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Annual Review of Immunology Volume 7 1989

How One Thing has Led to Another George Klein and Eva Klein.Vol. 7: 1–34

Decay-Accelerating Factor: Biochemistry, Molecular Biology, and Function D M Lublin, and J P Atkinson. Vol. 7: 35–58

Heterogeneity of Mast Cells and Phenotypic Change Between Subpopulations Y Kitamura. Vol. 7: 59–76

The Cellular Basis of T-Cell Memory J C Cerottini, and H R MacDonald. Vol. 7: 77–89

Microanatomy of Lymphoid Tissue During Humoral Immune Responses: Structure Function Relationships A K Szakal, M H Kosco, and J G Tew. Vol. 7: 91–109

Cells and Molecules that Regulate B Lymphopoiesis in Bone Marrow P W Kincade, G Lee, C E Pietrangeli, S I Hayashi, and J M Gimble. Vol. 7: 111–143

TH1 and TH2 Cells: Different Patterns of Lymphokine Secretion Lead to Different Functional Properties T R Mosmann, and R L Coffman. Vol. 7: 145–173

The Structure, Function, and Molecular Genetics of the gamma/delta T Cell Receptor D H Raulet. Vol. 7: 175–207

V-Region Connectivity in T Cell Repertoires P Pereira, A Bandeira, A Coutinho, M A Marcos, M Toribio, and C Martinez-A. Vol. 7: 209–249

The Immune System of Xenopus L D Pasquier, J Schwager, and M F Flajnik. Vol. 7: 251–275

Molecular Genetics of Chronic Granulomatous Disease S H Orkin. Vol. 7: 277–307

Cell Biology of Cytotoxic and Helper T Cell Functions: Immunofluorescence Microscopic Studies of Single Cells and Cell Couples A Kupfer, and S J Singer. Vol. 7: 309–337

The Leukocyte Common Antigen Family M L Thomas. Vol. 7: 339–369

T Cell Receptors in Murine Autoimmune Diseases H Acha-Orbea, L Steinman, and H O McDevitt. Vol. 7: 371–405

Manipulation of T-Cell Responses with Monoclonal Antibodies H Waldmann. Vol. 7: 407–444

Clonal Expansion Versus Functional Clonal Inactivation: A Costimulatory Signalling Pathway Determines the Outcome of T Cell Antigen Receptor Occupancy D L Mueller, M K Jenkins, and R H Schwartz. Vol. 7: 445–480

Immunogenetics of Human Cell Surface Differentiation W J Rettig, and L J Old. Vol. 7: 481–511

Probing the Human B-Cell Repertoire with EBV: Polyreactive Antibodies and CD5+ B Lymphocytes P Casali, and A L Notkins. Vol. 7: 513–535

Stable Expression and Somatic Hypermutation of Antibody V Regions in B-Cell Developmental Pathways C Kocks, and K Rajewsky. Vol. 7: 537–559

T-Cell Responses and Immunity to Experimental Infection with Leishmania Major I Muller, T Pedrazzini, J P Farrell, and J Louis. Vol. 7: 561–578

The Biologic Roles of CD2, CD4, and CD8 in T-Cell Activation B E Bierer, B P Sleckman, S E Ratnofsky, and S J Burakoff. Vol. 7: 579–599

Antigen Recognition by Class I-Restricted T Lymphocytes A Townsend, and H Bodmer. Vol. 7: 601–624

The Biology of Cachectin/TNF -- A Primary Mediator of the Host Response B Beutler, and A Cerami. Vol. 7: 625–655

The T-Cell Receptor Repertoire and Autoimmune Diseases V Kumar, D H Kono, J L Urban, and L Hood. Vol. 7: 657–682

T-Cell Recognition of Minor Lymphocyte Stimulating (MLS) Gene Products R Abe, and R J Hodes. Vol. 7: 683–708

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Annual Reviews

Annual Reviews Ann. Re~. lmmunol. 1989. 7: I 33 Copyright © 1989 by Annual Rev&wsInc. All rights reserved

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HOW ONE THING HAS LED TO ANOTHER George Klein

and Eva Klein

Department of TumorBiology, Karolinska Institutet, S-104 01 Stockholm, Sweden, and Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, Jerusalem, Israel GEORGE

KLEIN

WRITES:

Dawn This story starts on the 10th of January, 1945, when I emerged from a cellar on the outskirts of Budapest where I had been hiding, with false papers, during the last weeks of the Germanoccupation. With a totally newfeeling about the sunshine that was floating over the snow, the ruined houses, the dead and frozen soldiers, civilians, and horses, I suddenly realized, with a mixture of surprise, guilt, and delight, that I had survived in spite of an 80%chance that I wouldend my19 years in the gas chambers or in a military slave labor camp. After a few quick walks in the newly liberated area of the still besieged capital, I decided that it was time to start mymedical studies, already delayed by almost two years. During the first year after mygraduation from middle school, it was impossible for a Jewish boy to enter medical school. After the Germanoccupation nothing mattered except survival. Wewere free at last, but it was a complicated freedom. After a few more days, the Eastern side of the city, Pest, was all in Russian hands. I moved around relatively freely but I was caught twice, like other young menwho were automatically regarded as disguised soldiers. In comparison with my earlier escape from a Nazi labor camp, it was an easy matter to run away from the improvised, loosely organized Russian patrols. It was a wise move. Several friends of mine whowent out to get a loaf of bread returned years later from Russia. As soon as the streets were open, I walked to the University to see 1 0732--0582/89/04104)001 $02.00

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whether it would open its doors for me now. I found deserted buildings, broken windows, and dead soldiers. Together with a friend we therefore decided that we should try to reach Szeged. The journey of less than 300 kmtook more than five days. Wewalked long stretches, hitched on horsedrawn carriages and every other vehicle that we could get on, including a Russian military truck. Wearrived in Szeged on February 4. It was a cold and beautiful morning. The city was intact, and we were admitted to the University on the same day. It was a strange place. All the professors had fled to the West. An assistant professor of forensic medicine with a Christlike head and very sad eyes was teaching anatomy, pathology, and forensic medicine all by himself. Students kept arriving from all former theaters of war, labor camps, and illegal hiding. Cadavers were abundant. The large dissection hall of the AnatomyDepartment was crowded. The smell of formalin, the half dissected or fully prepared body parts, and even the continually tipsy attendant appeared to meas parts of a magic, enchanting landscape, a previously forbidden paradise that was nowall mine. Twoyears passed as a single wave of febrile activity. I finished three terms during three months in Szeged and returned to Budapest when the university reopened there. I wanted to start research work, but the departments were still paralyzed. They had no resources and the routine workconsumed the energyof all staff. Still, I got a first decisive inspiration from the professor of histology, Tivadar Huzella, one of the few internationally knownscientists in Hungaryand als O one of the few true liberals amongthe medical professors of his generation. In spite of his consistent anti-Fascist stance, and his strong opposition to any form of discrimination during the war, he became a suspected person in the eyes of the new rulers. His uncompromisingindividualism and his democratic value system invited the enmity of the political opportunists whowanted to see a more compromisingperson in his position. His arch-enemy, the professor of anatomy, a political opportunist and a scientific nonentity who had resented Huzella’s international fame for manyyears, delivered a list of accusations against him to the "people’s court." The sympathies of all the students were on Huzella’s side. The crucial trial, where all the absurd accusations--exemplified by the charge that Huzella ate eggs ordered for tissue culture--were readily dismissed, ended in tragedy when the presiding lay judge asked whether Huzella still believed a sentence he wrote during the war. Huzella had stated (an act of great courage at the time) that Hitler, Stalin, and Salazar were equally abominable dictators. If he would have been willing to exempt Stalin and admit his "mistake," he would have been cleared. But he stuck to his words and was summarily dismissed. He died a few years later. Today he has been "rehabilitated."

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HOWONE THINGHAS LED TO ANOTHER 3 His homeand laboratory are kept as a public memorial. They also house the leading immunological laboratory of Hungary. Huzella had an exceptional ability to convey his own deep interest in biology to his students. He was convinced that the time had come when biology could be converted from "metaphysical speculation" into a natural science with precision and dignity similar to those of chemistry and physics. He believed that the biology of the interstitial space would turn into detailed biochemistry in a few decades but that the cell interior would remain a black box during the rest of the century. Before blaming him for a lack of foresight, we must realize that most biologists of the time were unwilling to accept his "optimistic" view even about the connective tissue. I learned some tissue culture, but my practical experience remained rudimentary, and I compensatedonly slightly by avid reading in the still quite deficient library. After Huzella’s removal, I realized that I could not learn more in the nowlargely nonfunctional department, and so I movedto Pathology. After a few weeksI found myself totally immersedin autopsies. There was a great abundance of cadavers here and very few pathologists. The large postwar classes of medical students had to be taught quickly. I greatly enjoyed the double task of teaching the little I knewand trying to explain to the rushed and often very nervous clinicians what their patients had died of. In the early spring of 1947, one of "my"students approached me after an autopsy. He said something appreciative about my demonstration and asked whether I would be interested to visit Swedenwith a student group. I was amused by his naivet6. Whowould not like to visit Sweden? But were we not all aware of the fact that foreign travel was the exclusive privilege of important functionaries and people with much moneyand many good connections? He replied that he was currently organizing a trip for students and that he would include me. Hungarystill had an elected coalition government at this time. It was possible to get a passport, but this was not sufficient to leave the country. A special exit permit had to be issued by the "Allied" forces, i.e. the Soviet Army.It was very difficult to get this permit, and it was nearly impossible to obtain foreign currency. I mailed mypapers to my student who was interested in Sweden and totally forgot about our conversation. Decisive

Summer

In June 1947 myboss, Professor Balr, told me that I would be responsible for the autopsies during the comingmonth, virtually alone. I was happy, proud and frightened. I was not yet 22, far from being an MD,but the night’s sleep of a professor in surgery could depend on what I was going

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to find. The combined feeling of responsibility and awe turned every autopsy into an exciting detective story. During myminimal "spare hours" I also started myfirst attempts to do someexperiments. I was sitting in a corner of the laboratory with a small water bath and a stalagrnometer, trying to follow a lead that had been opened up by mychief. The most important messengers of my future destiny appeared in the shape of two house painters in the middle of July. They had been ordered to repaint the laboratories. I was chased from room to room with my water bath, but I refused to give up. Finally, I was squeezed into a small corner in a tiny windowless alcove that I refused to leave. The painters complained to Professor Balr. With an irritated "you can take two weeks vacation for once" he ordered meto leave myparadise. A senior colleague was to take care of the autopsies. I was angry and disappointed. Whatwas I to do during two whole weeks? By coincidence I learned that some fellow students, two couples from the Pharmacology Department, were planning to spend the forthcoming weekat the Lake Balaton. I was also told that they had invited someother friends and that I was welcometo join them. Wewere allowed to use the terrace of a bombedsummerhouse and were going to sleep on mattresses, spread out on the terrace. It was quite warmduring the first week in August, and we would have a roof over our head. After considerable hesitation, I decided to join them, but I felt ambivalent and uninterested. The place was unexpectedly pleasant and myfellow students were much nicer in private life than at the University. On the second day, the two other boys went down to the train to meet another student from the Pharmacology Department, who was to join us. I did not know who it was, and since the Hungarian language does not dist.inguish between he and she, I did not even knownwhether we were expecting a boy or a girl. After a while I saw them walking up the hill with the new guest: a dark girl with a strange, breathtaking beauty. I perceived a most unusual combination of hilarity and sorrow, seriousness and play in her eyes. It was Eva, myfuture wife and colleague until this day. I had seen her before at the university, but myobsessive preoccupation with work prevented me from giving her or any other girl muchattention. Still, I could remembervery well how I met her the first time. On the second day of mymedical studies in Szeged, I was standing in the Dean’s office, to get mypapers. She entered, dressed in a skiing outfit, having arrived in the city after a long and adventurous trip from Budapest, like myown. She asked me howto get papers. I saw that she was very beautiful. Her direct way of talking to a strange boy--very unusual for a girl in Hungary at the time--struck me as original and sympathetic. During the forthcoming weeks I saw her at some lectures, but then she disappeared.

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HOWONE THING HAS LED TO ANOTHER 5 Later I saw her name on the posters of the city theater. She was playing small roles in Pirandello and Moli&eplays. Halfa year later I saw her again in Budapest. She had returned to medical studies and came sometimes to my autopsy demonstrations. I knew that she belonged to the same group of students in the Pharmacology Department as mymarried friends and temporary hosts. Their "gang" treated me with friendly tolerance, and even with a trace of respect for my"knowledge"--in spite of their "objections" to the "dead morphology"that pathology represented in their eyes. I respected their intelligence and their dynamicexperimentation and could therefore forgive their blatant ignorance of pathology and clinical medicine. But this time everything was different. There was one table but only three intact chairs in the ruined villa, and we were six. Wehad to place a board on each chair to hold two. Eva and I were placed on the same board and had to coordinate our movementsto prevent each other from falling down. This trivial problem initiated a contact that metamorphosedafter only a few hours into a passion that conquered myentire consciousness with the force of an elementary power. All other interests and problems vanished as if they had never existed. I spent eight days at the lake, intoxicated, overwhelmed,cut-off from all earlier reality. An unexpected telegram arrived on the seventh day. Everything was settled for the trip to Sweden!Myformer pathology student or, as we were soon to call him, Our Leader, had succeeded against all odds. He had pursued his plan with obstinate ingenuity and obtained all the exit permits for a group of seventeen students selected by himself with the arbitrariness of a sovereign. Wecamefrom different faculties and were to visit Stockholm and Gothenburgas the guests of the Jewish Student Club there, in order to see a country that was saved from the war. NowI did not have the slightest wish to go. I felt very bitter about having to leave the person who had becomemore important than anything else in mylife so far. The week at the Balaton appeared as an eternity; everything before was unreal. But vague feelings 6f responsibility and premonition commandedme to go. I left at dawn on a Sunday morning. Eva told me later that she heard the train whistle while half asleep and thought that a beautiful summerepisode was nowover. She did not believe that I would ever come back from Swedenor that she would see me again. Cell Biology

1947

The first International Congress of Cell Biology had just terminated when I arrived in Stockholm. I was told that Torbjrrn Caspersson was one of the most important figures at the Congress. His recent development of ultraviolet microspectrophotometryon fixed cells created muchattention.

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The method was based on his doctoral thesis, written in 1936 in German and largely unavailable to English speaking readers during the war years. It was the first major attempt to combine morphologyand cytochemistry. Cells were photographed in monochromatic UVlight under standardized conditions. A semiquantitative method was developed to map the localization of nucleic acids and proteins in different cell types. Jack Schultz, one ofJ. H. Morgan’slast disciples, was the first Americangeneticist who saw the potentialities of the new approach. He traveled to Stockholm to work with Caspersson shortly before the outbreak of the war. He brought genetic thinking to the biophysically oriented group. His studies with Caspersson on the banding patterns of polytenic insect salivary gland chromosomesgave the first information about the distribution of nucleic acids and chromosomal proteins and set the conceptual basis for the development of the chromosome banding technique by Caspersson and Zechthree decades later. The chemistry of the genetic material was still unknownat the time of the Cell Biology Congress in Stockholm. Most biologists believed that only proteins could provide the necessary diversity. Nucleic acids were considered as repetitive, boring molecules. Levene and Bass pronounced the death sentence on the coding capacity of the nucleic acids already in the 1930s. The mistaken analogy between the "4-letter alphabet" of the nucleic acids and the phonetic alphabet served as a roadblock: howcould one build a language from four letters? Caspersson’s semiquantitative measurementsof nucleic acids and proteins in different cell organelles led him to conclude that there was a definite relationship betweennucleic acid and protein synthesis and that the former might actually govern the latter. This visionary insight was widely disbelieved, however. The idea that nucleic acids might carry genetic information that could be translated into proteins was totally foreign, even to Caspersson. The fundamental discovery of Avery, McLeod, and McCarthy on DNA-mediated transformation in Pneumococcus, published in 1944, was widely ignored or discarded as an artefact. The Cell Research Department of Karolinska Institute had just moved to the newly built campuson the northern edge of the city; there I was to spendall myscientific years, up to the present day. I visited it first in the middle of August, 1947, the peak of the vacation season and soon after the Congress participants had left town. Membersof the Department who happened to be in town were frantically trying to get settled in the new building. As I mademyentry, tall, blond, 37-year-old Torbjorn Caspersson was lying under a large instrument in a blue overall, trying to fix the wires. I thought that he was an electrician or a technical assistant. His identity was not revealed to me and I was not introduced to him. After I had

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HOWONE THING HAS LED TO ANOTHER 7 learned the difficult art of protecting him from uninvited visitors a few years later, I could understand the reasons. In 1947, I was desolate when I had learned the next day that he had left fo~ the USA.Only after a long series of complications did I get in touch with him, several weeks later. But my first conversation with him was decisive. Thanks to the rudimentary and largely theoretical knowledgeof tissue culture, acquired in the Huzella laboratory two years earlier, I got the best-paid job of mylife (if the importanceof the salary is considered). I was employedas a junior research assistant, on 500 SwCrs (about US$100) per month. I still rememberthe mixture of ecstatic happiness and enormousanxiety. Mysituation appeared totally hopeless. I knew virtually nothing. I was halfway through mymedical studies, still far removedfrom an MD.I was desperately in love with a girl whomI had only knownduring a summer vacation of eight days and who was on the other side of an increasingly forbidding political barrier. I did not knowa word of Swedish. Still, I was firmly decided to resist the more comfortable possibility of continuing my studies in Hungary. Mymotivation was reinforced by a series of articles that kept appearing in the major Swedish daily, Da#ens Nyheter, translated for me by my temporary host. The Prime Minister of Hungary, Ferenc Nagy (not to be confused with Imre Nagy) of the Smallholder’s Party has just fled to the West, and he gave a series of interviews to the Swedishpaper. In contrast to the rosy optimism that prevailed amongmy friends in Budapest who hoped that Hungary would become a democratic country, Nagy’s statements had an ominous ring. He said that the influence of the Communist Party was increasing continuously behind the scenes. The Stalinist party leader, Rfikosi, was acting under the protection of the Russian forces. The politicians of the other parties were frightened. Several of their leading representatives were arrested on false charges and deported to unknown destinations. Those whoremained were increasingly inclined to give in. The police were infiltrated by party members. Nagy did not have the slightest doubt that a Communisttakeover was imminent. Similar signals reached me indirectly from one of myteenage idols, Nobel Prize winning biochemist Albert Szent-Gy6rgyi. He was still holding manyhigh posts in Hungary at the time, but he had told his nephew, who was a friend of mine, that the days of freedom were numbered. If you were young and wantedto have a future in science, you should get your degree as soon as possible and leave the country. Farewell,

My Native

Land

In mid-September,I decided to go back to Budapest and try to get out for good. Mymost important acquisition was safely tucked awayin mybreast

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pocket: a re-entry visa to Sweden and a labor permit for continued work in Caspersson’s department. Mypassport was still valid for a few months. The reunion with Eva confirmed what we both knew already: we wanted to live and work together. The day after myarrival, someof our friends gathered at myhome to hear the latest news from the "great world." I told them about Nagy’s report and the iron curtain that was about to descend over Hungary. The reaction was mixed. Those who were already preparing to leave believed me. Others wanted to stay and hoped that my report was exaggerated. One of them--still a good friend today--declared that I was probably right, and for that reason, he was going to break all further contact with me. This was his country, Hungarianwas his language, his historical roots were here. I should leave, if I felt so inclined, but he had to stay and do the best he could. Today he is the foremost medical historian of Hungary. I had none of his historical perspectives. I had only one goal, to get married and leave the country. But howto get married? It had to be in secret, because nobody would understand why two 22-year-old students who had known each other for only a short time and had no income would want to get married. And how could myfuture wife join me? She had no passport and the difficulties in getting one were now increasing day by day. Weagreed that I would go back to Stockholm before my own passport expired and try to obtain letters of invitation for Eva that could help her to get a passport. The last weekday before my trip was a Friday. Eva and I met outside the pharmacological institute to go to the day’s lecture. I suggested that we should go to the prefecture instead and ask howone gets married. We got a list of the manydocumentsyou needed. It looked hopeless. It would take months to get them. I suggested that we ask for the first document, a certificate to showthat we had no police records. Wewent to the police station. "It takes at least three weeks." SuddenlyI acted on impulse. I had always heard others tell of such things but I myself had neither seen nor done it. I pulled a fairly modest bill out of mypocket and put it in the policeman’s hand. "Pardon me, how muchtime was it, you said? .... I’ll go and get it at once," he answered. It was now11 Ara. Wecontinued from office to office. Everywherethe same answer: one week, four weeks, six weeks. A little bill in the hand--the certificate was completed within a few minutes. I was amazed to find that the shyness I usually exhibited before persons of authority vanished completely. I learned a lesson about the importance of motivation and the unsuspected possibilities it mayopen to surpass one’s limitations.

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HOWONE THING HAS LED TO ANOTHER

9

By 3 PMonly one document was missing: a medical certificate that neither of us had venereal disease. The tests would take several weeks. What to do now? Wewent to a slightly older colleague who had recently finished his medical studies. He had just started his first assignment in the Children’s Hospital. Wetold him, in the strictest confidence, about our situation. He had a good laugh and wrote the certificate on the hospital stationary. By 4 PMwe were at the prefecture again. Wehad all the papers and wanted to get married that second. Twoother friends, swornto the highest secrecy, camealong as witnesses to the wedding.The official had just finished the day’s work and had taken off the broad Hungarian tricolor from his corpulent chest when we rushed in. Weheard him telling his wife on the phone that he was on his way homefor dinner. Marry us at this time of day? Not a chance! Comeback on Monday! I started to appeal to his humanfeelings. I had to leave the country on Sunday. Howcould 1 leave myyoung bride alone if we didn’t get married? He was noticeably irritated and doubted that we had all the papers. While leafing through the documents,he caught sight of the doctor’s certificate that had been drawnup at the Children’s Hospital. He laughed until tears ran downhis cheeks. This was the funniest thing he had seen during his whole time in service. Nowhe was in splendid spirits. The flag resumedits place on the large body. Wepromised to love one another til death us did part. Afterwards we ate our wedding dinner on the hall bench together with our witnesses. There was only one dish: mymother’s carefully packed goose liver sandwiches. In the evening we went back to our parents’ homes where no one suspected anything. That SundayI returned alone to Stockholm. Eva joined me, after many complications, in March 1948, after the Iron Curtain had already descended over the country. GEORGE

AND EVA

The Genetics

WRITE:

Congress

In August 1948, several months after we were happily settled in our rented room and Eva had also started to work in Caspersson’s department, the International Congress of Genetics took place in Stockholm. The presidential address of J. H. Muller was a scathing denunciation of the abuse of genetics in the Soviet Union. The scientific world was still largely unawareof the fact that the "theories" of a charlatan, Lysenko, had been declared "official" by the Central Committee of the CommunistParty, meaning that it became essentially illegal to do any scientific work in

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genetics. Mullerhimselfhadbeenthe first to introduceDrosophilagenetics into Russia, and he wasstill a member of the Soviet Academy of Sciences at this time. He called Lysenko"a paranoic and half educated young demagoguewhohad done some work in raising plants but whowas in fact ignorant of scientific principles andincapableof understandingthem." He added that many of the outstanding Russian geneticists had disappeared,and somehad lost their lives in unexplainedways.His speech ended with his resignation from the Soviet Academy.The reaction from Moscow camethe day after. Theyrefused to accept his resignation and expelled him. Onthe last day of the congress, the Bulgarian delegate asked to make a statement at the concludingplenary session. Speakingin the nameof the delegates from Bulgaria, Roumania,Poland, and Czechoslovakiahe delivered a strong protest against Muller’s introductory speechthat was "ill-suited to favor international understanding."His protest wastaken to the protocol. After the session wasclosed, the representative of Hungarycameto us. Hedid not understand English well, and wehad previously helped him. Hewantedto knowwhat the Bulgarian delegate said. Whenhe heard our interpretation he becameextremelyupset. It wastypical for the Slavic delegates to leave out the Hungarians!Hehad to join the protest, he had to think of his family! Howcould he return without havingsigned it! But he wasout of luck, the congresswasover, nothing could be donefor him. His panic showedus howthe fear imposedby Stalinism had descendedon the country wehad left only a few monthsearlier. It wasalso a reminder of the eternal strife amongthe nations that have risen from the ruins of the Hapsburgmonarchy. Duringthe Congresswelearned about the startling progress in microbial genetics. Bacteriologyhad been the last citadel of Lamarckism.At this time, whenproteins wereregardedas the vehicles of genetic information, whennotions about a bacterial nucleuswereregardedwith great suspicion, inducedenzymeadaptations and drug resistance werewidelyattributed to the inheritance of acquired characteristics. But the rapidly growingevidenceof clonal variation and Darwinianselection wasnowdefinitely gaining ground,integrating microbiologywith the rest of biology. Ourknowledgeabout cancer was rudimentary,but westarted nevertheless wondering whether the population dynamicsof microorganismsand the phenomenon Of cancer might share some commondenominator(s). Cancer cells are resistant against growth control of the organism. Canthey be compared to drug resistant microorganisms? Couldcancers also arise by a series of mutations?Theseseeminglypuerile notions wereto play an importantrole for our worklater on.

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The Cell

Research

11

Department

Back at the laboratory, we found ourselves in an exciting environment but facing another impossible situation. Wewere still medical students in midcourse. Wewere struggling hard to get into a Swedish medical school and finish our studies. At first, this looked impossible but eventually we succeeded, one by one, taking turns between the school and the lab work. Worsethan that, the project given us turned out to be quite unmanageable. Caspersson’s methodologywas based on the absorption of monochromatic ultraviolet light in fixed cells. Shortly before our arrival, it was heavily criticized by Barry Commonerand other biophysicists. They suggested that the loss of UVlight registered by Caspersson’s optical system was not due to absorption but to light scattering from the denatured proteins. Due to this artefact, part of the nonabsorbed light would never reach the objective, leading to false conclusions about the localization of nucleic acids and proteins. Their distribution in living cells could be totally different from the pattern suggested by Caspersson’s measurements. Our task was to measure light absorption in living cells. But this was more easily said than done. Tissue culturing of the times followed the dogmaslaid downby Alexis Carrell. The plasma clot and the embryonic extract were regarded as essential substrates. Nobodyin his fight mind wouldhave thought of culturing cells directly on glass, even less on quartz slides. The plasma clot was not transparent to UV. It turned out that mysudden and unexpected employmentafter my first conversation with Caspersson was due to the fact that I had someexperience of growing cells on collagen, Huzella’s favorite method. Collagen is poor in aromatic amino acids, and it was therefore expected to provide less of a problem for UVmicroscopy. Westruggled frantically to obtain someresults. Wehad no experience, no assistance, and virtually no apparatus. The large UV-equipmentwas not suitable. The cells were killed by UVlong before we could take a picture. Washingand sterilization of the glassware, preparation of the embryonicextract, and most difficult of all, collecting plasma from the carotid of our single rooster with a primitive paraffin oil canule were a neverending struggle. At last, we managedto take a few pictures before the cells died, but we were still far from the number of monochromatic exposures needed for a spectrum. Our future looked dim. Salvation came in the form of two unexpectedevents. The first was a lecture given by Hans Lettr6 of Heidelberg on the Ehrlich ascites tumor, which he used for biochemical studies. Eva immediately pointed out that we might obtain homogenouspopulations of living cells from the peritoneal cavity of the mouse, without having to do any tissue culture at all! Our first attempt to

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propagate the tumor, kindly sent by Lettr6 in the form of a single mouse, ended in total failure, however.The first of our inoculated mice developed a nice round belly that turned out to carry a lovely litter of eight, instead of the expected tumor cells. But the second mousedeveloped a tumor, and we were in business. But as we were getting ready for the UVpictures, a paper was published by Brumberg & Larionov in the USSR.They used a new, reflecting optical system that avoided the killing of the cells during UV-exposure. They had done all the experiments that we had planned and showed that Caspersson was right and the critiques were wrong. UVmicroscopy did measure nucleic acids, and they were localized exactly in the organelles where Caspersson had found them in fixed cells. Our project had becomeobsolete overnight. What were we going to do? Weexpected the worst, but Caspersson suggested that we continue to work with ascites tumors and try to formulate our ownproject. The early experience of the Genetics Congress came to our rescue. Whywas the Ehrlich ascites carcinoma unique? Whycould other tumors not be propagated in this freely dissociated "fluid" form? Did most tumor cells require a solid substrate and/or the microenvironmentof a solid tissue? Wehad some ideas about how to start looking at this, but our mouse and tumor facilities were very limited. Inbred mice were totally unknownin Sweden at this point. Salvation came again unexpectedly. In the summer of 1950 we participated in the International Cancer Congress in Paris. The week was occupied by frantic and hopeless efforts to get acquainted with the entire cancer field, interspersed with meetings with old friends who had left Hungaryafter us. At the end of the week, we felt definitely reassured about two earlier, disparate but equally important, conclusions: (i) It was very fortunate that we had left Hungaryin time, for the Stalinistic system nowhad a firm grip, and (ii) tumor cells could be definitely regarded genetically heterogeneous populations with extensive subclonal variation. Wealso felt proud as contributors to the Congress. George lectured in broken and very slow English in the 32nd parallel section, the late afternoon of the last day, with six persons in the audience (1). It turned out later, however, that this was an extremely important event. One of Otto Warburg’sassistants had been in the audience and brought homethe great news that the mouseascites tumor cell is an equally good tool for largescale experimentation on such relatively homogeneouscell populations as the famous Chlorella algae of the Great Master. Warburg immediately requested the cells and was later very helpful in supporting us. In a letter written in 1956, he stated that we had madea very important contribution, because we had sent him the cells that made it possible for him to solve the cancer problem. All bureaucrats were deeply impressed!

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HOWONE THING HAS LED TO ANOTHER 13 During the Congress week, we also enjoyed frantic, colorful, decadent, exhilarating, and slightly putrescent Paris. Sitting at a cafe on Boulevard St. Michel the evening of Bastille Day we exclaimed: "Howwonderful! howcrazy! how can one possibly live in a sterile country like Sweden?" Rattling home a week later in a third-class wagon across strike-tom Belgium we said: "Howmarvelous that we can return to quiet, boring, aseptic, polite Swedenwith its thousands of lakes, endless forests, and luminescent nights!" Wehad hardly opened the door to our rented room in Stockholm when I saw the new miracle: an express letter in Caspersson’s ownhar~dwriting. "Get in touch with me immediately on arrival." One of the main private research foundations in Sweden, established in the memory of Knut and Alice Wallenberg, had asked Caspersson to choose two young men for an urgent mission. They were to go to the United States for several months and report about recent advances in cancer research. Caspersson chose one of myolder colleagues and myself. But Eva had to stay home--there was not enough money. Mycolleague was to travel around from center to center. Mytask was to work with Jack Schultz at the Institute for Cancer Research in Fox Chase, Philadelphia, on myownproject, and to make short visits to some of the major centers in the neighborhood. Wereceived the newswith a mixture of joy and sorrow. It was a fantastic opportunity. But the sorrow and anxiety of being separated again from myyoung wife, and for quite some time, were further aggravated by the sudden outbreak of the Korean war with its forebodings of a possible world war. Weshared our vision of an approaching Apocalypse with most other survivors of the Second WorldWarand the Holocaust. Our officially stateless status addedfuel to the nightmares. Still, I knewI had to go. The Statue

of Liberty

The Institute for Cancer Research has developed from a small private research group at LankenauHospital, due to the great foresight of Stanley Reimann.WhenI got there, they had just finished a major expansion from a small group of scientists to a large research center in a magnificent new building. Several prominent biologists had joined the laboratory. The leitmotiv was to look at the cancer problem from the biological point of view. Myownboss was Jack Schultz, a lively little manin his sixties. Jack exuded boundless curiosity, joy of life, and great humanwarmth. He received me as if I were his long lost, finally recovered son. During my stay he often gave me a lift from myrented room to the laboratory. Most of what I knowabout genetics can be traced to those car tides. But the trip was not over when we arrived. Jack’s office was at the far end of a

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long corridor. Walking down the hallway he would stick his head into every lab and stop and talk with people on the way. He asked them about everything, the health of their kids, mother’s broken leg, the weekend excursion, but first and foremost about the latest experiment. The people brightened visibly when they saw him and were always ready to stop for a chat or to ask him to come in and look into the microscope, at a bacterial plate or at a Drosophilaprogeny. Jack looked, listened, discussed, interpreted, proposed new experiments. Under his arm he carried his briefcase with all the papers he planned to finish during the day. Sometimes half a day passed before we arrived at his office where his secretary waited in despair! I visited Jack’s office 25 years later, long after his death. It has been refurnished as a conference room. It bears Jack’s name. A silver plate on the wall reminds us of the unselfish inspiration he provided to everybody in his environment. Jack succeeded in communicating the notion that biology is the most exciting science. He told me about whole worlds I had never heard about. Barbara McClintock’s discovery of transposons in maize was one of them. Jack was one amongthe dozen or even fewer geneticists who understood what McClintock was talking about. He already knew, 10-15 years before most others, that her findings were going to revolutionize biology. Jack’s corridor was a wonderland for me at the age of 25. Briggs and King experimented with nuclear transplantation to enucleated frog’s eggs. The question was whether the nuclei remained totipotent during differentiation. This was also relevant for cancer research. Could cancer cells contain a totipotent nucleus? The question was answered several decades later, by Beatrice Mintz at the sameinstitute--at least answeredso far as diploid teratoma cells were concerned. A cartoon appeared on the wall of the same corridor where Jack and I so often walked in the morning. Two mice were talking to each other. One of them said: "Myfather was a cancer, what does your father do for a living?" The Mintz experiment is still unique in showing that at least some cancers can develop by epigenetic changes. The majority are no doubt due to changes at the DNAlevel, however. Myother important master at the ICR in Philadelphia was the mouse geneticist Theodore Hauschka. Through him I became acquainted with the inbred mouse. He had also taken a direct interest in myexperiments. He gave me myown room in the perfectly organized mouse colony where I was in full operation for days on end. I comparedthe ability of different solid tumors to grow in the fluid form in the abdominal cavity. Whenthey were reluctant to behave according to mywishes, I tried to select variants that would. At the same time I began to wonder whether the "histo-

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compatibility genes" that were shown to govern the transplantability of tissues might provide me with the right system to substantiate our speculations on variation and selection within populations of tumor cells? Despite myloneliness and separation from Eva, alleviated only somewhat by the letters I mailed her daily, I enjoyed being in America. In addition to the positive attitude of Schultz and Hauschka,the environment of the whole laboratory was highly supportive for a young man. There was a wholesome difference compared to European laboratories, particuarly with regard to teacher-student relationships. It can best be summarized by a statement of the Danish biochemist, Lindestrrm-Lang: "The greatest accomplishmentof the Americanrevolution was to establish the right of youngstudents to ask foolish questions." During mystay in the United States I lost part of myemigrant complex. Hungarian emigrants in a comparable situation commentedlater that my initial shyness has turned into its opposite in the Americansetting. It may have seemedso. I was no longer afraid to ask questions, to inject myself into the conversation of learned professors, to speculate, and to risk makinga fool of myself. I began to feel that this was not only mynatural right but myresponsibility.

TumorProgression by Variation and Selection The work on the conversion of solid into ascites tumors turned out to be quite interesting (2). Lymphomas and leukemias often converted immediately, while carcinomas and sarcomas refused to grow in the ascites form at first. Someof them could be converted gradually, however, by passaging the few desquamated,freely floating tumor cells in the peritoneal fluid. Using a simplified form of the Luria-Delbrfick fluctuation analysis, I could show that this conversion was due to the selective enrichment of a small number of spontaneously occurring variants. After four months in the United States, I returned to Swedenwith 200 mice, anxiously guarded in myNewYork hotel room overnight and during the plane trip of more than 24 hours, to the great displeasure of myfellow passengers. Back in Stockholm, the ascites tumor variants turned out to be stable. Theyretained the ability to growin the peritoneal fluid immediatelyafter inoculation, even after reconversion to the solid form and subcutaneous propagation over extended periods of time. The ascites adapted tumors were also more metastatic, less adhesive, and had a higher surface charge than their original nonadapted counterparts (3, 4). A comparison of our findings with Leslie Foulds’ (5) work on tumor progression and Jacob Furth’s studies (6) on the change of hormone dependent to autonomous tumors convinced us that we had hit an unusually well-defined case of

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progression (7). It appearedto have a certain clinical relevance, at least the conceptual level, because it showedt.hat tumor cell populations were heterogeneous, and subpopulations could differ in their metastatic properties. But where did we go from here?

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Tumor

Immunolofy

To study variation and selection in tumorcell populations, it was obviously necessary to study variation first. Wewere looking for cellular markers, determined by knowngenes that could be detected at the cellular level. Wefound them in the recently discovered H-2 antigens of the mouse. George Snell has just started to distribute his first H-2 congenic mouse strains. Wehad induced tumors in H-2 heterozygous but otherwise congenic F1 hybrids and isolated haplotype-loss variants by transplantation to the parental strains (8). Single haplotype-loss variants could be readily obtained, but in frequencies that varied widely between different tumors, even if they had been induced by the same agent and in the same host genotype. This biological variability was no longer a surprise to us, after the variations in ascites convertibility that we had encountered previously. Double H-2 haplotype losses were extremely rare. Around this time, in the mid-1950s, a former colleague from medical school started to makeextravagant claims concerning the prospects for the immunological prevention and cure of humancancer. He had immunized a horse with pooled tumor tissue and was firmly convinced that his serum reacted with a universal tumor antigen. He advocated immediate vaccination against cancer. The newspapers made a big splash. He was supported by some of the most powerful professors of microbiology and virology whohad no experience in cancer. He injected himself with a HeLa cell derived "cancer vaccine" on TV. The public regarded him as a hero, particularly since the newspapers started to accuse the "cancer establishment" of lacking any concern for preventing cancer, due to vested interests. The few of us who actually worked with cancer cells were profoundly sceptical. This could just not be true. But what was the real situation? Wouldtumors elicit immunityin their own inbred strain of origin? Myprevious work with Hauschka left me imbued with a healthy scepticism against most earlier research in tumor immunology.The field was dominated by misinterpreted artefacts of experimentation with noninbred mice. The confusion between transplantation immunology and tumor immunologyprevailed during the entire first part of the century. Only several decades after the developmentof the inbred mice by Little, Strong, Tyzzer, McDowelland others, and after the formulation of the "transplantation laws" by George Snell, was it gradually realized that the so-

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called "transplantable tumors" violated histocompatibility barriers because serial homografting had selected them to outpace the rejection response. If the balance was tilted in favor of the host, e.g. by preimmunizationwith attenuated tumor cells, it could reject the tumor. This easily won immunitycould not be reproduced with tumors that had arisen in homozygousmice and were tested within their own strain. But what would happen at a more modest level of ambition? Could immunityprotect the syngeneic host against near-threshold numbersof tumor cells? Clinicians and pathologists have always maintained that only a small proportion of disseminated tumor cells could grow into metastases in the humanpatient. Could an immuneresponse that fell short of protecting the host against an established tumor still reject disseminated cells, in analogy with concomitant immunityin antiparasite responses? Just as we started to think about these matters, Foley (9) and Prehn Main (10) suggested that chemically induced mouse sarcomas, but not spontaneous mammarycarcinomas, could elicit a state of immunity in syngeneic mice. The data were persuasive but still not fully convincing. Did chemically induced tumorcells really possess a distinct antigenicity of their own, or did these experimentsmerely reflect a residual heterozygosis in the inbred strains? It was obvious that the question could be decisively settled if it could be shown that the primary host could be immunized against its owntumor. Using a combined scheme of tumor induction, operative removal, immunization with irradiated autologous tumor cells, and challenge with graded numbers of viable cells, we could show that methylcholanthreneinduced sarcoma cells were indeed capable of inducing true rejection reactions in the original host (11). Different tumors varied in their immunogenicity over a 5 log range of cell doses, required to break the state of immunity. Another and even more striking manifestation of biological individuality concernedthe individual distinctness of the tumor antigens, also noted by Prehn, Baldwin, and Old (12-14). Each tumor could only immunizeagainst itself. Cross-reactions were rare and irregular. The total number of possible specificities is still not known.Wefound no crossreactions among more than 20 tumors. Hellstrrm could not immunize mice against MC-carcinogenesis by using pools of a dozen tumors for immunization, while Old reported a certain preventive effect after the use of nonspecific immunomodulatorsthat acted presumably by boosting the host’s own responsiveness. The nature of the carcinogen was not immaterial in determining the immunogenicity of the chemically induced tumors. Amongthe aromatic hydrocarbons, MC, BP, and DMBAinduced sarcomas with decreasing immunogenicity, in that order. Sarcomas induced by the implantation of

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cellophane film were hardly immunogenicat all (15). In the rat, Baldwin found that most azo dye-induced tumors were highly immunogenic, whereas acetylaminofluorene-induced tumors and spontaneous fibrosarcomas were not immunogenicat all (16). Several decades have passed since these findings, but the nature of the TSTA(tumor specific transplantation antigen) of the chemically induced tumorsis still a mystery.

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Antigenicity of Virus-Induced Tumors In 1958 I went to the Canadian Cancer Conference, in Honey Harbor, Ontario. Stewart and Eddy’s pioneering work on the polyoma virus was still very new. Most participants were flabbergasted by the number and variety of the tumors that arose after the inoculation of the virus into newborn mice. Burnet was one of them. "Sir Mac" had recently shifted from virology to immunologyand had developed a very negative view of the role of viruses in cancer in the course of this transition; he considered all virus-induced tumors as laboratory artefacts. Viruses were essentially cytopathic, and he saw no place for any true tumor inducing effect. Confronted with the polyoma story, he formulated immediately a new hypothesis. It was based on the only observation of Stewart and Eddy that turned out to be incorrect. They claimed that polyoma tumors were not transplantable. This was due to the accidental use of heterozygous mice, however. Burnet suggested that polyoma virus may destroy some unknown, systematic "growth-controlling center," a possible "hypothalamus-like" homeostatic regulator of cell renewal in manydifferent tissues. This would explain the ability of the virus to cause tumors in manydifferent tissues. These tumors would not be transplantable to mice that have not been similarly conditioned by polyomavirus. Hans-OlofSjrgren had just started to work with us at this time. Stimulated by Burnet’s idea, I asked him to test the transplantability of polyoma tumors in unmanipulated and polyoma-infected syngeneic mice. The result was the exact opposite of what was predicted by Burnet’s hypothesis: the tumors were readily transplantable to untreated mice, while small, graded numbers of cells were rejected by virus-inoculated syngeneic mice (17). The resistance of the virus-infected mice could be transferred adoptively with lymphocytes but not with serum. Both Karl Habel and our group later showedthat antiviral immunitywas neither necessary nor sufficient to induce rejection. Polyoma-inducedtumors or transformed cells induced rejection, whether they released virus or not. All polyoma-inducedtumors were rejected by the immunizedmice, irrespective of tissue origin, but they did not reject tumors induced by other viruses or by chemical agents. We

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HOWONE THING HAS LED TO ANOTHER 19 have therefore developed the concept ofa polyoma-specific transplantation antigen (TSTA)that was present in all tumors induced by. polyoma, but not in tumors induced by other agents. Weand others later found that similar group-specific rejection-inducing antigens were present.on other virus-induced tumors (18). The retrovirus induced leukemias were particulady useful for the study of both humoraland cell-mediated reactions, as was shownby Old et al (19), and by our group. Moloneyvirus-induced lymphomaswere particularly useful for these studies, since they gave a brilliant membranefluorescence reaction with the sera of preimmunized, syngeneic animals. Nevertheless, it was not possible to distinguish the rejection inducing antigen from the viral glycoprotein that accumulated on the surface. Different Moloneylymphomas induced in the sameinbred strain differed in their rejection inducing potential. This system has permitted a distinction between immunogenicity and immunosensitlvity, and we could show that they were independent variables. The former correlated with virus release, while the latter did not. The Department

of Tumor Biology

During the years of our transition from H-2 antigens to tumor immunology, our department developed rapidly. It was formally established in 1957, against all odds. Previously, both George and Eva had becomeassistant professors in Caspersson’s Department of Cell Research (in 1951 and 1955, respectively), but our appointments were limited to maximum of 6 years. Unless one acquired a tenured position, one was out of the research system. But no tenured positions were available in our field, which had not been previously represented at the Swedishuniversities. To circumvent the inflexibility of the university system, a numberof"personal professorships" had been established for individual scientists, but some years before this time, the governmentdecided to stop creating new positions of this type. Science was too expensive already for a country of eight million, they said; and it was also undesirable to continue the traditional recruitment of medical students into research. There was a shortage of doctors that madethe authorities very sensitive about this, particularly since all higher education was financed by the taxpayers, and there was heavy competition for admission to the medical schools. I had a good offer from the ICR in Philadelphia, and we seriously considered movingto the United States. Meanwhile,the Karolinska Insti~ tute, the Medical Research Council and the SwedishCancer Society joined forces to initiate a parliamentry move,requesting the establishment of a Department of TumorBiology with George Klein as its first head. This move was supported by representatives from the four major political

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parties, but it failed to convince the Government.Decisions about budgetary matters rest with the Parliament, however. A parliamentary committee dealt with the matter on April 30, 1957. The odds were against us. The committee had 13 members of the ruling Labour Party and 12 members. from the three major opposition parties. It was expected that the movewouldfail with a majority of at least one vote. In fact, the opposite happened. One Labour Party member(unknown to us) decided to vote with the opposition parties and the Departmentwas established, as of July 1, 1957. Numerousmedical and PhDstudents interested in research joined our group. With the support of the National Institutes of Health of the United States and the Swedish Cancer Society, the department expanded rapidly. The accumulation of married couples who pursued research together was a peculiar feature of the lab that has remained with us ever since. In the early years, the Hellstrrms, the Mfllers, the Sjfgrens, the Nordenskjflds, the Nadkarnis, and the Ozers were some of the examples. At one point we had seven married couples working at the lab at the same time, surely a world record. The problem of space became overwhelming in the late 1950s. Again, there was no provision or precedent for the type of support that was needed. Wewere facing the possibility of having to return the first major NIHgrant we had received under the Virus Cancer Program. I turned to the SwedishCancer Society, although with little hope since the statutes of this essentially private organization explicitly discouraged the support of building facilities. But the Chairman,Professor Hilding Bergstrand, drove through a positive resolution against all odds. A new laboratory building was constructed in 1961. It houses the Department even today. Burkitt’s

Lymphoma

Sometimein the mid-1960s, Eva suggested that we should use our experience on virus-induced murine lymphomas to examine a human lymphoma with a presumptive viral etiology. Could we detect group specific antibody responses that might be helpful in tracing a virus? Burkitt’s lymphoma (BL) was the obvious choice. The recent description of the highly endemic occurrence of the African form which is climate-dependent strongly supported the idea of a possible viral etiology. I wrote letters to numerous hospitals in Africa and to international organizations, explaining our project and asking for tumor, blood, and serum. I received somepolite letters in reply, promises of material, and lovely stamps which made myson happy. But the material was not forthcomingat all, apart from an occasional shipment that arrived broken or infected. Then somebody--I have forgotten who--advised me to write to Peter Clifford, ENTsurgeon at the Kenyatta National Hospital in Nairobi.

