoclonalalloantibodiesspecitie for the constantregionof T ceil antigenreceptors. £ Exp. Med. 153:888-897 63. Lonai,P., Bitton,S., Savelkoul,H.F. J., Purl, J., Hammerling, G. J. 1981. Two separategenesregulate self-Ia andearrier recognitionin H-2restricted helper factors secreted by hybridoma cells. ,L Exp. MecL154:1910-1921

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76. Infante, A. J., Atassi, M.Z., Fathman, C. G. 1981. T cell clones reactive with sperm whale myoglobin:isolation of clones with specificity for individual determinants on myoglobin. J. Exp. Med. 154:1342-1346 77. Fathman, C. G., Kimoto, M. 1981. Studiesutilizing murineT cell clones:Ir genes, Ia antigens and MLR determinants. Immun.Rev. 54:57-80 78. Fathman,C. G., Infante, P. D. 1979. HybridI region antigen and I region restriction of recognitionin MLR. Immunogenetics8:577-581 79. Pierres, M., Devaux,C., Dosseto, M., 67. Kanagawa, O., Louis, J., Cerottini, J. Marctetto, S. 1981. Clonalanalysis of 1982.Frequency and cross reactivity of B- and T-cell responsesto Ia Antigens. kI. Topologyof epitope regions in I-A cytolytic T lymphocyte precursors reacting against maleantigen. J. Imand I-Ek molecules analyzed with 35 raunoL128:2362-2366 monoclonalalloantibodies. Immuno68. Braciale,T. J,, Andrew, M.E., Braciale, genetics 14:481-495 V. L. 1981.Simultaneousexpressionof 80. Leruer,E. A., Marls,L. A., Janeway,C. H-2-restrictedandalloreactiverecogniA. Jr., Jones, P. P., Schwartz,R. H., tion bya clonedline of influenzavirusMurphy,D. B. 1980. A monoclonalanspecific cytotoxic T lymphocytes.Z tibody against an immune response(Ir) Exp. Med. 153:137-13761 gene product? J. Exp. Me~152:108569. Sredni,B., Schwartz,R. 1981.Antigen1101 specific, proliferating T lymphocyte 81. Sherman,L. A. 1982. Recognition of clones. Methodology, specificity, MHC conformationaldeterminantson H-2by restriction andalloreactivity. Imrauno. cytolytic T lymphocytes.Nature 297: Rev. 54:187-224 511-513 70. Schwartz,R. H., Sredni, B. 1982. AI82. Wylie,D. E., Sherman,L. A., Klinman, loreactivity of antigen specific T cell N. R. 1982.Participation of the major clones. In Isolation, Characterization histocompatabilitycomplexin antibody andUtilization of T Lymphocyte Clones, recognitionof viral antigensexpressed ed. C. G. Fathman,F. W.Fitch, pp. on infected cells. J. Exp. Med.155:403375-384. NewYork: Academic 414 71. Janeway,C. A. Jr., Conrad,P. J. 1983. 83. Sarmiento, M., Glasebrook, A. L., Degeneracy in the responseof individFitch, F. W.1980. IgG or IgMmonoual T cells to non-self majorhistocomclonalantibodiesreactive withdifferent patibility complexantigens. Behrin. determinantson the molecularcomplex InsL Rex Corarr~In press bearingLyt-2 antigen blockT cell me72. Sherman,L. A. 1980. Dissectionof the diated cytolysis in the absenceof com~ cytolytic B10.D2anti H-2K T lymponent. J. lmmunoL125:2665-2672 phocyte receptor repertoire. J. Exp. Med. 151:1386-1397 84. MacDonald,H. R., Thieruesse, N., Cerottiul, J.-C. 1981. Inhibition of T 73. Nairn, R., Yamaga,K., Nathenson,S. cell-mediatedcytolysis by monoclonal G. 1980.Biochemistry of the geneprodantibodies directed against Lyt-2: ucts from murine MHC mutants. Ann. heterogeneityof inhibition at the clonal Rev. Genet. 14:241-277 level. J. Imraunol.126:1671 74. Endres,R. O., Grey, H. M.1980. AntigenrecognitionbyT cells. I. suppressor 85, Golstein, P., Goridis, C., Schmit-Verhulst, A., Hayot,B., Pierres, A., et al. T cells fail to recognizecross-reactivity 1982. Lymphoid cell surface I interacbetweennative and denatured ovalbution structures detectedusingcytolysis rain. J. ImmunoL125:1515-1520 inhibition monoclonalantibodies. Im75. Maizels,R. M., Clarke, J., Harvey,M. muno. Rev. 68:5-42 A., Miller,A., Scrcarz,E. E. 1980.Epi86. Loken, M. R., Fitch, F. W. 1982. tope specificity of the T Cell proliferaCloned T lymphocytesand monoclonal tive responseto lysozyme:proliferative antibodies as probes for cell surface T cells react to predominantly different molecules active in T cell mediated determinantsfromthose recognizedby B cells, gur. J. Immunol.10:509-515 cytolysis. Irnrauno.Rev. 68:135-170

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87. Dialynas, D. P., Loken,M. R., GlaseBarr virus129:1446-1450 B lymphoblastoidcells. Y. Immunol. brook, A. L., Fitch, F. W.1981. Lyt2/Lyt-3variants of a clonedcytolytic T 97. Fathman,C. G., Hengartner,H. 1979. cell line lack an antigenreceptor funcClonesof ailoreactiveT ceils. II. Crosstional in eytolysis. Y. Exp. Med.153: reactive MLR determinants recognized 595-604 by cloned alloreactive T cells. Proc. 88. Giorgl,J. V., Zawadzki, J. A., Warrer, Nat~/lcad. Sci. USA.76:5863-9 N. L. 1982. Cytotoxic T lymphocyte 98. Shigeta, M., Fathman,C. G. 1981. I lines reactive against murineplasma region genetic restriction imposedupon cytomaantigens: dissociation of cytothe recognition of KLHby murine T cell clones. Immunogenetics14:415toxicity andLyt-2expression.Eur.,1.. Immuno.12:831-837 422 89. Infante,A.J., Infante,P. D., Glllis, S., 99. Silver, J., Swain,S. L., Hubert,J. J. Fathman,C. G. 1982. Definition of T 1980. Small subunit of I-A subregion cell idiotypesusinganti-idiotypicantiantigensdeterminesthe ailospecificity sera producedby immunizationwith T recognized by a monoclonalantibody. cell clones. ,/. Exp. Mea~ 155:1100-1107 Nature 286:272-274 90. Asano, Y., Shigeta, M., Fathman,C. 100. Jones, P. P., Murphy,P. B., McDevitt, G., Singer, A., Hodes,IL J. 1982.Role H. O. 1978. Twogene control of the expression of a murineIa antigen. Y. .of the majorhistocompatibilitycomplex mT cell activation of Bcell subpopulaExp. Med. 148:925-939 tions. Asingle monoclonal T helper cell 101. Beck,B. N., Frelinger, J. G., Shigeta, population activatesdifferentB cell subM., Infante, A.J., Cummings, D., et al. populations by distinct pathways. Z 1982.T cell clones specific for hybrid Exp. Med. 156:350-360 I-A molecules. Discrimination with 91. Lamb,J. R., Eckels,D., Lake,P., Johnmonoclonalanti I-Ak antibodies, j.. son, A. H., Hartzman,R. J., Woody,J. Exp. Med. 156:1186-1194 N. 1982. Antigen-specific humanT 102. Frelinger,J. G., Shigeta,M., Infante,A. lymphocyteclones: induction, antigen J., Nelson,P., Pierres, M., Fathman,C. G.1983.Multiplerestriction sites per Ia Suflpecenzaificity, and MHC restriction of invirus-immuneclones. J. Immoleculerecognizedby T cell clones. In munoL128:233-238 lr genes~"Past, PresentandFuture,ed. 92. Lamb,J. R., Woody,J. N., Hartzman, C. W.Pierce, S. E. Cullen, J. A. Kapp, J., Eckels, D. 1982.In vitro influenza B. D. Schwartz,and D. C. Shreflter. virus specific antibody productionin Clifton, NJ: Humana. In press man: antigen specific and HLA- 103. Nelson,P. A., Beck,B. N., Fathman,C. restricted inductionof helper activity G. 1983. Functional evidence for two mediated by cloned human T lymantigen presentationsites for a single phocytes. ,£ ImmunoL129:1456-1470 I-A molecule.In Ir genes:Past, Present 93. McKean, D. J., Infante, A. J., Nelson, andFuture,ed. C. W.Pierce, S. E. CulA., Fathman, C. G., Walker, E., len, J. A. Kapp,B. D. Schwartz,and D. Warner,N. 1981. MHC restricted antiC. Shrefller. Clifton, NJ: Humana. In genpresentation to antigen reactive T press cells by lymphocyte tumorcells. Z Exp. 104. McNicholas,J. M., Murphy,D. B., MaMed. 154:1419-1431 tis, L. A., Schwartz,R. H., Lemer,E., 94. Glimcher,Z. H., Kim,K. J., Green,I., et al. 1982.Immune responsegenefuncPaul, W.E. 1982. Ia antigen bearingB tion correlateswiththe expressionof an cell tumorlines can present protein anIa antigen.L Preferentialassociationof tigen and alloantigen in a majorhiscertain At and E, chains results in a tocompatibilitycomplex restricted fashquantitative deficiencyin expressionof ion to antigen reactive T cells. Z Exp. an AtE~complex. Y.. Exp. Med.155: Med. 155:445-459 490-507 95. Kappler, J., White, J., Wegmann, P., 105. Matis, L. A., Jones, P. P., Murphy,D. Mustain,E., Marrack,P. 1982. Antigen B., Hedrick,S. M., Lerner,E. A., et al. presentation by Ia + B ceil hybridomas 1982. Immuneresponse gene function to H-2Nat~ restricted T ceilUS~4 hybridomas. correlates with cell surface expression Proc. ~4caRSci. 79:3604of an Ia antigen.IL A quantitativedefi3607 ciency of an AtE~complexexpression 96. Issekutz, T., Chu, E., Geha,R. 1982. causesa corresponding defect in antigen Antigenpresentation by humanB ceils: presenting ceil function. Y. Exp. Med. T cell proliferation inducedby Epstein155:508-523

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