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I got no letter and no stamps in reply, but the material started comingin a continuous flow. It arrived with chronometric precision on the single direct flight from Nairobi, late Tuesday afternoon. Large dry ice boxes carried hundreds of sera, and a special wet ice package contained fresh biopsy material. There was always a long list in Clifford’s ownhandwriting with all the essential details and a brief "good luck" message. Weworked together with Peter over a period of more than 10 years. Wehave published 45 joint papers, the first in 1966(20), the last in 1974 (21). Weworked and published together for several years before we had a chance to meet in person. This taught us a new lesson. For collaborative studies, we tried to find a colleague who was motivated to study the problem and to collaborate with us, no matter where he or she resided. But we hasten to add that we have never encountered another clinical collaborator like Peter Clifford. He had a profound interest in BL, ever since he introduced chemotherapy in the treatment of the disease and became fascinated by the remarkably good regression in most of the patients. Their long-term survival eventually turned out to be complete cure in 15-20%of patients, including those whohad only received incomplete chemotherapy. This was quite different from the effect of chemotherapy on other types of B-cell lymphomas.Clifford was convinced that the immunologicalresponse of the patient was decisive. If it was effective, even incomplete chemotherapy could induce total and long-lasting remission. If it was not, even more effective forms of chemotherapywere ultimately unsuccessful. Peter hoped that we would find evidence for an antitumor response in his patients. We changed our working habits. Every Tuesday night was "Burkitt night." Wemade living cell suspensions from the fresh tumors, reacted them with the patient’s own serum and other sera, and tried to read the tests immediately to obtain clues for the continued work. It was not difficult to motivate our personnel to work through the night every Tuesday. Eventually, numerous other laboratories requested material, in the United States, England, and Japan, and some of them became engaged in collaborative projects. Wecould identify a membraneantigen (MA)that was expressed in some Burkitt lymphoma~terived cultures, but not in others (20). WhenI presented these data at an ACSConference in Rye, NewYork, in 1967 (22), WernerHenle gave a talk in the same session. reported his results, obtained with an immunofluorescencetest on fixed BL cells that he and Gertrud Henle had recently developed, later known as the VCA(viral capsid antigen) test (23). They already knew that reaction was due to structural antigens of a newly discovered herpes virus, first seen by Epstein, Barr, and Achongin the electron microscope. Henle

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showedthat it was antigenieally distinct from previously knownherpes viruses (24). Wedecidedto call it EBV. The Henles’ VCAtest and our MAtest showeda certain concordance. Thesamelines appearedto react or failed to react in both tests. At the Ryemeetingweagreed to collaborate. This initiated a highly productive association that has lasted for 20 years, terminatedonly by WernerHenle’s death in 1987. Alreadyin the beginningof this workweobtaineddefinite evidencethat MAwasencodedby EBV(25). It is nowknownas one of the viral envelope glycoproteins. It assembleswithin the membrane of the virus-producing ceils, andafter virus release, it can also attach to other cells in the same culture if they carry EBV-reeeptors.WithJondal, Yefenof,and Oldstone, welater identified the B-cell specific C3d(CR2)receptor as the attachment site of the viral glyeoprotein(26, 27). By1970, it was clear that Epstein, the Henles, and ourselves had only seen the top of the iceberg whenwelookedat viral particles, VCA or MA. Theyonly appear in virus-producingcell lines, and only in someof the cells. With Harald zur Hausen, we found in 1970, however, that more than 90%of the African BLsand all low differentiated or anaplastie nasopharyngeal carcinomas (NPC)contained multiple EBV-genomes r cell, no matter whetherthey producedvirus or not (28). In 1973I [GK] have found with Beverly Reedmanthat 100%of the cells in EBV-DNA positive BLbiopsies and cell lines contained an EBV-encoded nuclear antigen, which wedecided to call EBNA (29). Todaywe knowthat EBNA consistsof a familyof at least six different proteins(30). Several important discoveries have been madeby others in the meanwhile. The Henles, Pope et al, and Nilsson et al found that EBVcould readily immortalize normal B cells in vitro (31-33). Departing from serendipitous observationon a laboratory assistant, the Henlesdiscovered (75) that EBVis the causative agentof infectious mononucleosis (IM). Svedmyr,wecould readily detect EBNA-positive cells in the peripheral blood of mononucleosispatients (34), and the Henles and GeorgeMiller foundthat the saliva of these patients containedtransformingvirus. Transformation was thus a natural property of the virus, not a laboratory artefact due to the accidental isolation of a defective strain, as our colleaguesin the lytic herpesvirus fields initially surmised.Miller &Epstein have also shownthat EBVcan cause lethal lymphoproliferativedisease in immunologicallynaive marmosetand owl monkeys(35, 36). Mononucleosis appearedas an acute rejection reaction of the "immunologically prepared" humanhost, selectively conditionedby a nearly symbiotic relationship with EBVover millions of years, against the virally transformedB cells. Wefound that the peripheral blood of the acute IM

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patient contains activated killer cells that can lyse EBV-carrying and other target cells (37, 38). Moreover,autologous mixedlymphocytecultures between EBV-transformedB-cell lines and T cells of the same normal donorgenerated a proliferative and cytotoxic response equally as strong as that of MHC-incompatible allogeneic MLC(39). Later, Rickinson, Mossand Pope showedthat the autologous mixed cultures generated specific MHC class 1-restricted CTLsby repeated stimulation (40). Eva’s group, Sigurbj6rg Torsteinsdottir, and MariaGrazia Masucciin particular, showedthat theCTLresponse was heterogenous, directed against different target epitopes (41). Thenature and specificity of the relevant targets have not beenclearly defined yet in terms of the knownvirally encodedproteins, althoughcurrent evidenceby Mosset al and by ThorleyLawson,respectively, indicates that both EBNA-2 and LMP epitopes may servein this capacity(42, 43). Since the work of Townsendet al (44, 45) has shownthat MHC class I-associated peptides of processedendogenous or viral proteins can serve as immunogens and CTLtargets, it wouldnot be surprising if even more amongthe seven knowngrowth transformation-associated EBVproteins could serve as CTLtargets. A similar reasoning can be applied to the polyomavirus-induced TSTA,discussed above. The recent work of Dalianis et al in our laboratory suggests that all three polyomaencoded T-antigenscan elicit rejection responsesof the TSTA-type. Thehypothesisthat T cell-mediatedresponsesinhibit the proliferation of EBV-carryingB cells in healthy seropositives and in IMpatients was reaffirmed whenwefound with DavidPurtilo (46) that most and perhaps all lymphoproliferativediseases that appear in congenitally or iatrogenically immunodefective patients, like children with the X-linkedlymphoproliferative syndromeor organ transplant recipients, carry EBV-genomes. Hanto, Ho, and others have later shownthat these initially polyclonal immunoblastie proliferations mayprogress to monoelonal lymphoma (47, 48). Whilethe tumorigenicpotential of EBVwasclearly established by these and related findings, its lifelong, innocuouslatent presencein morethan 80%of all humanpopulations has also suggested that disease occurs only as an accident. Even mononucleosisappears as an "accident" of civilization. Modernhygienicconditions haveapparently interfered with the normal,disease-free ecologyof the virus-host relationship, with its predominantsymptom-freeearly childhood infection. The "accident" of the EBV-associatedtumors has nowbeen largely clarified for Burkitt’s lymphoma,as described below, while the pathogenesis of nasopharyngealcarcinoma, the most regularly EBV-carrying humantumor, is still not understood.

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Oncogene Activation

by Chromosomal Translocation

By 1970, it was clear that some important element was missing from the BLscenario. EBVhas clearly contributed to the genesis of the high endemic form of the disease, since 97%of the African BLs carried the viral genome, whereas non-BLlymphomasdid not (49). Moreover, the prospective study of Geser and de The (reviewed in 50) showed that children with a high EBV-loadare at a greater risk to develop BLthan are their brothers and sisters with a low EBV-load,as indicated by the antibody titers. Since the numberof EBV-infected B cells represents only a minor fraction of the total B-cell population even in persons with a high EBV-load, the presence of the virus in the majority of the African BLs can only be interpreted to mean that an EBV-carrying B cell runs a greater risk of turning into a BL cell under the conditions prevailing in the "high BL belt" of Africa than does its EBV-negativecounterpart. This is to say that EBVcontributes to the etiology of the tumor. But this is still not a satisfactory explanation; someessential element is obviously missing. BLs differ from the true EBV-inducedlymphoproliferative diseases like fatal mononucleosisor the immunoblastic iymphoproliferative diseases in organ transplant recipients, with regard to their cellular phenotype (51). The latter resemble the EBV-transformedB-cell lines of nonneoplastic origin (LCLs). LCLsare permanently growing immunoblasts that express a set of activation markers but not CALLAor BLA. BL cells, on the other hand, carry surface antigen and glycoprotein markers that resemble resting B-cells, rather than immunoblasts (52, 53). They express CALLAand BLAbut no activation markers (unless they drift to a more LCL-like phenotype during prolonged cultivation). Recently, Gregory et al found normal B cells with a corresponding phenotype in tonsil germinal centers (54). For the understanding of BL pathogenesis, it is also important to remember that approximately 3% of the African BLs, and 80% of the sporadic BLs that oc~:ur all over the world are EBV-negative. Amongthe recent, AIDS-associated BLS, the incidence of the EBV-carrying form is currently estimated as 40-50%. The discovery of the "missing factor" in the "Burkitt equation" started when Manolovand Manolova reported in 1972 (55) that a 14q + chromosomal marker was present in about 80% of the tumors. The Manolovs came from Sofia, Bulgaria, to work with us in 1970, at the time whenthe chromosomebanding technique was discovered by Caspersson and Zech. I suggested that they apply the banding technique to the cytogenetically unexplored BL that kept coming in from Clifford every Tuesday in excellent condition. Theyagreed rather reluctantly since they had hoped to learn

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some immunology. But their cytogenetic work soon picked up momentum, particularly after Albert Levan agreed to consult and guide them. When George Manolovshowed me the extra band that he found attached to the distal part of the long arm of one chromosome14 in a BLbiopsy, I first suspected sometrivial reason, perhaps a constitutional variation (isochromosome),and suggested that the Manolovsshould take a look at the fibroblasts of the patient. So they did, but they found that the anomaly was totally restricted to the clonal tumor. After the Manolovsreturned to Bulgaria, we continued the work with Lore Zech. She soon showed that the "extra piece" was derived from chromosome8; the 14q + marker was thus a product of a reciprocal 8; 14 translocation (56). Several groups found subsequently that approximately 20% of the BLs that had no 14q+ marker carried one of two variant translocations instead (for review, see 57). Chromosome 8 broke at the same site (8q24) and entered into a reciprocal translocation either with the short arm of chromosome2 or with chromosome22. All BLs were found to carry one of the three translocations, no matter whether they were high endemic or sporadic, EBV-positive or negative. The same translocations were only exceptionally found in non-BL-lymphomas, although 14q+ markers are quite common;they usually arise by reciprocal translocations between chromosome 14 and some other chromosome, with 11 and 18 as the most frequent participants. But BL-typetranslocations were also found in the form of B cell-derived ALLthat resembles Burkitt lymphomacells phenotypically and is often called Burkitt leukemia. Meanwhile,another, quite independent cytogenetic study, entirely confined to mousetumor cells, was progressing in our laboratory. It started when the Hungarian-Rumanian pathologist, Francis Wiener joined our group in 1970. He is still one of our closest coworkers. Wiener became interested in the role of chromosome15 trisomy in mouseT-cell leukemia, and he was also the main cytogeneticist in the somatic hybrid studies, together with Henry Harris, mentioned below. In the late 1970s Wiener examined a series of pristane oil-induced mouse plasmacytomas (MPCs); he was working together with a Japanese guest worker, Shinsuke Ohno, and in collaboration with Michael Potter’s group at the NIH. Our 1979 Cellpaper described the MPC-associatedtypical (12; 15) and variant (6; translocations (58). Mouse plasmacytomas are very different from Burkitt lymphomas. The only commondenominator is that both originate from cells of the Blymphocyte series. Wenever expected to find anything in commonbetween the two. Therefore, the fact that two apparently unrelated research projects, carried out by different cytogeneticists, led to the discovery of a commonpathogenetic mechanism, based on almost exactly homologous

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chromosomaltranslocations, was one of the greatest and most pleasant surprises of myentire scientific career. It was even more surprising that the highly speculative workinghypothesis, formulated to explain the mechanism wherebythe translocations contribute to the tumorigenic process in such a decisive fashion, turned out to be essentially correct. The hypothesis was built on the fact that the recipient murine chromosomes of the dislocated fragment from chromosome 15 were known to carry the IgH (chromosome 12) and the kappa (chromosome 6) gene, respectively. Likewise, human chromosome 14 was known to carry the IgH cluster. Wehave therefore speculated that a proto-oncogene and probably the same proto-oncogene could be localized at the breakpoint of the murine chr 15 and the humanchr 8. Accidental translocation of the putative gene to one of the immunoglobulin loci might have led to the constitutional activation of the gene, in analogy with the retroviral activation of the c-myc gene by the insertion of an ALV-derivedLTRin the chicken bursal lymphoma,as described by Haywardet al. I started to expose the hypothesis to the test of peer criticism in 1979. An outstanding molecular biologist, a good friend of mine, called it the "most hair-raising extrapolation from the centimorgans to the kilobases." It was. Still, the hypothesis was published in Nature in 1981 (59), but was not fully convinced of it myself, until the critical momentduring the summerof 1981, when I was waiting for a plane at Washingtonairport to take me to Tokyo. The waiting hall was full of people, mostly Japanese. There were only two telephones on the other side of the security check. They were busy most of the time. The plane was called up. Finally, one of the telephones was free. I tried to get hold of Philip Leder at the NIH. I wanted to hear whether he knew anything about the chromosomallocation of the immunoglobulin light chain genes in humans. Leder came to the telephone. No, he hadn’t heard anything; it was still unknown.But one of his colleagues had just come back from the recently held HumanChromosome Mappingmeeting in Oslo. If I waited, he would try to ask if the colleague had heard anything. "Final call." The last Japanese walked aboard, and I had to leave. At the momentwhen I was about to hang~up the phone, Leder’s voice came back: Yes, there were two small reports in Oslo. An English group had found that kappa is on chromosome 2. An American group had proved that lambda is on chromosome22. I ran on board. It was an intoxicating feeling! I knew for certain that the hypothesis was correct. The molecular confirmation and clarification camein a virtual avalanche during 1982. Taking off from quite different points, Jerry Adamswith Susan Cory in Australia and Kenneth Marcu in New York showed for

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MPC,and Carlo Croce and Phil Leder for BL, that the translocations resulted in the juxtaposition of donor chromosomederived sequences and immunoglobulin gene sequences. Michael Cole’s group has identified the transposed gene as c-myc (for review, see 60). The subsequent development has led to many new insights, but it has also created some puzzles and paradoxes with regard to myc-regulation, constitutive activation, and certain details of the timing and regulation of Ig-gene rearrangement (for review see 65). With Francis Wiener and Janos Siimegi, we have also found a third Ig/myc translocation system (61-63), the spontaneous immunocytoma of the Louvain rat (RIC), developed by Herv~ Bazin. A comparison of the translocations in MPC, RIC, and BLat the molecular level reveals more similarities than differences. In fact, it wouldbe hard to find a comparablesituation in cancer biology where three pathogenetically different tumors that arise from the same cell lineage in three different species show a similarly close pathogenetic mechanismat the molecular level. The causal, i.e. rate limiting, involvementof constitutive rnyc activation in the genesis of the three tumors was deduced from the regularity of the Ig/myc juxtaposition that extended to cryptic translocations and complex rearrangements, where two or three successive genetic events had occurred (61, 64). Further confirmation came from recent facsimile experiments. Michael Potter and Francis Wiener showed (66) that introduction of activated rnyc gene within a retroviral (J3) construct into pristane oiltreated Balb/c mice induced plasmacytomasthat did not carry any translocations, provided they expressed the inserted (v-myc) gene. Meanwhile, Adams& Cory’s group generated transgenic mice that carried the mycgene coupled to the IgH enhancer (67). The mice developed more than 90%pre-B- or B-cell-derived lymphomas.Using the Australian transgenic mice, Francis Wiener recently found that Abelson virus infection, already knownto increase the incidence and shorten the latency period of pristane oil-induced mouse plasmacytoma, has led to the appearance of plasmacytomas in the Emu-myctransgenic mice. The virus has obviated the pristane requirement and lifted the genetic restrictions to MPCsusceptibility. These plasmacytomaswere also translocation free. That introduction of an activated myc construct was tumorigenic for B cells and obviated the need for the translocations could be only interpreted to mean that the naturally occurring constitutive activation of myc by the Ig-translocations provided an essential, rate-limiting step within the carcinogenic process. But it is not the only step. All tumors were monoclonal, even in the transgenic mice where myc was activated in all B and pre-B cells. Sequential activation of several oncogenesor, alternatively, loss of suppressor genes mayprovide additional steps. Feedbackinhibition

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by the clone that happensto get the upper hand first wouldbe another alternative. The Burkitt lymphoma story has also developedfurther in the meanwhile and has posed somenewfascinating questions. Wehave suggested, for both conceptualand factual reasons, that the BLprogenitoris a longlived B-memory cell. In this scenario, antigenicallystimulatedB-cell clones that have previously expandedas immunoblasts,were in the process of switching their phenotype to CALLAand BLA-positive, activation markernegative memory cells when,uponthe waningof the antigenic stimulus, the translocation accident occurred. Dueto the linking of rnyc to a constitutively active Ig-locus, the cells were unableto leave the cycling compartment,however.It could be shownthat the translocation carrying "suspendedresting cell" had several additional phenotypicproperties that could facilitate its evasion from immunologicalcontrol. Certain MHC elass I polymorphic specificities weredown-regulated in the BLcells, compared to EBV-transformed B-cell lines of normal origin. The BLcells also failed to express certain adhesionmoleculespresent on the LCLsor expressed them at a low level. Even the EBV-encoded,growth-transformation associated nuclear and membraneantigens were down-regulated in the BLcells, with the exceptionof EBNA-1. This wasparalleled by a relative resistance of the BLcell to CTL-mediated lysis (56). It thus appears that the myc/Ig translocation promotesthe malignant growthof the BLcell by several mechanisms.This mayexplain the extraordinaryregularity of its presencein all typical BLsso far studied. Tumor Suppressor

Genes

I haverecently reviewedthis field in somedetail (68) and concludedthat weare probably approaching an era whenthe study of genes that can antagonizetumorigenicbehaviorwill be equally as, if not morerewarding than, the study of the oncogenes.Ourowncommitment to this field started with a decadeof another long distance collaboration, initiated by Henry Harris in 1969(69). Wehave inoculated a large numberof somatic cell hybrids, derived from the fusion of high malignantwith normalor with low malignantcells, into genetically compatibleand/or immunosuppressed mice. The hybrids were generated by Harris, and Wienerexaminedtheir chromosomes.These studies have firmly established the notion that tumorigenicityis suppressedby fusion with normalcells. It reappearsafter somecritically important chromosomes, contributed by the normalcell, have been lost. Others have extended this work to human/human hybrids morerecently and obtained similar results. Chromosomes that carry tumor suppressor genes have been identified by Stanbridge, Klinger, Sager and their associates (70-72). Thefield is nowmovingtowardsa morereduc-

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tionistic analysis where microcell hybrids are taking the place of whole cell hybridization and c-DNAtransfections are initiated to identify the suppressor genes and their products. Meanwhile,evidence for tumor antagonizing genes has also emerged from the study of revertants and particularly from the rapidly movingfield of "recessive cancer genes" that contribute to tumorigenesis by their loss (73, 74). It is not clear if or what extent there is a relationship betweenthe genes identified by these three approaches. Annu. Rev. Immunol. 1989.7:1-34. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Whither Tumor Immunology? It is often asked if or to what extent the spectacular developmentof the oncogene field during the last decade may provide some new handles for targeting the antitumor response. The answer may differ in relation to oncogenes activated by regulatory or by structural changes, respectively. Up-regulation of a structurally normaloncoprotein is less likely to provide a rejection target than oncoproteins activated by structural changes, e.g. the products of the ras-mutations or the truncated growth factor receptors, exemplified by the tumorigenic variants of erbB or fins. Following Townsend’s discovery that intracellular, endogenousproteins can be processed to peptides that combinewith class I or class II molecules and can then serve as immunogensand/or as CTLtargets, the structurally changed oncoproteins deserve serious consideration. Progress will depend on the expression of mutation-activated (compared to normal) oncogenes in nonimmunogenic tumor cells--of which there are many--followed by the assessment of their immunogenicityand rejectability in syngeneic hosts.

Epilo#ue As each of us is moving towards the approaching darkness, the sun is never setting over the vast oceans of science. It has been a rare privilege to live and work through the times whenthe genetic material turned from protein to DNA,when adaptive changes in cell populations--including antibody production--were unmaskedas Darwinian variations and selection, when GODbecame the rearrangement of immunoglobulin genes, violating the dogmathat all somatic cells have the same DNA.Another central dogma was abolished when the RNAtumor viruses became DNA proviruses. Followingclosely in the wakeof this discovery, the enthusiastic retrovirologists, searching for the universal cause of cancer, permitted the great cuckoo egg, the oncogenes, to hatch--almost imperceptibly at first, but with a rapidly increasing crescendo, towards the triumphant emphasis on the regulatory genes of the cells and their dysfunction as the key factor in the oncogenic process. Departing from even greater obscurity, the MHC system, once the esoteric pet of a few mousegeneticists, nowoccupies a

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central place in virtually every area of immunology.It was a great time, and it still is, but it is only the stumbling, stuttering, premature foreshadowingof what lies ahead. Wehave barely scratched the surface.

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Literature Cited 1. Klein, G., Klein, E. 1951. The nucleic acid content and the growth rate of mouse ascites tumor cells. Acta Union Int. Contra Cancrum7:376-85 2. Klein, G. 1951. Comparative studies of mouse tumors with respect to their capacity for growth as "ascites tumors" and their average nucleic acid content per cell. Exp. Cell. Res. 11:518-73 3. Purdom, L,, Ambrose, E. J., Klein, G. 1958. A correlation between electrical surface charge and some biological characteristics during the stepwise progression of a mouse sarcoma. Nature 181:1586-87 4. Ringertz, N., Klein, E., Klein, G. 1957. Histopathological studies of peritoneal implantation and lung metastasis at different stages of the gradual transformation of the MCIMmouse sarcoma into ascites form. J. Natl. CancerInst. 18:173-99 5. Foulds, L. 1954. The experimental study of tumor progression: A review. Cancer Res. 14:32%39 6. Furth, J. 1953. Conditioned and autonomous neoplasms: A review Cancer Res. 13:477-92 7. Klein, G., Klein, E. 1957. Evolution of independence from growth stimulation and inhibition... Symp. Soc. Exp. Biol. I1:305 8. Klein, G., Klein, E. 1958. Histocompatibility changes in tumors. J. Cell Comp.Physiol. Suppl. 1, 52:125-68 9. Foley, E. J. 1953. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer Res. 13:835-37 10. Prehn, R. T., Main, J. M. 1957. Immunity to methylcholanthrene-induced sarcomas. J. Natl. Cancer Inst. 18:769-78 11. Klein, G., Sj6gren, H. O., Klein, E., Hellstrrm, K. E. 1960. Demonstration of resistance against methylcholanthrene-induced sarcomas in the primary autochthonous host. Cancer Res. 20: 1561-72 12. Baldwin, R. W. 1955. Immunity to methylcholanthrene-induced tumors in inbred rats following atrophy and regression of implanted tumors. Br. J. Cancer 9:652-57 13. Old, L. J., Boyse, E. A., Clarke, D. A.,

Carswell, E. A. 1. 1962. Antigenic properties of chemically induced tumors. Ann. NY Acad. Sci. 10l: 80-106 14. Prehn, R. T. 1962. Specific isoantigenicities among chemically induced tumors. Ann. NY Acad. Sci. 101: 10713 15. Klein, G., Sj6gren, H. O., Klein, E. 1963. Demonstration of host resistance against sarcomas induced by implantation of cellophane films in isologous (syngenic) recipients. Cancer Res. 23: 84~92 16. Baldwin, R. W. 1973. Immunological aspects of chemical carcinogenesis. Adv. Cancer Res. 18:1-75 17. Sjrgren, H. D., Hellstrrm, I., Klein, G. 1961. Transplantation ofpolyoma virusinduced tumors in mice. Cancer Res. 21: 329-37 18. Klein, G. 1966. Tumor antigens. Ann. Rev. Microbiol. 20:223-52 19. Old, L. J., Boyse, E. A., Stockert, E. J. 1963. J. Natl. Cancer Inst. 31:97786 20. Klein, G., Clifford, P., Klein, E., Stjernsw/ird, J. 1966. Search for tumor specific immunereactions in Burkitt lympfioma patients by the membrane immunofluorescence reaction. Proc. Natl. Acad. Sci. USA 55:1628-35 21. Gunven, P., Klein, G., Clifford, P., Singh, S. 1974. Epstein-Barr virus-associated membrane-reactive antibodies during long term survival after Burkitt’s lymphoma.Proc. NatL Acad. Sci. USA 71:1422-26 22. Klein, G., Klein, E., Clifford, P. 1967. Search for host defenses in Burkitt lymphoma: Membrane immunofluorescence tests on biopsies and tissue culture lines. Cancer Res. 27:2510-20 23. Henle, G., Henle, W. 1966. Immunofluorescence in cells derived from Burkitt’s lymphoma.J. BacterioL 91: 124856 24. Henle, G., Henle, W. 1967. Irnmunofluorescence, interference and complement fixation technics in the detection of herpes-type virus in Burkitt tumor cell lines. Cancer Res. 27:2442-46 25. Klein, G., Pearson, G., Nadkarni, J. S., Nadkarni, J. J., Klein, E., Henle, G., Henle, W., Clifford, P. 1968. Relation between Epstein-Barr viral and cell

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membraneimmunofluorescenceof Bur- 35. Epstein, M. A., Hunt, R., Rabin, H. kitt tumorcells. I. Dependence of cell 1973.Pilot experimentswith EBvirus in membraneimmunofluorescenceon presowl monkeys(aortus-trivigatus). Reticulo proliferative disease in an ence of EB virus. J. Exp. Med. 128: 1011-20 inoculated animal. Int. J. Cancer12: 26. Jondal, M., Klein, G., Oldstone, M.B. 309-18 A., Bokish,V., Yefcnof,E. 1976.Surface 36. Shope, T., De Chiaro, D., Miller, G. markers on human B and T lympho1973. Malignant lymphoma in cottoncytes. VIII. Association betweencomtop marmosetsafter inoculation with Epstein-Barr virus. Proc. Natl. Acad. plementand Epstein-Barr virus recepSci. USA70:2487-91 tors on human lymphoidcells. Scand. J. Immunol. 5:401-10 37. Adams,J. M., Cory, S. 1985. Myconco27. Yefenof,E., Klein, G., Jondal, M., Oldgene activation in B and T lymstone, M.B. A. 1976.Surfacemarkerson phoid tumours. Proc. R. Soc. London humanB and T-lymphocytes. IX. TwoSer. B 226:59-72 color immunofluorescence studies on the 38. Svedmyr,E.,Jondal, M.1975. Cytotoxic association betweenEBVreceptors and effectorcells specificfor Bcell linestranscomplementreceptors on the surface of formedby Epstein-Barrvirus are present lymphoidcell lines. Int. J. Cancer17: in patients with infectious mono693-700 nucleosis. Proc. Natl. Acad. Sci. USA 28. zur Hausen, H., Schulte-Holthausen, 72:1622-26 H., Klein, G., Henle, W., Henle, G., 39. Svedmyr,E. A., Deinhardt, F., Klein, G. 1974.Sensitivity of different target Clifford, P., Santesson, L. 1970. EBVDNA in biopsies of Burkitt tumorsand cells to the killing action of peripheral anaplastic carcinomas of the nasolymphocytesstimulated by autologous pharynx. Nature 228:1056-58 lymphoblastoid cell lines. Int. J. Cancer 29. Reedman,B. M., Klein, G. 1973. Cellu13:891-903 lar localization of an Epstein-Barrvirus 40. Rickinson, A. B. 1966. Cellular (EBV)-associatedcomplement-fixing animmunological responses.In The Epsteintigen in producer and non-producer BarrVirus: RecentAdvances,pp. 77-125. lymphoblastoid cell lines. Int. J. Cancer London: HeinemannMedical 11:499-520 41. Torsteinsdottir, S., Masucci, M. G., 30. Ricksten,A., Kallin, B., Alexander,H., Ehlin-Henriksson, B., Brautbar, C., Dillner, J., F~hraeus,R., Klein,G., LerBen-Bassat, H., Klein, G., Klein, E. 1986. Differentiation-dependent senner, R., Rymo,L. 1988. The BAMHIE region of the Epstein-Barrvirus gesitivity of human B-cell derivedlines to nomeencodes three transformationmajor histocompatibility complexassociatednuclear proteins. Proc.Natl. restricted T-cellcytotoxicity.Proc.Natl. Acad. Sci. USA85:995-99 Acad. Sci. USA83:5620-24 31. Henle, W., Diehl, V., Kohn, G., zur 42. Moss,D. J., Misko,I. S., Burrows,S. R., Burman,K., McCarthy,R., Sculley, Hausen, H., Henle, G. 1967. Herpestype virus and chromosomemarker in T. B. 1988. CytotoxicT-cell clones disnormal leukocytes after growth with criminatebetweenA- andB-typeEpsteinirradiated Burkitt cells. Science 157: Barr virus transformants. Nature331: 1064-65 719-21 32. Nilsson, K., Klein, G., Henle, W., 43. Thorley-Lawson, D. A., Israelsson, E. S. Henle, G. 1971. The establishment of 1987.Generationof specific cytotoxic T lymphoblastoid lines from adult and cells with a fragmentof the Epstein-Barr fetal humanlymphoidtissue and its virus-encoded p63/latent membrane dependenceon EBV.Int. J. Cancer8: protein. Proc. Natl. Acad.Sci. USA84: 443-50 5384-88 33. Pope, J. I-I., Hornc,M. K., Scott, W. 44. Townsend,A. R. M., McMichael,A. J., 1968. Transformationof foetal human Carter, N. P., Huddeston, J. A., leukocytes in vitro by filtrates of a Brownlee,G. G. 1984. CytotoxicT-cell humanleukaemia cell line containing recognition of the influenza nucleoherpes-likevirus. Int. J. Cancer3: 857protein and hemagglutininexpressedin 66 transfected mouseL-cells. Cell 39: 1334. Klein, G., Svedmyr,E., Jondal, M., 25 Persson, P. O. 1976. EBVdetermined 45. Townsend,A. R. M., Rothbard, J. M., nuclear antigen (EBNA)-positive cells Frances, M., Gotch, G., Bahadur,J., the peripheral bloodof infectious monoWrath,D., McMichael,A. J. 1986. The nucleosispatients. Int. J. Cancer17: 21epitopes of influenza nucleoproteins 26 recognized by cytotoxic lymphocytes

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can be definedwith short synthetic peptides. Cell 44:959~58 46. Purtilo, D. T., Klein, G. 1981. Introduction to Epstein-Barr virus and lymphoproliferative diseases in immunodeficient individuals. Cancer Res. 41:4209 47. Hanto,D. W.,Frizzera, G., Gajl-Peczalska, K.J., Sakamoto, K., Purtilo, D. T., Balfour, H. H., Simmons, R. L., Najarian,J. S. 1982.Epstein-Barrvirusinduced B-cell lymphoma after renal transplantation. N. Engl. J. Med.306: 913 18 48. Ho,M., Jaffe, R., Miller, G., Breining, M. K., Dummer,J. S., Makowka.,L., Atchison, R. W., Karrer, F., Nalesnik, A., Starzl, T. E. 1988.Thefrequencyof Epstein-Barrvirus infection andassociated lymphoproliferativesyndromeafter transplantation andits manifestationin children. Transplantation45:719-27 49. Klein, G. 1975. Studies on the EpsteinBarr virus genomeand the EBV-determinednuclear antigen in humanmalignant disease. Cold Spring HarborSymp. Quant.Biol. 39:783-90 50. De The, G. 1980. Role of Epstein-Barr virus in humandiseases: Infectiousmononucleosis, Burkitt’s lymphoma andnasopharyngeal carcinoma.In Viral Oncoloyy, ed. G. Klein, pp. 769-97.NewYork: Raven 51. Nilsson, K., Klein, G. 1982. Phenotypic andcytogeneticcharacteristics of human B-lymphoid cell lines andtheir relevance for the etiology of Burkitt’s lymphoma. Adv. CancerRes. 37:319-80 52. Ehlin-Henriksson,B., Klein, G. 1984. Distinction betweenBurkitt lymphoma subgroups by monoclonalantibodies: relationships between antigen expression and type of chromosomal translocation. Int. J. Cancer33:459-63 53. Rowe, M., Rooney, C. M., Edwards, C. F., Lenoir, G. M., Rickinson,A. B. 1966.Epstein-Barrvirus status and turnour cell phenotypein sporadic Burkitt’s lymphoma. Int. J. Cancer37:367-73 54. Gregory, C. D., Tursz, T., Edwards, C. F., Tetaud, C., Talbot, M., Caillou, B., Rickinson,A. B., Lipinski, M.1987. Identification of a subset of normalB cells with a Burkitt’s lymphoma (BL)like phenotype.J. Immunol.139:313-18 55. Manolov,G., Manolova,Y. 1972. Marker band in one chromosome14 from Burkitt lymphomas.Nature 237:33-34 56. Zech, H., Haglund, U., Nilsson, K., Klein, G. 1976. Characteristic chromosomal abnormalities in biopsies and non-Burkitt lymphomas,lnt. J. Cancer 17:47-56

57. Bernheim,A., Berger, R., Lenoir, G. 1981. Cytogenetic studies on African Burkitt’s lymphoma cell lines: t(8; 14), t(2; 8) andt(8; 22) translocations. Cancer Genet.Cytogenet.3:307-15 58. Ohno, S., Babonits, M., Wiener, F., Spira, J., Klein, G., Potter, M. 1979. Nonrandom chromosome changes, involving the Ig gene-carryingchromosomes 12 and 6 in pristane-induced mouseplasmacytomas.Cell 18:1001-7 59. Klein, G. 1981. Therole of genedosage and g.enetic transpositions in carcinogenesis. Nature294:313-18 60. Klein, G. 1983. Specific chromosomal translocationsandthe genesis of B-cellderived tumorsin mice and men. Minireviews. Cell 32:311-15 61. Pear, W. S., Wahlstrrm, G., Nelson, S. F., Axelson,H., Szeles, A., Wiener, F., Bazin,H., Klein,G., Sumegi,J. 1988. 6-7 chromosomal translocation in spontaneously arising rat immunocytomas: evidencefor c-mycbreakpointclustering and correlation betweenisotypie expression andthe c-myctarget. MoLCell. Biol. 8:441-51 62. Sumegi,J., Spira, J., Bazin,H., Szpirer, J., Levan,G., Klein, G. 1983.Rat c-myc oncogene is located on chromosome-7 and rearranges in immunocytomas with t(6; 7) chromosomal translocation. Nature306: 497- 98 63. Wiener, F., Babonits, M., Spira, J., Klein, G., Bazin, H. 1982. Nonrandom chromosomal changes involving chromosomes-6 and 7 in spontaneous rat immunocytomas_._~/_nt~_J. Cancer29:43137 64. Fahrlander,P. D., Sumegi,J., Yang,J. Q., Wiener,F., Marcu,K. B., Klein, G. 1985. Activation of the c-myc oncogene by the immunoglobulin heavy-chain gene enhancer after multiple switch region-mediated chromosome rearrangements in a routine plasmacytoma. Proc. NatLAcad. Sci. USA82: 3746-50 65. Klein, G., Klein, E. 1985. Myeflgjuxtaposition by chromosomal translocations. ImmunoLToday 6:208-15 66. Potter, M., Mushinski,J. F., Mushinski, E. B., Brust, S., Wax,J. S., Wiener,F., Babonits, M., Rapp, U. R., Morse, H. C. III. 1987. Avian v-myc replaces chromosomaltranslocation in murine plasmacytoma-genesis. Science 235: 787-89 67. Adams,J. M., Hard.s, A. W., Pinkert, C. A., Corcoran, L. M., Alexander, W.S., Cory,S., Palmiter,R. D., Brinster, R. L. 1985. Thec-myc oneogenedriven by immunoglobulinenhancers induces

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68. 69.

70.

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71.

lymphoid malignancy in transgenic nfice. Nature 318:53938 Klein, G. 1987. The approaching era of the tumor suppressor genes. Science 238: 1539-45 Harris, H., Miller, O. J., Klein, G., Worst, P., Tachibana, T. 1969. Suppression of malignancy by cell fusion. Nature 223:363~8 Klinger, H. P. 1982. Suppression of tumorigenicity. Sixth International workshop on human gene mapping. Cytogenet. Cell Genet. 32:68-84 Sager, R. 1985. Genetic suppression of tumor formation. Adv. Cancer Res. 44:

33

43~8 72. Stanbridge, E. J. 1987. Genetic regulation of tumorigenic expression in somatic cell hybrids. Adv. Viral Oncol. 6:83-87 73. Benedict, W. F. 1987. Recessive human cancer susceptibility genes (retinoblastoma and Wilms’loci). Adv. Viral Oncol. 7:19-34 74. Knudson, A. G. 1987. A two-mutation model human cancer. Adv. Viral Oncol. for 7:1-17 75. Henle, W., Henle, G. 1973. Epstein-Barr virus and infectious mononucleosis. New En,ql. J. Med. 288:263-64

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Ann. Rev. Immunol. 1989. 7:35-58 Copyright © 1989 by Annual Reviews Inc. All rights reserved

DECAY-ACCELERATING FACTOR: BIOCHEMISTRY, MOLECULAR BIOLOGY, AND FUNCTION Douglas M. Lublin* and John P. Atkinson~f Departmentsof Pathology* and Medicine*’~and the HowardHughes MedicalInstitute Laboratoriest, WashingtonUniversity School of Medicine,St. Louis, Missouri 63110 INTRODUCTION Complement componentsregularly becomeanchoredto host cells as well as to microbes.Thesecomponents mustbe allowedto promotethe reaction cascadeon microbesbut mustbe inhibited on self-tissue. Thus,it is critical for the host cell to downregulate the complementpathwayon its own membrane.To accomplishthis, cells possess several membraneproteins that can modulate complementcomponentsdeposited on their surface (reviewedin 1). Thestep at whichmuchof this control is directed is the formationof the enzymesthat cleave C3(so-called C3convertases). These bimolecular enzymecomplexesconsist of a cell-bound component to which a serine protease is noncovalentlyattached. Oncelarge amountsof C3 fragmentsare deposited, then the cell or microbemaybe ingested through interaction with phagocyticcells bearing C3receptors or lysed through engagement of the terminal (membraneattack complex) components. Consequently,muchof the regulatory activity is directed at modulating the C3-cleavingenzymes. Decay-acceleratingfactor (DAF)is one of these regulatory membrane proteins of the complement system. As its namestates, it can dissociate (decay-accelerate) both the classical and alternative pathwayC3convertases, as well as serve to preventtheir assembly.It is a widelydistributed membrane glycoproteinthat wasfirst describedas a functional entity over 35 0732-0582/89/0410-0035502.00

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20 years ago(2, 3), althoughit wasnot isolated andpurified until 1981(4, 5). DAF is of special clinical interest becauseit is deficient in paroxysmal nocturnal hemoglobinuria (PNH)(6, 7), an acquired donal hemolytic disorder of man.Theincreased sensitivity to complement-mediated lysis of erythrocytes of PNHpatients is causally related to DAFdeficiency and is partially corrected by supplying the cells with DAF(8). Further, DAF has recently been shownto be a glycophospholipid-anchoredmembrane protein (9, 10), and this interesting structural feature mayfacilitate movement in the membraneand thereby permit DAFto downregulate complementactivation moreeffectively. In this chapter wereviewthe structure of DAFat the protein and DNA levels, and highlight its role in protecting cells fromdamageby.autologous complement.

COMPLEMENT SYSTEM Complement is a major effector system of the humoralimmuneresponse. This group of 20 plasma proteins and over a half-dozen cell membrane receptor and regulatory proteins serves for clearance or lysis of foreign particles or cells (reviewedin 11, 12). . Complement can be activated by either of two pathways.The classical pathwayis triggered by antigen-antibodycomplexes,specifically IgMand IgG. Thealternative pathwayis activated by foreign surfaces including bacteria, fungi, viruses, and tumorcells, as well as by immune complexes containing IgG, IgA, and IgE. Activation by either pathwayleads to production of a bimolecularcomplexdesignated C3convertase, C4b2afor the classical pathwayand C3bBbfor the alternative pathway,whichhas the ability to cleave C3 to C3b and C3a. Hence, the two pathways of activation convergeat the C3step. Thefact that C3is itself a component of C3convertase in the alternative pathwayresults in an amplification loop. At this C3step of the complement cascade, manyof the effector functions of the systemare brought into play. The released C3a, as well as C4aand C5a, are anaphylatoxins that serve as important mediators of inflammation.C3bis covalently boundto the target membrane or immune complex,promotingits clearance by phagocyticcells bearing C3breceptors (CR1). Finally, the terminal membrane attack complexcan be assembled on the target membrane,with the formation of transmembranechannels that can lead to cell death. This destructive potential of the complement systemrequires tight control so that host tissues are not damaged. In particular, there is a constant, low-level activation or tick-over of the alternative pathway,leading to

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deposition of C3b on all surfaces. Activators such as bacteria cannot control the formation of C3 convertase, whereas host cells and other nonactivators rapidly inactivate the C3 convertase. The plasma proteins factor I (I), factor H (H), and C4-binding protein (C4bp) function to end. Additionally, cells possess a numberof membraneproteins to regulate complementthat is deposited on their surfaces; the largest group, focused on C3 convertases, includes CR1 (13), DAF, and membrane cofactor protein (MCP)(14). These plasma and membraneproteins inactivate convertase by dissociating its two components(decay-accelerating activity) or by serving as a eofactor for the proteolytic inactivation of C3b or C4b by plasma factor I (cofactor activity). Thus, the complementsystem remains focused on its proper (foreign) target.

DECAY-ACCELERATING FACTOR (DAF) Identification and Purification In 1969 Hoffmannreported that a substance remaining in the aqueous phase of an extract of humanerythrocyte stroma with n-butanol inhibited the complement-mediated hemolysis of antibody-coated sheep erythrocytes (2). He further showedthat this inhibition involved an acceleration in the decay of EAC14b2ato EAC14b(3). Over a decade later NicholsonWeller and colleagues purified an intrinsic membraneglycoprotein from guinea pig and human erythrocyte (E) stroma by butanol extraction, followed by sequential chromatography on DEAE-Sephacel, hydroxylapatite, phenyl-Sepharose, and trypan blue-Sepharose (4, 5). The protein was purified during this schemeby monitoring its ability to accelerate the decay of the classical pathway C3 convertase; hence it was named decayaccelerating factor. The purified componentwas a single chain membrane protein, DAF, with a Mr of 60,000 (guinea pig) or 70,000 (human) SDS-PAGEunder reducing conditions. Staining of human DAFwith periodic acid Schiff reagent demonstratedthat it is a glycoprotein. Biochemical

Activities

and Physiological

Roles

Several lines of evidence indicate that DAFprotects cells from damageby autologous complementproteins deposited on their surfaces. Specifically, DAFprevents the assembly of the C3 and C5 convertases of the classical or alternative pathways, which act as amplification steps in the complement cascade. DAF(all references are to humanDAFunless otherwise noted) was initially purified based on its ability to accelerate the spontaneous decay of the preformed classical C3 convertase, C4b2a(5). Blocking with antibodies to DAFshowed that it was responsible for this function on

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intact E, for both the classical andalternative pathways,but that it lacked any cofactor activity for I-mediatedcleavageof C4bor C3b(7). Pivotal insights into the role of DAFcame from studies using DAF reincorporated into sheep E (15). These studies demonstratedthat DAF inhibits the formationof the C3andC5convertases;this effect wasreversible, as DAFdid not affect the structure of C4bor C3b.In addition, DAF only exerted this effect intrinsically, i.e., on C3convertasesassembledon the samecell as the DAF.Thefunctional activity of DAFis schematically shownin Figure 1. Anothergroup of investigators narrowedthe site of action further by showingthat DAFdoes not prevent the initial binding of C2or B to the cell (containing C4bor C3b,respectively), but that rapidly dissociates C2aor Bbfromtheir bindingsites, thus preventingthe assemblyof the C3convertase (16). The precise mechanismunderlying this interference with the C3convertase,and the specific bindingsites for DAFon the C3 convertase, are still unclear. One group used a homobifunctional cross-linking reagent to showan endogenousassociation of DAFwith C4b and C3bon the cell surface (17). Anotherinvestigation

DAF

Bb

Bb

DAF

Figure 1 Functional decay-accelerating activity as demonstrated by DAF.C3b is shown covalently attached to a cell surface through an ester or amide bond to a glycoprotein or glycolipid. DAFis present in the membrane,anchored by a glycophospholipid structure;, the four short consensus repeat domainsof DAFare shownby striped circles. (See later sections for details of these structures.) (Top) DAFbinds to a C3band prevents formation of the C3 convertase, C3bBb. This might be the most important role of DAFon a cell. (Bottom) DAFdissociates a preformed C3 convertase. It is not knownwhich short consensus repeat domain(s) binds to C3b. DAFshows the same activities with Cgb and the classical pathway C3 convertase, C4b2a.

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39

using fluid-phase competitive inhibition suggested that the primary interaction of DAFwith C3 convertases is with the C2a or Bb components (18). The experimental systems of these two groups are quite different, and the discrepancy in their results has not been resolved.

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Biosynthesis

and Glycosylation

of DAF

DAFundergoes several posttranslational modifications to attain its final overall structure in the cell membrane.These have been elucidated for DAFby studying the biosynthesis of DAFin tissue culture and by chemical and enzymatic analysis of purified DAF. Weanalyzed the oligosaccharide structure of DAFby endo- and exoglycosidase digestions. The 74,000 Mr DAFfrom E was lowered ,-~ 3000 by endoglycosidase F, whereas endoglycosidase H had no effect, indicating one N-linked complex-type oligosaccharide (Figure 2) (19). Treatment with endo-~-N-acetylgalactosaminidase to remove O-linked oligosaccharides dropped the apparent Mr on SDS-PAGE by ,-~26,000, with two thirds of the shift due to sialic acid (Figure 2/. Thus, E DAFpossesses multiple, highly sialylated O-linked oligosaccharides (19). Similar results were obtained for DAFfrom peripheral blood granulocytes and cell lines such as HL-60, except for partial resistance to enzymatic removal of Olinked oligosaccharides. This suggests that the higher Mrof DAFon white blood cells versus E (see below) might arise from differences in O-linked glycosylation, but this point requires further investigation. A study of biosynthesis in the HL-60 cell line demonstrated two DAFintracellular species of 43,000 and 46,000 Mr(Figure 3) 09). Both species possess N-linked high-mannose unit, added cotranslationally, but no O-linked oligosaccharides. The lower Mrform is only seen in brief pulse labelings of 5-10 min; longer biosynthetic labelings only reveal the 46,000 Mrspecies and the mature form of DAF. Pulse-chase experiments indicate that the 43,000 Mr species is the earliest biosynthetic form of DAF,and that it undergoes an early posttranslational modification to a 46,000 Mrspecies (19). This change occurs before DAFenters the central region of the Golgi and does not appear to involve N- or O-linked glycosylation. The nature of this modificationis still unknown,as is its possible relation to the other major known DAFposttranslation modification, addition of a glycophospholipid anchor (see below). The 46,000 Mr species of DAFproceeds through the Golgi, where the one N-linked oligosaccharide is modified to a complex type, and the multiple O-linked oligosaccharides are added to produce the mature form of the protein seen on the cell surface. All forms of DAFhave a slower migration on SDS-PAGE under reducing, compared to nonreducing, conditions; this indicates the presence of intrachain disulfide bonds.

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Similar results were found in a study of DAFbiosynthesis in the HeLa epithelial cell line (10). A single DAFprecursor of 48,000 Mr (equivalent to the 46,000 Mr DAFspecies discussed above) was identified using biosynthetic labelings of 30 min or greater. This DAFprecursor incorporated ethanolamine, a componentof the glycophospholipid anchor (10). This discussed later in more detail.

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Glycophospholipid

Anchor

of Membrane DAF

Whenpurified DAFfrom the E membraneis added back to a cell suspension, it reincorporates in the membrane,apparently as an integral membrane protein, and displays functional activity (15). This property prompted an examination of the membrane-anchoring domain of DAF by two groups. DAFwas found to belong to a recently described class of membraneproteins (reviewed in 20, 21) whose carboxy terminus covalently attached to a glycophospholipid containing phosphatidylinositol (PI) inserted in the outer leaflet of the lipid bilayer. This anchoring was first shown by Davitz and colleagues, who demonstrated the release of DAFfrom peripheral blood cells following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) (9). Specifically, 60-80% of membrane DAFwas released from leukocytes by PI-PLC, although only 10% of E membrane DAF was removed. This partial resistance to PI-PLC has been found in other glycophospholipid-anchored proteins, and it mayrepresent structural modifications in the PI (22). These investigators and others found that DAFreleased by PI-PLChad lost its hydrophobiccharacter and its ability to reincorporate into cell membranes, and thus it could not intrinsically inhibit assembly of the C3 convertase on the cell surface (9, 10). However,this hydrophilic form of DAFcould still accelerate the decay of preformedC4b2aon a surface, albeit at a much reduced efficiency (10). A different hydrophilic fragment of DAF,also lacking the glycophospholipid anchor, demonstrated equal efficiency to purified membraneDAFin dissociating the fluid-phase C3 convertases

Figure 2 N- and O-linked oligosaceharide structure of erythrocyte membrane DAF. Erythrocytes were prepared from peripheral blood of a healthy humandonor and then were surface-labeled with ~zsI. DAFwas immunoprecipitated from detergent lysates and then was divided into equal aliquots for treatment with enzymes. The samples were analyzed by SDSPAGE(on a 9% gel run under reducing conditions) and autoradiography. Enzymetreatments are neuraminidase to remove sialic acid (lane 2), endo-ct-N-acetylgalactosaminidase remove O-linked oligosaccharides (lane 3), endoglycosidase H to remove high-mannose linked oligosaccharides (lane 4), endoglycosidase F to remove high-mannose and complex N-linked oligosaccharides (lane 5), or buffer alone (lanes 1 and 6). Reprinted from (19), permission.

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Annual Reviews 42 LUBLIN & ATKINSON

Annual Reviews DECAY-ACCELERATING FACTOR O Prot ein~)-~-NH-CH~,~CH~ 0 "O-,P=O 0

~

an~l-2Man-l,

43

I-IO~-x OH "1£01"~

~Mandl-4GIcNHa~l-O~-~ OH

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9 R~ R~ Figure 4 Common backbone structure of glycophospholipid anchor from trypanosomc variant surface glycoprotein (VSG)andrat brain Thy-1 glycoprotein. Thecompletestructure of the anchors from VSG(24) ~ad Thy-I (25) has been dcte~ined, and they contain common structure shownhere. The~-carboxy group of the carboxy-te~inal aminoacid of the proteia is linked,to aa ethanolaminephosphate.This is then attach~ to a glycan group containing three mannose(Man) residues and a glucosamine (GlcNH~) and ends in inositol phospholipiS.R~and R2arc fatty acids that form a di(acyl/alkyl)glyccrol moiety whichis inserted in the lipid bilaycr. Theglycangroup hasvarying additional substituents in the VSGand Thy-1 anchors. TheDAFglycophospholipid anchor has not beencompletely dete~ined,but the data (10, 26) are consistent with this structure. Figure is basedon data

in(24,25). (23). Thusthe functional site on DAFis separate from the glycophospholipidanchor. Thecompletestructureof the glycophospholipid anchorhas beendeterminedfor the trypanosomevariant surface glycoprotein (24) andfor Thy-1antigenfromrat brain (25), andthese anchorsshowan identical backbonewith variation in the side chain groups(Figure 4). Chemical analysis of the anchorfromE DAF,thoughless detailed, is consistent with these structures. Thesestudies by Medofandcoworkers(10, 26) demonstrated the presenceof ethanolamine andglucosamine(1.8 and0.8 Figure 3 Biosynthetic labeling of DAFin HL-60cells. HL-60cells (differentiated for 48 hr with vitamin D to increase DAFexpression) were biosynthetically labeled with [35S] methionine during a 10-rain pulse (P) followed by a 60 min chase (C) with unlabeled methionine. The detergent lysate from each condition was divided in half and was immunoprecipitated with either anti-DAF antibody (~DAF)or nonspecific (NS) control nonimmune rabbit Ig, and then was analyzed by SDS-PAGE (under reducing conditions) and fluorography. Another aliquot of HL-60cells was ~25I surface labeled, was immunoprecipitated with anti-DAF antibody and was analyzed by SDS-PAGEand autoradiography (lane 5). An arrow marks the position of mature DAF(80,000 Mr), and open and solid arrowheads mark the positions of the DAFspecies of 43,000 and 46,000 Mr, respectively. Reprinted from (19), with permission.

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moles per mole of DAFprotein, respectively) in the carboxy terminus of the protein, as well as inositol (0.7 mole) and a mixture of saturated and unsaturated fatty acids (0.7 and 1.2 moles, respectively). In addition, analysis by thin layer chromatography of labeled anchor fragments released by nitrous acid deamination (which cleaves at the nonacetylated glucosamine)revealed the presence ofinositol phospholipids other than PI. This could explain the partial resistance to PI-PLCdescribed previously. The presence of the glycophospholipid anchor in DAFmight be expected to bestow some advantage in its functional role. Measurement of the lateral mobility of DAFin HeLacells using the fluorescence photobleaching recovery technique gave a mean diffusion coefficient of 1.61 +0.17 × 10-9 emE/s (27). This mobility is close to that exhibited membranelipids and an order of magnitude higher than most cell surface proteins. Physiologically, this increased mobility could enhancethe ability of a limited number of DAFmolecules to contact a large number of C3b and C4b fragments on the cell surface. Other possible roles for glycophospholipid anchors include serving as a means to release the protein from the cell membraneand transducing an intracellular signal (reviewed in 20, 21). AlthoughDAFis found in plasma and other body fluids, it is not knownwhether it arises from the membrane form via endogenousphospholipases. It has been found that antibodies to DAFinduce activation of human T cells, and removal of DAFby PIPLCabrogated the response (28). This has also been noted with two glycophospholipid-anchored murine proteins, Thy-1 (29) and T-cell-activating protein (30), but the role of the anchor itself in these processes not known. Sites

of Expression

and Alternate

Forms of DAF

DAFis present on virtually all peripheral blood cells: E, granulocytes, T and B lymphocytes, monocytes, and platelets (31, 32). The DAFmolecule from leukocytes has a 3000-9000 higher Mr than E DAF. Quantitation by iinmunoradiometric assay demonstrated 3000 DAFmolecules per E, 85,000 per neutrophil, 68,000 per monocyte, 33,000 per lymphocyte (B cell > T cell), and 2000 per platelet (31). Surface expression of DAF neutrophils can be doubled within minutes of exposure to activators such as N-formyl-methionyl-leucyl-phenylalanine; this occurs by translocation of an intracellular pool to the surface (33). Interestingly, DAFis absent on natural killer cells (34). DAFhas also been found on bone marrow mononuclear cells and erythroid progenitors (35). It is present on the epithelial surface of cornea, conjunctiva, oral and gastrointestinal mucosa, exocrine glands, renal tubules, ureter and bladder, cervical and uterine mucosa, and pleural, pericardial, and synovial serosa (36), as well as

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FACTOR

45

cultured umbilical vein endothelial cells (37). It is clear that DAFis widely distributed, and this supports its important role in controlling the complement system. Soluble forms of DAFhave been found in extracellular fluids and tissue culture supernatants. With the use of a two-site immunoradiometricassay (36), DAFantigen was detected in plasma, urine, tears, saliva, synovial fluid, and cerebrospinal fluid, with levels ranging from 40-400 ng/ml. Analysis by immunoprecipitation and Western blotting showed that the DAFfrom plasma, tears, and saliva had an apparent Mr of ~ 100,000, whereas that in urine had a Mr of 67,000, slightly lower than E membrane DAF. Urinary DAF was less hydrophobic than membrane DAF and did not inhibit the intrinsic assemblyof C3 convertases on the cell surface, but it could accelerate the decay of preformed C4b2awith an efficiency comparable to C4bp. Urinary DAFis thus similar to DAFreleased from membranesby PI-PLC (10). A species similar in size to urinary DAFwas also detected in the culture supernatants of the HeLaepithelial cell line (36), prompting the suggestion that urinary DAFis synthesized by the adjacent urethelium. Alternate forms of the membrane DAFmolecule have also been described. A larger variant, designated DAF-2, was detected on E membranes by Western blotting (38). DAF-2 possesses a 140,000 Mr and represents less than 10%of membraneDAF.This variant accelerates the decay of C3 convertase and shares with DAFthe ability to reincorporate into E membranes, suggesting the presence of the glycophospholipid anchor. The apparent Mr of DAF-2raises the possibility that it is a dimer of DAF, although neither reduction with 2-mereaptoethanol nor denaturation in SDS could separate DAF-2 into two components. The structure of DAF-2thus remains unexplained. Degradation fragments of membraneDAFhave been produced in vitro by treatment of DAFwith PI-PLC(9, 10, 23), a PI-specific phospholipase D from serum(39), or papain (10, 23). Interestingly, incubation of surfacelabeled E with leukocytes led to release of a fragment of equal size to a papain-derived fragment (23). It is unknownwhether any of these degradative processes are relevant in vivo. Blood

Group Antiyens

on DAF

It was recently demonstrated that the Cromer-related humanblood group antigens Cr" and Tc" reside on the DAFmolecule (40). Antibodies to ~ and Tc" recognized purified DAFon Western blots, and these antisera had reduced or absent reactivity with PNHE that lack DAF(see below). Moreover, cells of the rare Cromer-related null phenotype Inab did not react with antiserum to DAFby direct binding or Western blotting. The

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LUBLIN& ATKINSON

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reason underlying lack of expression of DAFin this null phenotypeis unknown. DAF In Other Species DAF’srole in protecting host tissues leads to the expectationthat a DAF or DAF-like molecule would exist in any species with a complement system. To date, only the DAFfromguinea pig (4) and rabbit (41, 42) have been isolated. The guinea pig DAFwas actually purified before humanDAF(4); the same investigators then used this schemeto isolate humanDAF(5). The guinea pig DAFhas a Mr of 60,000 on SDS-PAGE under reducing conditions. Decay-acceleratingactivity wasalso found on rabbit erythrocytes (41, 42). The rabbit DAFwas purified, yielding protein with a Mr of 66,000 on SDS-PAGE under nonreducingconditions (42). Rabbit DAFhas an amino acid composition resembling human DAF,and it can spontaneouslyand selectively reincorporate into sheep E, whichsuggests that it possessesa glycophospholipidanchorsimilar to human DAF. Cloning

of DAF eDNA

Twogroups have independently cloned DAFcDNAs(43, 44). Both used oligonucleotide probes based on the amino-terminalsequenceof immunoaffinity-purified E DAF.The clones were derived from libraries constructed with mRNA from either the HeLaepithelial cell line (43, 44) or the HL-60promyelocyticleukemiacell line (43). Thenucleotide and derived aminoacid sequencesfor DAFare shownin Figure 5. There is a single long open reading frame beginning with an initiation methionine codonand extending 1143bp. This is surroundedby 5’- and 3’-untranslated regions, the latter endingin a poly(A)track. Thededucedaminoacid sequencepredicts a protein of 381 aminoacids including a 34 aminoacid signal peptide. Starting at the aminoterminusof the matureprotein, there are four contiguous short consensus repeat (SCR)units of ~ 60 amino acids (Figure 6). EachSCRcontains four cysteines, .as well as conserved residues of proline, tryptophan, glycine, and several other aminoacids, and are homologousto domains found in other complementregulatory proteins, including CR1,CR2,C4bp, and H, as well as in several noncomplementproteins (reviewed in 45). The SCRsare followed by a 70aminoacid region that is rich in serine and threonine residues (45%). similar serine- and threonine-richregion, located just extracellular to the plasmamembrane,is the site of clustered O-glycosylation in the lowdensity lipoprotein receptor (46), with single O-linkedunits located other regions of the protein. This is consistent with the large amountof O-linkedoligosaccharide previously identified in DAF(19); the deduced

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47

G~TGCGACTCGGCGGAGTCCCGGCGGCGCGTCCTTGTTCTAACCCGGCGCGCCATGACCGTCGCGCGGCCGAGCGTGCCCGCGGCGCTG 89 MTVARPSVPAAL-23 °34 ~~cCT~cTcGGGGAG~TGC~CCGGCTGCTG~TGCTGGTGCTGTTGTG~CTG~CGGCCGTGTGGGGTGA~TGTGGC~TT~CCCCAGATGTA 179 PLLG~LPRLLLLVLLCLpAVWGOCGLPPDV

Cc TGGCGAGAAGGACTCAGTGATCTGCC TTAAGGGCAGTC AATGGTCAGATATTGAAGAGTTC TGGAATCGTAGCTGCGAGGTGCCAACA P G E K D S V I C L K G S Q W $ D I E E F C N R S C E V P T AGGCT A AATTC TGCATCCCTCAAACAGCCT TATA TCACTCAGAATTATTTTCCAGTCGGTACTGTTGTGGAATATGAGTGCCGTCC^GGT 44~ R L N $ A S L K O P Y I T O N Y F P V G T V V E Y E C R P G

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~"AC AGA AGAGAACC T TCTCTATCACCAAAACTAACT TGGCTTCAGAATTTAAAATGGTCCACAGCAGTCGAATTT TGTAAAAAGAAATCA $39 ¥ R R E P $ k $ P K k T C k O N L K W 8 T A V E F G K K K S 128 829 1~ GGGTA~AAATTATTTGG~T~~A~TTCTAGTTTTTGT~TTATTT~AGG~AG~T~TGT~~AGTGGAGTGA~~CGTTGCcAGAGTGcAGAGAA 71 g G Y K L F G $ T $ $ F C L I S G 8 8 V ~ W ~ D P L P E G R E 133 ATT T ATTGT~CAGCA~~A~CA~AAATTGACAATGGAATAATT~AAGGGGAACGTGA~~ATTATGGATATAGACAGTCTGTAACGTATGCA 809 ~ Y C P A P P O I D N G I I 0 G E R D H Y G ¥ FI O $ V ’r Y A 213 T GT AA T AAAGGA T TCACCA TGATTGGAGAGCACTCTA TTT ATTGTACTGTGAATAATGATGAAGGAGAGTGGAGTGGCCCACCACCTGAA 899 248 C N K O F T M I G E H $ I Y G T V N N D E G E W 8 Q P P P E TGCAGAGGAAAATCT~TAA~TT~~AAGGTC~~A~CAA~AGTT~AGAAA~~TAC~A~AGTAAATGTT~CAA~TACAGAAGT~T~A~cAACT 989 C R G K S L T $ K V P P T ¥ Q K P T T V N ¥ P T T E V S P T 278 T C T C AG A A AACCACCAGAA AA ACCACCACACCAAATGCTCAAGCAACACGGAGTACACCTGT TTCCAGGACAAGCAAGGATTTTCATGAA ’t079 $ Q K T T T K T T T P N A O A T R $ T P V 8 R T T K H F H E 30E ACA ACCCCAAA TAAAGGAAGTGGAACCAC TTCAGGTACTACCCGTGTTCTATCTGGGCAC AGGTGTTTCACGTTGAC AGGTTTGCTTGGG1 139 338 T T P N K G S G T T 8 G T T R |L L S (3 H T C F T L T G L L A CGCTAG T A ACCATGGGC T TGCTGACTTAGCCAAAGAAGAGTTAAGAAGAAAA TACACACAAGTATACAGACTGTTCCTAGTTTC TTAGA 125~ T L V T M (3 k L T I ° 347 1~49 C T T A T C T GCAT A T TGGATA A.A.A.T.A.A..ATGC^^T TGTGCTC T TCAT T TAGGATGC TTTCATTGTCTTTA^GATGTGTTAGGAATGTCAAC^G AGCAAGGAG^^^^^^GGGA~T~~T~GA^T~^~^TTGTT^G¢^~^~~TA~A~~T~TTGAA^ATAG^AGA^GTTG~^G^ATTG^G^~TGATT 1438 CC T T TCCT A A AAGTGTAAGAAAGC AT AGAGAT TTGTTCGT ATT TAGAATGGGATCACGAGGAAAAGAGAAGGAAAGTGAT TT TTTTCGAC 152~ A AGATC T GTAATGTTATTTCGAC TTATAAAGGA.A.A.T.A.A.A.A AATGAAAAAGAT TATTTGGATATCAAAAGGA.A.A..T.A.A.AAAGGGAATTGAGT 1§19 C TG T TC T AAGCAAAAT TGCTAAAGAGAGATGAACGACATTATAAAGTAATCTTTGGCTGTAAGGCA TTTTCATCTTTCCTTGGGGTTGGG 170~ A AA A T AT T TT AAAGGTAAA ACATGGTGGTGAACCAGGGGTGT TGATGGTGATA ^GGGAGGAATATAGAATGAAAGAGTGAATGTTCGTTT 1799 1089 GT TGCAC A_A.A.T.A~G_AGT TTGGAAAAAGGC TO TQAAAGGTGTCTTC TT TGACTTAATQTCTTTAAAAGTATCCAGAGATACTACAATATTAA CATAAGAAAAG^TTATATATTAT TTCTG^ATCGAG^TGTCC^TAGTCAAATTTGTAAATCTTATTCTTTTGTAATATTTATTTATATTT^ 1979 T T T ATGACAGTGAACATTCTGATTTTACATGT~AAAcAAGAAAAGTTGAAGAAGATATGTGAAGAAAAATGTATTTTTCCTAAATAGAA-A20~ " A_A_A_TGATCCCATTTTTTGGTAAAAAAAAAAA

2101

Figure 5 Nucleotide and derived amino acid sequences of DAFeDNA. The nucleotide sequence is numbered from the most 5’ nucleotide, and the derived amino acid sequence, numberedfrom the first aminoacid of the mature protein, is shownbelow, using single-letter codes with an asterisk denoting the stop codon. The single N-glycosylation site is marked with an arrow, an S/T-rich region (probable site of O-linked glycosylation) is markedwith an underline, a carboxy-terminal hydrophobic region (replaced posttranslationally with glycophospholipid anchor) is boxed, and potential polyadehylation signals are markedwith dashed lines. Data from (43; D. M. Lublin, unpublished).

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protein sequence also showsone site for N-linked glycosylation, again as expected (19). The deduced protein structure ends in a hydrophobic 24-amino acid segment. The series of basic residues (that act as a stop anchor sequence) and the cytoplasmic tail that are present in polypeptid¢-anchored membrane proteins are not seen in DAF. However, this carboxy-terminal hydrophobic peptide is similar to extension peptides encoded by the cDNAsfor other glycophospholipid-anchored membraneproteins such as the trypanosome variant surface glycoprotdns (47) and Thy-1 (48). These extension peptides of 17-31 amino acids are removedposttranslationaily and the carboxy-terminal glycophospholipid anchor is attached (reviewed in 20, 21). A similar processing presumably leads to the attachment of the DAFglycophospholipid anchor, perhaps with the hydrophobic extension peptide acting as a transient membraneanchor in the endoplasmic reticulum. Studies in HeLacells demonstrated that the major intracellular DAFprecursor of 48,000 Mr incorporated ethanolamine, a component of the glycophospholipid anchor (10). In addition, partitioning of proteins into the detergent phase of Triton X-114 (49) was used to show that treatment of this DAFprecursor species with PI-PLC removedits hydrophobic domain (D. M. Lublin, unpublished). These results demonstrate that the glycophospholipid anchor is already attached to this DAFprecursor. Each of the SCRdomains in f12 glycoprotein I (50) and several that have been studied in C4bp(51) have the four cysteine residues disulfide bonded as cys 1-cys 3 and cys 2-cys 4. This pattern most likely holds for the SCRsin DAF. Secondary structure predictions for DAFbased on the methods of Chou-Fasman(52) or Robson (53) indicate predominantly structure. Similar predictions for many of the SCRsalong with spectroscopic data on H, suggest that the SCRforms a compact domain with antiparallel fl-sheets (54). At this point the structure of DAFat the cDNAand protein level can be summarized. The cDNAsequence shown in Figure 5 is organized into structural regions in Figure 7. Translation of this sequence into protein, coupled with the co- and posttranslational modifications discussed previously, results in a structural model of the membraneDAFglycoprotein (Figure 7). The signal for attachment of the glycophospholipid anchor to a given protein is still unclear. However, two groups have made and expressed mutant cDNAscontaining the carboxy-terminal segment of DAFattached to the amino-terminal segment of another protein (55, 56). These mutant cDNAs,whenexpressed in transfected cell lines, led to the production of a membrane protein that was anchored by a glycophospholipid, thus

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& ATKINSON

Consensus Repeats

s’--I

I 1 I 2 I 3 I

! Signal Peptide

Ser- and ThrRich Region

4 I

II

l HydrophobicRegion

A n

NH 2

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4 Repeat Domains

O-Linked Carbohydrate Domain

CHO-O~ CHO-O.O-CHO CHO-O,~O-CHO , -O-CHO

Glycophospholipid Anchor

~

Membrane

Figure 7 Structure of DAF cDNA and DAF membrane glycoprotein. (Top) This DAF cDNA structure corresponds to the sequence in Figure 5. Coding regions are shown by boxes, and 5’- and 3’-untranslated regions by lines. The ser-and thr-rich region is the probable site of most of the extensive O-linked glycosylation of DAF. The carboxy-terminal hydrophobic region of DAFis replaced posttranslationally with a glycophospholipid anchor. (Bottom) This model of the membrane DAF glycoprotein is based on the above cDNA and the biochemical in the text.

studies

of DAF glycosylation

and the glycophospholipid

anchor discussed

establishing that the carboxy-terminal aminoacids (either 37 [55] or 91 [56] aminoacids) containthe signal for attachment of a glycophospholipid anchor.Thenatureof that signal is still unknown. TheeDNA for DAFdetects several bandson Northernblot analysis of mRNA fromvarious cell lines. The majorspecies are reported as 2.0 and2.7 kb (43) or 1.5 and2.2 kb (44), apparentlysimplyreflecting standardizationdifferences. Thesetwo species of mRNA are productsof alternative polyadenylation(44). Relative levels of DAFmRNA in the HeLa,HL-60,and HSB-2cell lines correlated with the levels of DAF proteindetectedby immunoradiometric assay (43), suggestingtissue-specific transcriptionalcontrol of DAF expression. One group has found a second class of DAFcDNAclones which containeda 118 bp insertion near the endof the codingregion(44). The resulting frameshiftpredicts a longer encodedprotein (440 aminoacids

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including signal peptide) that wouldhave a hydrophilic carboxy terminus. The authors speculate that the 118 bp represents an unspliced intron. A probe based on this sequence detected a minor species of DAFon Northern analysis of HeLacell RNA.Transfection of these two types of cDNAinto Chinese hamster ovary (CHO)cells resulted in the production of DAF immunoreactive material, but only the cDNAending in the hydrophobic extension peptide produced surface DAF.It was suggested that the spliced and unspliced species of cDNAencode membrane and secreted DAF species, respectively. However, subsequent work has cast doubt on this hypothesis (D. M. Lublin, unpublished; V. Nussenzweig, personal communication). CHOcells transfected with the spliced (regular or hydrophobic) DAFcDNAproduce both DAFattached to the membrane by glycophospholipid anchor and a secreted form of DAFapproximately 5000 lower in Mr. Antibodies raised against the carboxy-terminal hydrophilic peptide (encoded only by the alternate, unspliced cDNA)did not recognize the soluble DAFspecies in HeLacell culture supernatants. The physiological relevance of this alternate DAFcDNAspecies, along with the origin of the secreted form of DAF,remains unclear. DAF Gene Southern blot analysis of human DNAshows that the DAFgene is~ approximately 35 kb in length (57-59). The relatively simple pattern generated from restriction digests suggests that DAFis a single copy gene, and this was supported by hybridizations with DAF-specific oligonucleotide probes (59). The structure and organization of the DAFgene is not yet known. Three RFLPshave been identified in the DAFgene: two for the enzyme Hind III and one for BamHI (58, 59). All are located in the noncoding region of the gene. The chromosomallocation of the DAFgene is on the long arm of humanchromosome1, band q3.2. This was derived from analysis of a panel of hamster x humansomatic cell hybrids and by in situ hybridization of the DAFcDNAto human metaphase cells (57). The same result was obtained by segregation analysis of the DAFRFLPs in families that are informative for segregation of alleles at the CR1, C4bp, and H loci (58). The latter three complementproteins were already known to be located at thg regulator of complementactivation (RCA)gene cluster at lq3.2, so the DAFgene is added to this group. Furthermore, recombinations within the RCAlocus demonstrated that DAFmaps closer to the CR1/C4bploci than to the H locus. Subsequent detailed mapping of the RCAgene cluster by pulsed-field gel electrophoresis has shown the order of the genes to be MCP-CR1-CR2-DAF-C4bp, located within an 800-kb segment of DNAon the long arm of chromosome1 (60, 61; N. S. Bora, J. P. Atkinson, submitted).

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PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (I’NJ) The physiological role of DAFhas been elucidated by studies of the disease paroxysmal nocturnal hemoglobinuria (PNH), an acquired clonal disorder of hematopoietic stem cells (reviewed in 62). Circulating blood cells that arise from the affected pluripotent stem cell show increased complement sensitivity, leading to episodes of hemolysis that are a hallmark of PNH. By use of a quantitative complementlysis sensitivity assay (63), three populations of blood cells are detected in the circulation of PNHpatients: PNHI cells with normal sensitivity, PNHII cells with 3-5-fold increased sensitivity, and PNHIII cells with 15-25-fold increased sensitivity to lysis by complement. PNHII and PNHIII cells show increased C3b uptake (64); in addition, PNHIII cells showincreased susceptibility to bystander or reactive lysis by the terminal complement pathway components C5b-9 (65). Twogroups of investigators found that the affected E of PNHpatients lack DAF(6, 7). This was also found for PNHleukocytes and platelets (31, 66). Furthermore,this defect was causally related to increased sensitivity to complement, since reincorporation of purified DAFinto these E normalized their C3b uptake and partially corrected the sensitivity (8). Specifically, incorporation of DAFinto PNHII cells completely corrected their complement sensitivity, whereas PNHIII cells with DAFincorporated, although taking up C3b normally, still had markedly increased susceptibility to reactive lysis (67). NormalE possess a membraneprotein, homologousrestriction factor (HRF) (68, 69), which can inhibit transmembrane channel formation by the terminal complement components. PNHE have been shown to lack HRF, and thus to have increased susceptibility to reactive lysis, which can be corrected by reincorporation of purified HRFinto the cell membranes (70). Thus, both DAFand HRF appear to be critical in vivo for protection of host cells from damageby autologous complement. Investigations of DAFhave also shed light on the underlying lesion in PNH. Southern and Northern analysis utilizing leukocytes from PNH patients revealed a normal DAFgene and mRNA transcripts (59). Indeed, affected cells of PNHpatients lack not only DAF, but also acetylcholinesterase (71), alkaline phosphatase (72), lymphocyte functionassociated antigen 3 (73), Fc receptor type III (74), and HRF(70). these proteins except HRFhave been directly shown to be anchored to the cell membraneby a glycophospholipid anchor [Table 1 in reference (21); for FcRIII see 74-76]. Purified HRFcan spontaneously reincorporate into cell membranes(70), suggesting that it also possesses this form

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membraneanchor. The fact that these otherwise unrelated proteins are all absent in a clonal disorder strongly suggests that the lesion.in PNHmust involve their only commonelement, the glycophospholipid anchor (9). The nature of this defect in the pathwayfor biosynthesis or attachment of the anchor structure is unknown,as is the reason for the existence of more than one type of affected cell, PNHII versus PNHIll, in this clonal disorder.

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CONCLUSIONS The work reviewed here has provided a picture of DAFwith its polypeptide backbone, extensive glycosylation, and glycophospholipid anchor (Figure 7). The bulk of the extracellular part of the protein is organized as four contiguous short consensus repeat domains, thus putting DAFin a family of complement (and noncomplement) proteins that share this 60 amino acid structural unit. Furthermore, DAFis genetically linked to a subgroup of these (CR1, CR2, H, C4bp, and MCP)that are all C3 regulatory receptor proteins located on the long arm of humanchromosome1, band q3.2, at the regulator of complementactivation locus. This group probably arose by a process of gene duplication from an ancestral C3 binding protein. The study of the organization and expression of the DAFgene will help in understanding the evolution of this gene family and the role that DAFplays in inflammation. The glycophospholipid anchor, which is unique to DAFamong this group, might help DAFserve its function by increasing its mobility in the cell membrane.Future workproviding further characterization of the fine structure of the glycophospholipid anchor in DAFand assessing its contribution to the function of DAFwill be important not only for an understanding of the role of DAFin the complement system, but also for elucidating the disease PNHin which there is a defect in the synthesis or attachment of this anchor. DAFdown regulates complement activation by preventing C3 convertase formation on cell surfaces as well as by dissociating preformed convertases. The physiological importance of DAFis suggested by its wide tissue distribution and is further highlighted by the increased complement sensitivity and lysis of cells from PNHpatients, which lack DAF.DAFis present on the membranesof invading inflammatory cells and on the tissue at sites of inflammation, and it protects both from bystander lysis due to complementactivation. More generally, there is a constant low-level activation of the alternative pathway of complement; this nonspecific initiation is targeted to and amplified on foreign surfaces and avoids host tissues because the latter can control complementon their surfaces. Although the plasma proteins H and C4bp can control the C3 convertase,

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this appearsto be importantmainlyfor the fluid phase, whereason cell surfaces the membrane proteins are critical. DAFdoes not permanently modifythe C3bor C4battachedto the cell surface, so they can reforman active C3convertase.Thus, membrane proteins that can act as cofactors for the permanent inactivationof the C3convertaseby the serine protease factorI also play a role in protectinghost cells. Theimportant proteinin this regardis probablymembrane cofactor protein (MCP) (14, 77), which sharesthe widetissue distribution of DAF,whereasthe principalrole of CR1,whichhas both decay-acceleratingactivity andcofactor activity, probablyinvolves processing immune complexes.In this way, DAFand other complement regulatoryproteins not only protect host tissues from inflammation initiated by antibody,but also serve to target the constant low-level activation of the alternative pathwayof complement awayfrom self-tissues andtowardnonself(78). ACKNOWLEDGMENT The authors

thank Avtar Khalsa for excellent

secretarial

assistance.

Literature Cited 1. Holers, V. M., Cole, J. L., Lublin, D. M., Seya, T., Atkinson, J. P. 1985. Human C3b- and C4b-regulatory proteins: A new multi-gene family. Immunol. Today 6:188-92 2. Hoffmann, E. M. 1969. Inhibition of complement by a substance isolated from humanerythrocytes. I. Extraction from human erythrocyte stromata. Immunochemistry 6:391M03 3. Hoffmann, E. M. 1969. Inhibition of complement by a substance isolated from humanerythrocytes. II. Studies on the site and mechanism of action. Immunochemistry 6:405-19 4. Nicholson-Weller, A., Burge, J., Austen, K. F. 1981. Purification from guinea pig erythrocyte stroma of a decay-accelerating factor for the classical C3 convertase, C4b,2a. J. Immunol. 127: 203539 5. Nicholson-Weller, A., Burge, J., Fearon, D. T., Weller, P. F., Austen, K. F. 1982. Isolation of a humanerythrocyte membrane glycoprotein with decay-accelerating activity for C3convertases of the complement system. J. Immunol. 129: 184-89 6. Nieholson-Weller, A., March, J. P., Rosenfeld, S. I., Austen, K. F. 1983. Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria

are deficient in the complement regulatory protein decay-accelerating factor Proc. Natl. Acad. Sci. USA 80:5066-70 7. Pangburn, M. K., Schreiber, R. D., Miiller-Eberhard, H. J. 1983. Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria. Proc. Natl. Acad. Sci. USA 80:5430-34 8. Medof,M. E., Kinoshita, T., Silber, R., Nussenzweig, V. 1985. Amelioration of lytic abnormalities of paroxysmal nocturnal hemoglobinuria with decay-accelerating factor. Proc. Natl. Acad. Sci. USA 82:2980-84 9. Davitz, M. A., Low, M. G., Nussenzweig, V. 1986. Release of decay-accelerating factor (DAF)from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein. J. Exp. Med. 163: 115061 10. Medof, M. E., Walter, E. I., Roberts, W. L., Haas, R., Rosenberry, T. L. 1986. Decay accelerating factor of complementis anchored to cells by a C-terminal glycolipid. Biochemistry 25:6740-47 11. Mfiller-Eberhard, H. J., Miescher, P. A., eds. 1985. Complement.Berlin: SpringerVerlag 12. Ross, G. D., ed. 1986. lmmunobiologyof

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DECAY-ACCELERATING FACTOR the ComplementSystem. An Introduction for Research and Clinical Medicine. London: Academic Press 13. Fearon, D. T. 1980. Identification of the membraneglycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte and monocyte. J. Exp. Med. 152:20-30 14. Seya, T., Turner, J., Atkinson,J. P. 1986. Purification and characterization of a membraneprotein (gp45-70) which is cofactor for cleavage of C3b and C4b. J. Exp. Med. 163:837-55 15. Medof, M. E., Kinoshita, T., Nussenzweig, V. 1984. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J. Exp. Med. 160:1558-78 16. Fujita, T., Inoue, T., Ogawa, K., Iida, K., Tamura, N. 1987. The mechanismof action of decay-accelerating factor (DAF). DAFinhibits the assembly C3 convertases by dissociating C2a and Bb. J. Exp. Med. 167:1221-28 17. Kinoshita, T., Medof, M. E., Nussenzweig, V. 1986. Endogenousassociation of decay-accelerating factor (DAF)with C4b and C3b on cell membranes. J. Immunol. 136:3390-95 18. Pangburn, M. K. 1986. Differences between the binding sites of the complement regulatory proteins DAF, CR1, and factor H on C3 convertases. J. Immunol. 136:2216-21 19. Lublin, D. M., Krsek-Staples, J., Pangburn, M. K., Atkinson, J. P. 1986. Biosynthesis and glycosylation of the human complement regulatory protein decay-accelerating factor. J. Immunol. 137:1629-35 20. Low, M. G. 1987. Biochemistry of the glycosyl-phosphatidylinositol membrane protein anchors. Biochem. J. 244: 1 13 21. Low, M. G., Saltiel, A. R. 1988. Structural and functional roles of glycosylphosphatidylinositol in membranes. Science 239:268 75 22. Roberts, W. L., Kim, B. H., Rosenberry, T. L. 1987. Differences in the glycolipid membrane anchors of bovine and human erythrocyte acetylcholinesterases. Proc. Natl. Acad. Sci. USA84: 7817-21 23. Seya, T., Farries, T., Nickells, M., Atkinson, J. P. 1987. Additional forms of human decay-accelerating factor (DAF). J. Immunol. 139:1260~57 24. Ferguson, M. A. J., Homans, S. W., Dwek, R. A., Rademacher, T. W. 1988. Glycosyl-phosphatidylinositol moiety

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that anchors trypanosoma brucei variant surface glycoprotein to the membrane. Science 239:753-59 25. Homans, S. W., Ferguson, M. A. J., Dwek, R. A., Rademacher, T. W., Anand, R., Williams, A. F. 1988. Complete structure of the glycosyl phosphatidylinositol membrane anchor of rat brain Thy-1 glycoprotein. Nature 333:269-72 26. Walter, E. I., Roberts, W. F., Rosenberry, T. L., Medof, M. E. 1987. Analysis of fatty acids and inositol in the membrane anchor of human erythrocyte decay accelerating factor (DAF). Fed. Proc. 46:772 (Abstr.) 27. Thomas, J., Webb, W., Davitz, M. A., Nussenzweig, V. 1987. Decay accelerating factor diffuses rapidly on HeLaAEcell surfaces. Biophys. J. 51: 522a (Abstr.) 28. Ritter, A. R., Davis, L. S., Patel, S. S.~ Atkinson, J. P., Lipsky, P. E. 1988. An antiserum to decay-accelerating factor (DAF) activates human T cells. Fed. Proc. 47:A871 (Abstr.) 29. Gunter, K. C., Malek, T. R., Shevach, E. M. 1984. T cell activating properties of an anti-Thy-I monoclonal antibody. Possible analogy to OKT3/Leu-4. J. Exp. Med. 159:716-30 30. Rock, K. L., Yeh, E. T. H., Gramm,C. F., Haber, S. I., Reiser, H., Benacerraf, B. 1986. TAP,a novel T-cell-activating protein involved in the stimulation of MHC-restricted T lymphocytes. J. Exp. Med. 163:315-33 31. Kinoshita, T., Medof, M. E., Silber, R., Nussenzweig, V. 1985. Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J. Exp. Med. 162: 7592 32. Nicholson-Weller, A., March, J. P., Rosen, C. E., Spicer, D. B., Austen, K. F. 1985. Surface membraneexpression by humanblood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system. Blood 65:1237~,4 33. Berger, M., Medof, M. E. 1987. Increased expression of complement decay-accelerating factor during activation of humanneutrophils. J. Clin. Invest. 79:214-20 34. Nicholson-Weller, A., Russian, D. A., Austen, K. F. 1986. Natural killer cells are deficient in the surface expression of the complement regulatory protein, decay-accelerating factor (DAF). Immunol. 137:1275-79 35. Moore, J. G., Frank, M. M., Miiller-

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Eberhard, H. J., Young,N. S. 1985. organization, and regulation of the Decay-acceleratingfactor is present on complementgenes. Ann. Rev. Immunol. paroxysmal nocturnal hemoglobinuria 6:161-95 erythroid progenitors and lost during 46, Davis, C. G., Elhammer,A., Russell, erythropoiesisin vitro. J. Exp.Med.162: D. W., Schneider, W.J., Kornfeld, S., 1182-92 Brown,M. S., Goldstein, J. L. 1986. 36. Medof,M. E., Walter, E. I., Rutgers, Deletion of clustered-O-linked carboJ. L., Knowles, D. M., Nussenzweig, hydratesdoes not impairfunction of low V. 1987. Identification of the compledensity lipoprotein receptor in transment decay-accelerating factor (DAF) fected fibroblasts. J. Biol. Chem.262: on epithelium and glandular cells and 2828-38 in bodyfluids. J. Exp. Med.165: 848- 47. Boothroyd,J. C., Paynter,C. A., Cross, G. A. M., Bernards,A., Borst, P. 1981. 37. Asch,A. S., Kinoshita,T., Jaffe, E. A., Variant surface glycoproteins of TryNussenzweig, V. 1986. Decay-accelpanosomabrucei are synthesized with erating factor is present on cultured cleavable hydrophobicsequencesat the humanumbilical vein endothelial cells. carboxy and amino termini. Nucleic J. Exp. Meal. 163:221-26 Acids Res. 9:4735-43 38. Kinoshita,T., Rosenfeld,S. I., Nussen- 48. Tse, A. G. D., Barclay,A. N., Watts,A., zweig, V. 1987. ,6 high m.w. form of Williams, A. F. 1985. A glycophosphodecay-acceleratingfactor (DAF-2)exhilipid tail at the carboxylterminusof the bits size abnormalities in paroxysmal Thy-1 glycoprotein of neurons and nocturnal hemoglobinuriaerythrocytes. thymocytes.Science 230:1003-8 J. Immunol.138:2994-98 49. Bordier, C. 1981. Phase separation of 39. Davitz, M. A., Hereld, D., Shak, S., integral membrane proteins in Triton XKrakow,J., Englund, P. T., Nussen114 solution. J. Biol. Chem.256: 563zweig, V. 1987. A glycan-phospha67 tidylinositol-specific phospholipaseDin 50. Lozier, J., Takahashi, N., Putnam,F. humanserum. Science 238:81-84 W.1984. Completeaminoacid sequence 40. Telen, M.J., Hall, S. E., Green,A. M., of humanplasma fl2-glycoprotein I. Moulds,J. J., Rosse,W.F. 1988.IdentiProc.Natl. Acad.Sci. USA8 I: 3640-44 fication of humanerythrocyte blood 51. Janatova,J., Reid, K. B. M., Willis, A. group antigens on decay-accelerating C. 1988. Involvementof the disulfide factor (DAF)and an erythrocyte phenobonds in the structure of complement type negative for DAF.J. Exp. Med. regulatory protein C4bp.Fed. Proc. 47: 167:1993-98 A1832(Abstr.) 41. Horstmann,R. D., M/Jller-Eberhard,H. 52. Chou,P. Y., Fasman,G. D. 1978. PreJ. 1986. Demonstration of C3breceptordiction of the secondary structure of like activity and of decay-accelerating proteins fromtheir aminoacid sequence. factor-like activity on rabbit erythroAdv. Enzymol. 47:45-148 cytes. Eur. J. ImmunoL 16:1069-73 53. Gamier,J., Osguthorpe,D. J., Robson, 42. Sugita, Y., Uzawa, M., Tomita, M. B. 1978. Analysis of the accuracy and 1987. Isolation of decay-accelerating implications of simple methodsfor prefactor (DAF)from rabbit erythrocyte dicting the secondarystructure of globumembranes.J. Immunol. Methods104: lar proteins. J. Mol.Biol. 120:97-120 123G0 54. Perkins,S. J., Haris, P. I., Sim,R. B., Chapman, D. 1988.A study of the struc43. Medof,M.E., Lublin, D. M., Holers, V. M., Ayers,D. J., Getty, R. R., Leykam, ture of humancomplementcomponent J. F., Atkinson,J. P., Tykocinski,M.L. factor H by fourier transform infrared 1987. Cloning and characterization of spectroscopy and secondary structure cDNAsencoding the complete sequence averaging methods.Biochem.27: 4004of decay-accelerating factor of human 12 complement.Proc. Natl. Acad.Sci. USA 55. Caras, I. W.,Weddell,G. N., Davitz, M. 84:2007-11 A., Nussenzweig,V., Martin, D. W.Jr. 44. Caras, I. W., Davitz, M. A., Rhee,L., 1987. Signal for attachment of a Weddell,G., Martin,D. W.Jr., Nussenphospholipid membrane anchor in zweig,V. 1987.Cloningof decay-acceldecay-acceleratingfactor. Science238: eratingfactor suggestsnoveluse of splic1280-83 ing to generatetwo proteins. Nature325: 56. Tykocinski, M. L., Shu, H. K., Ayers, 54%49 D.J., Walter,E. I., Getty,R. R., Groger, 45. Campbell,R. D., Law,S. K. A., Reid, R. K., Hauer, C. A., Medof,M. E. 1988. K. B. M., Sim, R. B. 1988. Structure, Glycolipid reanchoringof T-lymphocyte

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DECAY-ACCELERATING FACTOR surface antigen CD8using the 3’ end sequence of decay-accelerating factor’s mRNA.Proc. Natl. Acad. Sci. USA 85: 3555 59 57. Lublin, D. M., Lemons, R. S., LeBeau, M. M., Holers, V. M., Tykocinski, M. L., Mcdof, M. E., Atkinson, J. P. 1987. The gene encoding decay-accelerating factor (DAF)is located in the complement-regulatory locus on the long arm of chromosome 1. J. Exp. Med. 165: 1731-36 58. Rey-Campos,J., Rubinstein, P., Rodriguez de Cordoba, S. 1987. Decay-accelerating factor. Genetic polymorphism and linkage to the RCA(regulator of complementactivation) gene cluster in humans. J. Exp. Med. 166:246-52 59. Stafford, H. A., Tykocinski, M. L., Lublin, D. M., Holers, V. M., Rosse, W. F., Atkinson, J. P., Medof, M. E. 1988. Normal polymorphic variations and transcription of the decay-accelerating factor gene in paroxysmal nocturnal hemoglobinuria cells. Proc. Natl. Acad. Sci. USA 85:880-84 60. Rey-Campos,J., Rubinstein, P., Rodriguez de Cordoba, S. 1988. A physical mapof the humanregulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP. J. Exp. Med. 167:664-69 61. Carroll, M. C., Alicot, E. M., Katzman, P. J., Klickstein, L. B., Smith, J. A., Fearon, D. T. 1988. Organization of the genes encoding complement receptors Type 1 and 2, decay accelerating factor, and C4-binding protein in the RCA locus on humanchromosome1. J. Exp. Med. 167:1271-80 62. Rosse, W. F., Parker, C. J. 1985. Paroxysmal nocturnal haemoglobinuria. Clin. Haematol. 14:105~5 63. Rosse, W. F., Dacie, J. V. 1966. Immune lysis of normal human and paroxysmal nocturnal hemoglobinuria (PNH) red blood cells. I. Thesensitivity of PNH red cells to lysis by complementand specific antibody. J. Clin. Invest. 45:736-48 64. Logue, G. L., Rosse, W. F., Adams, G. P. 1973. Mechanismof immunelysis of red cells in vitro. Paroxysmalnocturnal hemoglobinuriacells. J. Clin. Invest. 52: 1129-37 65. Packman, C. H., Rosenfeld, S. I., Jenkins, D. E. Jr., Thiem, P. A., Leddy, J. P. 1979. Complementlysis of human erythrocytes: Differing susceptibility of two types of paroxysmal nocturnal hemoglobinuria cells to C5b-9. J. Clin. Invest. 64:428-33 66. Nicholson-Weller, A., Spicer, D. B.,

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Austen, K. F. 1985. Deficiency of the complementregulatory protein, "decayaccelerating factor," on membranesof granulocytes, monocytes, and platelets in paroxysmal nocturnal hemoglobinuria. N. Engl. J. Med. 312:1091-97 67. Medof, M. E., Gottlieb, A., Kinoshita, T., Hall, S., Silber, R., Nussenzweig,V., Rosse, W. F. 1987. Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complementpathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes. J. Clin. Invest. 80:165-74 68. Zalman, L. S., Wood, L. M., MfillerEberhard, H. J. 1986. Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels. Proc. Natl. Acad. Sci. USA 83:6975-79 69. Sch6nermark, S., Rauterberg, E. W., Shin, M. L., L6ke, S., Roelcke, D., H~inch, G. M. 1986. Homologousspecies restriction in lysis of humanerythrocytes: a membrane-derivedprotein with C8-binding capacity functions as an inhibitor. J. Immunol. 136:1772 76 70. Zalman, L. S., Wood,L. M., Frank, M. M., Miiller-Eberhard, H. J. 1987. Deficiency of the homologousrestriction factor in paroxysmal nocturnal hemoglobinuria. J. Exp. Med. 165:572 77 71. Auditore, J. V., Hartmann, R. C. 1959. Paroxysmal nocturnal hemoglobinuria II. Erythrocyte acetylcholinesterase defect. Am. J. Med. 27:401-10 72. Lewis, S. M., Dacie, J. V. 1965. Neutrophil (leucocyte) alkaline phosphatase in paroxysmal nocturnal haemoglobin~ uria. Br. J. Haematol. 11:549-56 73. Selvaraj, P., Dustin, M. L., Silber, R., Low, M. G., Springer, T. A. 1987. Deficiency of lymphocyte functionassociated antigen 3 (LFA-3) in paroxysmal nocturnal hemoglobinuria. J. Exp. Med. 166:1011-25 74. Selvaraj, P., Rosse, W. F., Silber, R., Springer, T. A. 1988. The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal nocturnal haemoglobinuria. Nature 333:565~i7 75. Simmons, D., Seed, B. 1988. The Fcv receptor of natural killer cells is a phospholipid-linked membraneprotein. Nature 333:568~0 76. Huizinga, T. W. J., van der Schoot, C. E., Jost, C., Klaassen, R., Kleijer, M., von demBorne, A. E. G. Kr., Roos, D., Tetteroo, P. A. T. 1988. The PI-linked

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receptor FcRIII is released on stimu(MCP):Evidence for inclusion in the lation of neutrophils. Nature333: 667multi-gent family of complement-regu69 latory proteins. J. Exp. Med.168: 18177. Lublin, D. M., Liszewski,M. K., Post, 94 T. W., Arce, M. A., Le Beau, M.M., 78. Atkinson, J. P., Farries, T. 1987. Rebentisch,M.B., Lemons,R. S., Seya, Separationof self from non-self in the complementsystem. Immunol.Today 8: T., Atkinson,J. P. 1988.Molecularcloning and chromosomallocalization of 212-15 human membrane cofactor protein

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Ann. Rev. Immunol. 1989. 7: 59-76 Copyright © 1989 by Annual Reviews Inc. All rights reserved

HETEROGENEITY OF MAST CELLS AND PHENOTYPIC CHANGE BETWEEN SUBPOPULATIONS Yukihiko Kitamura Division of Cancer Pathology, Biomedical Research Center, Osaka University Medical School, Nakanoshima4-3-57, Kita-ku, Osaka, 530 Japan

INTRODUCTION Mast cells are not seen in routine histological sections stained with hematoxilin and eosin, but a considerable numberof mast cells are found in various tissues that are properly fixed and stained with dyes such as toluidine blue and Alcian blue. The substances in granules that stain specifically with these dyes are proteoglycans, whichare negatively charged and thought to form complexes with positively charged proteases and histamine. Mast cells have high affinity IgE receptors on their surface, and the immunologicalactivity of mast cells is mediated through these IgE receptors (1). Binding of antigens to IgE molecules results in the formation of linkages between IgE receptors, and then the release of the granules themselves or chemical mediators in the granules (2). This process constitutes an important step in the immediatehypersensitivity reaction that occurs in allergic diseases such as urticaria, bronchial asthma, and allergic rhinitis. In addition to havinga role in allergic diseases, mastcells have a physiological role as an effector of host defense mechanismsin intestinal helminth infection (3-5) and dermaltick infestation (6, Although some of cytochemical and functional characteristics of mast cells are commonalso to basophils, these two types of cells can be distinguished with an electron microscope(8). Moreover,their differentiation 59 07324)582/89/04104)059502.00

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processesare different, in spite of the fact that both mast cells and basophils originate from multipotential hematopoietic stem cells. Like neutrophils and eosinophils, basophils complete their differentiation within the bone marrow,then circulate in the blood, and finally function and die in tissues (8-10). In contrast, undifferentiated precursors of mast cells leave the bone marrow, migrate in the blood, invade tissues, and then proliferate and differentiate into mast cells (9, 10). The life span of at least somemast ceils appears to be muchlonger than that of basophils. In contrast with basophils which lose proliferative potential after differentiation, some morphologically differentiated mast cells can proliferate extensively (9, 10). Mastcells in various tissues differ in their phenotype. The heterogeneity has either biological or clinical significance, and it has been investigated from morphological, biochemical, immunological, and functional points of view (reviewed in 11-14). In the present review, I describe the heterogeneity and the differentiation processes of mast cells and attempt to interpret the heterogeneity from the perspective of their unique differentiation process. HETEROGENEITY Morphology Maximow (15) was probably the first to recognize that certain mast cells in the rat intestinal mucosawere atypical in their staining characteristics and differed from those of the mast cells observed in other anatomical sites. Enerbfick (16) greatly extended these observations and defined conditions of fixation and histochemical staining that discriminated between such atypical or mucosal mast cells (MMC) and the connective tissue-type mast cells (CTMC)of the skin, peritoneal cavity, and muscularis propria of the digestive canal, amongother sites. In the mid-1960swhen Enerb~ck started his investigations, the concentration of mast cells in the intestinal mucosa was considered to be very low (16), since granules of MMC are not fixed with the most commonlyused fixative, 10%formalin. However, after adequate fixation and staining, Enerb~ick found that in rats the gastrointestinal mucosais one of the tissues richest in mast cells. Carnoy’s solution (containing methanol, chloroform, and acetic acid) or a combination of 0.6%formaldehyde and 0.5% acetic acid is necessary for the fixation of MMC (16). Whenappropriately fixed sections are stained, rat MMC stain blue with Alcian blue, while the granules of rat CTMC stain red with safranin. MMC are smaller than CTMC,are more variable in shape, and as a rule contain fewer granules of more variable size and shape. Although MMC are never found in the epithelium in normal rats,

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numerous MMC are observed between the individual epithelium cells in infections of intestinal parasites (16). Berberine sulfate is a fluorescent dye suitable for identification of CTMC in rats and mice. In these animals this dye forms a strong fluorescent complexwith heparin in CTMC granules (17, 18). The prior digestion with heparinase abolished the fluorescence, but digestion by chondroitinase ABCdid not (19). Since the proportion of CTMC of which granules stain red with safranin is lower in mice than in rats, I prefer berberine sulfate to safranin for staining mouseCTMC.In spite of the presence of heparin, granules of humanmast cells do not stain with berberine sulfate. In addition to tissues from which mast cells are harvested, the species and age of animals also influence the phenotypesof mast cells. Even within the peritoneal cavity of rats, the morphologyof mast cells changes with age (20). The nomenclature of MMC and CTMC is based on observations of rat tissues, but the samecriteria are applicable to mast cell populations of mice. Althoughthe heterogeneity of mast cells is detectable in humans, the distinction between MMC and CTMC is not so clear as that observed in rats and mice. Therefore, I will hereafter confine the terms, MMC and CTMC,to mast cell populations of’rats and mice. Cultured

Mast Cells

The characteristics of individual mast cells can be investigated with morphological techniques. However,for biochemical and functional investigations, it is necessary to obtain pure suspensionsof mast cells belonging to each subpopulation. Both CTMCand MMC can be purified in rats, but purification of MMC from mouse intestinal mucosa has not been accomplished. For this reason mice have not been used for the study of mast cell heterogeneity until recently. Developmentof a simple and easy technique to culture mast cells from mouse hematopoietic cells changed the situation. Lzrge numbers (> 108) of bone marrow~lerived cultured mast cells (BMCMC) can be generated as virtually homogeneous populations or as clones. Attempts to culture mast cells have been carried out at least since 1963. Ginsburg & Sachs (21) cultured thymus cells on a feeder layer composed of mouseskin fibroblasts and observed the development of mast cells. Ishizaka et al obtained similar results by using rat thymus, and they demonstrated the presence of histamine and IgE receptors in mast cells developing in such a system (22, 23). About20 years after the first paper of Ginsburg&Sachs (21), six groups of investigators independently reported that mast cells developed when hematopoietic cells of mice were cultured in suspension with growth factors (24-29). Five of these six groups obtained the growth factor from stimu-

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lated T cells, whereas Nagaoet al (26) used the culture mediumof a mouse myelomonocytic leukemia cell line (WEHI-3) as the source of growth factor. Ihle et al (30) purified a mast cell growth factor from the culture medium of WEHI-3cells and designated it interleukin 3 (IL-3). Fung et al (31) isolated cDNAencoding IL-3 by using WEHI-3cells, whereas Yokota et al (32) isolated cDNAof a mast cell growth factor from a mouse T cell line stimulated by concanavalin A and demonstrated that this mast cell growth factor was identical to IL-3. Smith &Rennick (33) identified another mast cell growth factor in the culture mediumof the same T-cell line that was used for the isolation of cDNAencoding IL-3; Lee et al (34) isolated and characterized a cDNA clone that encodedthis mast cell growth factor. Unexpectedly, the nucleotide sequence of the cDNAwas identical with that of cDNAencoding the IgG-l-inducing factor isolated by Nomaet al (35). Moreover, the terminal amino acid sequence of the peptide inferred from this cDNAwas in agreement with the amino acid sequences of B-cell stimulating factor-1 (BSF-1), which were partially determined by Grabstein et al (36) and Paul, Ohara and coworkers (37). Nowthis peptide is designated as BSF-1/IL-4 (hereafter IL-4). Pure mast cell suspensions can be obtained from rat hematopoietic cells by a similar method (38). However, when humanhematopoietic cells are cultured in the presence of humanIL-3, basophils but not mast cells develop (39). Mediators PROTEOGLYCANS Differences

in histochemical reactions of mast cell granules are attributed to differences of their proteoglycans. Proteoglycans consist of a protein core and attached side chains of glycosaminoglycans. Although CTMC in the peritoneal cavity of rodents (rats and mice) and mast cells in the humanlung contain heparin proteoglycans, the relative molecular mass (Mr) of heparin proteoglycans is considerably different (600,000 to 1,000,000 in rodents and 60,000 in humans)(11-14, 40). Somesubpopulations of mast cells don’t synthesize heparin proteoglycan but do synthesize chondroitin sulfate proteoglycans. Rat MMC purified from the small intestine infected with a helminth, Nippostrongylus brasiliensis (41, 42), and rat BMCMC (43) incorporate 35S into a highly sulfated chondroitin sulfate. The type of proteoglycan contained in or synthesized by mouseMMC has not been determined, since it is difficult to collect enough of these cells for chemical analysis. However, mouse BMCMC have been shown to incorporate 3~S into an oversulfated chondroitin sulfate (44).

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Synthesizing a type of proteoglycan does not necessarily meanthat the cell contains only this type of proteoglycan. Although rat peritoneal CTMC incorporate 3~S exclusively into heparin proteoglycan, staining by a monoclonal antibody showed that someof the cells contained chondroitin sulfate as well (45). PROTEASES Apparent heterogeneity of proteases has been observed in rat mast cell populations (ll-14). Rat CTMC contain a chymotrypsin-like neutral protease (rat mast cell protease I, RMCP-I)and carboxypeptidase A, an enzyme that cleaves C-terminal aromatic amino acids, whereas MMCand BMCMC of rats contain another chymotrypsin-like neutral protease (rat mast cell protease II, RMCP-II)(43, 46, 47). Although RMCP-!and RMCP-II have substantial homology in their amino acid sequences, antibodies to each of them do not cross-react (48). During infection by intestinal parasites, the serumlevel of RMCP-IIrises remarkably (49). Carboxypeptidase A is detectable in both CTMC and BMCMC of mice, but the concentration is more than 100 times higher in CTMC than in the BMCMC(50). AMINES Histamine is contained in mast cells of all the mammalianspecies so far examined. However, the concentration of histamine differs among mast cell subpopulations (11-14). Rat peritoneal CTMC contain 15 pg histamine per cell whereas rat MMC contain only 1-2 pg per cell. Mouse peritoneal CTMC also contain l0 pg of histamine per cell, and mouse BMCMC 0.1 pg per cell (19). Moreover, a significant difference in histamine concentration is detectable among peritoneal CTMCharvested from different inbred strains of mice (T. Nakano, U. Waki, J. Fujita, A. Yamatodani, H. Asai, Y. Kitamura, unpublished data). Unlike histamine, serotonin showsa remarkable species difference (1114). Both CTMCand MMCof rats contain serotonin, and CTMCand BMCMC of mice contain serotonin as well. In contrast, serotonin is not present in mast cells isolated from humanlung. ARACHIDONIC ACIDMETABOLITES Stimulated mast cells produce pharmacologically active metabolites from arachidonic acid. Since arachidonic acid metabolites are produced by various types of cells other than mast cells, purification of mast cells is a prerequisite for the chemical analysis. Purified rat peritoneal CTMC almost exclusively metabolize arachidonic acid through the cyclo-oxygenase pathway; the chief product is prostaglandin D2 (13, 51). On the other hand, rat MMC produce leukotriene C4, leukotriene B4, and prostaglandin D2 (51). Arachidonic acid metabolism in mouse BMCMC is similar to that in rat MMC,that is, they

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generate and release leukotriene C4 along with some leukotriene B4 and small amounts of prostaglandin D2 (52).

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Surface

Antigen

Katz et al developed rat monoclonal antibodies that are useful for distinguishing mast cell subpopulations of mice (53). One antibody, designated B1. l, recognizes a neutral glycosphingolipid, globopentaosylceramide (Forssman glycolipid), on the surface of mouse peritoneal CTMC, but fails to detect this glycosphigolipid in mouse BMCMC. Since mouse BMCMC express the direct precursor of globopentaosylceramide, namely, globotetraosylceramide (globoside) (54), Katz et al proposed mouse BMCMC differed from mouse CTMCin either lacking or having an inactive form of the glycosyltransferase nccdcd to synthesize the more complex globopentaosylceramide 02). Functional

Heterogeneity

Antigens and anti-IgE antibodies that aggregate between IgE molecules induce histamine release from both CTMCand MMC of rats. Whereas the secretagogue compound48/80 does induce histamine release from rat CTMC,it fails to induce release from rat MMC.Similarly, compound 48/80 activates mouse CTMCbut does not activate mouse BMCMC (13, 14). Inhibitors of release of mast cell mediators have practical significance as antiallergic drugs. Theophylline and disodium cromoglycate prevent antigen-stimulated histamine release by rat peritoneal CTMC but not by rat MMC (13, 14). Heterogeneity

in Human Mast Cells

Staining with dyes is not so useful for discriminating between human mast cell subpopulations as it is in the cases of rats and mice, but the immunohistochemicalstaining of tryptase (trypsine-like neutral protease) and chymase(chymotrypsin-like neutral peptidase) distinguishes two mast cell populations in humantissues (55). Mastcells in the skin contain both tryptase and chymase(TC-positive), whereas mast cells in the lung and the intestinal mucosacontain only tryptase (T-positive) (56, 57). Functional heterogeneity is also found between TC-positive and T-positive mast cells. TC-positive mast cells purified from the skin release histamine in response to compound48/80, morphine, and substance P (58, 59), but T-positive mast cells from the lung and intestinal mucosado not (60). Extensive characterization of humanmast cells purified from various tissues has been performed. Characteristics first reported in rodent mast cells have been found in human mast cells as well. For example, the

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presence of chondroitin sulfate proteoglycan in purified humanlung mast cells was recently demonstrated (61, 62). Although the terms CTMC and MMC are not suitable for humanmast cell populations, heterogeneity does exist amongthem.

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ORIGIN

AND DIFFERENTIATION

The generation of mast cells from hematopoietic cells was first shownby using giant granules of beige (C57BL/6-bg-’/bg~, Chediak-Higashi syndrome) mice as a marker. Transplantation of bone marrow cells from C57BL/6-bg~/bg~ to irradiated normal (C57BL/6-+/+) mice resulted development of beige-type mast cells with giant granules (63). Twoother mutant mice are also useful for investigations of mast cell differentiation. v (64) or WCB6Ft-SI/SId (65) are deficient Mice of either WBB6F~-W/W in mast cells. In the first description, only CTMC were counted, but later the depletion of MMC was also reported (66). The absence of mast cells ~ mice and to is attributed to a defect of precursor cells in WBB6F1-W/W a defect of the tissue environment necessary for differentiation of mast d mice (64, 65). cells in WCB6F~-SI/SI There are various types of cells in the bone marrow. In mylaboratory, we demonstrated that mast cells are the progeny of multipotential hemato~ mice as recipients of the cells poietic stem cells by, using WBB6F~-W/W to be tested. In the first experiment, we enucleated spleen colonies which were produced by individual stem cells in irradiated mice. The intravenous transfer of cell suspensions from a single spleen colony resulted in the ~ mice (67). development of mast cells in tissues of WBB6F1-W/W the second experiment, various in vitro hematopoietic cell colonies were produced from the bone marrow of normal WBB6F~-+/+mice, and then cell suspensions from individual colonies were directly injected into the ~ mice (68). The appearance of a mast cell colony skin of WBB6Fl-W/W at the injection site was evidence for the presence of mast cell precursors in the injected cell suspension. More than 40%of the mixed colonies tested, which contained erythroblasts, megakaryocytes, neutrophils, and macrophages,contained mast cell precursors as well (68). Wedid not find morphologically identifiable mast cells in the mixed colonies. However, Nakahata & Ogawa(69) reported mixed colonies that did contain mast cells. This observation also indicates that mast cells can originate from the multipotential hematopoietic stem cells. Most of the progeny of multipotential stem cells such as erythrocytes, platelets, neutrophils, eosinophils, and basophils leave the hematopoietic tissue after they differentiate. However,mast cells do not complete their differentiation in hematopoietic tissue. Nomast cells are detectable in the

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blood, but when blood mononuclearcells are plated in methylcellulose in the presence of IL-3, large mast cell colonies containing more than 500 mast cells appear (70, 71). The mast cell precursors that produce such large colony (CFU-Mast) resemble lymphoid cells, by light microscopy (71). Although the density of mouse peritoneal CTMC is significantly greater than that of small lymphocytes, the density of CFU-Mastis comparable to that of small lymphocytes(71). CFU-Mastinvade the connective or mucosal tissues, proliferate, and differentiate into morphologically identifiable mastcells (72, 73). Since the increase and the subsequent decrease of MMC at the site of helminth infection occur within three weeks(16, 49), the life span of most MMC appears to be limited to 1 or 2 weeks. In contrast, CTMC in the skin (74) and peritoneal cavity (71) of mice have a long life (probably than one year), and some humanlung mast cells produce granules again after degranulation (75). Although most progenies of multipotential hematopoietic stem cells lose the proliferative potential whenthey differentiate fully, some morphologically identifiable CTMC have an appreciable proliferative potential. Sonodaet al (76) identified murine peritoneal CTMCunder the phase contrast microscope, picked up a single CTMC vW/W with the micromanipulator, and injected it into the skin ofWBB6F1mice. Mast cell colonies containing about 2000 mast cells developed in 10 of 168injection sites (76). Proliferation of peritoneal CTMC was also confirmed by using an in vitro culture technique. Nakahata et al (77) and Hamaguchiet al (78) plated purified peritoneal CTMC of mice in methylcellulose; about 20% to 50%CTMC showed clonal growth. In this condition, colonies produced by differentiated CTMC are smaller than colonies produced by CFU-Mast (71, 77). Although CFU-Mastrequired only IL-3 for colony formation (77), both IL-3 and IL-4 are necessary for development of colonies from morphologically identifiable CTMC (78). Colony-forming activity is not limited to peritoneal CTMC; the recent result of Kanakuraet al suggests that CTMC in the skin of mice can also produce mast cell colonies (79). REGULATION OF DIFFERENTIATION

PROLIFERATION/

IL-3 elaborated by helper/inducer T cells appears to induce the mast cells in the intestinal mucosaof mice and rats. Such a mast cell accumulation does not occur in T cell depleted mice (80) and rats (81). On the other hand, a significant increase of MMC (82) and subsequent repulsion of helminth, Strongyloides ratti (83), occurred whenT cell~tepleted nude mice were injected with IL-3. Since IL-4 stimulates the proliferation of both

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mast cells and B cells and the synthesis of IgE (37), it is possible that newly formed mast cells may be armed by lgE antibodies synthesized in their vicinity.

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Fibroblast

Dependent Proliferation

Although the proliferation and differentiation mediated by IL-3 and IL-4 is the only regulatory mechanismunderstood at the molecular level, other regulatory mechanismsless clearly defined may also influence the proliferation and differentiation of mast cells. For example, the concentration of CTMC in the skin of genetically T cell-depleted nude mice is comparable to that of normal congenic mice, implicating the presence of a regulatory mechanismwithout T-cell involvement (84, 85). Fujita et al (86) recently investigated the role offibroblasts on the proliferation of mast cells. Without the addition of IL-3 and IL-4, mouse BMCMC may continue to proliferate on a monolayerof the NIH-3T3fibroblast cell line. Since the fibroblasts synthesize neither IL-3 nor IL-4, and since direct contact of mast cells with fibroblasts is necessary for the proliferation, the supportive effect of fibroblasts appears to be mediated neither by knowngrowth factors (such as IL-3, IL-4) nor by unknowndiffusible substances (86). Fujita et al analyzed the mechanismof mast cell deficiency in WBB6F1W/V~ (86, 87) and WCB6FI-SI/SU (J. Fujita, H. Onoue, Y. Ebi, H. Nakayama, Y. Kanakura, Y. Kitamura, unpublished data) mice by cocul~ turing of BMCMC and fibroblast cell lines. T cells of both WBB6F~W/W and WCB6F~-SI/SUmice may produce IL-3 and IL-4 (J. Fujita, H. Onoue, Y. Ebi, Y. Kitamura, unpublished data). BMCMC develop when bone ~ or marrow cells or blood mononuclear cells of either WBB6F~-W/W WCB6F~-SI/SU mice are cultured in the mediumcontaining IL-3. Therefore, IL-3 and IL-4 do not appear to be involved in the actions of the W and S1 mutantgenes. In addition, several fibroblast cell lines were screened; all six fibroblast cell lines derived from mouseembryos (including the NIH-3T3 cell line) supported the growth of BMCMC derived from WBB6Ft-+/÷ mice. In contrast, none of these mouse embryo-derived v mice (87). fibroblast cell lines supported BMCMC of WBB6F~-W/W synchronizing BMCMC at the G~ phase of the cell cycle, the defect of ~ BMCMC W/W was further characterized as a failure to transit G~ and enter the S phase upon contact with fibroblasts. This suggests that W gene product expressed on the surface of BMCMC is mandatory for the fibroblast-dependent proliferation (87) (Figure BMCMC derived from WCB6F~-SI/SIa mice were maintained as well by the NIH-3T3 cell line as were BMCMC of WCB6F~-+/+mice, indicating the normal function of SI/SU BMCMC. Then, 3T3 fibroblast cell ~ and the control WCB6F lines were established from WCB6F1-SI/SI ~-+/+

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KITAMURA

(+)

Proliferation (-)

(-)

= W Gene Product

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- SI Gene Product Figure i Scheme showing possible expression of W gene product on the surface of BMCMC and SI gene product on the surface of fibroblasts. These molecules are indispensable for flbroblast-dependent proliferation of BMCMC.

embryos. The 3T3 cell lines derived from +/+ embryos induced the G~ to S transition in synchronized ÷/+ BMCMC upon contact, but the 3T3 cell lines derived from Sl/Sl a embryosdid not. This suggests that Sl gene product expressed on the surface of 3T3 fibroblasts is indispensable for the fibroblast-dependent proliferation of BMCMC (J. Fujita, H. Onoue, Y. Ebi, H. Nakayama, Y. Kanakura, and Y. Kitamura, unpublished data) (Figure 1). Suppression

of Differentiation

Unpleasant symptoms accompany overproduction of mast cells such as are observed in patients with urticaria pigmentosa, a benign skin tumor of mastcells. This raises the possibility that suppressionas well as induction of mast cell differentiation may be an important normal regulatory mechanism. Recently Kanakura et al (71) investigated a mechanism for the suppression of mast cell differentiation. Peritoneal CTMC of mice were eradicated by intraperitoneal injection of distilled water, and the regeneration process was analyzed by estimating the changes in numbers of CFU-Mastand morphologically identifiable mast cells. CFU-Mastincreased after the injection of distilled water. Whenpurified peritoneal CTMC were injected two days after the water injection, the increase of CFU-Mastdid not occur (71). In the peritoneal cavity of WBB6F~+/ mice that had been lethally irradiated and rescued by bone marrowinjection of C57BL/6-bg~/b9~ mice, CFU-Mastwere of the bg~/b9J type, but the morphologicallyidentifiable mast cells were of + / + type. Injection of distilled water into the radiation chimeras resulted in development of bg~/bgS-type CTMC with giant granules. The presence of morphologically identifiable CTMC appears to suppress the invasion of CFU-Mastfrom the blood and to inhibit the differentiation of CFU-Mastinto morpho-

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69

logically identifiable mast cells (71). The concentration of CTMC in tissues is too low for the CTMC to be in direct contact with each other. Therefore, the inhibitory effect of differentiated CTMC on invasion and differentiation of CFU-Mastis considered to be mediated by diffusible substance(s) rather than the direct contact. PHENOTYPIC

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Experiments

CHANGES Using

WBB6F~- W/Wv Mice

As already mentioned, mast cell populations are heterogeneous. However, the interrelation between subpopulations had not been systematically investigated until Nakanoet al (19) demonstrated the phenotypic change from BMCMC to CTMC.BMCMC were used since they can be obtained as a homogeneouspopulation and since some of the characteristics of BMCMCare shared by MMC. Nakano et al (19, 88) cultured BMCMC from the bone marrow WBB6F~-+/+mice and transferred them into the peritoneal cavity of v mice. At various times after genetically mast cell~deficient WBB6FIW/W the intraperitoneal transfer, mast cells were recovered from the peritoneal cavity. The density of the original BMCMC is significantly less than that of CTMCharvested from the peritoneal cavity of WBB6F~-+/+mice, but the density increased and becamecomparable to that of the peritoneal CTMC 10-30 weeks after the transfer (88). The recovered mast cells acquired the electron microscopic features of CTMC (19). Furthermore, the histamine content increased more than 20-fold after the transfer. Although the starting BMCMC did not stain with berberine sulfate, the recovered mast cells stained with this fluorescent dye. This suggests that BMCMC acquired the ability to synthesize and store heparin proteoglycan after the intraperitoneal transfer (19, 88). Recently, the change from BMCMC to CTMCwas confirmed by biochemical and immunochemical criteria (40). BMCMC derived from WBB6F~-+/+mice synthesized 350,000-Mr proteoglycan which contained 55,000-Mr chondroitin sulfate glycosaminoglycans. Fifteen weeks after the intraperitoneal transfer, transferred mast cells were recovered ~ mice; these from the peritoneal cavity of the recipient WBB6F~-W/W mast cells synthesized 650,000-Mr proteoglycan containing 105,000-Mr heparin glycosaminoglycans. Although globopentaosylceramide ~Forssman glycolipid) recognized by the B I.I rat monoclonal antibody was weakly expressed on the surface of BMCMC, it was strongly evident 15 ~ mice weeks after the transfer into the peritoneal cavity of WBB6FI-W/W (40). The phenotypic change occurs in the opposite direction as well (89).

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70 KITAMURA Whenpurified peritoneal CTMC of WBB6F~+ / + mice were cultured in methylcellulose with IL-3 and IL-4, about 25%of the CTMC formed colonies, all of whichcontainedboth berberine sulfate-positive and berberine sulfate-negative mast cells. Whenthese mast cells weregrownin suspensionculture, they generated populations that were 100%berberine sulfate-negative, and that synthesized predominantlychondroitinsulfate proteoglycans (89). Whenthese MMC-like cultured mast cells derived from WBB6F~-÷/+ peritoneal CTMC were injected into the peritoneal v mice, the adoptively transferred mast cell popucavity of WBB6F~W/W lation became100%berberine sulfate-positive. In methylcelluloseculture, these "second generation peritoneal CTMC" formedclonal colonies containing both berberine sulfate-positive and -negative mast cells. Thus, clonal mastcell populations, initially derived from a single peritoneal CTMC, exhibited multiple and bidirectional alterations between CTMClike and MMC-like phenotypes(89). The fate of CTMC derived from WBB6F~-+/+ mice was investigated v mice (90). After the injection of in the stomachwall of WBB6F~-W/W single CTMC, mast cells mayappear both in the mucosaand the muscularis propria. Mastcells that appearedin the mucosashowedthe histochemical and electron microscopic features of MMC, whereas the cells that appeared in the museularis propria showedthe features of CTMC (90). In Vitro Experiments Galli et al (91) attempted to induce a phenotypic change in murine BMCMC by adding sodium butyrate to the culture medium.The electron density of cytoplasmicgranules greatly increased, and the histaminecontent increased by up to 50-fold. However,the BMCMC treated with sodiumbutyrate incorporated 35S into chondroitin sulfate proteoglycan but not into heparin proteoglycan (91). So far no one has succeeded switchingthe type ofproteoglycansynthesis by simpleaddition of a defined substance to the medium of a suspensionculture. Stevens and coworkers cultured mouse BMCMC with the Swiss albino mouse-skin~lerivedfibroblast cell line and demonstratedthat the BMCMC acquired CTMC-likephenotype (92). The cells becamesafraninpositive, showedincreased amountsof histamine and carboxypeptidaseA, and synthesized considerably moreheparin proteoglycan (50, 92). These investigators also comparedthe arachidonic acid metabolites produced by BMCMC before and after the coculture with the fibroblasts. When stimulated with antigen, BMCMC cocultured with fibroblasts produce more leukotriene B4 and prostaglandin D2 than did the starting BMCMC (52). Theseresults cannot be explained by the selective expansionof

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particular subpopulation on the fibroblasts, since comparableresults have been obtained by using cloned BMCMC as well (93). Stevens and coworkers added IL-3 to their coculture system (92, 93). Mouse embryo-derived cell lines support the proliferation of BMCMC without IL-3, whereas somefibroblast cell lines derived from the tissues of adult mice do not (87). It is possible that the fibroblast cell line used Stevens and coworkers (which was derived from the skin of Swiss albino mice) does not support BMCMC by itself. However, the presence of IL-3 is not mandatoryfor the phenotypic change, since Fujita et al reported a similar phenotypic change when mouse BMCMC were cocultured with the NIH-3T3 fibroblast cell line in the absence of IL-3 (86). CONCLUSION Heterogeneity of mast cells is found in rats, mice, and humans. The difference between CTMC and MMC observed in rats and mice is a good exampleof such heterogeneity, but likely additional differences are present amongmast cell populations. For example, electron microscopic differences are detectable amongthe CTMC population in the skin of rats (94) and mice (95). The presence of heterogeneity in mast cell populations maybe explained by their unique differentiation process. Mast cell precursors (CFU-Mast) migrating in the blood do not appear to be committed to any subpopulations. Since CFU-Mast differentiate after invading particular tissue, the phenotype of mast cells is influenced by the tissue environment in which differentiation occurs (Figure 2). The mechanismby which the phenotype is determinedby tissue factor(s) remains to be clarified. The phenotypic change between subpopulations is possibly due to the other uniquecharacteristics of mastcells, that is, the extensiveproliferative potential of differentiated mast cells. Somedifferentiated mast cells can BMCMC CFUMast / (~)

~

MMC (~

#~7~ (a)

Environment Suspension Culture Mucosa

(b)

k~_~ CTMC

Muscularis

propria

Peritoneal

Cavity

Skin

Figure 2 Phenotypic changes between subpopulations. Proliferation does not appear necessary for the phenotypic change in direction (a) but it appears necessary for the phenotypic change in direction (b).

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KITAMURA

proliferate in an environmentthat is different from the original environment. The phenotype of the resulting progeny is determined by the tissue environment in which the second differentiation occurs. In other words, some mast cells can experience cycles of proliferation and phenotypic changedue to their extensive proliferative potential (Figure 2).

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ACKNOWLEDGMENTS

The studies described here were supported by grants from the Ministry of Education, Science, and Culture, the Ministry of Health and Welfare, the Mitsubishi Fou!adation, the Asahi Fund for Science and Culture, and the HoanshaFoundation, and the Cell Science Foundation. The author thanks his scientific colleagues whoaided in the preparation of this review by providing preprints and allowing him to quote from their unpublished works, and Drs. Stephen J. Galli and Jun Fujita for reviewing the manuscript.

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of cultured mast cells. J. Parasitol. 73: 155-60 8. Galli, S. J., Dvorak, A. M., Dvorak, H. F. 1984. Basophils and mast cells: morphologicinsights into their biology, secretory patterns, and function. Pro#. Allergy 34:1-141 9. Kitamura, Y., Sonoda, T. 1985. Differentiation of mast cells and basophils. In Hematopoietic Stem Cells, ed. D. W. Golde, F. Takaku, pp. 65-80. New York: Marcel Dekker. 379 pp. 10. Kitamura, ¥., Kanakura, Y., Fujita, J., Nakano, T. 1987. Differentiation and transdifferentiation of mast ceils; a unique memberof the hematopoietic cell family, lnt. J. Cell Clon. 5:108 21 11. Barrett, K. E., Metcalfe, D. D. 1984. Mast cell heterogeneity: evidence and implications. J. Clin. Immunol. 4: 25361 12. Katz, H. R., Stevens, R. L., Austen, K. F. 1985. Heterogeneity of mammalian mast cells differentiated in vivo and in vitro. J. Allergy Clin. ImmunoL76: 25059 13. Lee, T. D. G., Swieter, M., Bienenstock, J., Befus, A_ D. 1985. Heterogeneity in mast cell populations. Clin. Immunol. Rev. 4:14349 14. Pearce, F. L. 1986. Onthe heterogeneity of mast cells. Pharmacology.32:61-71 15. Maximow,A. 1906. Ober die Zellformen des lockeren Bindesgewebes. Arch. Mikrosk. Anat. 67:68(~757

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MAST CELL DIFFERENTIATION 16. Enerb~ick, L. 1986. Mast cell heterogeneity: the evolution of the concept of a specific mucosalmast cell. In MastCell Differentiation and Heterogeneity, ed. A. D. Befus, J. Bienenstock, J. A. Denburg, pp. 1~6. NewYork: Raven. 426 pp. 17. Enerb/ick, L. 1974. Berberine sulfate binding to mast cell polyanions: a cytofluorometric method for the quantitation of heparin. Histochemistry 42: 30113 18. Dimlich, R. V. W., Meineke, H. A., Reilly, F. D., McCuskey,R. S. 1980. The fluorescent staining of heparin in mast cells using berberine sulfate: compatibility with papaformaldehyde or ophthalaldehyde induced-fluorescence and metachromasia. Stain Technol. 55:212 23 19. Nakano, T., Sonoda, T., Hayashi, C., Yamatodani, A., Kanayama, Y., Yamamura T., Asai, H., Yonezawa,T., Kitamura, Y., Galli, S. J. 1985. Fate of bone marrow-derivedcultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically v mast cell-deficient W/W mice: evidence that cultured mast cells can give rise to both "’connective tissue type" and "mucosal" mast cells. J. Exp. Med. 162: 1025-43 20. Combs,J. W., Lagunoff, D., Benditt, E. P. 1965. Differentiation and proliferation of Biol. embryonic mast cells of the rat. J. Cell 25:57792 21. Ginsburg, H., Sachs, L. 1963. Formation of pure suspension of mast cells in tissue culture by differentiation of lymphoid cells Inst. from 31 the: 1-39 mouse thymus. J. Natl. Cancer 22. Ishizaka, T., Okudaira, H., Mauser, L. E., Ishizaka, K. 1976. Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells. J. Immunol. 116:747-54 23. Ishizaka, T., Adachi, T., Chang, T. H., Ishizaka, K. 1977. Developmentof mast cells in vitro. II. Biologic function of cultured mast cells. J. Immunol. 118: 211-17 24. Hasthorpe, S. 1980. A hemopoietic cell line dependent upon a factor in pokeweedmitogen-stimulated spleen cell conditioning medium.J. Cell. Physiol. 105: 379-84 25. Schrader, J. W., Lewis, S. J., ClarkLewis, I., Culvenor, J. G. 1981. The persisting (P) cell: histamine content, regulation by a T cell-derived factor, origin from a bone marrow precursor, and relationship to mast cells. Proc. NatL Acad. Sci. USA 78:323~7 26. Nagao, K., Yokoro, K., Aaronson, S. A.

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1981. Continuous lines of basophil/mast cells derived from normal mouse bone marrow. Science 212:333-35 27. Nabel, G., Galli, S. J., Dvorak, A. M., Dvorak, H. F., Cantor, H. 1981. Inducer T lymphocytes synthesize a factor that stimulates proliferation of cloned mast cells. Nature 291:332-34 28. Tertian, G., Yung, Y. P., Guy-Grand, D., Moore, M. A. S. 1981. Long-term in vitro culture of murinemast cells. I. Description of a growth-factor dependent culture technique. J. Immunol.127: 78894 29. Razin, E., Cordon-Cardo, C., Good, R. A. 1981. Growth of a pure population of mousemast cells in vitro with conditioned medium derived from concanavalin A-stimulated splenocytes. Proc. Natl. Acad. Sci. USA 78:2559~61 30. Ihle, J. N., Keller, J., Oroszlan, S., Henderson, L. E., Copeland, T. D., Fitch, F., Prystowsky, M. B., (3oldwasser, E., Schrader, J. W., Paraszynski, E., Dy, M., Lebel, B. 1983. Biologic properties of homogeneousintcrleukin 3. I. Demonstration of WEHI-3growthfactor activity, mast cell growth-factor activity, P cell-stimulating factor activity, colony-stimulating factor activity and histamine-producing cell-stimulating factor activity. J. lmmunol. 131: 28~87 31. Fung, M. C., Hapel, A. J., Ymer, S., Cohen, D. R., Johnson, R. M., Campbell, H. D., Young, I. G. 1984. Molecular cloning of cDNAfor murine interleukin-3. Nature 307:233 37 32. Yokota, T., Lee, F., Rennick, D., Hall, C., Arai, N., Mosmann,T., Nabel, G., Cantor, H., Arai, K. 1984. Isolation and characterization of a mousecDNAclone tttat expresses mast-cell growth-factor activity in monkeycells. Proc. Natl. Acad. Sei. USA 81:1070-74 33. Smith, C. A., Rennick, D. M. 1986. Characterization of a murine lymphokine distinct from interleukin 2 and interleukin 3 (IL-3) possessing a T-cell growth factor activity and a mast-cell growth factor activity that synergizes with IL-3. Proc. Natl. Aead. Sci. USA 83:1857-61 34. Lee, F., Yokota, T., Otsuka, T., Meyerson, P., Villaret, D., Coffman, R., Mosmann, T., Rennick, D., Roehm, N., Smith, C., Zlotnik, A., Arai, K. 1986. Isolation and characterization of a mouse interleukin cDNAclone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cellstimulating activities. Proc. Natl. Acad. Sei. USA 83:2061-65

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35. Noma,Y, Sideras, P., Naito, T., Bergstedt-Lindquist, S., Azuma,C., Severinson, E., Tanabe,T., Kinashi, T., Matsuda, F., Yaoita, Y., Honjo,T. 1986. Cloning of eDNAencoding the murine IgG1induction factor by a novel strategy using SP6 promoter. Nature 319: 640-46 36. Grabstein, K., Eiseman,J., Moehizuki, D., Shanebeek,K., Conlon, P., Hopp, T., March,C., Gillis, S. 1986. Purification to homogeneity of B cell stimulating factor. Amoleculethat stimulates proliferation of multiple lymphokinedependentcell lines. J. Exp. Med.163: 1405-14 37. Paul, W.E., Ohara, J. 1987. B-cell stimulatoryfactor-l/interleukin 4. Ann. Rev. Immunol.5:429-59 38. Haig, D. M., MeKee,T. A., Jarrett, E. E. E., Woodbury,R., Miller, H. R. P. 1982. Generationof mucosalmast cells is stimulatedin vitro by factors derived from T cells of helminth-infectedrats. Nature 300:188-90 39. Saito, H., Hatake, K., Dvorak,A. M., Leiferman, K. M., Donnenberg,A. D., Arai, N., lshizaka,K., lshizaka,T. 1988. Selective differentiation and proliferation of hematopoietiecells inducedby recombinanthumaninterleukins. Proc. Natl. Acad. Sei. USA85:2288-92 40. Otsu, K., Nakano, T., Kanakura, Y., Asai, H., Katz, H. R., Austen, K. F., Stevens,R. L., Galli, S. J., Kitamura,Y. 1987. Phenotypicchangesof bone marrow-derived mast cells after intraperitoneal transfer into W~Wv micethat are geneticallydeficientin mastcells. J. Exp. Med. 165:61:%27 41. Enerbfick,L., Kolset, S. O., Kusche,M., ~jerpe, A., Lindahl, U. 1985. Glycosaminoglycans in rat mucosalmast cells. Biochem.J. 227:66168 42. Stevens,R. L., Lee, T. D.G., Seldin, D. C., Austen,K. F., Befus,A. D., Bienenstock, J. 1986. Intestinal mucosalmast cells from rats infected with Nippostrongylusbrasiliensis containproteaseresistant chondroitin sulfate di-B proteoglycans. J. Immunol.137:291~5 43. Jarrett, E. E. E., Haig, D. M. 1984. Mucosalmastcells in vivo andin vitro. Immunol. Today 5:115 19 44. Razin, E., Stevens, R. L., Akiyama,F., Schmid,K., Austen,K. F. 1982. Culture from mousebone marrowof a subclass of mastcells possessinga distinct chondroitin sulfate proteoglycanwith glycosaminoglycansrich in N-acetylgalactosamine-4,6-disulfate.J. Biol. Chem. 257:7229-36 45. Katz, H. R., Austen, A. F., Caterson,

B., Stevens,R. L. 1986. Secretorygranules of heparin-containingrat serosal mastcells also possesshighly sulfated chondroitin sulfate proteoglycans. J. Biol. Chem.261:13393-96 46. Lagunoff,D., Pritzl, P. 1976. Characterization of rat mast cell granule proteins. Arch. Biochem.Biophys. 173: 554-63 47. Woodbury,R. G., Gruzenski, G. M., Lagunoff, D. 1978. Immunofluorescent localization of a serine proteasein rat small intestine. Proc. Natl. Acad.Sci. USA 75:2785-89 48. Woodbury,R. G., Everitt, M. T., Neurath, H. 1981. Mast cell proteases. MethodsEnzymol. 80:588-609 49. Woodbury,R. G., Miller, H. R. P., Huntley, J. F., Newlands, G. F. J., Palliser, A. C., Wakelin,D. 1984. Mucosal mastcells are functionallyactive during spontaneousexpulsionof intestinal nematodeinfections in rat. Nature312: 450-52 50. Serafin, W.E., Dayton,E. T., Gravallese, P. M., Austen,K. F., Stevens, R. L. 1987. CarboxypeptidaseA in mouse mastcells: identification, characterization, anduse as a differentiation marker. J. lmmunol.139:3771-76 51. Heavey,D. J., Ernst, P. B., Stevens, R. L., Befus, A. D., Bienenstock, J., Austen, K. F. 1988. Generationof leukotriene C4, leukotriene B4, and prostaglandin D2by immunologically activated rat intestinal mucosa mastcells. J. lmmunol. 140:1953-57 52. Levi-Schaffer, F., Dayton, E. T., Austen,K. F., Hein,A., Caulfield,J. P., Gravallese, P. M., Liu, F. T., Stevens, R. L. 1987. Mousebone marrow-derived mast cells cocultured with fibroblasts: morphologyand stimulation-induced release of histamine, leukotriene B4, leukotriene C4,and prostaglandinD2. J. Immunol. 139:3431-41 53. Katz, H. R., LeBlanc,P. A., Russell, S. W.1983.Twoclasses of mousemastcells delineated by monoelonalantibodies. Proe. Natl. Acad. Sci. USA80:5916-18 54. Katz, H. R., Austen,K. F. 1986. Plasma membrane and intracellular expression of globotetraosylceramide (globoside) mousebone marrow-derivedmast cells. J. Immunol.136:3819-24 55. Irani, A. A., Sehechter,N. M., Craig, S. S., DeBlois,G., Schwartz,L. B. 1986. Twotypes of humanmast cells that have subsets with distinct neutral protease compositions. Proc. Natl. Acad. Sci. USA 83:4464-68 56. Schwartz,L. B., Irani, A.A., Roller, K., Castells, M.C., Scheehter,N. M.1987.

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MAST CELL DIFFERENTIATION Quantitation of histamine, tryptase, and cttymase in dispersed truman T and TC mast cells. J. Immunol. 138: 261115 57. Irani, A. A., Craig, S. S., DeBlois, G., Elson, C. O., Schechter, N. M., Schwartz, L. B. 1987. Deficiency of the tryptase-positive, chymase-negativemast cell type in gastrointestinal mucosaof patients with defective T lymphocyte function. J. Immunol. 138:4381 86 58. Benyon, R. C., Lowman, M. A., Church, M. K. 1987. Humanskin mast cells: their dispersion, purification, and secretory characterization. J. Immunol. 138:861~7 59. Lawrence, I. D., Warner, J. A., Cohan, V. L., Hubbard, W. C., Kagey-Sobotka, A., Lichtenstein, L. M. 1987. Purification and characterization of human skin mast cells: evidence for humanmast cell heterogeneity. J. Imrnunol. 139: 3062-69 60. Befus, A. D., Dyck, N., Goodacre, R., Bienenstock, J. 1987. Mastcells from the humanintestinal lamina propria: isolation, histochemical subtypes, and functional characterization. J. Immunol.138: 2604-10 61. Thompson,H. L., Schulman, E. S., Metcalfe, D. D. 1988. Identification ofchondroitin sulfate E in humanlung mast cells. J. Immunol. 140:2708-13 62. Stevens, R. L., Fox, C. C., Lichtenstein, L. M., Austen, K. F. 1988. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of humanlung mast cells. Proc. Natl. Acad. Sci. USA85: 2284-87 63. Kitamura, Y., Shimada, M., Hatanaka, K., Miyano, Y. 1977. Development of mast cells from grafted bone marrow cells in irradiated mice. Nature 268: 44243 64. Kitamura, Y., Go, S., t-latanaka, K. 1978. Decrease of mast cells in °W/W mice and their increase by bone marrow transplantation. Blood 52:447-52 65. Kitamura, Y., Go, S. 1979. Decreased production of mast cells in Sl/Sl d anemic mice. Blood 53:492-97 66. Crowle, R. K., Reed, N. D. 1984. Bone marroworigin ofmucosal mast cells. Int. Archs. Allergy Appl. lmmun. 73:242-47 67. Kitamura, Y., Yokoyama, M., Matsuda, H., Ohno, T., Mori, K. J. 1981. Spleen colony-forming cell as common precursor for tissue mast cells and granulocytes. Nature 291 : 159~50 68. Sonoda, T., Kitamura, Y., Haku, Y., Hara, H., Mori, K. J. 1983. Mast cell precursors in various haematopoietic

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colonies of mice producedin vivo and in vitro. Br. J. Haematol. 53:6ll-20 69. Nakahata, T., Ogawa, M. 1982. Identification in culture of a class of hemopoietic colony-formingunits with extensive capability to self-renew and generate multipotential hemopoietic colonies. Proc. Natl. Acad. Sci. USA 79:3843-47 70. Kobayashi, T., Nakano, T., Nakahata, T., Asai, H., Yagi, Y., Tsuji, K., Komiyama, A., Akabane, T., Kojima, S., Kitamura, Y. 1986. Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W~Wv mice: evidence that a commonprecursor can give rise to both "connective tissue-type" and "mucosal" mast cells. J. Immunol. 136: 1378-84 71. Kanakura, Y., Kuriu, A., Waki, N., Nakano, T., Asai, H., Yonezawa, T., Kitamura, Y. 1988. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of miceafter injection of distilled water: evidencethat mast cells suppress differentiation of bone marrow-derived precursors. Blood 71:573-80 72. Kitamura, Y., Matsuda, H., Hatanaka, K. 1979. Clonal nature of mast cell clusv mice after bone ters formed in W/W marrow transplantation. Nature 281: 154-55 73. Hatanaka, K., Kitamura, Y., Nishimune, Y. 1979. Local development of mast cells from bone marrow-derived precursors in the skin of mice. Blood53: 142-47 74. Sonoda, T., Tsuyama, K., Kitamura, Y., Tanooka, H. 1982. Different effects of dimethylbenz(~)anthracene and tetradeeanoylphorbol acetate on differentiation of mast cells in the skin of mice. Am. J. Pathol. 106:312-17 75. Dvorak, A. M., Schleimer, R. P., Lichtenstein, L. M. 1988. Humanmast cells synthesize new granules during recovery from degranulation. In vitro studies with mast cells purified from human lungs. Blood 71:76-85 76. Sonoda, T., Kanayama, Y., Hara, H., Hayashi, C., Tadokoro, M, Yonezawa, T., Kitamura, Y. 1984. Proliferation of peritoneal mast cells in the skin of ~ W]W mice that genetically lack mast cells. J. Exp. Med. 160:138-51 77. Nakahata, T., Kobayashi, T., Ishiguro, A., Tsuji, K., Naganuma,K., Ando, O., Yagi, Y., Tadokoro, K., Akabene, T. 1986. Extensive proliferation of mature connective-tissue type mast cells in vitro. Nature 324:65q57

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78. Hamaguchi, Y., Kanakura, ¥., Fujita, J., Takeda, S., Nakano, T., Tarui, S., Honjo, T., Kitamura, Y. 1987. Interleukin 4 as an essential factor for in vitro clonal growth of murine connective tissue type mast cells. J. Exp. Med. 165: 268-73 79. Kanakura, Y., Sonoda, S., Nakano, T., Fujita, J., Kuriu, A., Asai, H., Kitamura, Y. 1987. Formation of mast cell colonies in methylcellulose by mouse skin cells and development of mucosallike mast cells from the cloned cells in the gastric mucosa of W/W"mice. Am. J. Pathol. 129:168-76 80. Ruitenberg, E. J., Elgersma, A. 1976. Absenceof intestinal mast cell response in congenitally athymic mice during Trichinella spiralis infection. Nature 264: 258~50 81. Mayrhofer, G., Fisher, R. 1979. Mast cells in severely T-cell depleted rats and the response to infestation with Nippostrongylus brasiliensis. Immunology37: 145-55 82. Abe, T., Ochiai, H., Minamishima, Y., Nawa, Y. 1988. Induction of intestinal mastocytosis in nude mice by repeated injection of interleukin-3. Int. Archs. Allergy Appl. lmmun. 86:35~58 83. Abe, T., Nawa, Y. 1988. Wormexpulsion and mucosal mast cell response induced by repetitive IL-3 administration in Strongyloides ratti-infected nude mice. lmmunolo.qy 63:181 85 84. Viklick~, V., ~ima, P., Prichard, H. 1973. Onthe origin of mast cells in adult life. Folia Biol. 19:247-51 85. Keller, R., Hess, M. W., Riley, J. F. 1976. Mast cells in the skin of normal, hairless and athymic mice. Experientia 32:171 72 86. Fujita, J., Nakayama, H., Onoue, H., Kanakura, Y., Nakano, T., Asai, H., Takeda, S., Honjo, T., Kitamura, Y. 1988. Fibroblast-dependent growth of mousemast cells in vitro: duplication of mast cell depletion in mutant mice of v genotype. J. Cell. Physiol. 134: W/W 78-84 87. Fujita, J., Nakayama, H., Onoue, H., Ebi, Y., Kanakura, Y., Kuriu, A., Kita° mousemura, Y. 1988. Failure of W/W derived cultured mast cells to enter S phase upon contact with NIH/3T3 fibroblasts. Blood 72:463 68 88. Nakano, T., Kanakura, Y., Asai, H.,

Kitamura, Y. 1987. Changing processes from bone marrow-derived cultured mast cells to connective tissue-type mast cells in the peritoneal cavity of mastcell° mice: association of prodeficient W/W liferation arrest and differentiation. J. Immunol. 138:544~9 89. Kanakura, Y., Thompson, H. L., Nakano, T., Yamamura, T., Asai, H., Kitamura, Y., Metcalfe, D. D., Galli, S. J. 1988. Multiple bidirectional alterations of phenotype and changes in pro" liferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells. Blood 72:877 85 90. Sonoda, S., Sonoda, T., Nakano, T., Kanayama,Y., Kanakura, Y., Asai, H., Yonezawa, T., Kitamura, Y. 1986. Developmentofmt/cosal mast cells after injection of a single connective tissuetype mast cell in the stomach mucosa of genetically mast cell-deficient °W~W mice. J. Immunol. 137:131%22 91. Galli, S. J., Dvorak, A. M., Marcum,J. A., Ishizaka, T., Nabel, G., Der Simonian, H., Pyne, K., Goldin, J. M., Rosenberg, R. D., Cantor, H., Dvorak, H. F. 1982. Mast cell clones: a modelfor the analysis of cellular maturation. J. Cell Biol. 95:435-44 92. Levi-Schaffer, F., Austen, K. F., Gravallese, P. M., Stevens, R. L. 1986. Coculture of interleukin 3-dependent mousemast cells with fibroblasts results in a phenotypic change of the mast cells. Proe. Natl. Aead. Sei. USA83:6485-88 93. Dayton, E. T., Pharr, P., Ogawa, M., Serafin, W. E., Austen, K. F., LeviSchaffer, F., Stevens, R. L. 1988. 3T3 fibroblast induce cloned interleukin 3dependent mousemast cells to resemble connective tissue mast cells in granular consistency. Proc. Natl. Acad. Sci. USA 85:569 72 94. Aldenborg,F., Enerb~ick, L. 1988. Histochemical heterogeneity of dermal mast cells in athymic and normal rats. Histochem. J. 20:19-28 95. Yamamura, T., Nakano, T., Fukuzumi, T., Waki, N., Asai, H., Yoshikawa, K., Kitamura, Y. 1988. Electron microscopic changes of bone marrow-derived cultured mast cells after injection into the skin of genetically mast cell-deficient W/W~ mice. J. Invest. Dermatol.91 : 26973

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THE CELLULAR BASIS OF T-CELL MEMORY Jean-Charles Cerottini and H. Robson MacDonald Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland

INTRODUCTION One of the major characteristics of the immunesystemis its ability to react more quickly and more intensely on the second exposure to an antigen. This characteristic, usually referred to as immunologicmemory,was first observed in studies of antibody production in vivo (reviewed in 1). The secondary, or anamnestic, response to an antigen not only results in the production of increased levels of antibodies, but these antibodies also generally have a higher affinity for antigen comparedto those antibodies produced during the primary response (2). Memoryat the B-cell level generally acceptedas the result of selection and specific expansionof clones secreting high affinity antibody molecules (3, 4). Molecularstudies suggest that somatic hypermutation of antibody variable-region genes during the primary response maybe responsible for the occurrence of B cells expressing high affinity receptors (reviewed in 5). However,the differentiation pathway involved in the generation of memoryB cells is still poorly understood. In particular, it is not clear how, in the primary response, someof the B cells differentiate into terminal plasmacells and others into memorycells. While there is ample evidence that immunologicmemoryalso involves T cells, our understanding of the molecular and cellular basis of T-cell secondary responses is quite limited. Following early studies demonstrating that cytolytic T lymphocytes(CTL)are not necessarily terminal cells but maydifferentiate, at least in vitro, into cells exhibiting the expected properties of memorycells (6), attempts have been made to define quantitative and qualitative differences betweenmemory T cells and T cells that have not yet been stimulated by antigen (which are often designated as 77 0732~)582/89/0410~077502.00

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naive or virgin T cells). Until recently, detailed analysis of these differences has been hamperedby the lack of suitable surface markers allowing identification of memoryT cells. As discussed in this chapter, recent studies indicate that naive and memory T cells can now be distinguished phenotypically by surface markers.

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Muchof the information concerning the differentiation and ultimate fate of T cells in primary or secondary responses has been obtained in Studies + T cells appear to exhibit cytolytic potenconcerning CTL. As most CD8 tial, it is likely that the results of these studies are applicableto the majority of T cells expressing this surface phenotype. Muchless is knownon the properties of CD4+ T cells involved in memory. That some form of memoryexisted in CTLresponses was first indicated in studies concerning the formation of CTL in mice immunized with allogeneic tumor cells (reviewed in 6). The level of CTLmemoryresulting from primary immunization was further analyzed by measuring the development of CTLactivity in cultures of lymphoid cells from immunized mice restimulated with the appropriate antigens (7). Althoughaccelerated kinetics and higher peak levels of CTLactivity could be readily demonstrated in these secondary responses in vitro, these studies provided little insight into the cellular basis of T-cell memory.Withthe development of appropriate limiting dilution microculture systems (8), however, became possible to determine the frequencies of antigen-specific CTL precursors (CTL-P). Using this approach, direct evidence has been obtained that the frequency of CTL-Pagainst a given antigen is increased after primary immunization (9). In someinstances, this increase is quite substantial. For example, primary immunization of female mice with male cells bearing the H-Yminor transplantation antigen has been found to result in a > 20-fold increase in the frequency of H-Yspecific CTL-P.In most viral systems studied, primary immunization leads to a > 10-fold increase in CTL-Pfrequencies. In contrast, primary immunization against major histocompatibility complex (MHC)antigens generally results in modest( < 3-fold) increase in CTL-Pfrequencies. Whileit is clear that T-cell memorymayinvolve increased frequencies of antigen-specificT cells, there is also evidence,albeit indirect, for qualitative differences between memoryand naive T cells. For example, it has been shownthat the antigenic requirements for the generation of alloreactive CTLin vitro are quite different between normal and alloimmunelymphoid cell populations (6). Althoughthe molecular basis for these differences has yet to be defined, it is conceivablethat they are related, at least in part, to

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the affinities of the antigen receptors expressed by naive and memory T cells. Presently, there is no direct methodto measurethe affinities of Tcell receptors (TCR).However, relative affinities of TCRcan be indirectly comparedby varying the concentration of antigen/MHCcomplexon the stimulator cell (10, 11) or by neutralizing the avidity-enhancingfunction of accessorymoleculessuch as CD4or CD8(12, 13). Usingthis approach, it has beenfoundthat the proportionof alloreactiveCTL-P bearingrelatively high affinity TCRis increasedafter appropriateimmunization in vivo (12). As discussed below, there is evidence that memoryT cells express increased levels of the surface moleculesthoughtto be involvedin T-cell adhesion or activation (such as CD2,LFA-1,and LFA-3).The enhanced responsivenessof memory T cells to antigenic stimulation maythus involve several other properties, in additionto expressionof high affinity TCR.In this context, it is noteworthythat alloreactive or virus-specific memory CTL-P have beenreported to respondto activation by nonspeeific stimuli such as phorbol ester and calcium ionophore in conditions under which alloreactive CTL-Pfrom normal mice are nonresponsive(14-16). Moreover, humanmemory T cells appear to respond better than naive T cells to activation by anti-CD3antibodies, although both cell types express comparable amountsof CD3on the cell surface (17). As these latter results havenot beenconfirmedin similar studies performedin the mouse,further workis neededto ascertain whetherdifferentiation into memory cells is accompanied by qualitative or quantitative changesin the T-cell requirementfor activation. In addition to increased proliferation, secondaryT-cell responses to antigen maybe characterized by production of larger amountsof lymphokines. In most instances, it has beendifficult to distinguish whether this increased production can be accountedfor solely by the increased frequencyof antigen-specific T cells in immunizedlymphoidcell populations. As discussed below, there is nowevidence that memory T cells produce, on a per cell basis, moreIFN-yor IL-4 than do naive T cells uponstimulationwith specific or nonspecificstimuli. As sucha difference does not applyto the productionof IL-2, these results raise the possibility that the expression of the IFN-7and IL-4 genes can be modulateddifferently in naive and memory T cells and independentlyof the expression of the IL-2 gene. PGP-1 (LY24) AS MARKER OF MURINE MEMORY T CELLS Pgp-1wasoriginally described as a major integral membrane glycoprotein on murinefibroblasts andperitoneal phagocyticcells (henceits designation

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phagocyte glycoprotein 1) (18, 19). Also designated Ly24 (20), Pgpa mol wt of 80,000-95,000 and is encoded by a locus on chromosome2 (21). Twoallelic forms have been defined using mousealloantibodies (22). The glycoprotein has been identified in a wide variety of tissues including brain, liver, kidney, and lung (23). In hematopoietictissues, Pgp-1is found in highest amounts in the bone marrow. Whenbone marrow cells are stained with antibody to Pgp-1, mature myeloid cells stain very brightly, committed progenitors brightly, and pluripotent stem cells and lymphoid cells relatively weakly(23, 24). Whileit is expressed in most prothymocytes (25), only about 5%of adult thymocytes are Pgp-1+, at least in certain mousestrains (23). Early reports on Pgp-1 expression amongthymocytes suggested that the antigen was restricted to the minor (CD4- CD8-) subset knownto contain immature thymocytes (23, 26, 27). Unlike adult thymocytes, the great majority of fetal thymocytes in AKRmice are Pgp1+ + at day 13-14 of gestation (27). Thereafter, the proportion of Pgp-1 thymocytes declines, reaching adult levels by day 19 of gestation. On the basis of these findings, it has been proposed that at least someof the PgpI + cells within the thymus are progenitors of mature thymocytes (27). However, direct evidence for such a developmental relationship has not been obtained yet (28). It is nowclear that Pgp-1 + expression is not confined to subpopulations of immature thymocytes but can be found on a small percentage of mature (CD4+ CD8+ and CD4- CD8+) cells in the thymus (28-31). Moreover, peripheral T cells contain a significant proportion of Pgp-1 + ceils (29, 30, 32). It should be emphasizedthat the difference in Pgp-1 expression among peripheral T cells is quantitative rather than qualitative. Two-colorflow microfluorometry analysis of peripheral T cells reveals two well-defined subpopulations among CD4+ and CD8+ cells, i.e. a major subpopulation that stains weakly (designated Pgp-1 ) and a minor (Pgp-1 +) subpopulation that stains brightly. Pgp-1+ T cells have been identified in blood, spleen, and lymphnode (29). While there is no significant difference in the percentages of positive cells within these lymphoidcell compartments, it has been shownthat the relative numberof Pgp-1+ cells increases + and CD8 + subsets. This progressively as a function of age in both CD4 increase can be accelerated by surgical thymectomyof adult mice (29). A possible relationship between Pgp-1- and Pgp-1 + peripheral T cells was first suggested by the demonstration that Pgp-1- CD8+ lymph node cells became rapidly Pgp-1 + after mitogenic or antigenic stimulation in vitro (29). Moreover,analysis of Pgp-1expression by these stimulated cells revealed that this phenotype was stable for as long as the cultures could be viably maintained without restimulation (3 weeks). As it is well established that CD8+ CTLgenerated in allogeneic MLCcultures revert to

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small-sized quiescent lymphocytes under similar culture conditions (6), these findings suggested that Pgp-1 is a marker not only of recently activated T cells but also of the progeny of these activated cells, including memorycells. Direct evidence that Pgp-1 identifies recently activated T cells in vivo was obtained by examining the surface phenotype of the CTLgenerated in mice undergoingrejection of an intraperitoneal tumorallograft. In this + Pgp-1 + (29). It system, virtually all of the CTLwere found to be CD8 noteworthy that the CTLgenerated under these conditions represent a relatively homogeneous noncycling population of small-sized lymphocytes. These data thus suggest that Pgp-1 identifies recently activated T cells independent of the usual criteria of blast transformation and DNA synthesis. As discussed previously, immunization may result in increased frequencies of antigen-specific CTL-P.Several studies indicate that these cells are mainly found in the Pgp-1 + subpopulation (29, 32, 33). For example, the frequency of H-Yspecific CTL-Pin CD8+ spleen cells from female mice immunizedwith male cells has been found to be enriched 30-fold in + antigen-specific the Pgp-1 + subpopulation (29). Data pertaining to CD4 T cells are more limited at present; however, there is already evidence in several antigenic systems that the Pgp-1+ CD4+ subpopulation from immunizedmice is enriched in precursor cells directed against the immunizing antigen (33). Further evidence in favor of the expression of Pgp-1 by memoryT cells has been provided by studies of the surface phenotype of alloimmune CTL-Pbearing high affinity TCR.As mentioned previously, measurement of the resistance of CD8+ CTLclones to inhibition of cytolytic activity by anti-CD8 antibodies provides a way to indirectly assess the frequency of CTLwith high affinity TCR.Using this approach, it has been found that the CD8+ Pgp-I ÷ subpopulation from alloimmune spleen cells was highly enriched for such cells compared to the CD8+ Pgp-1- subpopulation (32). In contrast, no difference in susceptibility to inhibition by anti-CD8 antibodies was observed when Pgp-l + and Pgp-1- subpopulations from normal mice were analyzed by the same method. Taken together with earlier studies (12), these data strongly suggest that the Pgp-I + subset in immunizedmice is enriched for antigen-specific T cells bearing high affinity TCR. Evidence that affinity (rather than density) of TCRaccounts for this difference comes from the observation that CD3staining (and hence presumablyTCRdensity) is not significantly different in the Pgp-1 + versus + or CD8 + T cells (33). Pgp-l- subpopulations of either CD4 Additional functional differences between Pgp-1 + and Pgp-1- T cells have been revealed by studies of lymphokine production by these cells

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upon stimulation with nonspecific stimuli (33, 34). While IL-2 production was similar in the two subpopulations, Pgp- 1 ÷ cells (irrespective of their CD4or CD8phenotype) produced greater amounts of IFN-~, IL-4, and (to a lesser extent) IL-3 comparedto Pgp-1- cells. Since the difference lymphokine production was observed after stimulation with nonspecific stimuli, it is likely that these data reflect enhancedsecretion of IFN-7and IL-4 by individual Pgp-1 ÷ T cells. Moreover,these data indicate that there is no apparent relationship between the subpopulations of CD4÷ T cells ÷ defined by Pgp-1 and the recently described Thl and ThEsubsets of CD4 T cell clones (35) since IFN-~and IL-4, which are both producedin greater + Pgp-l + cells, are expressed by mutually exclusive subamounts by CD4 sets of T-cell clones. Taken together, the results discussed in this section indicate that + and CD8+ expression of Pgp-1 identifies discrete subpopulations of CD4 T cells that have both the quantitative and qualitative properties of memory T cells. Accordingly, a simplified modelfor the cellular differentiation pathway leading to the formation of memoryT cells can be proposed. Mature T ceils produced in the thymus are Pgp-l-. Whenthese (naive) cells seed the peripheral lymphoid tissues, they remain Pgp-1- until they are triggered by antigen. Those with sufficiently high affinity TCRto be triggered acquire Pgp-1expression as they differentiate into effector cells and (ultimately) memoryT cells. Once acquired, Pgp-1 expression would be stable for the lifetime of the cell. A prediction of this modelis that the Pgp-1+ subpopulation of peripheral T cells should include recently activated lymphocytes as well as memory + Pgp-1+ T cells cells. In this context, it is noteworthythat 10-20%of CD8 are medium- to large-sized lymphocytes, whereas the vast majority of + Pgp-1- T cells are small (29). However, most of the cells in both CD8 groups are not actively cycling. Whetherthe large Pgp-1+ T cells represent recently activated cells has yet to be determined. Finally, despite current lack of knowledgeof the function of the Pgp-1 glycoprotein, it is clear that Pgp-1is a useful markerfor the identification and separation of T lymphocytes in the mouse. It should be noted, however, that Pgp-1 expression by mature T cells exhibits considerable heterogeneity amongmousestrains. While the conclusions presented here regarding the usefulness of this marker apply to C57BL/6mice, it appears that, in certain other strains, expression of Pgp-I does not resolve T cells into two distinct subsets (28, 31). In these strains, further study is needed to identify other surface markers of memoryT cells. As discussed in the next section, a variety of such markers have been identified in humans. It is thus likely that, in the near future, the phenotypic identification of memoryT cells will be feasible in all mousestrains.

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Human studies Human T lymphocytesalso express the Pgp-1 glycoprotein (36). Although + and there is quantitative heterogeneity in Pgp-1 expression amongCD4 + T cells, the differencesare too small to allow definition of distinct CD8 subpopulations. However,two-color flow microfluorometryanalysis using antibodies against a variety of surface moleculesexpressedby T cells has revealed coordinate enhancedexpression of Pgp-1and five other markers (LFA-I, LFA-3, CD2, CDw29and UCHL1)on a subpopulation of cells (37). Amongthem, LFA-3and UCHL1 appear to discriminate two subpopulations better than the other markers because their level of expressionon adult T cells exhibits a clear bimodaldistribution. It should be pointed out that the subpopulationsdefined by these coor+ T cells, to dinately expressed moleculescorrespond, at least in CD4 the "helper-inducer" and "suppressor-inducer"T-cell subsets previously identified by the monoclonalantibodies 4B4(directed against CDw29) and 2H4(directed against CD45R),respectively (38, 39). Whileit has generallyassumed that these subsetsrepresent different lineagesof T cells, recent data strongly suggestthat they correspondto different maturational stages (17). Accordingto this newinterpretation, naive T cells are found + subset, whereasactivated and memory in the CD45R T cells are confined + to the CDw29subset. A major finding in favor of this contention is the demonstrationthat + subset convert to the phenotype peripheral blood T cells of the CD45R + of the CDw29subset upon stimulation in vitro, whereasconversion in the opposite direction is not observed.Thesechangesaffect in particular the expression of someof the CD45polypeptides. The CD45complex, previously knownas humanleukocyte common antigen or T200, consists of four molecularspecies of 220, 205, 190, and 180 kd (reviewedin 40). Geneticstudies indicate that a single geneproducesthe different formsof CD45by alternate splicing of RNA(41) and by posttranslational processing (42). The 205/220-kdmolecular species have been designated CD45R becauseof their restricted distribution on B cells, NKcells, and subsets of T cells. Whilesomemonoclonalantibodies recognizeepitopes on all forms of CD45,others react only with CD45R (such as 2H4)(38) or the 180-kdmolecular species (UCHL1) (43), respectively. Expression of CD45R and UCHL1 on T lymphocytesis mutually exclusive: while 40+ or UCHL1 50%of peripheral blood T cells are CD45R +, < 1%of these (small-sized)cells are positive for both markers.Moreover,clonal analysis + CD45R + T cells, after mitogenic stimuhas shownthat individual CD4

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lation, lose cell surface expression of CD45Rand acquire UCHL1 (and CDw29) with time in culture (44, 45). As is the case for Pgp-1 in the mouse, expression of UCHL1,once acquired, appears to be stable. The switch in + to UCHL1+ is prothe phenotype of stimulated T cells from CD45R gressive and unidirectional. For example, about 40%of the cells obtained + T cells with PHAwere found to be 3 days after stimulation of CD45R + +, CD45R UCHL1 compared to < 1% and < 10% at day 0 and day 7, respectively (45). In contrast, no double staining was observed in PHA+ cells which remained essentially "= after stimulated UCHLI CD45R extended culture in vitro. In line with these data is the observation that cord blood T cells consist + cells (which convert to the CDw29phenoalmost exclusively of CD45R type after in vitro stimulation with PHA)(37). Also, a significant fraction + cells (44). Interestingly, of mature thymocytes is represented by CD45R + + most, if not all, immature thymocytes are CD45R- UCHL1 CDw29 (44, 45), suggesting that several changes in the expression of these markers + CDw29 + thyoccur during T-cell development. Immature UCHL1 mocytes may lose these markers and gain CD45Ron maturation in the thymus. These mature nai’ve T cells, when they seed peripheral lymphoid + UCHL1-CDw29-until they are triggered by tissues, remain CD45R antigen. Uponactivation, these cells lose CD45Rand reexpress UCHL1 and CDw29,this time in a stable manner. + CDw29 + subset of peripheral blood Functional analysis of the UCHL1 T cells supports the notion that it contains memory(previously activated) cells (reviewed in 17). In agreement with the data obtained in the mouse, the frequencyof antigen-specific cells is enhancedin this subset. Similarly, enhanced IFN-y production has been observed after PHAstimulation, whereas IL-2 production appears not to be significantly different between + and CD45R + T cell subsets (37). Consistent with this latter UCHL1 finding is the observation that cord blood T cells (which are virtually + cells) produce significant amounts of IL-2, but little devoid of UCHL1 IFN-~, in response to PHAstimulation (46). Since differentiation from naive to memoryT cells appears to be accompanicdby increased expression of several surface molecules which are (presumably) involved in T-cell function (such as LFA-1, LFA-3 CD2), it has been proposed that such an increase mayrender memorycells moresusceptible to activation signals (37). As virtually all peripheral T cells are clonogenic under optimal stimulation conditions (47), this hypothesis maybe tested experimentally by determining the frequencies of T cells in the various subsets that proliferate in response to a limiting stimulus. It should be noted that the data presented in this section only concern T cells expressing tlae ~//3 TCRheterodimer. Little is knownabout the

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differentiation and fate of y/6 TCR-expressingT cells. Since they represent only a minor (1-5%) population of peripheral T cells, detailed analysis their surface phenotype is difficult. Moreover, the ability of y/6 TCRexpressing T cells to undergo clonal growth in vitro appears more limited than that of ~///TCR-expressing T cells (48). There is evidence in the mousethat ~/6 TCR-expressing T cells in lymph node are Pgp-1 ÷ (H. R. MacDonald,unpublished results). As reported recently, the majority of mouse T cells residing in intestinal epithelium express y/6 TCR(49). Although their Pgp-lphenotype is not known, it is noteworthy that most of the human T cells in normal gut mucosa have been reported to be ÷ (50). If these cells turn out to express y/~i TCR,it would CDw29 interesting to determine whether they are memorycells.

Rat Studies Rat CD45also exists in different molecular weight species (51, 52). monoclonal antibody, OX-22, has been shown to react with the higher molecular weight forms of rat CD45but not with the 180 kd-form (53). ÷ cells This antibody stains approximately two-thirds of peripheral CD4 + (51). Functional data support the view that OX-22-CD4 T cells in the ÷ CD4÷ T cells in humans. In rat are homologous to UCHL1÷ CDw29 particular, it has been shownthat, in immunizedrats, this subset was much ÷ CD4 ÷ subset in providing help for more potent than the reciprocal OX-22 B cells (54). Whileinitial studies suggested that alloreactive proliferating precursor cells were confined to the OX-22÷ CD4÷ subset, more recent work using different culture conditions shows no difference between the two subsets in this respect (D. W. Mason, personal communication). Moreover, a direct lineage relationship between the two CD4÷ T cell subsets has been demonstrated using in vivo transfer experiments. It is ÷ cells become OX-22- upon differentiation, now clear that OX-22+ CD4 ÷ whereas OX-22- CD4 cells remain OX-22-. While comparable studies concerning CD8÷ ceils have yet to be done, it is likely that a similar phenotypic change occurs amongthese cells, since rat CTLappear to be OX-22- (D. W. Mason, personal communication). Although many points remain to be established, the analogy betweenthe data obtained in the rat, ÷ T cells are naive the mouse, and the humanstrongly suggest that OX-22 ceils, whereas OX-22-T cells are memory(previously activated) cells.

CONCLUDING

REMARKS

Both B and T cells participate in the establishment of immunologic memory.Little is knownabout the differentiation pathwaythat results in the generation of memoryB cells, in addition to terminally differentiated

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plasma cells. In contrast, there is evidence, at least in vitro, that memory T cells are the progeny of antigen-induced effector cells. MemoryT cells in mice, rats, and humanscan be phenotypically distinguished from naive T cells. Expression of most of the surface markers that fulfill this purpose is low in naive cells but is rapidly enhancedupontriggering by antigen. In contrast to other surface activation antigens, which are only transiently expressed, the enhanced expression of these markers is stable. A particularly useful antigenic system in this respect is the leukocyte common antigen encoded by the CD45gene. Differentiation of naive T cells into activated (and memory)cells is accompanied by the progressive loss expression of higher molecular weight forms and gain of a lower molecular weight form. Antibodies against these markers define two subsets in per+ and CD8 + T cells. In contrast to a previous interpretation, ipheral CD4 these two subsets appear to correspond to different maturational stages rather than two different functional lineages. ~ Phenotypic identification of memoryT cells has allowed a more definitive analysis of their functional properties. It nowseemsclear that memory cells produce greater amountsof lymphokines (particularly IFN-7 and IL4) than their naive counterparts. This property of memorycells probably accounts (at least in part) for their enhancedability to help B cells and provoke inflammation in vivo. Furthermore, indirect evidence suggests that antigen-specific memorycells express higher affinity TCRthan most naive T cells. This finding (if confirmedat the molecularlevel) is a direct verification of the clonal selection theory. Further insights into the nature of T cell selection by antigen should be forthcoming with the availability of specific markers for activated and memoryT cells. ACKNOWLEDGMENT Wewish to thank Josiane Ducfor her excellent secretarial assistance. Literature Cited 1. Celada, F. 1971. The cellular basis of the immunologicmemory.Pro~7. Aller~Ty 15: 22347 2. Eisen, H. N., Siskind, G. W. 1964. Variations in affinities of antibodies during the immuneresponse. Biochemistry 3: 9961008 3. Andersson, B. 1970. Studies of the regulation of avidity at the level of the single antibody-formingcell. The effect of antigen dose and time after immunization. J. Exp. Med. 132:77-88 4. Davie, J. M., Paul, W. E. 1972. Receptors on immunocompetent cells. V.

Cellular correlations of the "maturation" of the immuneresponse. J. Exp. Med. 135:660-74 5. Mtller, G., ed. 1987. Role of somatic mutation in the generation of lymphocyte diversity. Immunol. Rev. 96:5-162 6. Engers, H. D., MacDonald,H. R. 1976. Generation of cytolytic T lymphocytes in vitro. In ContemporaryTopics in Immunobioloyy, ed. W. Weigle, pp. 145-90. New York: Plenum 7. Cerottini, J.-C., Engers, H. D., MacDonald, H. R., Brunner, K. T. 1974. Generation of cytotoxic T lymphocytes

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T-CELL in vitro. I. Response of normal and immunemousespleen cells in mixedlymphocyte cultures. J. Exp. Med. 140: 70317 8. Ryser, J.-E., MacDonald, H. R. 1979. Limiting dilution analysis of alloantigen-reactive T lymphocytes.III. Effect of priming on precursor frequencies. J. lmmunol. 134:128-32 9. MacDonald, H. R., Cerottini, J.-C., Ryser, J.-E., Maryanski, J. L., Taswell, C., Widmer,M. B., Brunner, K. T. 1980. Quantitation and cloning of cytolytic T lymphocytes and their precursors. Immunol. Rev. 51:93-123 10. Marrack, P., Endres, R., Shimonkevitz, R., Zlotnik, A., Dialynas, D., Fitch, F. W., Kappler, J. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. II. Role of the L3T4product, J. Exp. Med. 158: 107791 11. Shimonkevitz, R., Luescher, B., Cerottini, J.-C., MacDonald, H. R. 1985. Clonal analysis of cytolytic T lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for Lyt-2/3 function. J. Immunol. 135. 892-99 12. MacDonald, H. R., Glasebrook, A. L., Bron, C., Kelso, A., Cerottini, J.-C. 1982. Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes (CTL): possible implications for the affinity of CTLantigen receptors. Immunol. Rev. 68:89-115 13. Swain, S. L. 1983. T cell subsets and the recognition of MHC class. Immunol. Rev. 74:129-42 14. Truney, A., Albert, F., Golstein, P., Schmitt-Verhulst, A.-M. 1985. Calcium ionophore plus phorbol ester can substitute for antigen in the induction of cytolytic T lymphocytes from specifically primed precursors. J. Immunol.135: 2262q57 15. Isakov, N., Altman, A. 1985. Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes. J. Immunol. 135: 3674-80 16. Tabi, Z., Lynch, F., Ceredig, R., Allan, J. E., Doherty, P. C. 1988. Virus-specific memoryT cells are Pgp-1÷ and can be selectively activated with phorbol ester and calcium ionophore. Cell. Immunol. 113:268 77 17. Sanders, M. E., Makgoba,M. W., Shaw, S. 1988. Human naive and memory T cells: reinterpretation of helper-inducer

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and suppressor-inducer subsets. ImmunoL Today 9:195-98 18. Hughes, E. N., Mengod, G., August, J. T. 1981. Murine cell surface glycoproteins. Characterization of a major componentof g0,000 daltons as a polymorphic differentiation antigen of mesenchymalcells. J. Biol. Chem.256: 702327 19. Hughes, E. N., Colombatti, A., August, J. T. 1983. Murine cell surface glycoproteins. Purification of the polymorphic Pgp-I antigen and analysis of its expression on macrophagesand other myeloid cells. J. Biol. Chem.258: 101421 20. Morse, H. C. III, Shen, F.-W., H/immerling, U. 1987. Genetic nomenclature for loci controlling mouselymphocytes antigens. Immunogenetics 25:71-78 21. Colombatti, A., Hughes, E. N., Taylor, B. A., August, J. T. 1982. Gene for a major cell surface glycoprotein of mouse macrophages and other phagocytic cells is on chromosome2. Proc. Natl. Acad. Sci. USA 79:1926-29 22. Lesley, J., Trowbridge, I. 1982. Genetic characterization of a polymorphic murine cell-surface glycoprotein, lmmunogenetics 15:313-20 23. Trowbridge,I. S., Lesley, J., Schulte, R., Hyman, R., Trotter, J. 1982. Biochemical characterization and cellular distribution of a polymorphic, murine cell-surface glycoprotein expressed on lymphoid cells. Immunogenetics15: 299312 24. Bauman, J. G. J., Wagemaker, G., Visser, J. W. M. 1986. A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and committedprogenitors. J. Cell. Physiol. 128:133-42 25. Lesley, J., Hyman,R., Schulte, R. 1985. Evidence that the Pgp-1 glycoprotein is expressed on thymus-homing progenitor cells of the thymus. Cell Immunol. 91: 397-403 26. Trowbridge,I. S., Lesley, J., Trotter, J., Hyman, R. 1985. Thymocyte subpopulation enriched for progenitors with an unrearranged T-cell receptor/~chain gene. Nature 315:666-69 27. Lesley, J., Trotter, J., Hyman,R. 1985. The Pgp-1 antigen is expressed on early fetal thymocytes. Immunoyenetics 22: 149-57 28. Lynch, F., Ceredig, R. 1988. Ly-24 (Pgp1) expression by thymocytes and peripheral T cells. Immunol. Today 9:7-10 29. Budd, R. C., Cerottini, J.-C., Horvath, C., Bron, C., Pedrazzini, T., Howe, R. C., MacDonald,H. R. 1987. Distinc-

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tion of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation. J. Immunol.138:312029 30. Lynch, F., Chaudhri, G., Allan, J. E., Doherty, P. C., Ceredig, R. 1987. Expression of Pgp-I (or Ly24) by subpopulations of mouse thymocytes and activated peripheral T lymphocytes. Eur. J. Immunol. 17:137~,0 31. Lesley, J., Schulte, R., Trotter, J., Hyman, R. 1988. Qualitative and quantitative heterogeneity in Pgp-1 expression among murine thymocytes. Cell. Immunol. 112:40-54 32. Budd, R. C., Cerottini, J.-C., MacDonald, H. R. 1987. Phenotypic identification of memory cytolytic ÷ T lymphocytes in a subset of Lyt-2 cells. J. Immunol. 138:1009-13 33. MacDonald, H. R., Budd, R. C., Cerottini, J.-C. 1989. Pgp-1 (Ly24) as marker of murine memory T lymphocytes. In Curr. Top. Microbiol. Immunol. In press 34. Budd, R. C., Cerottini, J.-C., MacDonald, H. R. 1987. Selectively increased production of interferon-~, by subsets of Lyt-2 + and L3T4+ T cells identified by expression of Pgp-1. J. Immunol. 138:3583 86 35. Mosmann,T. R., Coffman, R. L. 1987. Twotypes of mousehelper T-cell clone. lmmunol. Today 8:223-27 36. Isacke, C. M., Sauvage, C. A., Hyman, R., Lesley, J., Schulte, R., Trowbridge, I. S. 1986. Identification and characterization of the human Pgp-I glycoprotein. Immunogenetics 23:326-32 37. Sanders, M. E., Makgoba, M. W., Sharrow, S. O., Stephany, D., Springer, T. A., Young, H. A., Shaw, S. 1988. Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-I) and three other molecules (UCHLI, CDw29,and Pgp-1) and have enhanced IFN-y production. J. Immunol. 140: 1401-7 38. Morimoto, C., Letvin, N. L., Distaso, J. A., Aldrich, W. R., Schlossman, S. F. 1985. The isolation and characterization of the humansuppressor inducer T cell subset. J. Immunol. 134:1508-15 39. Morimoto, C., Letvin, N. L., Boyd, A. W., Hagan, M., Brown, H. M., Kornacki, M. M., Schlossman, S. F. 1985. The isolation and characterization of tl~e human helper inducer T cell subset. J. lmmunol. 134. 3762~59 40. Beverley, P. C. L., Merkenschlager, M., Terry, L. 1988. Phenotypic diversity

41.

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43.

44.

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46.

47.

48.

49.

50.

51.

of the CD45antigen and its relationship to function. Immunology(Suppl.) 3-5 Ralph, S. J. M., Thomas, M. L., Morton, C. C., Trowbridge, I. S. 1987. Structural variants of humanT200 glycoprotein (leukocyte-common antigen). EMBOJ. 6:1251-57 Lefranqois, L., Puddington, L., Machamer, C. E., Bevan, M. J. 1985. Acquisition of eytotoxic T lymphocyte-specific carbohydrate differentiation antigen. J. Exp. Med. 162:1275-93 Terry, L. A., Brown, M. H., Beverley, P. C. L. 1988. The monoclonal antibody, UCHL1, recognizes a 180,000 MWcomponent of the human leucocyte-common antigen, CD45. Immunology 64:331-36 Serra, H. M., Krowka,J. F., Ledbetter, J. A., Pilarski, L. M. 1988. Loss of CD45R (Lp220) represents a postthymic T cell differentiation event. J. Immunol. 140:143541 Akbar, A. N., Terry, L., Timms, A., Beverley, P. C. L., Janossy, G. 1988. Loss of CD45R and gain of UCHLI reactivity is a feature of primed T cells. J. Immunol. 140:2171-78 Lewis, D. B., Larsen, A., Wilson, C. B. 1986. Reduced interferon-gamma mRNAlevels in human neonates. Evidence for an intrinsic T cell deficiency independent of other genes involved in T cell activation. J. E.~cp. Med. 163: 1018-23 Moretta, A., Pantaleo, G., Moretta, L., Cerottini, J.-C. and Mingari, M. C. 1983. Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity. J. Exp. Med. 157:743-54 Moretta, L., Pende, D., Bottino, C., Migone, N., Ciccone, E., Ferrini, S., Mingri, M. C. and Moretta, A. 1987. Human CD3÷4-8-WT31 - T lymphocyte populations expressing the putative T cell receptor y-gene product. A limiting dilution and clonal analysis. Eur. J. Immunol. 17:1229-34 Goodman, T., Lefran~;ois L. 1988. Expression of the y-6 T-cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature 333:855-58 James, S. P., Fiocchi, C., Graeff, A. S., Strober, W. 1986. Phenotypic analysis of lamina propria lymphocytes. Gastroenterology 91:1483-89 Wooltett, G. R., Barclay, A. N., Puklavec, M., Williams, A. F. 1985. Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thy-

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mocytes and T and B lymphocytes. Eur. J. Immunol. 15. 168-73 52. Barclay, A. N., Jackson, D. I., Willis, A. C., Williams, A. F. 1987. Lymphocyte specific heterogeneity in the rat leukocyte commonantigen (T200) is due to differences in polypeptide sequences near the NH2-terminus. EMBOJ. 6: 1259-64 53. Spickett, G. P., Brandon, M. R., Mason, D. W., Williams, A. F., Wollett, G. R. 1983. MRCOX-22, a monoclonal anti-

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body that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocytecommonantigen. J. Exp. Med. 158: 795810 54. Arthur, R. P., Mason, D. 1986. T cells that help B cell responses to soluble antigen are distinguishable from those producing interleukin 2 on mitogenic or allogeneic stimulation. J. Exp. Med. 163. 774-86

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Ann. Rev. lmmunol. 1989. 7.’91-109 Copyright © 1989 by Annual Reviews Inc. All rights reserved

MICROANATOMY OF LYMPHOID TISSUE DURING HUMORAL IMMUNE RESPONSES: Structure Function Relationships A. K. Szakal Department of Anatomyand Division of Immunobiology, MedicalCollege of Virginia, Richmond,Virginia 23298 M. H. Kosco and J. G. Tew Departmentof Microbiology and Immunology,Medical College of Virginia, Richmond,Virginia 23298 INTRODUCTION The complexcellular interactions involved in the regulation of immune responseshavegenerally beenstudied in vitro. Animplicit assumptionis that cells performthe sameroles in culture as they do in vivo. However, it shouldbe appreciatedthat secondarylymphoidtissues are highly organized and that this functional architecture is destroyedin the process of suspendingcells. The aim of this review is to relate novel structures observedin the lymphoidnoduleor follicle with inductionof the secondary antibody(Ab)response.Special attention is focusedon antigen (Ag)recognition, Agprocessing, and Agpresentation steps that appear to occur in the lymphoidnodule. In contrast with the primaryresponse, Abis present whenthe booster immunizationis given. This Ab binds the immunogen, and immunecomplexes are rapidly formed. These immunecomplexes localize in lymphoidnodulesin minutes, germinalcenters developrapidly, and Ab production and memoryB-cell production are accelerated. These 91 0732-0582/89/0410-0091 $02.00

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events are documented in draining.lymph nodeswith reference to other lymphoid tissue for comparison.

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KINETICS OF THE GERMINALCENTER REACTION Basedon histochemical observations of Ag localization and germinal center (GC)development in mouselymphnodes, the kinetics illustrated in Figure 1 emerge. Horseradishperoxidase (HRP)immunemice were boostedin the hind feet with HRP.Notethat the popliteal lymphnode morethandoubledin size in the first three days.Thisincreaseis largely due to development of the Ag-retainingreticulum(ARR)andedemawhich resolvesby day5. Byday10 the nodeis againenlarged,but this is dueto increasedcellularity attributablemostlyto a proliferativeexpansion of B cells in germinalcenters. TheAg-retaining reticulumrepresentssites of Ag localization on follicular dendritic cells (FDCs)in the light zone germinalcenters (Figure2a). Notethat Ag-retainingreticulumreachtheir maximum number(Figure 1) and size between days 1 and 3 after injection. Byday5 these parameters decreaseto a plateauof aboutfive ARR per node. Theincrease in Ag-retainingreticulumcoincides with the initial peakin lymphnodesize, andit is followedby a size reduction 1.60

10

T

111 Z

1.28-

-8

Z

"2

:~

0.96-

0.~4-

0.32"

0.00

÷

0 Ag 15’ D:I

.3

5

TIME AFTER ANTI(~EN INJECTION (rain.

1’0 & days)

Figure ] This chart illustrates size changesin pop]Jtea] lymphnodesas related to the kinetics of antigen retaining reticulum (ARR) and de novo germinal center (GC) development after antigen (Ag) injection in the hind feet of immune C57BL/6 mice. Note: lymph node volume reflects changes in ARRand PNA + GC numbers and that ARRdevelopment (Ag trapping) precedes B-cell proliferation (development of PNA + GC).

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Figure 2 Light micrographs of (a) an antigen retaining reticulum (ARR)and (b) a GCin the popliteal lymph node 5 days after Ag injection in immunemice. (a) Note the location of the ARRin the GCindicated by arrow heads. Faintly stained TBMare also visible below the ARR;(b) Observe the highly PNA÷cells in the GC. Note that the upper half stains darker than the lower half which also contain some PNA+ TBM(arrows). This staining is the reverse of the convention on light and dark zones of GCs. The ARRis located in the upper darker staining half. ( × 70.)

paralleling the drop in the number of ARR.Using HRP-labeled peanut agglutinin (PNA)to bind activated germinal center lymphocytes (1), novo germinal center development can be monitored (Figure 2b). Germinal center development, dependent on Ag trapping in the ARR(2) begins between the third and fifth day after challenge, but new germinal centers develop only after preexisting germinal centers dissociate (3). Note (Figure 1) that the maximumnumber of germinal centers corresponds with the maximumnumber of ARR.The number of germinal center B cells peaks about day 10 and then begins to decline (1).

KEY EVENTS IN THE SECONDARY RESPONSE In primed animals the immunogen encounters Ab and is rapidly complexed. These complexes are quickly trapped by macrophages and Ag transporting cells in the lymphatic vessels and lymphnode (4). Figure 3 a model relating events in the lymphoid nodule to the induction and maintenance of the secondary Ab response. These events are described in subsequent sections.

ANTIGEN TRAPPING

AND ANTIGEN TRANSPORT

In the secondary response, the majority of complexesare phagocytized by sinusoidal macrophages, degraded and eliminated from the lymph node by 24-48 hr. Someimmunecomplexesare transported via an "alternative" pathway to lymphoid nodules. Early studies noted an apparently "purposeful movement"of Ag-bearing cells from-the splenic red pulp to the

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ANTIGEN TRAPPING, TRANSPORT, PROCESSING,

IMMUNOLOGICAL EVENTS

PRESENTATION ANDFEEDBACK

-- ANTIGEN TRAPPING BY ATCAND~ ANTIGEN TRANSPORT ATCs ~ EDCs

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ANTIGEN PRESENTATION TO B-CELLS BY FDCs ICCOSOME DISPERSION __ ANDUPTAKE BY B-CELLS ANDTBM ANTIGEN PROCESSING BY -- GERMINAL CENTER B-CELLS, TBM.ANDPRESENTATION TO T-CELLS

]

’~ANTIBODY FEEDBACK REGULATION

Figure 3 Immunological events in the "alternative pathway" of the Ag-stimulated draining lymph nodes of immune mice. ATC, antigen transport cells; SS, subcapsular sinus; M, macrophage; FDC, follicular dendritic cell; ICS, immune complex coated body or "iccosome"; Bm, B memory cell; TBM, tingible body macrophage; Ab, antibody; pAg, processed antigen; Time designations: 1 r~irq-5 HR; DI, day 1; DI-3, days 1 to 3; D5, day 5; D14, day 14; --indicate events prominent at these times.

peripheral aspects of germinal centers (5). In 1983, based on light and studies, we described the transport of horseradish peroxidase in lymph nodes (4). The Ag was transported on the surface of cells of varying dendritic morphology, from the subcapsular sinus (SS) to lymphoid nodules. The Agin transit was recognizable as early as 1 min after injection (Figure 4a). Monocyte-like cells with immunecomplex coated veil-like processes and cells referred to as penetrating "frilly" or "veiled" cells (6) (Figure 4b) appeared to transport the immunecomplexes through pores in the floor of the subcapsular sinus. Werefer to these nonphagocyticcells collectively as Ag transport cells (ATCs). One min after HRPinjection, Ag-transport cells are found near one another below the subcapsular sinus floor with their processes extending through pores into the subcapsular sinus, forming a "mitten-like" dendritic configuration. These short processes, coated with immunecomplexes, appear to be in different stages of withdrawal from the sinus (Figure 4c). SomeAg-transport cells located below the subcapsular sinus had dendritic processes in contact with ATCs in transit through the floor of the subcapsular sinus and with processes of less differentiated FDCsdeeper at the periphery of lymphoid nodules (Figure 4d). Thus, a progression of the immunecomplexes, detectable

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Figure 4 Light (a) and electron microscopic (b-d)histochcmical illustration of the PO+ inverted "funnel-like" Ag transport site (a),extending from the SS toward the future site of

ARR in the lymphoid nodule. (b) and (c), ATC (arrow heads) in transit through a pore (arrows) in the floor of the SS. Note the immune complex coated surface (black, PO+) of convoluted dendritic cell processes (P) extending into the SS lumen. (d) A chain (arrow heads) of ATC (pre-FDC?) in the Ag transport site with PO+ immune complex coated dendritic processes. Note, only the surface of dendrites but not the surface of lymphocytes are P O + . ATC,antigen transport cell; CA,capsule; FL, SS floor; L, lymphocyte; P, P1, P2, ATC cell processes; G,Golgi; N, nucleus. Magnification: (a) x 225; (b) x 1500; (c) x 1990; (d) x 2250 (Szakal et al, in Ref. 4).

on the surface of a chain of ATCs and FDCs, forms the profile, light microscopically visible, of an inverted funnel between the subcapsular sinus and lymphoid nodule (Figure 4a). As Ag-retaining reticulum develop, Ag transport cells diminish and Ag transport paths disappear. By day 1 most of the transported Ag is retained on the dendrites of FDCs in the fully developed ARR. However, not all ARR develop simultaneously, and Ag transport may continue into the first day at some sites (7).

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Somestudies implicate lymphocytes in Ag transport (8-10), and immune complexes reportedly maydrain passively into lymphoid nodules (11). general, preformed immunecomplexes were used, and this may relate to differences in observations. Weinject Ag for booster immunization, and we routinely detect transport by Ag transport cells. The single exception is in old mice where the ATC-mediatedtransport is defective. In aged animals Ag persists on cells near the subcapsular sinus and never reaches the lymphoid nodule (7). ANTIGEN-RETAINING

RETICULUM

Onceit was accepted that Agis retained extracellularly on dendritic processes of nonphagocytic cells, "antigen retaining reticulum" (ARR)was proposed (12) to replace the term "phagocytic reticulum." The ARR refers to the three-dimensional network formedby the interdigitating, Agretaining dendritic processes of neighboring follicular dendritic cells as visualized by autoradiographic or histochemical detection of Ag (Figure 5a) (2, 7, 13). The de novo formation of Ag-retaining reticulum is clearly observable in lymph nodes 0f mice (Figure 1). The dendritic processes trapping Ag have been shownto belong to FDCs(14-16), and it is accepted that Ag is retained in the form of Ag-Abcomplexes(17-19). Descriptions of lymph node and splenic FDCsat the EMlevel agree about the cytological features (15, 20-23). Typically, follicular dendritic cells are described as having a cell body with an irregular, sometimes bilobed, euchromatic nucleus containing a distinct nucleolus. A few multinucleate cell bodies also exist (23). The scanty cytoplasm contains few profiles of mitochondria, RER,Golgi, and vesicles. Emanating from the cell body are the dendritic processes, some of which appear attenuated, with folds and intermittent thickenings forming pleiomorphic cytoplasmic extensions. Other dendrites form more uniform, highly convoluted, labyrinthine configurations. These dendritic processes interdigitate with those of neighboring FDCs and are the primary structures involved in the retention of Ag. Recent scanning EMon follicular dendritic cells identified two major FDCtypes (24, 25): (a) FDCswith filiform dendrites (Figure 5b), and FDCswith "beaded" dendrites (Figure 5d). In addition, intermediate forms (Figure 5c) between filiform and beaded type FDCsand FDCswith pleiomorphic thickening of dendrites and veil-like processes were seen. Culture studies indicated that the filiform dendrites mature into "beaded" dendrites. High resolution EMstudies revealed a spiraling periodicity (440490 ~) of immunecomplexes on FDCprocesses (25). Lymphocytes were seen

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Figure 5 The antigen retaining reticulum (ARR). and its component FDC types. (a) Light microscopic view of the Ag (HRP) positive ARR at day 1 after Ag challenge. Scanning electron microscopy shows two types of FDC (arrows): (b) FDC with filiform dendrites; (d) FDC with beaded dendrites, and (c) intermediate form FDC. Magnification: (u) x 140; (b) x 2000; (c) x 2000; (d) x 2800 (Szakal et al, in Ref. 25).

bind to immune complexes on FDC dendrites in vivo, but they did not sequester free complexes on their surface. The periodicity of immune complex binding may facilitate the interaction between B cells and immune complexes. This interpretation is consistent with the report that in order to bind and directly stimulate B cells, an immunogen (e.g. DNP-polymer) must have a certain number of epitopes spaced 120-670 8, apart (26). Phenotypic studies indicate that follicular dendritic cells are a distinct cell type. Monoclonal Ab that selectively bind FDCs include MRC Ox 2 (27, 28), and Ki-M4R in the rat (29) and R4/23 or DRC-I (30), and KiM4 (3 1) in the human. The follicular dendritic cells express class I and I1 MHC Ags as well as common leukocyte Ag and receptors for C3b, Ig,

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and Fc (32). However, they lack macrophageor T-cell Ags such as Mac1, -2, -3; F4/80; Thy-1, CD5,or CD8(28, 32).

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ICCOSOME FORMATION, DISPERSION, ATTACHMENT, AND ENDOCYTOSIS This year we reported the discovery of a 0.3~0.4/~m diameter spherical particle, or "immunecomplex coated body," which we termed iccosome (13). Iccosomes are produced by follicular dendritic cells between day and day 3 after secondary challenge. Their discovery by EMwas facilitated by the use of the Ag horseradish peroxidase which after histochemical developmentis seen to outline these particles (Figure 6a). The existence of iccosomes in developing germinal centers appears to be limited to a period of hours at an early phase of germinal center development. Since the development of one germinal center may lag behind another by 24 hr, their visualization by EMis somewhatfortuitous. In addition, Ag dose and the level of circulating Abs appear to influence iccosome production. The EMsuggests (13) that iccosomes are formed through interaction two FDCtypes. The first population with pleiomorphic dendrites binds large accumulations of immunecomplexes. "Beaded" dendrites of the second FDCtype bind to areas of sequestered complexes. This binding proceeds in a way similar to a receptor-mediated endocytosis, resulting in the formation of an immunecomplex layer between the membraneof the pleiomorphic dendrite surrounding the "bead" and the limiting membrane of the "bead." Thus, the "bead" becomescoated with a uniform thickness of immunecomplexes and also with a layer of the pleiomorphic dendrite (cytoplasmic layer) (Figure 6b). This cytoplasmic layer remains incomplete; therefore the process does not result in true endocytosis. Next the "bead" is apparently penetrated by small globules of immunecomplexes, seen as PO+ material in the "bead" (Figure 6c). Subsequently, the cyto-

Figure 6 Electron micrographs of iccosome formation and dispersion in developing GC. (a) Shows iccosomes (PO +, small immunecomplex coated bodies) dispersed amongthe lymphocytes; note edema; (b) an FDCbead embeddedin the pleiomorphic dendrite is in the early stage of the formation of the immunecomplex layer (arrow); (c) an iccosome in intermediate stage of formation. Note membraneslimiting the (black) immunecomplex layer (arrow heads), the globular immunecomplexes in the center and the cytoplasmic layer (arrow); (d) free iccosomeattaching to the surface of a lymphocyte;(e) a recently endocytosed iccosome in a GClymphocyte. Magnification: (a) ×4380; (b) × 108,100; (c) × 101,500; (d) × 103,500; (e) × 12,150.

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plasmic layer disintegrates, releasing the iccosome which is coated by and contains varying amounts of immune complexes (Figure 6 4 . Around day 3, iccosomes are dispersed in the developing germinal center, an event associated with edema (Figure 6a) that appears to facilitate iccosome dispersion. The free iccosomes become attached to germinal center B cells via their immune complex layer and are endocytosed (Figure 6e). By day 5, iccosomes are rarely visible in germinal centers, but the majority of the lymphocytes contain PO endocytosed HRP complexes (Figure 7a). These events culminate in the delivery of the Ag to germinal center B cells, for endocytosis, degradation (1 3), and, potentially, Ag presentation (35).

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+

ANTIGEN PRESENTATION BY GERMINAL CENTER B CELLS It is now well known that B cells can present Ag to T cells and that their specific surface Ig receptors make them remarkably efficient. The iccosome-derived Ag appears to be processed by germinal center B cells as indicated by the decreasing intensity of the HRP reaction product at the Golgi apparatus (Figure 7a). The Golgi is known to be associated with

Figure 7 Endocytosis and presentation of in vivo obtained Ag by GC B lymphocytes: (a) an electron micrograph showing the Ag, HRP in endocytic vesicles 5 days after Ag injection in immune mice. Note the reduced PO activily of Ag associated with the Golgi (arrow); (0) bar graph illustrating the results of the presentation of Ag (OVA) to 3DO-54.8 T cells in vitro. Note Ag presentation is maximal at day 5 when the majority of GC B cells endocytose and process Ag. Magnification: (a) x 20,600 (Szakal et al, in Ref. 13); (b) Kosco et al, in Ref. 35.

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production of plasma membrane and could serve as a mechanism for delivering the Ag-Ia complexto the cell surface (13). Wesought to determine whether the germinal center B cells were able to present the Ag they internalized in vivo to T cells that are knownto be in germinal centers (33, 34). Germinalcenter B cells were isolated using peanut agglutinin panning (35). The germinal center B cells were obtained at various times after Ag challenge and assessed for their ability to stimulate T cells to produce IL-2. The results of the Ag presentation experiments (Figure 7b) correlate with the kinetics of Aguptake in vivo (13). Shortly after challenge, whenfew iccosomes are available to germinal center cells (i.e. Day0, Day1), very little IL-2 production is elicited. At day 5, when the maximumamount of Ag appears to be processed by germinal center B cell in vivo, maximum IL-2 production is stimulated. Finally, as the response is winding downin vivo, the ability to stimulate T cells also declines. It appears that germinal center B cells can efficiently process and present Ag they obtained from FDCs. ROLE

OF

TINGIBLE

BODY MACROPHAGES

(TBM)

Tingible bodymacrophages(36) are large cells containing the characteristic "tingible bodies" of Flemming (37). Tingible body macrophages are thought to derive from macrophages entering the lymph node via the afferent lymphatics (38). Kinetic analysis at the light and EMlevels during de novo formation of germinal centers revealed that: (a) by day 1, close association with the antigen retaining reticulum a few tingible body macrophages were present with low numbers of endocytosed cells; (b) day 3, TBMwere seen endocytosing iccosomes and immune complexes from FDCdendrites (13); (c) by day 5, Ag+TBMcontaining increased numbers of endocytosed cells were present in large numbers in the dark zone of germinal centers (Figure 8a). The observed uptake of iccosomes suggested that tingible body macrophages maypresent Ag to germinal center T cells. This prompted tingible body macrophage isolation and phenotyping studies, and TBMwere found to be unique. In addition to macrophagemarkers (Ia, F4/80, Mac-l, Mac2) they were Tby-I positive (39). The significance of Thy-I (Figure 8c) unknown,although it may act as an adhesion molecule (39) to help bind cells for endocytosis. Tingible body macrophagecould endocytose T cells and thus dampen Ag presentation. Alternatively, TBMexpress Ia and could present Ag. Wehope to test these possibilities. Proliferation and cell death are features of germinal centers (40). Tingible body macrophagesappear to play a major role in disposing of dead cells or cells destined to die. Originally, Cottier proposed(41) that "tingible

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Figure 8 Illustrates the morphology of Ag (HRP), Thyl, and Ia positive tingible body macrophages (TBM). (a) TBM in a GC day 5 after Ag injection of immune mice. Note

the Ag+ TBM (black, PO+) associated with the PO+ ARR retaining HRP-anti-HRP complexes. TBM below the AKR are less positive (arrows). (b) An isolated TBM stained with methyl green to show nuclei of phagocytized GC lymphocytes; N, TBM nucleus; (c) electron (EM) and light (inset) micrographs of TBM showing membrane and cytoplasmic labeling for Thy-I with anti-Thy-1 and PO conjugate (EM) and glucose oxidase conjugate (inset); (d) Ia+ TBM labeled with anti-la and PO conjugate (EM) and anti-Ia and glucose oxidase conjugate (inset). Magnification: (a) x 95; (b) x 730; (c) x 3150; inset, x 570; ( d ) x 3038; inset, x 540 (Smith et al, in Ref. 39).

bodies” may originate from the breakdown of germinal center cells. DNA labeling studies (40, 41) indicated that “tingible bodies” derive from germinal center cells dying before prophase when intact “tingible bodies” show a 4n DNA content. Concurrent EM studies revealed that “tingible

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bodies" represent lymphocytesand differentiating plasma cells in various stages of degradation (36). The significance of tingible body macrophagesmayrelate to recent data on affinity maturation of B cells. In strain A mice, B cells use a single heavy-chain V-gene segment to make most p-azophenyl-arsonate (Ars)specific Ab (42). In the secondary response, B cells are present that have mutatedderivatives of this V region. Mostof these B cells have even higher affinity for Ars than do the original B cells. This phenomenonhas been explained by an affinity-based selection mechanism(42). Antigen directs both the expression of the immunerepertoire and the amplification of V region diversity by selecting mutated cells with the highest affinity. However,manymutations would result in surface Ig on B cells with lower affinity for the antigen. Since these cells wouldnot bind Ageffectively, a mechanismfor eliminating these cells wouldbe desirable. Germinalcenters represent an in vivo structure with features matching those needed for affinity maturation (43). These features include: (a) Availability of Ag follicular dendritic cells in the light zone for selection of appropriate B cells; (b) rapid proliferation of B cells in the dark zone allowing for expression of mutations; and (c) destruction of B cells in large numbers by tingible body macrophages. Phagocytosis by TBMwould provide a mechanismfor eliminating mutated cells with low affinity receptors that cannot successfully compete for Ag at low concentration. These B cells may be programmedto die in the absence of stimulation by antigen, and this would be consistent with the observation that TBMare phagocytosing dead or dying cells (39). Thus, affinity maturation could be explained terms of the germinal center microenvironment. GERMINAL ANTIBODY

CENTERS FORMING

AND THE CELLS

INDUCTION

OF

The lymphoid nodule is recognized as a center for production of memory B cells (1). However, several studies, including one of our own, have reported that cells of the plasmacyticseries are also within germinalcenters as is indicated in Figure 9 (21, 44~6). The number of antibody forming cells (AFC) peaks about 3 to 4 days after Ag challenge. Note that the amount of anti-HRP antibodies released into the endoplasmic cisternae increases with antibody forming cell maturity. The observation of antibody forming cells within the germinal center led us to investigate whether these cells were capable of secreting Ab. At various times after challenge, germinal center B cells were isolated and cultured. The germinal center B cells obtained 3 or 4 days after challenge spontaneously produced (no Ag added) high amounts of HRP-specific

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Flqure 9 Immunoperoxidase localization of anti-HRP antibodies (black) in cells of the GC plasmocytic series (see lower left corner and insets) and on the convolutions of FDC dendrites retaining HRP-anti-HRP complexes (see upper right: arrows). Note (lower left) arrows heads indicate the beginning differentiation of a plasmocytic cell with anti-HRP antibodies in its nuclcar cnvelopc (scc also insct a). Insets b and c show a proplasmocyte and a plasmocyte (G, Golgi) respectively. Magnification: x 3200; insets: x 4900 (Szakal et al, in Ref. 44).

IgG which could not be augmented by T-cell factors. In contrast, germinal center B cells obtained 7-12 days after challenge produced small amounts of anti-HRP. However, production of anti-HRP by the 7-12 day population was dramatically enhanced when Con A supernatant was added (44; M. H. Kosco, unpublished findings). Two distinct phases of the germinal center reaction exist. During the first phase, germinal center B cells receive signals needed to differentiate into antibody forming cells. In the second phase, which peaks about day 10, cell proliferation results in the restoration and expansion of the memory B-cell pool. We observe many PNA positive cells leaving germinal centers

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during the first phase. Recent data suggest that these germinal center cells home to the medullary cords and to bone marrow where they become mature plasma cells which produce much of the Ab associated with the anamnestic response (47).

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FEEDBACK REGULATIONIN MAINTENANCEOF THE ANTIBODY RESPONSE The role of follicular dendritic cells, persisting Ag, and specific Abin the maintenance and regulation of humoral Ab production was reviewed in 1980 (48) and is not covered in detail here. The concept is that in the maintenance phase of immuneresponses, multiple dynamicequilibria exist between persisting Ag on FDCs, free specific Ab, and Ag-Abcomplexes of various ratios. The immunogenicityof the complexesis directly related to the Ag-Abratio, and alterations in serum Ablevels result in formation or dissociation of these complexes. WhenAb levels in the circulation decline, Agis exposed, memoryB cells are stimulated, and a new cycle of Ab synthesis is induced. This newly produced Ab feeds back and terminates the immunogenicstimulus. Repetition of this cycle serves to maintain serum Ab levels within narrow limits. Evidence supporting the role of FDCsand associated Ag in the maintenance mechanismis summarized in Table 1.

Table 1 Evidence supporting a role for FDCsand retained antigen in the maintenance of serum antibody 1. Specific Abtiters "spontaneously" rebound to the previous levels after severe bleeding (49). 2. Specific antigen persists intact on FDCsfor years and retains its immunologicalspecificity

(50). 3. Persisting Agis restricted to draining lymphoidorgans. The further the organ is from the injection site the less Agit retains (50). Correspondingly,specific AFCare localized the draining organs. AFCare most numerous in organs which contain the most FDC associated Ag(51). 4. As Agis depleted, the AFCbecomemore restricted in distribution. One year after immunizationin the hind foot, the AFCare almost exclusively localized to the popliteal lymph nodes (51). 5. Removingpopliteal lymphnodes with their high levels of persisting Agfrom mice injected in the hind feet results in a markeddecrease in serumAb(48). 6. Removinglymph nodes from the Abrich environment in vivo and culturing results in a "spontaneous Ab response". This "spontaneous response" is subject to Abfeedback inhibition (48). The "spontaneous Abresponse" only occurs in nodes with retained

(48).

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SUMMARY

KOSCO

AND

& TEW

SIGNIFICANCE

Secondary responses require no more immunogen than do primary responses, and lower doses are often adequate. In view of the feedback inhibitory effects of specific Ab, one might predict that secondaryresponses would require more Ag. However, a special pathway is operative in immune animals for handling immunogen. This Ag pathway, which efficiently utilizes the immunogen,is described in this review. A brief summaryof key events in this Ag pathway is described below and illustrated in Figure 3. In immuneanimals, immunogenencounters specific Ab and is rapidly converted into immunecomplexes. Most of these complexes are quickly trapped, endocytosed, and catabolized by macrophagcs. This occurs quickly, and by 48 hr after immunizationvery little Agpersists in or on these macrophages. However, an alternative Ag pathway exists which tends to preserve the immunogen. Immunecomplexes in the alternative pathwayare trapped on the surface of nonphagocytic cells which transport the immunogento the follicular dendritic cells in the lymph node outer cortex. During the next 3 days FDCdendrites bead and form immune complex-coated bodies or "iccosomes." The iccosomes are dispersed in the developing germinal centers where they are trapped and endocytosed by germinal center B cells. The germinal center B cells process the iccosome derived Ag, and this Ag appears to be presented to T cells in the area. During this early period of iccosomerelease, endocytosis, and Agprocessing, the lymph node is swollen and edematous. The edema appears to facilitate movementof antibody forming cells and B cells which have developed and are leaving the germinal center microenvironment. These departing AFCand stimulated germinal center B cells appear to be going to the medullary cords, and some may migrate to the bone marrowwhere large amounts of specific Ab are produced. The edemain the lymph node resolves somewhatby day 5, but the germinal center reaction continues with a second lymph node enlargement apparent by day 10. This enlargement of the lymph node is associated with a proliferative expansion of germinal center B cells. These germinal center B cells are memorycells, and an Ag-basedselection of high affinity B cells is thought to be taking place. Cycles of Ab production regulated by an antibody feedback mechanism maintain circulating levels of specific Abfor monthsor years. Cyclic release oficcosomesmaybe responsible for the induction of specific Ab. Iccosomes have not yet been identified in the maintenanceof Ab production; however, the concept that a mechanisminvolving iccosomes is responsible for both the induction and maintenance of the secondary response is appealing.

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Literature Cited 1. Coico, R. F., Bhogal, B. S., Thorbecke, G.J. 1983. Relationship of germinal centers in lymphoid tissue to immunologic memory. VI. Transfer of B cell memory with lymph node cells fractionated according to their receptors for peanut agglutinin. J. Immunol. 131:2254-57 2. Klaus, G. G., Humphrey,J. H., Kunkl, A., Dongworth, D. W. 1980. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53:3-28 3. Hanna, M. G. Jr., Congdon, C. C., Wust, C. J. 1966. Effect of antigen dose on lymphatic tissue germinal centers. Proc. Soc. Exp. Biol. Med. 121:286-91 4. Szakal, A. K., Holmes,K. L., Tew, J. G. 1983. Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131: 1714-27 5. Nossal, G. J., Austin, C. M., Pye, J., Mitchell, J. 1966. Antigens in immunity. XII. Antigen trapping in the spleen. Int. Arch. Alleryy 29:368-83 6. Fossum,S. 1980. The architecture of rat lymph nodes. 1I. Lymphnode compartments. Scand. J. Irnmunol. 12:411-20 7. Szakal, A. K., Taylor, J. K., Smith, J. P., Kosco, M. H., Burton, G. F., Tew, J. G. 1988. Morphometry and kinetics of antigen transport and developing antigen retaining reticulum of follicular dendritic cells in lymphnodes of aging immunemice. Aging: Immunol. and Inf. Dis. 1: 7-22 8. Brown, J. C., Harris, G., Papamichail, M., Slijvic, V. S., Holborow,E. J. 1973. The localization of aggregated human gammaglobulin in the spleens of normal mice. Immunolo#y 24:955-1003 9. Gray, D., McConnell, I., Kumararatne, D. S., MacLennan, I. C., Humphrey, J. H., Bazin, H. 1984. Marginal zone B cells express CR1 and CR2 receptors. Eur. J. lmmunol. 14:47-52 10. Heinen, E., Braun, M., Coulie, P. G., Van Snick, J., Moeremans, M., Cormann, N., Kinet Denoel, C., et al. 1986. Transfer of immune complexes from lymphocytesto follicular dendritic cells. Eur. J. Immunol. 16:167-72 11. Kamperdijk, E. W. A., Dijkstra, C. D., Dopp, E. A. 1987. Transport of immune complexes from the subcapsular sinus into the lymphnode follicles of the rat. ImmunobioL 174:395~405 12. Mitchell, J., Abbot, A. 1965. Ultra-

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structure of the antigen-retaining reticulum of lymph node follicles as shown by high resolution autoradiography. Nature 208:500-2 Szakal, A. K., Kosco, M. H., Tew, J. G. 1988. A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigen-processing cells. J. Immunol. 140:341-53 Nossal, G. J., Abbot, A., Mitchell, J. 1968. Antigens in immunity. XIV. Electron microscopic radioautographic studies of antigen capture in the lymph node medulla. J. Exp. Med. 127:263-76 Szakal, A. K., Hanna, M. G. Jr. 1968. The ultrastructure of antigen localization and viruslike particles in mouse spleen germinal centers. Exp. Mol. Pathol. 8:75-89 Tew, J. G., Thorbecke, G. J., Steinman, R. M. 1982. Dendritic cells in the immuneresponse: characteristics and recommendednomenclature. J. ReticuloendotheL Soc. 31:371-80 Nossal, G. J., Ada, G. L., Austin, C. M., Pye, J. 1965. Antigens in immunity.VIII. Localization of 125-I-labelled antigens in the secondary response. Immunolo#y 9:349-57 Humphrey, J. H., Frank, M. M. 1967. The localization of non-microbial antigens in the draining lymphnodes of tolerant, normal and primed rabbits. Immunoloyy 13:87-100 Chen, L. L., Frank, A. M., Adams, J. C., Steinman, R. M. 1978. Distribution of horseradish peroxidase (HRP)anti-HRP immune complexes in mouse spleen with special reference to follicular dendritic cells. J. Cell Biol. 79:184-99 Nossal, G. J., Abbot, A., Mitchell, J., Lummus,Z. 1968. Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles. J. Exp. Med. 127: 277-90 Hanna, M. G. Jr., Szakal, A. K. 1968. Localization of ~25I-labeled antigen in germinal centers of mousespleen: histologic and ultrastructural autoradiographic studies of the secondary immune reaction. J. Immunol. 101:949-62 Chen, L. L., Adams, J. C., Steinman, R. M. 1978. Anatomyof germinal centers in mousespleen, with special reference to "follicular dendritic cells". J. Cell Biol. 77:148~4 Fossum, S., Vaaland, J. L. 1983. The architecture of rat lymphnodes. I. Combined light and electron microscopy of

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lymph node cell types. Anat. Embryol. 167:229.46 24. Schnizlein, C. T., Kosco, M. H., Szakal, A. K., Tew, J. G. 1985. Follicular dendritic cells in suspension: identification, enrichment, and initial characterization indicating immune complex trapping and lack of adherence and phagocytic activity. J. Immunol. 134:1360 68 25. Szakal, A. K., Gieringer, R. L., Kosco, M. H., Tew, J. G. 1985. Isolated follicular dendritic cells: cytochemicalantigen localization, Nomarski, SEM, and TEMmorphology. J. Immunol. 134: 1349-59 26. Dintzis, R. Z., Middleton, M. H., Dintzis, H. M. 1983. Studies on the immunogenicityand tolerogenicity of Tindependent antigens. J. Immunol. 131: 2196-2203 27. Barclay, A. N. 1981. The localization of populations of lymphocytes defined by monoclonal antibodies in rat lymphoid tissues. Immunology 42:593 600 28. Humphrey, J. H., Grennan, D. 1982. Isolation and properties of spleen follicular dendritic cells. Adv. Exp. Med. BioL 149:823~7 29. Wacker, H. H., Radzun, H. J., Mielke, V., Parwaresch, M. R. 1987. Selective recognition of rat follicular dendritic cells (dendritic reticulum cells) by a new monoclonal antibody Ki-M4Rin vitro and in vivo. J. Leukocyte. Biol. 41: 7077 30. Naiem, M., Gerdes, J., Abdulaziz, Z., Stein, H., Mason, D. Y. 1983. Production of a monoclonal antibody reactive with humandendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue. J. Clin. Pathol. 36:167-75 31. Parwaresch, M. R., Radzun, H. J., Feller, A. C., Peters, K. P., Hansmann, M. L. 1983. Peroxidase-positive mononuclear leukocytes as possible precursors of humandendritic reticulum cells. J. Immunol. 131:2719-25 32. Kosco, M. H., Tew, J. G., Szakal, A. K. 1986. Antigenic phenotyping of isolated and in situ rodent follicular dendritic cells (FDC)with emphasis on the ultrastructural demonstration of Ia antigens. Anat. Rec. 215: 201-13, 219-25 33. Nieuwenhuis, P., Opstelten, D. 1984. Functional anatomyof germinal centers. Am. J. Anat. 170:421 35 34. Rouse, R. ¥., Ledbetter, J. A., Weissman, I. L. 1982. Mouselymph node germinal centers contain a selected subset of T cells--the helper phenotype. J. Immunol. 128:2243M6 35. Kosco, M. H., Szakal, A. K., Tew, J. G.

1988. In vivo obtained antigen presented by germinal center B cells to T cells in vitro. J. Immunol. 140:354-60 D. C., Congdon, 36. Schwartzendruber, C. C. 1963. Electron microscope observations on tingible body macrophagesin mousespleen. J. Cell Biol. 19:641-46 37. Flemming,W. 1885. Studien uber regeneration der Gewebe. Arch. Mikrosk. Anat. 24:50 38. Hoefsmit, E. C. M., Kamperdijk, E. W. A., Balfour, B. M. 1980. Reticulum cells and macrophages in the immune response. In Mononuclearphagocytes: Functional aspects, Part II, ed. R. van Furth, pp. 1809-36. Boston: Martinus Nijhoff 39. Smith, J. P., Kosco, M. H., Tew, J. G., Szakal, A. K. 1988. Thy-1 positive tingible body macrophages (TBM)in mouse lymph nodes. Anat. Rec. 222: In press 40. Odartchenko, N., Lewrenze, M., Sordat, B., Roos, B., Cottier, H. 1966. Kinetics of cellular death in germinal centers of mouse spleen. In Germinal centers in immune responses, ed. H. Cottier, D. Odartchenko, R. Schindler, C. C. Congdon, pp. 212-17. New York: Springer-Verlag 41. Fliedner, T. M. 1966. On the origin of tingible bodies in germinal centers. See Ref. 40, pp. 218-21 42. Manser, T., Gefter, M. L. 1986. The molecular evolution of the immune response: idiotope-specific suppression indicates that B cells express germ-line encoded V genes prior to antigenic stimulation. Eur. J. Immunol. 16: 143944 43. MacLennan,I. C., Gray, D. 1986. Antigen driven selection of virgin and memory B cells. Immunol. Rev. 91:61-85 44. Szakal, A. K., Kosco, M. H., Burton, G. F., Tew, J. G. 1988. Germinal center antigen presenting B cells in the induction and maintenance of the secondary antibody response. Progr. Leuk. Biol. 7: 281-90 45. Sordat, B., Sordat, M., Hess, M. W., Stoner, R. D., Cottier, H. 1970.~ Specific antibody within lymphoid germinal center cells of mice after primary immunization with horseradish-peroxidase: a light and electron microscopic study. J. Exp. Med. 131:7741 46. Terashima, K., Imai, Y., Kasajima, T., Tsunoda, R., Takahashi, K., Kojima, M. 1977. An ultrastructure study on antibody production of the lymph nodes of rats with special reference to the role of germinal centers. Acta Pathol. Jpn. 27:1~4 47. Benner, R., Hijmans, W., Haaijman, J. J. 1981. The bone marrow: the major

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LYMPHOIDNODULESTRUCTUREFUNCTIONS 109 source of serum immunoglobulins,but Progress in Immunology,p. 628. New still a neglectedsite of antibodyformaYork: AcademicPress tion.. Clin. E,,cp. Immunol. 46:1-8 50. Mandel,T. E., Phipps,R. P., Abbot,A., 48. Tew,J. G., Phipps,R. P., Mandel,T. E. Tew,J. G. 1980.Thefollicular dendritic 1980. The maintenanceand regulation cell: long term antigen retention during of the humoralimmuneresponse: perimmunity. Immunol.Rev. 53:29-59 sisting antigenandthe role of follicular 51. Donaldson,S. L., Kosco,M. H., Szakal, antigen-bindingdendritic cells as accesA. K., Tew,J. G. 1986. Localizationof sory cells. Immunol.Rev. 53:175-201 antibody-formingcells in draining lym49. Bystryn,J. C., Schenkein,I., Uhr,J. W. phoid organs during long-term mainten1971.Amodelfor the regulationof antiance of the antibodyresponse.J. Leukobody synthesis by serumantibody. In cyte. Biol. 40:147-57

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Ann. Rev. Immunol. 1989. 7.’111~43 Copyright © 1989 by Annual Reviews Inc. All rights reserved

CELLS AND MOLECULES THAT REGULATE B LYMPHOPOIESIS IN BONE MARROW Paul W. Kincade, Grace Lee, Carolynn E. Pietrangeli, Shin-Ichi Hayashi and Jeffrey M. Gimble Oklahoma Medical Research Foundation, 825 Northeast Thirteenth Street, Oklahoma City, Oklahoma 73104

Introduction Mature and immature forms of eight different types of blood cells are tightly packed within the spaces of bone marrow, complicating investigation of howit is efficiently regulated. Hundredsof billions of cells of the various lineages are produceddaily in this vital organ and exported to other tissues via processes which are responsive to inflammation and other systemic events. Because of advances in cell separation and culture techniques, rapid progress is nowbeing made in resolving steps in each differentiation pathway. It is even moreinteresting that regulatory interactions betweencells are being appreciated and defined in molecular terms. This review focuses on cells of the humoral immunesystem and those steps involved in their formation that can be observed and manipulated in culture. Purified recombinantmolecules can nowbe used to elicit particular responses in cultured lymphocyte precursors, and the probable source of such regulatory substances is becoming clearer. Moreover, cells that comprise the inductive microenvironment of bone marroware themselves subject to exquisite regulation. In somecases, this occurs via factors they can themselves make, i.e. autocrine regulation. Information of this kind is relevant to the treatment of a numberof diseases. However, while cell culture modelshave madeit possible to identify manymolecules that affect proliferation and differentiation of B-cell precursors, they do not provide a completely accurate representation of intact bone marrow. For example, there are indications that Compensatorycircuits and "quality control" 111 0732-0582/89/0410-0111502.00

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mechanismsthat operate in vivo are not present in established tissue cultures. Thus,recognition of the deficiencies in experimentalapproaches is informative with respect to the complexityof bonemarrowand should highlightareas for future investigation. Organization Within Bone Marrow Transmissionand scanning electron microscopicstudies have provided a general picture of bonemarrowarchitecture and areas of hemopoiesis(1 5). Someof the space within bonesis occupiedby fat (yellow marrow) is not engagedin blood cell production. Active red marrowcan expand into such areas in circumstances of unusual demand, and a molecular explanation of this response maybe near (see below). Stem cells and hemopoieticprogenitor cells are in extravascular areas and tend to be concentratedin the subendostealarea, i.e. near the bone cortex (6, 7). Newlyformedblood cells leave the marrowafter traversing the endothelium of venoussinuses, whichare morecentrally located within bones

(8). Platelets are madeand released from megakaryocytesand are conspicuousbecauseof their large size in marrowsections, while erythrocytes are madein close association with a centralized macrophage-likecell in what is knownas an "erythroblastic island" (9). Thus, these two blood cell types are madein highly specialized locations. Theorigin of other bloodcells is moredifficult to discern andrequires extrapolationfromin vitro studies. Maturinggranulocyteshavebeenidentified in close association with large "adventicular reticular cells," whichradiate awayfrom venoussinuses (10). Early B-lineage lymphocytesare moreabundant the subendostealarea of marrow;however,areas of focal proliferation comparable to the tbllicles in the avian bursa of Fabricius are not readily apparent(l 1, 12). B-lineageand myeloidcells growin close association with large "stromar’cells in culture (see below),and it seemslikely that they wouldbe near the extendedmembranes of similar cells in situ. Theincidence of multipotential stem cells amongtheir differentiated progenyis extremelylow in bonemarrow(less than 0.1%), whereasnearly a quarter of the cells are B-lineagelymphocytes.Therefore,a considerable amountof division mustaccompany the transition of one cell type to the other. Indeed,the kinetics andextent of proliferation in B-cell precursors is well documented (13). Since large clusters of lymphocytesare not conspicuous, it seemslikely that differentiating cells must be continually -.moving,perhaps in a "conveyorbelt" relationship along the convoluted ¯ membranes of stromal cells. Directed migration of maturingB-lineage cells could be passive, e.g. with movement resulting frompulse pressure and modulationof adhesionmolecules,or alternatively, chemotacticprocesses

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1 13

might be involved. Further details of the three-dimensional structure of bone marrowshould be revealed when distinctive markers are found for critical components of the lymphopoietic microenvironment. Such tools are likely to be developed by exploitation of long-term cell culture techniques.

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Long-Term Bone Marrow Cultures Short-term culture systems madeit possible to conclude that close cellular interactions favor B lymphocyte formation, and a number of potential regulators of lymphopoiesishave been identified with this relatively simple approach (14, 15). However, more impressive advances in understanding bone marrow function followed the development of long-term bone marrow culture techniques (16). Dexter and colleagues first found conditions suitable for maintenance of multipotential stem cells and sustained granulopoiesis (17). Whitlock &Witte (18;-19) successfully adapted these methods for selective growth of B-lineage lymphocytes, and other innovations facilitated analysis of the cells involved (20, 21). Theseimportant technical advances have been reviewed elsewhere (15, 22). Only a brief account is given here, based primarily on our personal experiences. Hemopoietic cells from bone marroware maintained in long-term cultures by close physical association with a complexlayer of adherent cells. Usually a majority of the adherent cells are macrophages,whichare i~eadily distinguished on the basis of marker expression, phagocytosis, and uptake of acetylated low density lipoprotein (LDL)(23, 24). A number of stances that influence lymphopoiesis are elaborated by macrophages, and several potential regulatory mechanisms can be proposed that involve them. However, macrophage-depleted, long-term cultures sustain the growth of lymphocytes that have already beenadapted to growth, and this proliferation is supported by one or more types of "stromal cells" (see below). In some respects, lymphocytes propagated in culture resemble their normal counterparts in bone marrow. Despite a relatively high mitotic index, they are notably small to mediumin size and die within three days when removedfrom the adherent layer (18, 22, 23). Also, at least some cells taken from pooled cultures are capable of normal differentiation following transplantation (25-28). However,we have not consistently been able to elicit responses in long-term cultured lymphocyteswith a variety of stimuli knownto be effective with freshly isolated lymphocytes from bone marrow, or pre-B tumor cell lines. Pre-B cells maintained for extended periods in vitro often have unusual phenotypes. For example, the BP-1 antigen can be absent or expressed in abnormally high density (29; G. Lee et al, submitted) and,~unlike normal bone marrowpre-B cells,

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mayfail to stain with 14.8 antibody (23). It is possible that a majority the lymphocytesmaintainedfor an extended period in vitro are intrinsically defective, and studies with bone marrowfrom immunodeficient SCIDmice are consistent with this view (29, 30). That is, lymphocyteswith aberrantly rearranged immunoglobulingenes expand in culture, but not in vivo. Ceils growing in individual culture dishes seem to arise from a small numberof surviving lymphocytes, and rare abnormal cells might have a growth advantage (23, 31). Therefore, populations of cultured lymphocytes could become more abnormal with time. Whensteroids and/or horse serum are used in the culture mediumas originally described by Dexter et al (17), multipotential stem cells are maintained and granulocytes predominate. Reduction of incubator temperature to 33°C is also an advantage. Use of relatively low concentrations of selected fetal calf serumand addition of 2-mercaptoethanol, as described by Whitlock & Witte, favor lymphocyte growth (18, 19). Shifting established Dexter-type culture to Whitlock-Witte conditions results in a switch from myeloid to lymphoid cell production (20, 32). Additional procedures add flexibility to the use of long-term cultures. For example, hemopoietic cells can be selectively eliminated by treatment with mycophenolic acid or 5-fluorouracil, leaving a functional adherent layer (21, 33). Fresh adherent layers can also be established with an initial low numberof bone marrowcells (19). Cells that can readily form an adherent layer in culture can be dramatically reduced by passage of suspensions through G-10 Sephadex or nylon wool columns (21). However, it. should be cautioned that precursors of macrophagesand stromal cells are probably not removedby this treatment (see below). While there are limitations to the existing long-term culture methodology, these approaches have madeit possible to dissect an extraordinarily complex tissue and identify at least some of the cell types critical to lymphocyte formation. Moreover, lines established from these cultures provide a means to clone genes for unique regulatory molecules. Stromal

Cell

Clones

Adherent layers of primary long-term bone marrow cultures are complex and include macrophages,endothelial cells, fat cells, and fibroblasts, in addition to the lympho-hemopoieticcells that are in close physical association with them (15, 24, 34, 35). Manyinvestigators have sought to simplify this by establishing cloned cell lines that can be subcultured manytimes. These are then used to investigate which cells are responsible for specific microenvironmental functions. Salient characteristics of some clones known to influence B lymphopoiesis are summarized in Table 1. Considerable phenotypic and functional diversity has been found in adherent

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Table 1 Someroutine stromal cell clones that influence B lymphopoiesis

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MBA-2.4 Endothelial-like line; makesM-CSF; membrane associated factors stimulate a transformed pre-B line and inhibit plasmacytoma growth(36) B.AD Fibroblastoid,phagocytic,pre-adipocyteline; makesCSFbut not IL-3; requires close contact with target cells to supportcompactmultipotentcolonies, includinglymphocyte precursors (38-40) TC-1 Fibroblast/endothelialline; makesM-CSF anda high MW synergistic activity for stemcells, anda factor whichstimulates pre-B formation,but not IL-3 (41, 42) S 17 Clonedslromal line; allows nylon woolpassagedbone marrowto makemyeloidcells and support B lineage precursors, whichexpandunder Whitlock-Witteculture conditions; Cells are la-, Thy-1-, Mac-I- and BP-1-; producesa factor whichstimulates pre-B cell formation and IL-4 (43-45) AC 4 Endothelial-like, pre-adipocyteclone; maintainsgrowthof a clonedpre-Bline and sorted bonemarrow cells withoutterminal differentiation; these andrelated clonesexpressthe 6C3antigen, but not Thy-1and makeM-CSF,but not IL-3 (37) ALC Fibroblastoid,pre-adipocyteclone; supportspre-Bclones as well as cultured myeloidcells; makesa 30-40Kpre-B growth factor, M-CSF and G-CSF,but not GM-CSF or IL-3 (46) 30R Stromalcell clone; supports growthof a clonedpre-Blymphocyteline; has IL-4 receptors and 1-2%are 6C3+ (47, 48) IxN/A6 SV40transformedstromalcell clone; used to purify andclone IL-7 (49, 50) BMS-2 Pre-adipocytestromalcell line; supportsgrowthof five clonedlymphocyte lines; part of a series whosephenotypeswerecompared(Table 2) and studied with respect to factor responsiveness;cells are Thy-1÷ and makeM-CSF (33) Stromalcell clone; supports growthof Blineage progenitorsfromvery early (9.5 day) embryos(51)

cell clones, and this was particularly notable when large numbers were isolated and studied in a single laboratory (33, 36, 37; D. Rennick, personal communication). Although many morphologic designations have been used, the term "stromal cell" is convenient because it does not imply a known lineage derivation. Our cloned stromal cells, like others reported in the literature, all have a large oval nucleus, with one or more nucleoli.

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The cytoplasmcan be highly spread, giving a total cell diameter of at least 150 microns. Hemopoieticcells placed in low numberson a stromal cell clone survive and grow in a typical functional assessment. However,even when highly purified bone marrowstem cells are added, it should be rememberedthat a simple precursor-adherent cell interaction maynot be taking place. For example, myeloid progenitors rapidly give rise to macrophages in this situation, and the adherent layer thus includes them in addition to the cloned line. Manylaboratories have also isolated cloned lymphocytelines from longterm cultures and used them to define stromal cell functions (33, 37, 46, 47, 49, 52). In our experience, the characteristics of these clones are as unusual as lymphocytes in the long-term cultures from which they were derived. They display unusual phenotypes and have been unresponsive to inductive stimuli (G. Lee, unpublished observations). Nonetheless, they provide a more homogeneous tool for the assessment of cytokine production and other stromal cell activities. Our panel of stromal cell clones was namedon the basis of the tissue of origin and interactions with cloned lymphocytes (33). If the lymphocytes quickly died when placed on the adherent layer, we designated them "nonsupport," as opposed to "support" stroma. For example, BMNS1 was isolated from bone marrow and did not support the growth of a particular lymphocyte clone. Impressive proliferation of lymphocytes in liquid or methyl cellulose cultures occurred when "support" clones were used, and this was usually improved by prior irradiation of the adherent layer. Indeed, division of the stromal cell clones could be influenced by mesenchymalgrowth factors, and this tended to relate inversely to good lymphocyte growth support (33). Functional heterogeneity of our stromal cell clones becameapparent by use of multiple clones of lymphocytes as indicators (C. E. Pietrangeli, manuscript in preparation). For example, one stromal cell clone (BMS2) supported all of five lymphocyte clones and another (SNS1) supported none of them. However,other stromal cells efficiently supported the proliferation of only certain of the lymphocyteclones. This important observation indicates that multiple lymphocytegrowth stimuli potentially can be provided by stromal cells. Somelymphocyte clones need more than one of these, whereas others could have simpler requirements. An alternative explanation might be that particular clones make inhibitors that affect only certain of the lymphocyte clones. As previously noted by Whitlock (22), lymphocyte clones occasionally becomestromal cell independent and would presumably then be tumorigenic. This has also happened occasionally in our lab, particularly with clones derived from BALB/cmice.

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Thestability of stromalcell clonesis of practicalandtheoreticalinterest. Wehave preparednonsupportsubclones of BMS2 andexpect these variants will be veryvaluablefor associatinggeneexpressionwithfunctional capability. Certaincharacteristics of the stromalcells seemto change duringcloning (Table 2). For example,the Thy-1antigenwasnot demonstrable in primaryculturesbut is expressedby mostof our stromalcell clones. This mayindicate an intrinsic phenotypicinstability of stromal cells, whichis made obviousby cloning.Also, veryrarecells in the primary cultures mayhavebeenexpandedby cloning. However, a moreinteresting possibility is that their geneexpressionis regulatedby interactionwith macrophages andother cells in primarycultures. As is discussedin more detail below,stromalcell clonesare responsiveto a number of regulatory stimuli. Marrowsuspensions that have been passed through G-10 Sephadex columnsandplatedat low densities simplydie withoutestablishment of an adherentlayer. When placedon a supportstromalcell clone, hemopoiesis is initiated. Macrophages are alwaysproduced,andin manycases, verylarge colonies of themare noticeable. Granulocytopoiesis can also occur, even whenWhitlock-Witte conditions are used (C. E. Pietrangeli, manuscript in preparation).Publishedaccountsof other stromalcell clones suggest Table 2 Comparisonof clones to stromal cells in primarycultures

Markers

Primary culture

SSI

E Cadherin Lgp 100 Class II LFA1 Macl ...... 14.8 (CD45R) Ly5/T200 -dH-2D + _ BP 1 .... Actin + + N-CAM + + + M 1/69 + M 1/87 + -Mac 2 + Mac3 ++ Collagen (IV) + + + + Thy 1 + ++ Qa2

SNS1

BMS1

BMS2

BMNS1

...... ...... ...... ......

+ --

...... + _

+ + + + + +++ _+ ++ +

+ + + + + + + ++ + + + ++ +

+ _+ + + + + +++ + + + ++ +

-+ _ + + + +++ -_+ ++ +

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this is more noticeable whenDexter-type culture conditions are employed (37, 43, 46). With certain of our clones, under Whitlock-Witteconditions, B-lineage lymphocytesare produced, and there is only a minimal tendency for these cells to give rise to surface-Ig-bearing B cells. This is in accord with reports from other laboratories (46, 53) in that the support of terminal maturation is less markedthan stimulation of growth. While there is good reason to think that a single type of stromal cell can sustain growth of lymphoidand myeloid cells (37, 43, 46), we do not yet knowif this occurs simultaneously in vivo (see below).

Cytokines Madeby Stromal Cells At least eight regulatory macromolecules can be elaborated by cloned stromal cells under some circumstances. Macrophagecolony stimulating factor (M-CSF;CSF-1) is almost always produced. This has been detected by direct coculture of fresh bone marrowwith stromal cells, by assay of conditioned medium,by inhibition with specific neutralizing antibodies, and by analysis of messenger RNAs. Likewise production of G/M-CSF and/or G-CSFhas been demonstrated with bioassays and RNAanalysis (Table 1, and J. M. Gimble, manuscript in preparation). Wehave found that with somestromal cell clones, CSFcan be readily detected in culture supernates, whereas with others, close contact with the adherent cells is necessary (C. E. Pietrangeli, manuscript in preparation). This could be due to differential production of extracellular matrix componentsthat absorb such factors (54, 55, and see below). Interleukin 3 is a T cell-derived multipoietin which, together with IL-6, can stimulate multipotential stem cells and more mature progenitors of several lineages (56, 57). Therefore, it might be expected that stromal cells or some other component of bone marrowwould elaborate this cytokine. Thus far, no one has found evidence of IL-3 production in cloned stromal cells ~r implicated its function in typical long-term bone marrowcultures (Table 1; and 58). Our experience has been that this factor alone does not sustain proliferation of cells that are recognizablypart of this lineage (59). In addition, there is a marked genetic polymorphism of IL-3 responsiveness, and virtual nonresponder animals make adequate numbers of lymphocytes (see below). Therefore, this mediator mayonly affect hemopoiesis during unusual circumstances. A novel 25-kd factor, termed interleukin 7 (IL-7), has been purified and obtained in recombinant form from a transformed bone marrow stromal cell line (49, 50). Asis detailed below,IL-7 is a potent proliferative stimulus for large pre-B cells and maysignificantly contribute to lymphocytegrowth in long-term cultures. Indeed, IL-7 mRNA is detectable in our best support stromal cell clones, as are other growth factors. Althoughlong-term cul-

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tured lymphocytesproliferate in response to IL-7, it is rare for them to grow indefinitely (G. Lee, submitted, and A. Namen, personal communication). Together with other findings that indicate a complexpattern of lymphocyte-stromal cell interactions, this suggests that more than one replieative stimulus is usually required for lymphopoiesisin culture. Wehave detected mRNA for IL-6, TGF-/~, and neuroleukin in all of our stromal cell clones (J. M. Gimble, manuscript in preparation). IL-6 of interest for a numberof reasons. Amongeffects on manydifferent cell types, IL-6 prepares multipotential hemopoietic stem cells for responses to other stimuli (57, 60). As is detailed below, IL-6 appears to autoregulate its ownsynthesis in stromal cells. TGF-fl is madeby manycell types in an inactive precursor form (61, 62). Whenactivated by acidification, it is potent inhibitor of lymphopoiesis and hemopoiesis (see below). Neuroleukin, a B lymphocyte activator, has recently been shown to be highly homologousto glucose-6-phosphate isomerase (63, 64). Stromal Cell Participation Responses

in Autocrine

and Paracrine

Hemopoiesis is dramatically affected by inflammation and exogenous stimulation (65), and it is nowclear that several componentsof the bone marrow are potentially involved in such responses. Macrophages can respond to bacterial endotoxin by release of IL-1 and TNF, which can in turn stimulate endothelial cells to produce IL-1 (66-68). Moreover, these two cell types, when induced, make M-CSF, G-CSF, and G/M-CSF(66, 67, 69, 70). Similarly, stromal cells are capable of constitutive and induced CSFproduction (71, 72). Constitutive production of M-CSFseems to a commonfeature of stromal cell clones, whereas monocyteor endothelial ceil-derived IL-1 causes them to increase expression of G/M-CSFand GCSFgenes (71-73 and Table 1). Wehave monitored the relative abundance of various mRNAs in a bone marrowderived stromal cell clone exposed to a numberof stimuli (J. M. Gimble, manuscript in preparation). For example, while the cells contain low levels of IL-6 message, this is increased following exposure to LPS, IL-1, 1L-7, or tumor necrosis factor (TNF). This type of data, using multiple cDNAprobes, indicates that functional receptors for IL-6, TGFfl, IL-4, and IL-7 are expressed on stromal ceils. Experiments involving cyclohexamide treatment suggest that some of these mRNA transcripts maybe produced under the influence of labile repressors or subject to a rapid degradation pathway(66, 74, 75). Growthof stromal cells in culture can be enhanced by addition of epidermal cell growth factor (EGF) and inhibited by LPSor interferon y (33). The function and ability of stromal cells to accumulate fat is modulated by TGF-fl’s, cytokines also knownto

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affect synthesis of extracellular matrix constituents (76, 77; S.-I. Hayashi, manuscript in preparation). Other studies indicate that platelet-derived growth factor (PDGF) can also influence bone marrow microenvironmental cells (78). These observations suggest that paracrine regulatory networks could potentially regulate growth, differentiation, and function of bone marrow stromal cells (Figure 1). Activated T lymphocytes can make IL-6 and interferon ~, in addition to myeloidcolony stimulating factors (79). Macrophages are a potential source ofTNF,IL-1, and TGF-fl (66). T cell-derived IL-4 can directly influence hemopoietic progenitors (47, 80). Stromal cell

Fi#ure 1 Stromal cells play a pivotal role in regulating progression of stem cells down lymphoid and myeloid lineages. They are also involved in paracrine and autocrine interactions with T cells, macrophages,other stromal cells, and endothelial cells (not shown).

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clones display receptors for IL-4 and may also be influenced by this lymphokine (48, and K. Landreth, personal communication). Therapy with recombinant factors may soon become routine, and it is important to understand direct effects on stromal cells, as well as indirect responses achieved via a cytokine cascade (81). Information about the effects of hydrocortisone and other drugs on these cells is also being obtained with long-term culture approaches (82-85). Indirect evidence might be used to argue that stromal cells are regulated by neighboring cells in vivo. Stromal cell clones that support pre-B cells can be isolated from cultures of adult spleen (33). However,the spleen not an active site for B lymphopoiesis in normal adult mice. Stromal cell function could be markedly influenced by cytokines and close cellular interactions which together constitute "he, mopoietic inductive microenvironments"in discrete sites (86). Coordination of responses by cells in the bone marrowmicroenvironment may be achieved via an autocrine mechanism (J. M. Gimble, manuscript in preparation). This follows from findings that our stromal cell clone responds to factors that it can produce, including IL-6, TGF-fl, and IL-7. Precedence exists for autocrine regulation in studies of T cells, macrophages,endothelial cells, fibroblasts, and B cells (79, 87-91). Cells each of these lineages mayrespond to factors which they, or their cohorts, make. Such networks can recruit cells of the same type to participate in particular responses. Further study might also reveal sometype of feedback inhibition through which individual stromal cells are sensitive to the concentration of their soluble products. All stromal cell clones that support lymphopoiesis can, under some circumstances of culture, also stimulate myelopoiesis, and it is interesting to consider that these functions might be linked in vivo. B lymphocyte precursor production can be markedly dysregulated in cyclic neutropenia (92). Pre-B cell numbersincreased 50-fold while myeloid progenitors were low, consistent with a competition because these two lineages for some commonfeature. B lymphocyteproduction is also compromised with a granulocytosis-inducing tumor (93). Stromal cells could be a pivotal site for regulation in bone marrow, ensuring that appropriate numbersof each blood cell type are produced. For example, stromal cells actively engaged in lymphopoiesis might make little CSF, relative to IL-7, and we should soon knowbowexogenousstimuli such as IL-1 affect this balance (71, 94). This information could have implications for managementof a number of bone marrowdisorders. While it is not yet clear which elements of the bone marrow microenvironment are routinely established in transplant recipients, the feasibility of stromal cell engraftment has been experb ment~il!y, demonstrated(95, 96).

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Oriyin, Derivation, Stromal Cells

and Differentiation

Potential

of

The bone marrowstroma may be mesenchymalin origin, based on embryologic and other studies. During mammalian development, mesenchyme derived cells invade the marrowcavity (97). Withtime, these cells differentiate into osteoblasts that form a bone matrix. This step is then followed by the formation of the bone marrowstroma or adventitia and the appearance of lymphohematologicprecursor cells (97). In the adult, the bone marrowstroma consists of at least three morphologically distinct phenotypes: fibroblasts or endothelial-like cells, macrophages, and adipocytes. Transplantation, and immunochemical and mRNA studies performed on mixed and cloned stromal cell cultures, indicate that fibroblast/endothelial and adipocyte stromal cells are not derived from hematologic precursors (24, 33, 98, and J. M. Gimble, manuscript in preparation). Unlike macrophages, they do not express a number of commonleukocyte genes (24, 33). In our view, immunohistochemical analysis alone does not provide a sharp distinction between fibroblasts and endothelial cells. However,our clones lack scavenger receptors for acetylated low density lipoprotein and are similar to pericytes in expression of a smooth muscle isoform of actin (33, 99, 100, and unpublished observations with P. D’Amore).Thy-1 antigen expression is typical of our clones but was not found in primary cultures and is not on all stromal cell clones isolated by other investigators (Tables 1 and 2). It is interesting that a cloned epithelial component of the human thymus microenvironment expresses this marker (101). Additional evidence for the mesenchymalderivation of stromal cells comes from LTBMCstudies. Under Dexter conditions, two non-bone marrowderived cell lines can substitute as stroma to support myelopoiesis, the fibroblasts Swiss 3T3 and CH3/10T1/2(102, and J. M. Gimble, unpublished observations). Eachof these lines also has characteristics of multipotential mesodermalprecursors. Whentreated with azacytidine, either 3T3 or CH3/10T1/2can give rise to myocytes, adipocytes, and chondrocytes. Mechanisms regulating the differentiation of these fibroblasts during myogenesishave been attributed to the expression of a single gene (103105). That these cells can act as typical myeloid stroma in coculture experiments is consistent with a mesodermalorigin for the stromal cells. Manycloned bone marrow stromal cells are pre-adipocytes (106, 107, and Table 1). After a period of several weeksin culture, they can accumulate lipids and take on a fat cell morphology.The adipocyte differentiation process is markedly influenced in vitro by exogenousgrowth factors such as TGF-fl, an inhibitor of adipogenesis, and hydrocortisone, an enhancer

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B-CELLFORMATION 123 of adipogenesis (10g, 109, and S.-I. Hayashi, manuscript in preparation). However, unlike extramedullary adipocytes, stromal cells do not respond to insulin (108, 110). In vivo, the natural history of the bone marrow the distal extremities is a progression from "red" hematopoietic marrow to "yellow" adipocyte laden, nonproductive marrow. With conditions of anemia, this process can be altered, with the reversion of "yellow" to "red" marrow (l 1 l). Bone marrow stromal cells may thus represent a type of multipotent mesenchymalcells, capable of further differentiation into adipocytes and possibly osteoblastic cells. Moreover,this differentiation process, while influenced by the age of the animal, can be reversed under certain conditions of hematologic stress. Developmentof gene probes and antibodies that detect unique products of these cells should be informative with respect to their lineage derivation and mayprovide insight into their normal lifespan and functional capabilities. Cell

Adhesion

Molecules

Somelymphocytes crawl beneath the stromal cell layer, in a curious phenomenon known as "pseudo-emperipolesis," and are flattened into foci which have a cobblestone appearance (24, 112). Other lymphocytes adhere to the surface of stromal cells, with sufficient strength to withstand the pull of gravity in inverted cultures (P. W. Kincade, unpublishedobservations). There is somespecificity to this attraction, because lymphocytes never bind to macrophages, which comprise a majority of the adherent cells (24). Specific recognition and adhesion betweenparticular cells is essential to normal bone marrow function, and molecules that could be involved in such functions are now being defined. In one well-studied example, a numberof different N-CAM (neural cell adhesion molecules) glycoproteins are madefrom a single complexgene (113, 114). Selective use of exons the N-CAM gene leads to the production of at least seven different mRNA species, and someof the resulting proteins are tissue specific (115, 116). For example, embryonicor regenerating muscle cells express a unique form of N-CAM,which has a domain not found in other tissues, and neural cells express a type of N-CAM not found in muscle cells. N-CAM was the first known adhesion molecule to be demonstrated on stromal cells derived from long-term cultures (117). The extracellular portions of N-CAM molecules can be extensively modified by carbohydrate addition, altering the overall charge of the molecule and its potential interactions with adhesion molecules on other ceils (118). However, anchorage of the carboxy terminal portions of the molecules is of particular interest. Most cell surface glycoproteins have one or more stretches of hydrophobic amino acids that float in the phos-

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pholipids of the cell membrane.However,a subset uses a unique glycosylphosphatidylinositol (G-PI) linkage between the protein and cell (119). There is a great deal of flexibility in howcells use the G-PI anchor. In the case of N-CAM, only molecules derived from a particular exon become G-PI linked 020, 121). Others either have a short, or very long, C terminal intracellular domain. The latter might interact with cytoskeletal elements in cells, whereas G-PI linked N-CAM is subject to release via action of a specific enzyme. Phosphatidylinositol specific phospholipases (PI-PLC) have been isolated from bacterial, parasitic, and mammalian sources (122, 123). Experimentally, PI-PLCs can be used to remove G-PI anchored proteins selectively from cell surfaces 024). Such molecules might also be liberated from cells by endogenous phospholipases generated as a consequenceof particular stimuli (125). It seems possible that attachment, and subsequent release of adhering cells is controlled in part by this mechanism, and it is interesting that PI-PLC treatment causes many lymphocytesto detach from stromal cells in long-term cultures (24). N-CAM molecules are structurally related to other members of the "immunoglobulingene superfamily," which includes T cell receptors for antigen and manyother molecules knownto be involved in specific recognition functions (126, 127). It is thought that these all derived during evolution from a single primordial gene. Another gene family includes adhesion molecules that function in a quite different way 028-130). They are called cadherins because an extracellular domainrequires a divalent cation such as Ca++ or cell-cell recognition, and at least one memberof this family is expressed on B lineage lymphocytes (P. W. Kincade, unpublished observations). Still other receptors on pre-B cells mayrecognize fragments prepared from fibronectin 031). It is important to learn howall of these molecules participate in the orientation and movementof lymphocytes within the microenvironment. The association of granulocytes with stromal cells maybe mediated by a recently described adhesion molecule termed "haemonectin" (132). Immobilized

Cytokines

and the Extracellular

Matrix

Complexproteoglycans encompassall of the cells in bone marrowand, in addition to structural functions, these substances may play an important regulatory role, Heparan sulfate is knownto bind and stabilize a number ofmesenchymalgrowth factors, and this has recently been found to be true for GM-CSF (55, 133). Therefore, some of the most important regulatory molecules maynot alwaysact as diffusible soluble substances. Extracellular matrix components made by stromal cells or other specialized components of the marrowmay bind factors to achieve very high local concentrations in discrete microenvironments (54). There can also be transmembrane

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125

forms of some mediators (134-137), and this provides a mechanismwhereby cells that make a substance can directly present it to adjacent cells. Experimentally it is mucheasier to detect mediators that are soluble than those that are cell bound. However, studies employing permeable membraneseparation of cells, conditioned medium, or matrix extracts demonstrate the importance of positive and negative regulators of hemopoiesis that are closely associated with adherent cells (54, 138, 139).

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B-Lineage

Precursors

in the Mouse

The development ofmonoclonal antibodies and cell separation techniques considerably advanced our understanding of B-lymphocyte lineage progenitors, particularly the relatively abundant cells near the end of this differentiation pathway. Thoroughphenotyping has been done with large numbers of patient samples as well as established tumor lines, and the results are usually depicted by placing them into a single lineage (15, 140). However,it is still difficult to determine the degree of overlap and incidences of very small subsets of lymphoidprecursors. Webriefly summarize our experience with cells taken from murine bone marrow and advise somecaution in strict application of lineage diagrams that we and others have published. Anindirect subtraction technique was used to infer the composition of marrow from normal, young adult (B6xDBA2)F1mice as summarized Figure 2 (59). Samples were first depleted of mature B cells, and then negatively selected with one of several antibodies. The results of flow cytometric and microscopic analyses were then used along with absolute cell recoveries to calculate the incidences of cells bearing various markers. Essentially all precursors express Ly5-200 (equivalent to CD45Ron humans), Lym19, and J11D. Substantial display of class II antigen (Ia), M1/75heat stable, Mel 14, and Thy 1 antigens as well as receptors for transferrin and Fc~2b/7l receptor (2.4G2) was also documented. A very small subpopulation also appeared to bear IL-2 receptors. The BP-1 antigen is uniquely associated with late B-lineage precursors (141). The sizes oflymphocytes expressing various markers were also evaluated in terms of low angle light scatter by flow cytometry (59). Whilea majority of the Lyb-2÷ cells in marrowwere small, surface Ig populations bearing other markers generally included greater numbersof large cells (Figure 3). In another study, done with a different strain of mice, it appearedthat ThB was preferentially expressed on small lymphocytes (142). However, nearly half of the slg ,Th-B+ cells in our analyses have been large. Detailed kinetic studies have been done of large and small pre-B cells, cells containing terminal deoxynucleotidyl transferase (TdT), and cells bearing lineage antigens in rat and mouse bone marrow (143-148). Our flow

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OtherMariners: Qa2 (15%) tL2R(3%l LessIhan1%: Lyt2 L3T4(GK1,5) M1/87 F4/80 Mac1 Mac2 Mac3 Grnt.2

Figure 2 Numerousantigen and receptors characterize the final three stages of B cell formation in murine bone marrow. Percentages of cells (range) expressing certain markers are given to indicate a certain degree of population heterogeneity.

SIZE PROFILESOF s lg7 Ly 5 (220)+ADULTBM CELLS small cells

= large cells

small cells

lar0e cells

I

~T~

y 5 (220) total

ThB+ + .,.~= - ,\

+

BP-1 +

Forward Angle Light Scatter (FALS) Figure 3 Size heterogeneity of B lymphocyte lineage precursors expressing particular markers is reflected in forward angle light scatter. Cells lacking surface immunoglobulin were purified from bone marrow with monoclonal 14.8 antibody and evaluated with other antibodies as indicated.

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127

cytometric analyses of cellular DNAcontent have been consistent with those findings, i.e. small lymphocytes are noncycling, and most of the mitotic activity is associated with large B-lineagecells. Manymonoclonal antibodies have been prepared to epitopes on the Ly5 (CD45) commonleukocyte antigen family (15). The large literature involving these reagents provides an example of complex gene regulation in lympho-hemopoieticcells. Multiple mRNA species are transcribed from a single large gene, and expression is controlled in a tissue-specific manner (149 151). The function of the resulting transmembraneglycoproteins not known, but their importance is suggested by substantial conservation during evolution. As has been described for CD45Rin the human(152), our monoclonal 14.8 antibody detects virtually all B-lineage lymphocytes and a subset of peripheral T cells in mature mice 045, 153, 154). This includes all B-cell precursors that can quickly maturein culture, and most that can do so after transfer to immunodeficient recipients (153, 155). However,pre-B cells in early embryos, sometransformed B-cell lines, and some of the B lineage lymphocytes that grow for prolonged periods in culture lack the epitope detected by this antibody (23, 153, 156, 157). This illustrates the importance of using more than one marker and establishing a "composite" phenotype before positioning any given normal or transformedcell type within the lineage. It also demonstrates that a strictly ordered series of differentiation steps need not always be followed as the progeny of stem cells progress downthis pathway. Physical differences in bone marrowcells, such as buoyant density and size, have been exploited along with lectin receptors, drug sensitivity, and marker expression in attempts to obtain enriched preparations of stem cells (53, 158-161). With multiparametercell sorting and sequential separation with various monoclonal antibodies, it has now becomepossible to obtain multipotential stem cells in virtually pure form (162). This remarkable achievement should soon lead to definitive diagrams of early differentiation steps as the cells are placed into all of the available assays with recombinantfactors and cloned stromal cell lines. The possibility has often been raised of branchedand/or parallel lineages of B-cell differentiation that result in functionally restricted cells. Most recently this has followed intriguing findings with Ly-I(CD-5) bearing cells (163, 164). As far as we are aware, none of the available data lymphocyte subpopulations preclude that possibility in bone marrow, or any other tissue. Factors That Stimulate B Lineage Precursors in Culture Extraordinary progress is being madein defining eytokines that are potentially important for regulating B lymphopoiesis. Responses detected in

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culture studies suggest mechanisms through whichfactors might directly interact with specific receptors on B lineageprogenitors,or indirectly via effects on macrophages, endothelial cells, and stromal cells (Figure 1). Somecould be involved in normalsteady state B-cell production, whereas others maybe important only in situations such as obtain in trauma, inflammation, leukemia, or autoimmunity. Antagonists maybe no less importantthan agonists, and a numberof these have already been identified. It wouldbe surprising if these findings do not quicklylead to therapeutic applications, as is already occurring with a numberof cytokines

(81). Theactive substancesin "tumornecrosis serum," whichwas first found to influence B-cell formationin culture, are likely to be tumornecrosis factor (TNF)and interleukin 1 (IL-I) (15, 165). IL-1 induces kappa chain expression on a pre-B cell tumor line and maturation of normal precursors in cultures of B cell depleted bone marrow(166, 167). Recent findingssuggestthat the latter responsecan proceedat least partly via an indirect mechanism (45). IL-1 stimulated a cloned stromal cell line release IL-4, whichin turn inducedmaturationof small pre-Bcells. A low pHsensitive cytokine in T cell-conditioned mediumwhich had similar effects was subsequently demonstratedto be immuneinterferon (IFN-~) (166, 168-170).Selectivestimulation of cells near the end of the B lineage was also demonstratedwith factors present in the serumof youngNZB strain mice (171). Whiletwo of these substances have been purified homogeneity, their cellular origin is not known(172). Relatively early B lineage precursors in humanand murinebone marrow respondto substancesexcreted duringdiscrete intervals by cyclic neutropenic patients (173). A standardized assay for this type of activity was constructed with routine bone marrowdepleted of all mature B cells and most other lymphocyteswith monoclonal14.8 antibodies. The subsequent appearanceof large 14.8÷ cells and pre-B cells was then monitoredin short-term cultures of such cell suspensions.At least one substancewith similar activity wasdetectable in the supernatantsof a murinestromalcell clone and then obtained in recombinantform (44, and K. S. Landreth, personal communication).The factor has no homologywith previously publishedmediatorsand is designatedIL-? in Figure 1. A transformed stromal cell clone was used to isolate another unique factor, IL-7, whichdramaticallyeffected replication ofmurinecells in longterm bone marrowcultures (49, 50). Since such lymphocytes may abnormal in manyrespects, the effects of recombinant IL-7 were thoroughlystudied using normalbone marrowcells (G. Lee, A. G. Namen, S. Gillis, P. W.Kincade, submitted). A promptproliferation followed addition of IL-7 to wholebone marrowcultures, and a series of cell

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separation experimentsshowedthat the immediatelyrespondingcells were large B-cell progenitors. This property wasexploitedto construct a selective cloningprocedurefor pre-Bcells in agar. Alinear doseresponsecurve for colonies resulting fromtitrated numbers of sorted marrowcells suggests that IL-7 directly stimulateslarger pre-Bcells to produceclonesof as many as 2000cells withinfive days. In contrast, highly enrichedpreparationsof smallpre-Bcells died in IL7, and 14.8- bone marrowsuspensions did not respond until after a considerable delay. It is possible that early progenitors spontaneously acquire IL-7 receptors, or that small amountsof IL-7 eventually stimulate upregulation of receptors to a functional level. Experimentswere then done to monitor population changes in bone marrow cultures maintainedin IL-7. BP-1antigen expressionwasvery high on these cells, as is the case for most transformedpre-B lines (141). Significant numbers of cells were also found that expressedclass II and/or Thy-1antigens (G. Lee, A. E. Namen,S. Gillis, P. W.Kincade,submitted). IL-7 primarily a replication factor, and little or no progression towardsurface Ig expressionoccurredduring5 days of semisolidor several weeksof liquid culture. B-cell precursors normallyundergoonly a finite numberof divisions under the influence ofIL-7(G. Lee, A. E. Namen, S. Gillis, P. W.Kincade, submitted). AlthoughIL-7 dependentlymphocyteclones have been isolated from long-termbonemarrowcultures, they must represent extremely rare variants (49, and A. Namen,personal communication).The phenotypes of such clones are unusual; they are not responsive to normal inductive stimuli, and they can spontaneously becomefactor independent (G. Lee, K. Medina, unpublished observations). These might be pre-malignant cells that have lost some normal regulatory responses, and it will be extremelyinteresting to learn howhumancells grownin IL-7 relate to those involved in leukemias (140). Problems with production, or receptor recognition of IL-7, mightbe involved in suchdiseases. Potential Antagonists of B Lymphopoiesis Normalmouseserumblocks most responses of an inducible pre-B tumor cell line in culture (G. Lee, unpublishedobservations).However,TGF-fl’s providedthe first clear exampleof potential antagonists for B lymphopoiesis (174). Thesefactors selectively blockedkappaexpressionon maturing normalpre-Bcells, or certain inducedresponsesin a pre-Bcell line. ClassII expressionis upregulatedon pre-Bcells by IL-4, and this response is blockedby prostaglandinE, but not by TGF-fl’s(174-177). Surface expressioncan be inducedon pre-Bcells by several factors, but only some

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of these are completely blocked by TGF-~’s 074). For example, LPS induction, but not IFN-~ induction, was effectively inhibited at the ~c mRNA level. This correlates with findings that variant clones of a pre-B cell line are differentially responsive to these agents (168, 178). A kappa enhancer binding protein, NFg-b, is induced by LPS (179), but not IFN~ although transcription of g mRNA is caused by both types of stimuli (M. Briskin, R. Wah, submitted). This suggests that multiple transmembrane signaling pathways and second messengers can lead to expression of this critical gene and that each type of response can be independently controlled by particular antagonists. TGF-fl’s are knownto be made and released in an inactive precursor form (61, 62). However, it was surprising to find that immunohistochemically detectable quantities of TGF-//werepresent on the surface of manynormal pre-B cells (174, and G. Lee, unpublished observations). little as 10-~° Mconcentrations of the active factor should block maturation of these cells. The mechanismwherebylatent TGF-fl’s are activated in vivo is not known. Production of myeloid or lymphoid cells in long-term cultures was completely blocked by adding TGF-/~’s to the medium(S.-I. Hayashi, manuscript in preparation). While these factors can directly interact with hemopoieticprogenitors (180), the effect was mediated in part by effects the microenvironment(S.-I. Hayashi, manuscript in preparation). TFG-/~’s block adipogenesis in long-term cultures and are knownto regulate synthesis of collagen and other componentsof the extracellular matrix (76, 77, 109, 181). Determining thc site of action of a cytokine can thus be difficult whenit is addedto a complexculture system. Clonal analysis of bone marrowcells with IL-7 plus recombinantfactors revealed other potentially important antagonists (G. Lee, A. E. Namen, S. Gillis, P. W. Kincade, submitted). A subpopulation of IL-7 responsive cells were sensitive to TGF-~inhibition, but complete proliferation arrest was achieved with IL-1 or high concentrations of IL-2. Recombinant IL2 is already being employedin clinical trials with certain malignancies, and it is intriguing to speculate that such cytokines might have efficacy for treatment of B lineage leukemias. IL-7 induced proliferation was unaffected by interleukins, 3, 4, 5, and 6 as well as IFN-y, G-CSF,G/M-CSF,TNF, or NZBserum derived factors (G. Lee, A. E. Namen, S. Gillis, P. W. Kincade, submitted). Thus, replication of B lineage precursors is under discrete control of agonists and antagonists. Other molecules have been defined that may influence B lymphocyte formation. For example, there is evidence that IL-3 directly (182, 183) indirectly (184) influences replication of someearly precursors. However, this thoroughly studied multipoietin has not been detectable in long-term

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B-CELLFORMATION 131 culture systems, and we have other reasons for thinking that it is not essential for normal lymphopoiesis (see below). The bone marrowis a site not only for primary blood cell formation, but also for secondary immune responses(185). In this context, it is interesting that a critical growthfactor for antibody forming cells, IL-6, is made. by stromal cells (72, 186, 187, and J. M. Gimble, manuscript in preparation). IL-6 and other stimuli increased IL-6 mRNA levels in stromal cells and fibroblasts (72, 89, and unpublished observations). Thus, stromal cells might potentially contribute to "peripheral" as well as "central" lymphoid tissue microenvironments. Genetically Determined Abnormalities and Polymorphisms Reveal Additional Complexity in Bone Marrow Long-termculture models and their utility have been emphasized in this review. If these approaches provided a perfect representation of normal bone marrow, one would always expect to see a direct correlation between in vivo and in vitro studies. For example, if a genetic abnormality prevents lymphocyte formation in culture, it should have the same influence on B-cell numbers in the affected mice. Reciprocally, bone marrow from immunodeficient mice which have few lymphocytes should not be able to form them in culture. Wehave found exceptions to both of these types of predictions. Also, genetic polymorphismsthat have little or no effect on blood cell formation in intact animals can profoundly influence hemopoiesis in vitro. These findings suggest that intact bone marrowmayhave "fail safe" regulatory mechanismsnot always duplicated in vitro. However, the relative simplicity of our experimental approaches makes it possible to appreciate the delicate balance and redundancypresent in this organ. Mice with severe combined immunodeficiency disease (SCID) have normal numbersof stem cells, myeloid cells, and NKcells, although B and T lymphocytes are virtually absent (188-191). The molecular basis of this mutation is thought to involve improper recognition of appropriate sequences during rearrangement of Ig and T cell receptor genes (192). Althoughthe rearrangements occur, this only rarely results in functional lymphocytes. However, long-term lymphocyte cultures can be readily established with bone marrow from SCID mice and subsequently maintained for at least eight months (29, 30). Therefore, some component intact marrowmay be able to sense and eliminate intrinsically defective lymphocytes. Our long-term cultures must lack positive and negative selection mechanismswhich operate to achieve this type of "quality control." Lymphoid and myeloid cells are made in severely immunodeficient/autoimmune mice with motheaten mutation (193). However, addition of bone marrowcells from these animals to long-term cultures

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established with normal littermate cells prevented production of nonadherent cells under three different types of culture conditions (139, 194, 195). The mechanismof this suppression is not clear, but it seems to involve abnormalexpansion of macrophage-like cells in primary cultures. The imbalance was not reflected in the release of soluble inhibitory substances; motheaten cells had to be closely associated with normal cells to prevent lympho-hemopoiesis. Thus, an antagonist that is cell- or matrixbound could be abnormally active in long-term cultures of motheaten marrow. An understanding of this phenomenoncould be instructive with respect to a regulatory mechanism which is normally coordinated and potentially important in vivo. The X chromosome-linked immunodeficiency of CBA/Nmice does not influence the rate of pre-B cell formation in bone marrow (195, 196). However, this process is unusually dependent on T lymphocytes (197199). Athymic nude mice congenic for the Xid mutation have few lymphocytes of any kind, and the same is true of thymectomized recipients of (Xid) bone marrow. Cells with obvious T lineage markers are not demonstrable in our long-term cultures (23). Thus, one might predict that cultures established from Xid marrow would be poor lymphocyte producers. However, the mutation actually caused accelerated lymphopoiesis, and myelopoiesis in vitro (S.-I. Hayashi, P. L. Witte, P. W.Kincade, submitted). Xid positively affected an early step in culture formation, presumablyinvolving the establishment of an adherent layer. IL-3 has been shownto influence cells of manylineages (56), including early B lymphocyte precursors (182, 183, 200, 201). However, we found enormousvariation in responsiveness of bone marrowcells from d’,~,erent inbred strains to this lymphokine (59). Cells from mice that taave obvious difficulty with blood cell formation were virtually unresponsiveto IL-3 in several culture assays. Thus, this multipoietin is probably not a necessary part of the regulatory network. Otherwise, polymorphisms affecting IL-3 responses would lead to markeddifferences in blood cell numbers. One nonlymphoid cell line (WEHI-3) makes large amounts IL-3. However,this resulted from insertion of a transforming retrovirus into the IL-3 gene, and T helper cells are probably the normal source for this mediator (202). Sensitive bioassays and mRNA analyses have not revealed IL-3 production by stromal cells, or any other component of long-term cultures (58). However, strom~ cells can support the growth and differentiation of stem cells that have been maintained for a time in IL-3 containing medium(102, 203,204). All of these findings indicate that one or moreother factors duplicate the functions of IL-3 in vivo. They also suggest that responses to treatment with certain recombinant cytokines can vary substantially amongindividuals.

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B-CELLFORMATION 133 Other types of polymorphismsmarkedly influence the behavior of cells in long-term bone marrow cultures. For example, BALB/cbone marrow cells are superior to CBA/H derived cells in terms of the ease of culture establishment and continued lymphocyte production (S.-I. Hayashi, P. L. Witte, P. W.Kincade, submitted). Virtually all of the nonadherentcells in cultures initiated with equal part mixtures of the two marrowtypes were BALB/c,suggesting that their hemopoietic cells have an intrinsic proliferative advantage. Wedo not knowthe molecular basis for these differences, and similar influences on lymphopoiesis are not obvious in vivo. Again, this suggests that long-term culture modelslack the full complexity of intact bone marrow. However, there are circumstances when results of short- and longterm cultures correlate with those predicted from in vivo behavior. As mentionedabove, factors active on early B lineage precursors appeared in the urine of cyclic neutropenia patients just whennumbersof bone marrow pre-B cells were markedly elevated (92, 173). This made it practical induce formation of humanpre-B cells in culture, and the experimental approach led to the molecular cloning of a unique cytokine (44, and K. S. Landreth, personal communication). Likewise, lymphopoiesis is premature and exaggerated in embryonic and young NZBstrain mice. Cytokines that maycontribute to this dysregulated situation were purified from the serum of these animals, and evidence obtained that older NZBmice make autoantibodies to them (171, 172). As the animals mature, pre-B cells virtually disappear from the bone marrowin what could be a related phenomenon(205). Abnormalhemopoiesis of this strain of mice was also reflected in long-term bone marrowcultures (206). Concludin9 Remarks Our understanding of B lymphocyte progenitors should increase with concerted application of improvedcell sorting protocols, monoclonalantibodies, purified cytokines, and gene probes. The latter are becoming increasingly available through subtractive hybridization and screening techniques (207) and will considerably facilitate our understanding differentiation events. Similar experimental approaches should be informative about stromal cells and other microenvironmental elements, about which several questions seem crucial. Weneed to know how many cytokines they are capable of making, what physical form they have in vivo, and which ones are relevant to the B lymphocyte lineage. Positive and negative regulatory interactions betweencells in bone marrowcultures are amenable to study, and this information should be extended by injection of recombinant materials in vivo. Further technical advances might lead to better duplication of the bone marrowin culture, and this would have

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major implications for diagnosis as well as basic research. Bonemarrow transplantation has becomea therapeutic option for manydisorders. However, the molecular and cellular basis for diseases such as myelofibrosis, anemias, autoimmunediseases, and malignancies differ. It may someday be possible to engraft only the relevant components(96) or treat with the appropriate cytokines.

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ACKNOWLEDGMENTS

Our research is supported by grants AI 20069 and AI 19884 from the National Institutes of Health and a Special Fellowship (to Carolyn G. Pietrangeli) from the LeukemiaSociety of America. Weare grateful for expert technical assistance provided by Ms. Annette Dorheim, Anna Henley, Kay Medina, and Margaret Robinson.

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nary activity associated with cyclic neutropenia. J. Itnmunol. 134:2305-9 174. Lee, G., Ellingsworth, L. R., Gillis, S., Wall, R., Kincade, P. W. 1987. fl transforming growth factors are potential regulators of B lymphopoiesis. J. Exp. Med. 166:1290-99 175. Polla, B. S., Poljak, A., Ohara, J., Paul, W. E., Glimcher, L. H. 1986. Regulation of class II gene expression: Analysis in B cell stimulatory factor 1-inducible murine pre-B cell lines. J. Immunol. 137:3332-37 176. Polla, B. S., Poljak, A., Geier, S. G., Nathenson, S. G., Ohara, J., Paul, W. E., Glimcher, L. A. 1986. Three distinct signals can induce class II gene expression in a murine pre-B cell line. Proc. Natl. Acad. Sci. USA83: 487882 177. Polla, B. S., Ohara, J., Paul, W. E., Nabavi, N., Myer, A., Liou, H.-C., Shen, F.-W., Gillis, S, Bonventre, J. V., Glimcher, L. H. 1988. Differential induction of class II gene expression in murine pre-B-cell lines by B-cell stimulatory factor-1 and by antibodies to Bcell surface antigens. J. Mol. Cell. lmmunol. 3:363-73 178. Mains, P. E., Sibley, C. H. 1983. LPSnonresponsive variants of mouseB cell lymphoma,70Z/3: Isolation and characterization. Somatic Cell Genetics 9: 699-720 179. Sen, R., Baltimore, D. 1986. Inducibility of x immunoglobulin enhancer binding protein NF-xbby a post-transcriptional mechanism. Cell 47: 92128 180. Ohta, M., Greenberger, J. S., Anklesaria, P., Bassols, A., Massague, J. 1987. Two forms of transforming growth factor-/~ distinguished by multipotential haematopoietic progenitor cells. Nature 329:539-41 181. McKearn, J. P., McCubrey, J., Fagg, B. 1985. Enrichment of hematopoietic precursor cells and cloning of multipotential B-lymphocyte precursors. Proc. Natl. Acad. Sci. USA82: 741418 182. Kinashi, T., Inaba, K., Tsubata, T., Tashiro, K., Palacios, R., Honjo, T. 1988. Differentiation of an interleukin 3-dependent precursor B-cell clone into immunoglobulin-producing ceils in vitro. Proc. Natl. Acad. Sci. USA85: 4473-77 183. Paige, C. J. 1985. Analysis of the requirements for murine B cell differentiation. Lymphokines 10:143-63 184. Koch, G., Benner, R. 1982. Differential requirement for B-memory and T-

memorycells in adoptive antibody formation in mouse bone marrow. Immunoloyy 45:697-704 185. Muraguchi, A., Hirano, T., Tang, B., Matsuda, T., Horii, Y., Nakajima, K., Kishimoto, T. 1988. The essential role of B cell stimulatory factor 2 (BSF2/IL-6) for the terminal differentiation of B cells. J. Exp. Med. 167:332-44 186. Kishimoto, T., Hirano, T. 1988. Molecular regulation of B lymphocyte response. Ann. Rev. Immunol. 6:485 512 187. Bosma, G. C., Custer, R. P., Bosma, M. J. 1983. A severe combined immunodeficiency mutation in the mouse. Nature 301:527-30 188. Dorshkind, K., Pollock, S. B., Bosma, M. J., Phillips, R. A. 1985. Natural killer (NK) cells are present in mice with severe combined immunodeficiency (SCID). J. Immunol. 134:3789 189. Dorshkind, K., Keller, G. M., Phillips, R. A., Miller, R. G., Bosma, G. C., O’Toole, M., Bosma,M. J. 1984. Functional status of cells from lymphoidand myeloid tissues in mice with severe combined immunodeficiencydisease, J. Immunol. 132:1804-8 190. Bosma,G. C., Fried, M., Custer, R. P., Carroll, A., Gibson, D. M., Bosma, M. J. 1988. Evidence of functional lymphocytes in some(Leaky) scid mice. J. Exp. Med. 167:1016-33 191. Schuler, W., Weiler, I. J., Schuler, A., Phillips, R. A., Rosenberg, N., Mak, T., Kearney, J. F., Perry, R. P., Bosma, M. J. 1986. Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency. Cell 46:963-72 192. Schultz, L. D., Sidman, C. L. 1987. Genetically determined murine models of immunodeficiency. Ann. Rev. Immunol. 5:367-403 193. Greiner, D. L., Goldschneider, I., Komschlies,K. L., Medlock, E. S., Bollure, F. J., Schultz, L. 1986. Defective lymphophoiesis in the bone marrowof motheaten (me/me) and viable mothV) mutant mice. 1. Analyeaten (meV/me sis of the development of prothymocytes, early B lineage cells and terminal deoxynucleotidyl transferase-positive cells. J. Exp. Med. 164:1129-44 194. Medlock, E. S., Goldschneider, I., Greiner, D. L., Shultz, L. 1987. Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable v) mutant mice. II. motheaten (meV/me Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase - positive

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B-CELL FORMATION bonemarrow cells in vitro. J. lmtnunol. 138:3590 195. Kincade, P. W., Jyonouchi, H., Landreth, K. S., Lee, G. 1982. B-lymphocyte precursors in immunodeficient autoimmune and anemic mice. Immunol. Rev. 64:81-98 196. Reid, G. K., Osmond,D. G. 1985. B lymphocyte production in the bone marrowof mice with X-linked immunodeficiency (xid). J. Immunol. 135:2299-2302 197. Mond,J. J., Scher, I., Cossman,J., Kessler, S., Mongini,P. K. A., Hansen, C., Findelman,F. D., Paul, W.E. 1982. Role of the thymusin directing the developmentof a subset of B lymphocytes. J. Exp. Med.155:924-36 198. Sprent, J., Bruce,J. 1984.Physiology of B ceils in mice with x-linked immunodeficiency(xid). II1. Disappearance of xid B cells in double bone marrowchimeras. J. Exp. Med. 160:711-23 199, Wortis, H. H., Burkly,L., Hughes,D., Roschelle, S., Waneck,G. 1982. Lack of matureBcells in nude micewith Xlinked immune deficiency. J. Exp. Med. 155:903-13 200. Palacios, R., Henson,G., Steinmetz, M., McKearn,J. P. 1984. Interleukin3 supports growthof mousepre-B cell clones in vitro. Nature309:126 201. Spalding, D. M., Griffin, J. A. 1986. Differentpathwaysof differentiation of pre-Bcell lines are inducedby dendritic cells and T cells from different lymphoidtissues. Cell 44:507-15 202. Ymer,S., Tucker,W.Q. J., Sanderson,

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C. J., Hapel, J., Campbell, H. D., Young,I. G. 1985. Constitutive synthesis of interleukin-3byleukemiacell line WEHI-3B is due to retroviral insertion near the gene. Nature317:255-58 203. Spooncer,E., Heyworth,C. M., Dunn, A., Dexter, T. M. 1986. Self-renewal and differentiation of interleukin-3dependentmultipotent stem cells are modulatedby stromal cells and serum factors. Differentiation31:111-18 204. Anklesaria,P., Klassen,V., Sakakeeny, M.A., FitzGerald,T. J., Harrison,D., Rybak,M.E., Greenberger,J. S. 1987. Biological characterization of cloned permanent stromal cell lines from anemic S1/Sld mice and +/+ littermates. Exp. Hematol.15:636-44 205. Jyonouchi, H., Kincade, P. W., Landreth, K. S., Lee, G., Good,R. A., Gershwin,M. E. 1982. Age-dependent deficiencyof Blymphocyte lineage precursors in NZBmice. J. Exp. Med. 155:1665-78 206. Yoshida,S., Dorshkind,K., Bearer,E., Castles, J. J., Ahmed, A., Gershwin,M. E. 1987. Abnormalities of B lineage cells are demonstrable in long term lymphoid bone marrow cultures of NewZealand Black mice. J. Immunol. 139:1454-58 G. G., Eisenberg, D., Kin207. Hermanson, cade, P. W., Wall,R. 1988.A newmember of the immunoglobulin superfamily exclusivelyexpressedon Blineagecells. Proc. Natl. Acad. Sci. USA85: 689094

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Ann. Rev. Immunol. 1989. 7:145 73 Copyright © 1989 by Annual Reviews Inc. All rights reserved

TH1 AND TH2 CELLS: Different Patterns of Lymphokine Secretion Leadto Different Functional Properties T. R. Mosrnann and R. L. Coffman DNAX Research Institute, California 94304

901 California Avenue, Palo Alto,

Introduction Effector functions in the immunesystem are carried out by a variety of cell types, and as our understanding of the complexity of the system expands, the numberof recognized subdivisions of cell types also continues to increase. B lymphocytes, producing antibody, were initially distinguished from T lymphocytes, which provide help for B cells (1, 2). The T-cell population was further divided when surface markers allowed separation of helper cells from cytotoxic cells (3). Althoughthere were persistent reports of heterogeneity in the helper T-cell compartment (reviewed below), only relatively recently were distinct types of helper cells resolved. In this review we describe the differences betweentwo types of cloned helper T cells, defined primarily by differences in the pattern of lymphokinessynthesized, and we also discuss the different functions of the two types of cells and their lymphokines. Patterns of lymphokinesynthesis are convenient and explicit markers to describe T-cell subclass differences, and evidence increases that manyof the functions of helper T cells are predicted by the functions of the lymphokines that they synthesize after activation by antigen and presenting cells. The separation of manymouse helper T-cell clones into these two distinct types is nowwell established, but their origin in normal T-cell populations is still not clear. Further divisions of helper T cells may have to be recognized before a complete picture of helper T-cell function can be obtained. 145 0732-0582/89/0410-0 145502.00

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Lymphokine Activities--The Need for Monospecific Assays Duringthe last several years, our understandingof lymphokinestructure and function has progressedenormously.Dueto the availability of purified proteins, reco~nbinant cDNA clones and monoclonalantibodies, manyof the knownlymphokineactivities can nowbe unambiguously attributed to well-characterized proteins. All knownlymphokinesaffect morethan one cell type and often havediverse effects evenon cells of the samelineage (reviewedin 4, 5, 6). Further complexityis addedby the fact that each type of cell in the immunesystemresponds to morethan one lymphokine. Becauseof this multiplicity of lymphokineaction, monospecificbioassayshavebeendifficult to establish. Monoclonal antibodies(7, 8, 9, 10, 11) havebeenused to improvethe specificity of bioassays, and to measure lymphokines directly by ELISAassays (Table 1). For example,in the T-cell growthassay, both Interleukin 2 (IL-2) and IL-4 can cause proliferation mostT-cell lines, althoughthe dose-response relationships are different (6, 12, 13, 14, 15, 16). Usingmonoclonalantibodies that neutralize the biological activities of IL-2 (6) and IL-4 (9), these bioassays can be monospecificfor either lymphokine(11). TH1 and TH2 Lymphokine Secretion

Patterns

Whenstringent, apparently monospecificassays are used for evaluating lymphokine synthesis, mousehelper T-cell clones fall into twomaingroups. Earlyresults showed that in a panelof clones, eachclonesynthesizedeither IL-2 andInterferon 7 (IFNT),or IL-4 (6). Usinga further set of bioassays and particuarly by evaluating mRNA synthesis by hybridization, the differences in lymphokine synthesis were extendedto a numberof lympho-

Table 1 "Monospecific" assays for lymphokines aBioassays IL-2 IL-4 IL-5 IFN~/ IL-3 GM-CSF

HT2+anti-IL-4 (9) HT2+ anti-IL-2 (7)

MC/9 +anti-IL-4

bELISAs $4B6 (7)+rabbit alL-2 -TRFK2+TRFK5 (10) XMGI.2(1 l)+rabbit alFN3, 8FS+43D11 (8) c31 G6+ 22E9

"Proliferation assaysusingthe indicatedtarget cell line, andblockingmonoclonal antibodiesas indicated. bTwo-sitesandwichassays in whichthe first antibodyis boundto the plate, and the second usedin solution. cj. Abrams,personal communication.

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kines and other secreted proteins (11). Table 2 lists our current knowledge of the lymphokinepatterns of the two types of clone, TH1 and TH2. TH1 clones synthesize IL-2, IFNy, and lymphotoxin (LT), whereas these lymphokinesare not detectably expressed in TH2clones. Conversely, only TH2clones synthesize detectable amountsof IL-4, IL-5 (11), and probably IL-6 (F. Lee, T. Mosmann,unpublished). An additional marker for TH2 clones was obtained with the discovery of the induction-specific cDNA clone P600 in cDNAlibraries from an induced TH2done (K. D. Brown, S. M. Zurawski, T. R. Mosmannand G. Zurawski, submitted). The synthesis of IL-2, IFNy, IL-4 and IL-5 is tightly controlled, because induced supernatants of the appropriate cell type contain at least 2,00010,000-fold more of the lymphokine than do induced supernatants of the

Table 2 Properties of mouseT cell clones TH 1

CTL

TH2

Surface markers: LY1 L3T4 LYT2 aLymphokines: Interferon ), Interleukin 2 Lymphotoxin GM-CSF Tumornecrosis factor TY5 P500 H400 Interleukin 3 Met-enkephalin Interleukin 4 Interleukin 5 lnterleukin 6 P600 B cell help: IgM, IgGl, IgA IgG2a IgE Delayed type hypersensitivity: Macrophageactivation: a Lymphokineexpression was evaluated by bioassays, ELISAsand RNA hybridization. b Somebut not all CTLclones produceIL-2.

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other cell type. Several other genes were expressed by all clones tested. These included three lymphokines, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF) and IL-3, neuropeptide, preproenkephalin (ppENK), and three other inductionspecific genes of unknownfunction, TYS, PS00, and H400. Careful analysis of mRNA levels revealed that THl cells expressed relatively more GMCSF, TNF, TY5 (11), H400, and PS00 (K. D. Brown, S. M. Zurawski, T. R. Mosmannand G. Zurawski, J. Immunol. In press), whereas TH2 clones expressed relatively more ppENK(11). These moderate differences (approximatelyfive-fold in most cases) appear to be statistically significant, but we do not yet understand their biological importance. These patterns are characteristic of the majority of long-term T-cell ¯ Clones tested. Someexceptions have also been seen, e.g. expression of IFN7 by a clone that was originally a good example of a TH2. It is not yet knownif these exceptions could be due to aberrations arising in tissue culture. IL-2 synthesis maybe regulated independently of the synthesis of other lymphokines, since THI clones can often lose the ability to produce IL-2 after lo.n_g periods in culture, while the synthesis of other lymphokines appears to be more stable (11). This mayalso account for reports of number of T-cell clones that secrete IFN7 and LT, but not IL-2 (17). Recent evidence from clones that have been grown in culture for short periods (e.g. 4-8 weeks) suggests that there mayalso be other states of differentiation preceding the TH1and TH2phenotypes (T. R. Mosmann, N. Street, H. Bass~and.J. Schumacher,unpublished). As discussed below, the in vivo representation of THI, TH2, and possible precursor phenotypes remains to be established. Lymphokine

Synthesis

Patterns of Other T-Cell Types MouseT-cell clones of the Lyt2+, cytotoxic phenotypes also express a pattern of lymphokines that corresponds closely to the TH1pattern (18, 19; T. A. T. Fong, T. R. Mosmann,unpublished). The CTLclones that we have tested all showed good levels of~ IFN~, and TY5expression, and they produced moderate amounts ofGM-CSF, IL-3, LT and TNFmRNA. No IL-4 or IL-5 synthesis could be detected, and ppENKexpression was detectable at low levels in some clones. These mRNA results have been confirmed by lymphokine assays for IL-3, IL-4, IL-5, GM-CSF,and IFN~. Someclones synthesized moderate amounts of IL-2, whereas IL-2 protein or mRNA was undetectable in other clones. The variability in IL-2 synthesis has been.reported previously (17) and may be physiologically relevant, or it may"be an extreme manifestation of the in vitrolinstability of IL~’2~synthegisin THl.clones. A fourth type of T c~ll, the ~TCI~I+ cell, expresses the 76 form of the T-cell antigen/MHCreceptor and has the

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surface antigen phenotypeThyl+, L3T4-,Lyt2 . A subset of these T cells constitutes the dendritic T cells found in the epidermis (20), and one exampleof a dendritic TCRl÷ T-cell clone expressedthe THIpattern of lymphokines(T. R. Mosmann, R. E. Tigelaar, unpublished). The expression of the TH1pattern of lymphokinesynthesis in three different T-cell types and the stability of the TH1 andTH2phenotypesin culture suggest that these two patterns of lymphokinegene expression constitute two tightly controlled "cassettes" of regulation. However, there are examplesof T-cell clones, especiallyat early times after establishment in culture, that produce a mixture of TH1and TH2patterns (T. R. Mosmann, N. Street, H. Bass and J. Sehumacher,unpublished; A. Glasebrook, personal communication).These clones and manyhumanclones (21) suggestthat further patterns of lymphokine expressionexist. Hybrids between a TH2clone and a CTL(expressing IFNy)were able to produceboth IL-4 and IFNyin response to stimulation with the antigen recognizedby either parent (22). This suggests that the lack of synthesis of IL-4 in a TH1,or IFNyin a TH2clone, is not due to a suppressive mechanism, but rather is dueto the lack of positive induction. In addition, the data indicate that the phenotypefor secretion of lymphokines is not linked to a particular specificity of the antigenreceptor but is probablya functionof the cell’s state of differentiation. Functions of TH1 and TH2 Cells and Their Lymphokines a-CELLHEL~The ability of THclones to function as helpers for Bcell responseshas beenstudied in vitro using two different experimental strategies. Thefirst is the traditional hapten-carriersystemused for many years to characterize normal THpopulations, In this system, haptenspecific responsesare measuredin cultures containing primedor unprimed B cells, a carrier-specific THcloneandhaptenatedcarrier protein (or cells) as the antigen. In such cultures, the frequencyof B cells that respond specifically is low, typically 10-3 to 10-5 , althoughthe frequencycan be greatly enhancedusing hapten-enrichedB cells (23). Thesecondstrategy is to use THclones specific for antigens such as Ia, Mls, or H-Y(malespecific antigen) whichare expressedon mostor all B cells. In this way, mostB cells are, in essence, antigen-specific,antigen-presentingcells and appear to respond to cell-mediated and lymphokine-mediated signals in the samewayas do antigen-specific B cells in the hapten-carrier systems. Asignificant refinementof this strategy is to use THclones specific for rabbit IgGand rabbit anti-mouseIgMor IgD antibodies as the antigens (24, 25). This system dependsupon a specific interaction betweenthe antigen andIg on the surfaceof the B cells, andit requires processingand Ia-restricted presentationof the rabbit IgG. Amajority of splenic B cells

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can be activated and induced to proliferate and differentiate in this way (26). In this discussion, we refer to this secondstrategy as "polyclonal stimulation" and distinguish it from another form of polyclonal response, which we term "bystander stimulation" and which does not require a THB interaction (see below). The recent purification and gene cloning many lymphokines and the development of neutralizing antilymphokine antibodies have madeit possible at last to define whichT-cell products are important in regulating B-cell growth and differentiation and to study the specific functions of each of these products. TH2help for B cells It is generally agreed that murine TH2clones can be excellent helpers, both in antigen-specific (27, 28, 29; DeKruyff, submitted) and polyclonal (25, 30, 31, 32) in vitro cultures. TH2clones induce growth and Ig secretion by 50-80%of B cells in limiting dilution cultures (26) and can efficiently induce responses in populations of small, resting B cells (27, 30, 32). The activation of resting B cells by TH2clones appears to require at least three "signals." The first of these is provided by direct contact with the activated helper cell. A/though a few mouse TH2clones may secrete a soluble factor that can induce growth and differentiation of resting B cells (33, 34), in mostcases, this processrequires direct contact with the TH cell and cannot be achieved with TH2supernatants (35, 36, 37). Optimumproliferation and differentiation require both IL-4 and IL-5, in addition to this cell-mediated activation. This requirement has been defined in two types of experiments. In the first, the addition of neutralizing anti-IL-5 antibodies to cultures of B cells and TH2 clones causes a substantial inhibition of Ig production (10, 30, 37). The addition of anti-IL-4 antibodies also inhibits Ig production but to a lesser and more variable extent (28, 30, 31), whereas the combination of both antibodies inhibits Ig production almost totally. Similar conclusions have been reached in experiments in which TH2 products are used to induce the differentiation of B cells polyclonally activated (but not induced to differentiate) by direct interaction with a TH1 clone. In these experiments, both IL-4 and IL-5 are required for optimal proliferation and Ig production, and no other TH2product was active in this system (31; R. L. Coffman, J. Christiansen, B. Seymour, D. Hiraki, H. Cherwinski, R. Schreiber, M. Bond and T. Mosmann,in preparation). Thus, IL-4 and IL5 are the major "helper factors" produced by TH2cells, and both act to enhance the growth and differentiation of activated B cells. The important exceptionto this is the IgE response, for whichIL-4 but not IL-5 is essential (see below). Large B cells, unlike resting B cells, do not require TH-Bcontact and can proliferate and differentiate in response to TH2supernatants. The

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active componentin these supernatants has been shownto be IL-5 (38). This response can account for muchof the unlinked "bystander" response observed in TH2-stimulated cultures at high antigen concentrations (37). The response to IL-5, however, is much smaller than the response of the same large B-cell population to direct interaction with a polyclonally stimulating TH2clone; this suggests either that IL-5 alone is a weak stimulus or that it stimulates only a subpopulation of large B cells (R. L. Coffman, unpublished). Nevertheless, this bystander response may be quite significant in some situations. For example, very young NZB/NZW F 1 mice, whichlater in life develop a severe lupus-like autoimmune disease, appear to have a much higher p~oportion of IL-5-responsive B cells. Culture of these cells with IL-5 leads to substantially higher production of autoantibodies than culture of cells from nonautoimmunemice (38). TH1help for B cells The helper function of TH1cells is more uncertain since they have been shownto provide antigen-specific help in some, but not all, in vitro systems. Several groups have reported TH1clones that can help antigen-specific secondary responses in primed B-cell populations (39; DeKruyff, submitted), and the ability of one TH1clone to stimulate primary antihapten antibody responses in unprimed, hapten-purified Bcell populations has been characterized (29). However,Bottomly, Janeway and their colleagues have described manyTH1 clones that cannot provide help for a specific primary response to the phosphorylcholine hapten, although many of these clones can induce polyclonal proliferation and differentiation of B cells at high antigen concentrations (27, 28). However, these authors have presented evidence that some of these clones produce no detectable IL-2 (17, 28), so their results maynot reflect the activity IL-2-producing TH1 clones. Similarly, Abbas and colleagues report that TH2,but not TH1,clones specific for rabbit ~-globulin can induce polyclonal proliferation and Ig production from dense, resting B cells in the presence of rabbit anti-mouse Ig antibodies (30). In our hands, most TH1clones reactive with self- or allo-Ia or with Mls antigens, efficiently stimulate proliferation, but not Ig production, by B cells bearing the appropriate surface molecule (31). The defect in differentiation to Ig production, however, is not caused by an inherent inability of TH1products to induce differentiation, but by insufficient production of IL-2 in vitro. Thus, addition of exogenousIL-2 to such cultures enhances Ig production. This demonstrates that helper function is possible with products of only TH1clones and suggests that IL-2 is the most important helper factor made by THI cells (31). Further enhancement can often be achieved blocking part of the IFN-~activity with anti-IFN-~ antibodies. In other words, manyTHI clones can stimulate activation and proliferation of B

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cells in vitro and can also stimulate Ig secretion independently of the T-B contact-mediated activation signal if the levels of IL-2 and IFN-7 are optimized. It should be noted that the differentiation of THl-activated B cells can also be induced by TH2products, IL-4 and IL-5 (31). TH1cells can also suppress B-cell responses The assessment of the role of TH1cells in B-cell responses is complicated by two things: (a) the fact that high concentrations of IFN-7 can be quite generally immunosuppressive (40, 41, 42), whereas low concentrations can enhance certain types of responses (42), and (b) by the observations that most TH1clones are directly cytotoxic for activated B cells (43, 44). Not surprisingly, activated TH1clones have been shownto be directly suppressive in cultures optimally stimulated by TH2cells (45, 46, 47). The helper activity TH1clones in vitro appears to dominate at low antigen concentrations (suboptimal TH activation) or at low T:B ratios (<0.2) (31, 32, whereas the cytotoxic activity begins to dominate at ratios exceeding 1.0 (43). Isotype regulation by TH1 and TH2 cells One important difference between the helper function of the two THsubsets is in their ability to stimulate the production of certain Ig isotypes, most importantly IgE and IgG2a. In a panel of over 30 TH1and TH2clones, virtually all of the TH2 clones were capable of inducing significant IgE responses, whereas none of the TH1clones tested could induce detectable IgE production (R. L. Coffman, B. Seymour, H. Cherwinski, J. Christiansen, D. Parker, T. R. Mosmann,manuscript in preparation, summarized in 31). IgE production stimulated by TH2clones can be substantially or totally inhibited by either IFN-y or anti-IL-4. These two lymphokines have been shown to have the same effects in LPS-stimulated B-cell cultures (40, 48). Furthermore, vivo administration of either IFN-7 or anti-IL-4 can inhibit IgE responses in mice (49, 50). In fact, the induction oflgE by TH2,but not TH1,clones can be explained entirely by their differences in IL-4 and IFN-~, production. TH1clones can induce good IgE responses if IL-4 and anti-IFN-7 antibodies are added to in vitro cultures (R. L. Coffman, B. Seymour, H. Cherwinski, J. Christiansen, D. Parker, T. R. Mosmann,in preparation). IL-4 mediates the enhancementof IgE pro.duction by increasing the frequencyof isotype switching. This is shownby examinationof single B cells stimulated by TH2cells (26) and also in limiting dilution experiments.with LPS-stimulated B cells (51). The unique dependence of IgE responses on IL-4,. and the ability of relatively low concentrations Of IFN-~to inhibit this activity of IL-4 (40), suggest that TH1cells mayact as isotype-specific suppi’essor cells for IgE, quite possibly under conditions in which they act as helpers for responses

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of other isotypes. Ovary and his colleagues have described, in SJL mice, IgE-specific suppressor cells that are Lyl ÷, CD8-,and require an antigen for activation, but which suppress IgE in an isotype-specific, antigennonspecific fashion (52). It is tempting to speculate that these cells are, fact, TH1 cells. TH1clones, in contrast, induce substantially more IgG2a than TH2 clones (29, 31). Results for several alloreactive and rabbit IgG-specific TH1clones showthat IgG2atypically accounts for 5%to 10%of the total Ig response, whereas IgG2a usually accounts for 0.1%to 0.5%of the total response if the sameB-cell population is stimulated by TH2clones. Several lines of evidence, both in vitro and in vivo suggest that IFN-7is important for high levels of IgG2a production, but other factors may be involved, since neither the addition of anti-IFN-7 antibody to TH1-stimulated cultures nor the addition of IFN-~ to TH2-stimulated cultures causes much change in IgG2a responses (29). Another important observation made with TH-stimulated cultures is that IL-4 is much less important for the production of IgG1 than was predicted on the basis of its strong IgGl-enhancingactivity in LPS-stimulated cultures (53). The addition of IL-4 can enhance the ability of some TH1clones to induce an IgG1 response (29), but many other TH1clones induce large IgG1 responses (comparable to those induced by TH2clones) in the absence of IL-4 (31). This is consistent with observations that antiIL-4 antibody causes little or no inhibition of IgG1 responses either in vitro (30, 31) or in vivo (48) and suggests that substantial IgG1 responses can be induced by an IL-4 independent mechanism. HELPFORCYTOTOXIC T CELLSThe generation of mature cytotoxic T cells can be enhancedby helper T cells, although it is not yet clear whether TH1 and TH2cells are equally efficient. Several years ago IL-2 was recognized as a major helper factor (54), and recent evidence has shownthat IFN7 (55), IL-4 (56) and IL-5 (57) are also able to enhance the generation CTLs. Thus THI and TH2 clones each produce two lymphokines that induce CTLs, and so both can probably function as helpers. Since these four lymphokinesare effective in subtly different ways, their helper functions for CTLsmaybe operative in different situations, or result in CTL populations with different functional properties. DELAYED TYPEHYPERSENSITIVITY Delayed type hypersensitivity (DTH) an inflammatory reaction mediated by the products of T cells, mainly of the Lyl+ helper phenotype (58). Whenthe distinction between TH1and TH2clones was discovered, it became important to ask whether DTHwas a function of one or both subsets. In an experimental system in which T cells and antigen were injected directly into mousefootpads, only TH1

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clones were able to elicit antigen-specific swelling (59). In someexperimental systems, Lyt2+ cells account for substantial DTH(60), which consistent with the production of similar patterns of lymphokinesby TH1 cells and CTLs. Although the induction of the effector phase of the DTHreaction by TH1 but not TH2 clones appears to be clearcut, the DTHreaction is complex, and why TH2clones are unable to induce DTHis not known. In particular, it has not been proven that the TH2clones were activated in the footpad environment, which is especially important since TH1 clones with dual specificity for antigen/MHCor Mls only induce DTHin response to antigen/MHCstimulation (59). Thus, it is possible that TH2clones cannot induce DTH,because they do not produce essential mediators for the DTHreaction or because they are not activated by antigen-presenting cells in the footpad. Somesupport for the first possibility comes from recent evidence (T. A. T. Fong, T. R. Mosmann,unpublished) that IFN~ is responsible for part but not all of the swelling reaction in response to TH1clone activation, and also from evidence that IFN7 is chemotactic for at least someof the cells that migrate into the inflammatorysite during a DTHreaction (61). A normal DTHreaction may involve two phases (62); an initial activation signal results in recruitment of effector T cells into the site, after which these T cells arc activated to recruit the monocytesand granulocytes that mediate subsequent stages of the reaction. TH1clones can clearly mimicthe effector phase of the reaction, but the T cell responsible for the early phase is not yet identified. Differentiation

of TH1 and TH2 Cells

ARE RESTING T CELLS COMMITTEDTO THI/2 LYMPHOKINEPATTERNS? Although

the TH1and TH2patterns are distinct when long-term mouseT-cell clones are examined, we do not yet know if normal resting mouse lymphocytes are already committed to these patterns. Another important question is whether other lymphokine secretion patterns are exhibited by precursor stages in the development of the mature TH 1 and TH2 phenotypes. The latter may be final differentiation states analogous to the plasma cells producing different isotypes of antibody in the B cell lineage. If mixed spleen cell populations are polyclonally stimulated, they produce large amounts of ILo2, and low amounts of IFNv, IL-4, and IL-5 (N. Street, T. R. Mosmann,unpublished results). This pattern cannot be explained simply by some mixture of TH1, TH2, and CTLs. Either there are cells with other lymphokinesecretion phenotypes, or else differential regulation of lymphokinesynthesis must occur in the mixed population. Preliminary evidence suggests that such differential regulation occurs, but it does not

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explain the lymphokine differences between clones and normal lymphocytes (N. Street, T. R. Mosmann,unpublished). Recently we examined the lymphokine profiles of helper clones soon (e.g. 2 to 6 weeks)after establishment in culture. In someexperiments, the patterns are not recognizably TH1or TH2at early times, but they sometimes change into clearcut TH 1 or TH2phenotypes on continued growth in culture (T. R. Mosmann,H. Bass, N. F. Street, J. Schumacher,unpublished results). Similar results have been obtained in another laboratory (A. Glasebrook, personal communication), except that the time needed change phenotype could be several months. These results mayalso explain the puzzling observation that humanhelper clones do not fall cleanly into TH 1 and TH2 patterns. Although human TH 1 and TH2phenotypes have beer~ reported (63), there are also manyexamplesof clones that secrete mixture of the two patterns (21, 63). These results can be reconciled with the data from mice if it is assumed that in culture humancells tend to persist as the mixed phenotype, whereas clones from mice tend to differentiate more rapidly into TH1 and TH2. These experiments, and the lymphokinepatterns of total spleen cells, suggest that there are precursor stages in the developmentof the TH1 and TH2differentiation states. Figure 1 shows two possible models for the derivation of TH1and TH2: In modelA, a single precursor, the TH0cell, can give rise to either TH1or TH2cells depending on the antigen-presenting cell. ModelB proposes that the precursors of TH1and TH2(TH1P and TH2P) are already committed to their final lymphokine secretion phenotype before antigen stimulation and that they have different activation requirements. In both models, we propose that the precursor cells synthesize a set of lymphokines that fits neither TH1nor TH2patterns. From the results of stimulation of normal spleen lymphocytes, the precursors maysecrete IL-2, but little or no IFNv, IL-4 and IL-5. This is consistent with the results of Budd and coworkers (64), who found that memoryT cells, identified by the Pgpl cell surface marker, synthesized similar amounts of IL-2 but larger amounts of IFNv when compared to unprimedT cells. These results have also been extended to IL-3 and IL-4, which are also synthesized at higher levels by Pgpl÷ cells (R. C. Budd, J. H. Schumacher, T. R. Mosmann,unpublished). RELATIONSHIP

OF TH!

AND TH2

TO PREVIOUSLY

DESCRIBED

T-CELL

SUB-

÷ TYeESBottomly and coworkers described the division of a panel of CD4 T-cell clones into four groups, based on the patterns of help provided to B cells (27). These groups were later assessed for lymphokineproduction, and two of the groups appear to fit with the TH1 and TH2 lymphokine classification (84). Groups 1 and 2 were equivalent to TH2clones and

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A

IL2, IFH, LT

ANTIGEN

ANTIGEN

IL4, IL5

IL2, IFN, LT

B

~ IL4, IL5

Figure 1 Possible TH1 and TH2 differentiation pathways. Two possible pathways of differentiation are shown. Heavy arrows show the predominant effects that occur when a THl-preferential antigen is used. Both models show two different APCs with different preferential stimulation effects on TH1and TH2. The models could also be drawn with a single APCwhich is able to deliver different signals, depending on the physical nature of antigen.

differed from each other in their ability to provide help for the T15idiotype (84). This mayhave reflected a difference in specificity rather than. phenotype. Group 3 included TH1clones, and group 4 was another set of TH2 clones specific for self-antigens (27). Bottomly(84) has suggested tl~at THI cells should be called inflammatory T cells because of their role in inflammatoryprocesses, and that TH2cells be called T helper cells because they have stronger helper functions for B cells. Since TH1clones can also provide help for B cells, and probably T cells, we prefer the THI/TH2 nomenclature at present. Another division between T cells is the separation of helper T cells in vivo into the T1 and T2 sets (65, 66, 67, 68, 69, 70). T1 cells are shortlived (rapidly lost after adult thymectomy),relatively resistant to in vivo administration of anti-lymphocyte serum (ALS), probably not recirculating, and able to provide B-cell help, but with relatively slow kinetics. T2 cells are long-lived, sensitive to ALS,probably recirculating, able to provide B-cell help with rapid kinetics, and probably include the memory T-cell population. Recent results from Swain and colleagues (71) have shown that freshly isolated lymphocytes express IL-2 and IFN~, but not

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IL-4 or IL-5. After in vitro culture under defined conditions of stimulation, IL-4 and IL-5 can be synthesized. Adult thymectomyabrogates the ability of surviying T cells to produce IL-2, and after stimulation in culture, the resulting cells’ develop the ability to synthesize both |L-2 and IL-4. Based on these results, Swainet al (71) suggested that T1 cells are equivalent TH2, and T2 (long-lived) cells are the TH1population. A second group has also reported that IL-2 but not IL-4 can.be detected in supernatants from stimulated normal lymphocytes, either in bulk culture or in limiting dilution experiments (72). However,results fi’om our laboratory and others show that IL-4 and IL-5 can be readily detected in stimulated normal spleen cell supernatants, suggesting that patterns of lymphokinesynthesis may vary from one animal colony.to another. The T1 compartment can contain precursors of B helper cells, DTHeffectors (66, 67) and cells producing IL-2 and IL-4 (71). The T2 compartment can contain active helper cells (66, 67) and IL-2-producing cells (71), and T2 cells can derived from. the T1 compartment (68). From these observations, appears unlikely, that T1 andT2 are equivalent to the TH2 and TH1 subsets. Instead, it is muchmore likely that the short-lived T1 population includes the pregu.rsors of both THI and TH2cells (either TH0or THlP and TH2P, see Figure !), and that the long-lived T2 population includes mature TH1 and TH2 cells (Figure 2). Depending on the-background immunity or the antigenic stimulus used, the T2 population might preferentially contain TH1or TH2cells. This possibi!ity~may explain why some laboratories.produce mainly or entirely TH1clQnes, whereas others produce only TH2.In our ownlaboratory, we find that in different experiments, apparently identical methods, and strain combinations can lead to drastically different ratios of TH1and TH2clones. If the immunesystem of the mouseis in a constantly changing,state of balance between’the two types of response, this. could explain both. individual variation between experiments, and more systematic variations between investigators using different mouse colonies with, presumably, different ongoing immune responses. By this argument, t.he balance is more important than the degree of response, i.e. a mousecolony with a large numberof infectious agents could be biased in either direction, as could a ,’.’clean" or specific pathogenfree colony. Other types of helper T cell diy.e.~sitY have been described. In a series of papers, Kappler, Marrackand.c011eagues showed,that one type of he, lper could help in a linked manners-wl~ereas,another cot~ld.o,nly help bystander B cells (73). This heterogeneity, mgy~be:rel~t~d, to’the TH1 and TH2,_subsets, but thc connection is not yet clear: H~lper T cells were also heterogenous for expression of a determinant(s) recognized by an anti-I region allo. a~ntiserum (74). Waldmann & Lefkovits (75),also ~showed:e,vide_n_.ce

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HIGHLYMPHOKINE SYNTHESIS

ANTIGEN’-"

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~

IL2 IFNY LT

TH1

TH2

T1

PRECURSOR T CELLS

T2

ACTIVATED T CELLS MEMORY T CELLS T CELLCLONES

Figure 2 Possible relationships of THI/TH2and T1/T2 subsets. Stippled arrows show possible alternative pathways for the generation of mature TH1 and TH2 cells from precursor(s).

different helper cells by limiting dilution experiments, and Tada and colleagues (76) showedthat T cells with surface "Ia" determinants could separated by nylon wool columns and were functionally distinguishable from Ia- T helper cells. Janeway and colleagues described another type of diversity with the discovery of T helper ceils that appearedto be specific for the B-cell idiotype, rather than for the antigen (77). This second type of helper could synergize with conventional helpers for optimal responses (78). The relationship of each of these kinds of heterogeneity to each other and to the current TH1and TH2 types is still not clear. At the risk of accusations of avoiding the question, we suggest that these kinds of heterogeneity might not all be equivalent to the TH1/TH2separation but could represent additional diversity within the helper T cell compartment. CELL SURFACE ANTIGEN DIFFERENCES The leukocyte common antigen (LCA), expressed on the surface of most hemopoietic cells, has unusual genetic structure in which different exons are expressed near the N-terminus in various cell lineages and differentiation states (79, 80). Monoclonal antibodies have been produced against a particular determinant of the mouse, rat, and humanversions of this antigen, and the subsets of cells expressing this determinant appear to be related to the POSSIBLE

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÷ ceils produce the majority THI and TH2subsets. In rat T cells, OX22 of IL-2, whereas OX22-cells mediate the majority of helper activity for B cells (81). Human helper T cells can also be divided into subsets by antiLCAantibodies (82, 83), and recently antibodies have been identified that perform a similar function for mousehelper T cells (84; E. Pure, personal communication). The lymphokines secreted by the two separated mouse populations correlate to some extent with those produced by the TH1 and TH2subsets, but unfortunately, in all three species, the expression patterns are more complex, so that the LCAdeterminants may be both lineageand activation-specific markers (85; K. Bottomly, personal communication). The existence of antigenically distinct populations of normal T cells that produce particular lymphokinepatterns is difficult to reconcile with cloning experiments that suggest that the TH1and TH2lymphokine patterns sometimesdo not emerge until several weekshave elapsed. Limiting dilution experiments (72) were able to demonstrate only IL-2-producing clones unless the cells weretaken through an in vitro cycle of antigen stimulation. Thesevarious results are difficult to fit into a simple modelconsisting of only THIand TH2cells, and so it is likely that further differentiation states of helper T cells exist and that the numberof T cells in each differentiation state can vary widely betweendifferent laboratories and protocols. Interregulation

of TH1 and TH2 Responses

WHATDETERMINESTHE SELECTIVE ACTIVATION OR DIFFERENTIATION OF THI OR

"rH2 CELLS?The ratio of THI to TH2 cells produced in various immune responses appears to be tightly controlled, as assessed both by the types of clones generated in tissue culture, and by the characteristic responses elicited by particular antigens or modesof immunization. Whetheror not the responding T cells are already precommitted to the TH1 or TH2 patterns, mechanismsmust exist for selectively activating, expanding, or differentiating precursor T cells into TH1or TH2cells. The antigenpresenting cell (APC) is a good candidate for the cell influencing the TH1/TH2 ratio, and it has been proposed that TH1cells maybe selectively activated and expanded by B cells, whereas macrophages (producing ILl) cause clonal expansion of TH2cells (44). On the other hand, TH1cells are probably more effective at activating macrophages, whereas TH2cells are probably the major B-cell helper population, leading to the proposal that the most important interactions of TH1 and TH2cells are with macrophagesand B cells respectively (30). These two divergent views could represent a real dichotomy between the most advantageous T-cell-APC interaction for T-cell proliferation, as distinct from the optimal interaction for activation of the non-T cell partner. Alternatively, both views may be partially correct, since B cells can enhance antigen presentation to

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proliferating T cells (probably TH1) by direct presentation as well producing antibody that enh~inces the ability of non-B cells (probably macrophages) to present antigen (86). Conversely, TH2activation can mediated by macrophages, and by B cells if IL-1 is added to the culture or supplied by bystander macrophages(87). A single APCtype might also influence the TH1/2ratio by providing different accessory signals to TH cells, depending on the physical nature of the antigen encountered. For example, Janeway has suggested that TH1cells can only be activated by high antigen density on the surface of APC(44). TmANDTH2 HAVEDIFFERENT GROWTH RESPONSES Autocrine or paracrine growth responses? Both types of T helper cell produce a T-cell growth factor, either IL-2 or IL-4. This has led to suggestions that the growth of these T cells is regulated by an autocrine pathway(12, 88, 89). However,the production of lymphokinesoccurs for only a few hours after stimulation in vitro, while proliferation occurs for several days provided that IL-2 or IL4 is present. If this pattern occurs in vivo, then the rapid clearance of lymphokines in vivo (e.g. 90) would mean that both TH1and TH2cells wouldnormally proliferate in response to lymphokines (either IL-2 or IL4), produced by other T cells. Although both TH1and TH2clones respond to IL-2 and IL-4, somecomplexities exist in their growth requirements. The TH1growth response After activation by antigen and antigen-presenting cells, TH1clones respond strongly to IL-2, and weakly to IL-4 (89). Several days after antigen stimulation, the IL-2 response is somewhat lower, but the IL-4 response has disappeared (88, 89, 91), although IL-4 will still synergize with suboptimal amountsoflL-2 (88). The TH1 response to IL-4 cannot be enhanced by IL-I which is consistent with the lack of IL-1 receptors on TH1clones (88, 91). IFN7 does not have an effect the growth response of TH1clones to either IL-2 or IL-4 (89), as would be expected for a cell that produces high levels of IFN~. An unknownTH2 product inhibits the growthof at least TH1 cells (92), raising the possibility that each THtype can reciprocally inhibit the growth of the other type. The TH2growth response Antigen-stimulated TH2cells also proliferate strongly in response .to IL-2 and weakly in the presence of IL-4 (88, 89, 91). The response to IL-4 is maintained for a longer period than in TH1 clones, and so TH2 cells remain responsive to both lymphokines. The response to IL-4 after lectin stimulation requires the presence of IL-1, and this correlates with the presence of IL-1 receptors on TH2cells (88, 91). IFN~ inhibits the proliferative response of TH2clones to either IL-2 or IL-4 (89, 93). RECIPROCAL REGULATION OF TH1 AND TH2

Because of the growth properties

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of TH1and TH2clones summarizedabove, the T-cell type that is stimulated morestrongly during the initial stages of an immune responsewould be expectedto encouragesimilar responsesand inhibit the other type of response. Theseeffects mayexplain the dominanceof responsesin certain parasite-infected mice whereboth the parasite and bystander responses are biased towardsIgE and mayalso explain the reciprocal nature of DTH and antibody responses against the sameantigen (94, 95). Strong immune responsesof a particular type against certain adjuvantscould predispose the immune response against accompanying antigens in the samedirection as the adjuvant, re~sponse, e.g. towardsIgE in the case of alumand/or Bordetella pertussis immunizations. This mutuallyinhibitory regulation of the twoT-cell types couldaccount for the bias in immune responsescausedby strong antigens, but it raises the question as to howthe systemreturns to a state of balance after an infection has beencured. Antibody could be the signal that tends to reverse the trend late in an immuneresponse, since IgG2aantibodies (enhanced by IFN7 and henceTH1 s) are inhibitory for DTH reactions and enhancing for antibody reactions, whereasIgG1 antibodies (enhancedby IL-4 and henceTH2s)havethe oppositeeffect (96, 96a, 97, 97a). Thenature of link betweenantibody subclass and T-cell regulation is unknown.The lymphokine cross-inhibitory effects wouldbe expectedto take effect early in the immune response, since lymphokinesare secreted in the first few hours after T-cell activation, whereasantibodyeffects wouldbe expected to reach a peak a few days later, whenserumtiters have built up. The combinationof these two mechanisms wouldthen account for a transient ~,bias of the general immunesystemin the samedirection as that induced by a major immunizingantigen, followed by a resetting of the system towardsa state of balance(Figure 3). BecauseCTLssynthesize a pattern of lymphokinesvery similar to that of TH1 cells, activation of CTLsmightalso be expectedto shift the balance of helper cells in favor ofTH1 cells. Sinceviral infectionswouldbe expected to result in greater CTLactivation than immunizationwith protein antigens, it is possible that viral infections wouldresult mainlyin TH1activation (Figure 4). This wouldbe consistent with the findings of vanSnick and colleagues (98) that viral infections resulted in higher IgG2a/IgG1 ratios than protein immunization,whichcould be explainedby the preferential enhancementof IgG2asynthesis by the TH1 product IFNT. Expected Roles of TH1 and TH2 Cells in Normal Responses THETHIRESPONSE A predominantlyTH1response is expectedto result in enhancementof several cytotoxic mechanisms(Figure 5). IFN7and activate macrophages, resulting in increasedkilling of intracellular para-

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IgG2a,~,-.IFN IL2

~

EARLY

? IL4,=.,.~,IgG1

LATE

~

IgG2a

IgG1

~,~

INDUCTION

~>

INHIBITION

Figure 3 Inter-regulation of THI and TH2. Heavy arrows show the predominant effects expected during the early and late phases of responses dominated by either TH 1 or TH2 responses. The lymphokinedesignated by "?" is possibly the TH2-derivedinhibitor described by Horowitzet al (92).

sites and tumor cells (99, 100), and increased expression of Fc receptors for IgG2aantibodies (101). These receptors could then bind the increased IgG2a levels produced in response to IFN~ (42), leading to increased antibody-dependent macrophage cytotoxicity. Lymphotoxin and IFNy synergize in the killing of target cells (102), and IgG2acan kill target cells by complementlysis. TH1clones also cause effective DTHreactions. All of these effector mechanismsare appropriate for dealing with intracellular (viral and parasite) infections. In general, a strong TH1response in the absence of any TH2response might be expected to result in DTHbut little or no antibody. THETH2RESPONSE Preferential activation of TH2cells should lead to high general antibody levels. IL-4 should cause increased IgE production, as well as increased levels of IgE Fc~ receptor on B cells (103) and Ia antigens on macrophages (104). IL-3 and IL-4 would be expected to result mucosalmast cell proliferation (6), and IL-5 wouldcause proliferation eosinophils (105). Thus, several features of an allergic response are increased by TH2activation (Figure 6). In contrast, TH1clones inhibit the pathway by decreasing TH2growth and inhibiting IgE production by B cells.

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GM, LT,~ IL2, IFN’¢ ~

163

ENVIRONMENT FAVORABLE FOR TH1 RESPONSES

ELL II C FOR MHC

IgG2a Figure 4 Immuneresponses against viral antigens. Someof the ex aected features of the immuneresponse during a viral infection are shown. Compared to a response against a protein antigen, the anti-viral response should produce more IFN,~ and LT because of the activation of CTLs. This should then lead to an enhancement of the THl-like side of the response, resulting in a bias towards IgG2aproduction.

MIXEDTHI ANDTH2 RESPONSES Although strongly biased TH1 or TH2 responses would be expected to result in clearly distinguishable immune responses, as described above, many normal responses may involve a mixture of the two types of cell, especially in cases where the response is neither strong nor prolonged. Under these conditions, we might expect that IgE would not be produced due to the dominant suppression by IFN~,, and a DTHresponse might also not occur because of possible inhibition of DTHby a TH2 response (94; T. A. T. Fong, T. R. Mosmann,unpublished). Since both THtypes can activate B cells, which are then responsive to lymphokines produced by either THtype, antibody responses would be strongly supported in a mixed TH1and TH2response. The isotype patterns may depend on the ratio of TH1and TH2 activation, with a THI bias giving preferentially IgG2a, and a TH2preference resulting in more IgG1. These expected properties of a mixed THl and TH2response are clearly compatible with the majority of immuneresponses, i.e. variable IgG isotype responses, without significant IgE or DTHreactions. The

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~

IgM, IgG1, etc.

ANTIGEN+ MNC

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DTH

LYMPHOKINE" MEDIATED CYTOTOXICITY

~

~

)

A~TIBODY-DEPENDENT CEI~L-MEDIATED CYTOTOXICITy, PHAGOCYTOSIS, INTRACELLULAR KILLING

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COMPLEMENTMEDIATED CYTOTOXICITY

functions.

patterns of normal immuneresponses could be even more complexwhen it~ is realized that TH1-biasedor TH2-biased responsescouldtheoretically occur simultaneouslyin different anatomicallocations, providedthat the responseswere not strong enoughto provok~systemic regulatory effects. Immune Responses in Which TH1 and TH2 Ratios May Be Important Several antigens, particularly with certain adjuvants, characteristically induce particular classes of immuneresponse, e.g. alum adjuvant, especially with Bordetellapertussis, provokesgoodIgE responses, whereas completeFreund’sadjuvantgives high antibodylevels but not IgE. Several other biased immune responsessuggest selective activation of the T~I~or TH2pathways(reviewedin.reference 44). A notableexampleis’th~ i.mrnune.,: response to collagen type IV, whichproduces an apparent T~f response in H2~ mice and a TH2response in other mice (106). Several infectious agents mayalso induce biased responses, such as the antiviral response discussed above, and ~t:numberof protozoanand metazoanparasites. NIPPOSTRONGYLUSBRAS1LIKNSIS Mosthelminth parasite infestations induce significant IgE responses, often accompanied by substantial productionof

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ANTIGEN + MHC

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~,~

Eosinophils~ANTIGEN

MEDIATOR RELEASE

IFNY~

~-~ Nast cells

MEDIATOR RELEASE

Figure 6 TH1 and TH2regulation of IgE. Stippled arrows indicate inhibitory effects, and solid arrows showstimulatory effects.

polyclonal IgE. In addition to the high IgE levels, these infestations are often associated with eosinophilia and intestinal mast cell hyperplasia. A well-studied exampleis Nippostrongylus brasiliensis (Nb, 107, 108, 109), in w.hiqh, these effects can be exp!a~_nedby the preferential activation of TH2cells (Table 3). The high IgE levels can be inhibited by in vivo administration of anti-IL-4 antibody (110), and the eosinophilia can inhibited by anti-IL-5 antibody (R. L. Coffman, D. M. Rennick, unpublished). Since t.lae~ T-cell response(possibly irtyplving mast cells) has been implicated in expulsion of wormsfrom the..gut (111), the TH2response can be considered appropriate for this parasite. Wehave recently studied the lymphokinepatterns synthesized by spleen and lymph node cells from Nb-infected mice, and we find that IL-2 and IFN7~Ie)~els are suppressed ,below normal levels, a~d..t~, at IL-4 and IL-5 levels are greatly elevated. , This is probably due both~to selective,amplification of TH2. cells, and regulation of activation of THI and TH2’(N..Stre,et, T.~.R. Mosmann, unpublished). LEISHMANIA ~ Inf_egtion of.~i~ce by Leishmania major ~(Lm)results~in one

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Table 3 Immuneresponses against parasites Nippostrongylus brasiliensis High IgE levels (polyclonal) Eosinophilia Mast cell hyperplasia High IL-4 levels High IL-5 levels LowIL-2 levels Low IFN7 levels Leishmania major in mice High IL-4 Low IFN? Balb/c High IgE levels ~High antibody No DTH Low IL-4 High IFN2 C57B1/6 Low IgE Low antibody Strong DTH

Progressive, fatal disease

Limited disease, cure

Nippostrongylus eliminationis associatedwith a TH2-1ike pattern. Leishmaniaeliminationis associatedwith a TH1response.

of two responses (Table 3). In susceptible strains, such as Balb/c, the response has the features expected of TH2activation, such as high antibody levels (including IgE; M. Sadick, R. L. Coffman, unpublished), high IL-4 and low IFN~, expression, and no DTH.The Balb/c mice develop a severe and progressive disease and die (112). In contrast, resistant strains such as C57B1/6, develop strong DTH,low antibody levels with no elevation of serum IgE, high IFN~,, and low IL-4 expression, and the infection is local and ultimately is cured (112). Theseresults and others suggest that a TH1response is effective in eliminating this parasite (an intracellular parasite in macrophages), possibly because of the ability of IFN~to activate macrophages. The most direct evidence for the TH1requirement has recently been obtained by Scott et al (113), who have prepared TH1and TH2cell lines and clones specific for Lmantigens. Whenthese cells are injected back into Lm-infected mice, the TH1line completely cures the infection, whereas the TH2line actually exacerbates the course of the disease. Thus for Lminfection, the THI response is the appropriate response that leads to elimination of the parasite. The Lmsystem in mice is particularly interesting since Leishmania donovani infection in humans also produces two alternative forms of the immuneresponse, either DTH leading to local containment of the infection and elimination of the

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parasite, or the Kala-Azarresponse in which high antibody levels are associated with low DTHreactions and a severe, disseminated disease (114). It remainsto be seen whetherthese responses are linked to TH1and TH2responses in humans. Concludin 9 Remarks Althoughwe still lack conclusive proof that TH1and TH2cells exist in vivo in the mouse,and especially in humans,the weight of evidence now suggests that, at least in the mouse,these two types of helper cell exist, and because of their profoundlydifferent functions, they are important regulators of the class of immuneresponse. Several major immune responses, especially against parasites, showa remarkably goodfit with the features expected of either TH1 or TH2responses. Becausethe appropriate response(i.e. the responsethat eliminates the infection) can be either TH1 or TH2,dependingon the infectious agent, it is obviously importantto consider the interregulationof these types of cell wheninducingtherapeutic immuneresponses. In closing, someof the outstandingquestions in this area are these: Are TH1and TH2cells found in vivo? Whatare the precursors of TH1and TH2cells? Whatare the lymphokinesecretion patterns of the precursors? Is the commitmentto one of the two lymphokinesecretion patterns made + cells have a similar before or after exposure to antigen? Do humanCD4 dichotomyof lymphokinesynthesis and function? Whichcell regulates the preferential activation of TH1 or TH2by certain antigens, andhowis this achieved? Howis the regulation of TH1 and TH2activation connected to the ongoing pattern of response? Since excellent tools are nowavailable to explore these possibilities further, we look forwardto answersfor many of these questionsin the next few years.

Literature Cited 1. Claman, H. N., Chaperon, E. A., Triplett, R. F. 1966. Thymus-marrow cell combinations. Synergism in antibody production. Proc. Soc. Exp. Biol. Med. 122:1167-71 2. Mitchell, G. F., Miller, J. F. A. P. 1968. Cell to cell interaction in the immune response. II. Thesource of hemolysinforming cells in irradiated mice given bone marrowand thymus or thoracic duct lymphocytes. J. Exp. Med. 128: 821-37 3. Cantor, H., Boyse, E. A. 1975. Functional subclasses of T-lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-

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Bond, M. W., Giedlin, M. A., Coffman, R. L. 1986. Twotypes ofmurine h.e!per T cell clone: I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136: 2348-57 8. Abrams, J. S., Pearce,. M. K. 1988. Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro. J. Immunol. 140:131-37 9. Ohara, J., Paul, W. E. 1985. Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1. Nature 315:333 36 10. Schumacher, J. H., O’Garra, A., Shrader, B., van Kimmenade, A., Bond, M. W., Mosmann,T. R., Coil’man, R. L. 1988. The characterization of four monoclonalantibodies specific for mouse IL-5 and development of mouse and human IL-5 enzyme-linked immunosorbent assays. J. Immunol. 141:1576-81 11. Cherwinski, H. C., Schumacher, J. H., Brown, K. D., Mosmann,T. R. 1987. Twotypes of mousehelper T cell clone: 3. Further differences in lymphokine synthesis between TH1 and TH2 clones revealed by RNAhybridization, functionally monospecific bioassays and monoclonal antibodies. J. Exp. Med. 166:122944 12. Lichtman, A.H., Kurt-Jones, E. A., Abbas, A. K. 1987. B-cell stimulatory factor 1 and not interleukin 2 is the autocrine growth factor for some helper T lymphocytes. Proc. Natl. Acad. Sci. USA 84:824-27 13. Fernandez-Botran, R., Sanders, V.M., Oliver, K. G., Chert, Y. W., Krammer, P. H., Uhr, J. W., Vitetta, E. S. 1986. Interleukin 4 mediates autocrine growth of helper T cells after antigenic stimulation. Proc. Natl. Acad. Sci. USA 83:9689-93 14. Grabstein, K., Eisenman, J., Mochizuki, D., Shanebeck, K., Conlon, P., Hopp, T., March, C., Gillis, S. 1986. Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines. J. Exp. Med. 163:1405-14 15. Smith, C. A., Rennick, D. M. 1986. Characterization of a murine lymphokine distinct from IL-2 and IL-3 possessing a TCGFactivity and an MCGF activity that synergizes with IL-3. Proc. Natl. Acad. Sci. USA 83:1857-61 16. Mosmann,T. R., Bond, M. W., Coffman,R. L., Ohara, J., Paul, W. E. 1986.

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T cell and mast cell lines respond to B cell stimulatory factor-1. Proc. Natl. Acad. Sci. USA 83:5654-58 Woods, A., West, J., Rasmussen, R., Bottomly, K. 1987. Granulocytemacrophage colony stimulating factor produced by cloned L3T4+, class IIrestricted T cells induces HT-2cells to proliferate. J. lmmunol. 138:4293-97 Prystowsky, M. B., Ely, J. M., Belier, L. E., Goldman, J., Goldman, M., Goldwasser,E., Ihle, J., Quintans, J., Remold,H., Vogel, S. N., Fitch, F. W. 1982. Alloreactive cloned T cell lines. Multiple lymphokineactivities secreted by helper and cytolytic lymphocytes. J. Immunol. 129:2337-44 Kelso, A., Glasebrook, A. L. 1984. Secretion ofinterleukin 2, macrophageactivating factor, interferon and colony-stimulating factor by alloreactive T lymphocyteclones. J. Irnmunol. 132: 2924-31 Bonyhadi, M., Weiss, A., Tucker, P. W., Tigelaar, R. E., Allison, J. P. 1987. The y/6 antigen receptor of Thy- 1 + dendritic epidermalcells: Identification of ~ as the Cx gene product. Nature 330:574-76 Paliard, X., de WaalMalefijt, R., Yssel, H., Blanchard, D., Chretien, I., Abrams,J., de Vries, J., Spits, H. 1988. Simultaneous production of IL2, IL4, ÷ and IFN~, by activated human CD4 and CD8÷ T cell clones. J. Immunol. 141:84%55 Havran, W., Fitchg F. W. 1987. Characterization of murine cytolytichelper hybrid T cell clones. Nature 325: 65 67 Vitetta, E. S., Bossie, A., FernandezBotran, R., Myers, C. D., Oliver, K. G., Sanders, V. M., Stevens, T. M: 1987. Interaction and activation of antigenspecific T and B cells. Immunol. Rev. 99:193-239 Tony, H.-P., Parker, D. C. 1985. Major histocompatibility complex-restricted B cell responses resulting from helper T cell recognition of anti-immunoglobulin presented by small B lymphocytes. J. E:~-p. Med. 161:223-41 Tony, H.-P., Phillips, N. E., Parker, D. C. 1985. Role of membrane immunoglobulin (Ig). cross-linking membrane Ig-mediated major histocompatibility-restricted T cell-B cell cooperation. J. Exp. Med. 162: 16951708 Lebman, D. A., Coffman, R. L. 1988. Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal.B cell cultures. J. Exp. Med. .168: 853-62

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TH1 AND TH2 HELPER CELLS 27. Kim, J., Woods, A., Becker-Dunn, E., Bottomly, K. 1985. Distinct functional phenotypes of cloned Ia-restricted helper T cells. J. Exp. Med. 162:188-201 28. Killar, L., MacDonald,G., West, J., Woods, A., Bottomly, K. 1987. Cloned, Ia-restricted T cells that do not produce interleukin 4 (IL-4)/B cell stimulatory factor 1 (BSF-1) fail to help antigenspecific B cells. J. Immunol.138: 167479 29. Stevens, T. L., Bossie, A., Sanders, V. M., Fernandez-Botran, R., Coffman, R. L., Mosmann,T. R., Vitetta, E. S. 1988. Subsets of antigen-specific helper T cells regulate isotype secretion by antigen-specific B cells. Nature. In press 30. Boom,W. H., Liano, D., Abbas, A. K. 1988. Heterogeneity of helper/inducer T lymphocytes. II. Effects of interleukin 4- and interleukin 2-producing T cell clones on resting B lymphocytes. J. Exp. Med. 167:1352~53 31. Coffman, R. L., Seymour, B. W., Lebman, D. A., Hiraki, D. D., Christiansen, J. A., Shrader, B., Cherwinski, H. M., Savelkoul, H. F. J., Finkelman, F. D., Bond, M. W., Mosmann, T. R. 1988. The role of helper T cell products in mouseB cell differentiation and isotype regulation. lmmunol. Rev. 102:5-28 32. Tite, J. P., Kaye, J., Jones, B. 1984. The role of B cell surface Ia antigen recognition by T cells in B cell triggering: Analysis of the interaction of cloned helper T cells with normal B cells in differing states of activation and with B cells expressing the xid defect. Eur. J. Immunol. 14:553 61 33. Leclercq, L., Bismuth, G., Theze, J. 1984. Antigen-specific helper T cell clone supernatantis sufificient to induce both polyclonal proliferation and differentiation of small resting B lymphocytes. Proc. Natl. Acad. Sci. USA 81:6491-95 34. Roth, C., Moreau, J.-L., Korner, M., Jankovic, D., Theze, J. t988. Biochemical characterization and biological effects of partially purified B cell-activating factor (BCAF).Eur. J. lmmunol. 18:577-84 35. Snow, E. C., Noelle, R. J. 1987. Thymus-dependent antigenic stimulation of hapten-specific B lymphocytes. Immunol. Rev. 99:173-92 36. Julius, M. H. 1987. Reciprocity in lymphocyte interactions. Immunol. Rev. 95:177-94 37. Rasmussen, R., Takatsu, K., Harada, N., Takahashi, T., Bottomly, K. 1988.

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T cell-dependent hapten-specific and polyclonal B cell responses require release of interleukin 5. J. lmmunol. 140:705-12 38. Herron, L. R., Coffman, R. L., Bond, M. W., Kotzin, B. L. 1988. Increase autoantibody production by NZB/ NZW B cells in response to interleukin 5. J. ImmunoL 141:84248 39. Giedlin, M. A., Longenecker, B. M., Mosmann, T. R. 1986. Murine T cell clones specific for chicken erythrocyte alloantigens. Cell. Immunol. 97:357-70 40. Coffman,R. L., Carty, J. 1986. AT cell activity that enhances polyclonal IgE production and its inhibition by interferon-~. J. Immunol. 136:949-54 41. Reynolds, D. S., Boom, W. H., Abbas, A. K. 1987. Inhibition of B lymphocyte activation of interferon-y. J. lmmunol. 139:76%73 42. Snapper, C. M., Paul, W. E. 1987. Interferon-3, and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944-47 43. Tite, J. P., Janeway, C. A. Jr. 1984. Cloned helper T cells can kill B lymphomacells in the presence of specific antigen: la restriction and cognate vs. noncognate interactions in cytolysis. Eur. J. Immunol. 14:878-86 44. Janeway, C. A. Jr., Carding, S., Jones, B., Murray, J., Portoles, P., Rasmussen, R., Rojo, J., Kaizawa, K., ÷ T West, J., Bottomly, K. 1988. CD4 cells: Specificity and Function. lmmunol. Rev. 101:39-80 45. Bottomly, K., Kaye, J., Jones, B., Jones, F. III, Janeway, C. A. Jr. 1983. Acloned, antigen-specific, la-restricted Lyt-l÷,2 - T cell with suppressive activity. J. Mol. Cell lmmunol.1: 4249 46. Friedman, S., Sillcocks, D., Rao, A., Faas, S., Cantor, H. 1985. A subset of Ly-I inducer T cell clones activates B cell proliferation but directly inhibits IgG secretion. J. Exp. Med. 161:785 804 47. Asano, Y., Hodes, R. J. 1983. T cell regulation of B cell activation. J. Exp. Med. 158:1178-90 48. Coffman, R. L., Ohara, J., Bond, M. W., Carty, J., Zlotnik, A., Paul, W. E. 1986. B cell stimulatory factor-1 enhances the lgE response of lipopolysaccharide-activated B cells. J. Immunol. 136:453841 49. Finkelman, F. D., Katona, I. M., Mosmann, T. R., Coffman, R. L. 1988. Interferon-7 regulates the isotypes of immunoglobulin secreted during in vivo humoral immune responses. J. Imrnunol. 140:1022-27

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50. Finkelman, F. D., Katona, 1. M., Urban, J. F., Snapper, C. M., Ohara, J., Paul, W. E. 1986. Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine B cell stimulatory factor-1. Proc. Natl. Acad. Sci. USA83: 967578 51. Savelkoul, H. F. J., Lebman, D., Benner, R., Coffman, R. L. 1988. Increase of precursor frequency and clonal burst size of murine immunoglobulin E-secreting cells by interleukin-4. J. Immunol. 141:749-55 52. Itaya, T., Ovary, Z. 1979. Suppression of lgE antibody production in SJL mice. IV. Interaction of primed and unprimed T cells. J. Exp. Med. 150: 507-16 53. Vitetta, E. S., Ohara, J., Myers, C., Layton, J., Krammer, P. H., Paul, W. E. 1985. Serological, biochemical and functional identity of B cell-stimulatory factor-I and B cell differentiation factor for IgG1. J. Exp. Med. 162:1726-30 54. Erard, F., Corthesy, P., Nabholtz, M., Lowenthal, J. W., Zaech, P., Plaetinck, G., MacDonald, H. R. 1985. Interleukin 2 is both necessary and sufficient for the growth and differentiation of lectin-stimulated cytolytic T lymphocyte precursors. J. Immunol. 134: 1644-52 55. Simon, M. M., Landolfo, S., Diamantstcin, T., Hochgeschwender, U. 1986. Antigen- and lectin-sensitized murine cytolytic T lymphocyte-precursors require both interleukin 2 and endogenously produced immune (gamma) interferon for their growth and differentiation into effector cells. Curr. Top. Microbiol. Immunol. 126: 173-85 56. Pfeifer, J. D., McKenzie,D. T., Swain, S. L., Dutton, R. W. 1987. B cell stimulatory factor 1 (interleukin 4) sufficient for the proliferation and differentiation of lectin-stimulated cytolytic T lymphocyteprecursors. J. Exp. Med. 166:1464-70 57. Takatsu, K., Kikuchi, Y. H., Takahashi, T., Honjo, T., Matsumoto, M., Harada, N., Yamaguchi, N, Tominaga, A. 1987. Interleukin 5, a T cell derived B cell differentiation factor also induces cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 84:4234-38 58. Vadas, M. A., Miller, J. F., McKenzie, I. F., Chism,S. E., Shen, F. W., Boyse, E. A., Gamble, J. R., Whitelaw, A. M. 1976. Ly and Ia antigen phenotypes of T cells involved in delayed-type hyper-

sensitivity and in suppression. J. Exp. Med. 144:10-19 59. Cher, D. J., Mosmann,T. R. 1987. Two types of murine helper T cell clone: 2. Delayed-Type Hypersensitivity is mediated by TH1 clones. J. Immunol. 138:3688-94 60. Zinkernagel, R. 1976. H-2 restriction of virus-specific T-cell-mediated effector functions in vivo. II. Adoptive transfer of delayed-type hypersensitivity to murine lymphocytic choriomeningitis virus is restricted by the K and D regions of H-2. J. Exp. Med. 144:776-87 61. Issekutz, T. B., Stoltz, J. M., van der Meide, P. 1988. Lymphocyte recruitment in delayed-type hypersensitivity. The role of IFN),. J. Immunol. 140: 2989-93 62. Van Loveren, H., Kato, K., Meade, R., Green, D. R., Horowitz, M., Ptak, W., Askenase, P. W. 1984. Characterization of two different Lyt-I ÷ T cell populations that mediate delayed-type hypersensitivity. J. lmmunol. 133: 2402-11 63. Maggi, E., Del Prete, G., Macchia, D., Parronchi, P., Tiri, A., Chretien, I., Ricci, M., Romagnani,S. 1988. Profiles of lymphokine activities and helper function for IgE in humanT cell clones. Eur. J. lmmunol. 18:1045-50 64. Budd, R. C., Cerottini, J.-C., MacDonald, H. R. 1987. Selectively increased production of interferon 7 by _ subsets of Lyt2 + and L3T4+ T cells identified by expression of Pgp-1. J. lmmunol. 138:3583-86 65. Raft, M. C., Cantor, H. 1971. Subpopulations of thymus cells and thymus-derived lymphocytes. In Proc. First Int. Congr. lmmunol. Washington, DC. New York: Academic 66. Kappler, J. W., Hunter, P. C., Jaeobs, D., Lord, E. 1974. Functional heterogeneity among the T-derived lymphocytes of the mouse.I. Analysis by adult thymectomy. J. Immunol. 113:27-38 67. Araneo, B. A., Marrack (Hunter), P. C., Kappler, J. W. 1975. Functional heterogeneity among the T-derived lymphocytes of the mouse. II. Sensitivity of subpopulations to antithymocyte serum. J. Immunol. 114: 747-51 68. Araneo, B. A., Marrack, P., Kappler, J. W. 1977. Functional heterogeneity among the T-derived lymphocytes of the mouse. VII. Conversion ofT1 cells to T2 cells by antigen. J. Immunol.119: 765 71 69. Simpson, E., Cantor, H. 1975. Regu-

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lation of the immuneresponse by subBoyse, E. 1987. Alternative use of 5’ classes of T lymphocytes. The effect of exons in the specification of Ly-5 isoadult thymectomy upon humoral and forms distinguishing hematopoietic cell cellular responses in mice. Eur. J. lineages. Proc. Natl. Acad. Sci. USA Immunol. 5:337~13 84:5364-68 70. Janeway, C. A. Jr. 1975. Cellular co81. Arthur, R. P., Mason, D. 1986. T cells operation during in vivo antihapten that help B cell responses to soluble antibody responses. The effect of cell antigen are distinguishable from those number on the response. J. Immunol. producing interleukin 2 on mitogenic 114:1394-1401 or allogeneic stimulation. J. Exp. Med. 71. Swain, S. L., McKenzie,D. T., Dutton, 163:774-86 R. W., Tonkonogy,S. L., English, M. 82. Morimoto, C., Letvin, N. L., Boyd, 1988. The role of IL4 and IL5: A. W., Hagan, M., Brown, H. M., Characterization of a distinct helper T Kornacki, M. M., Schlossman, S. F. cell subset that makes IL4 and IL5 1985. The isolation and characteriza(TH2) and requires priming before tion of the humanhelper inducer T cell induction of lymphokine secretion. subset. J. Immunol. 134:3762-69 Immunol. Rev. 102:77-105 83. Morimoto, C., Letvin, N. L., Distaso, 72. Powers, G. D., Abbas, A. K., Miller, J. A., Alrich, W. A., Schlossman,S. F. R. A. 1988. Frequencies of IL2 and 1985. The isolation and characterIL4-secreting T cells in naive and ization of the human suppressor inantigen-stimulated lymphocyte popuducer T cell subset. J. lmmunol. 134: lations. J. Immunol. 140:3352 57 1508-15 73. Marrack, P. C., Kappler, J. W. 1975. 84. Bottomly, K. 1988. A functional diAntigen-specific and nonspecific medichotomy in CD4 T lymphocytes, lmators of T cell/B cell cooperation. 1. munol. Today 9:268-74 Evidence for their production by dif85. Powrie, F., Mason, D. 1988. Phenoferent T ceils. J. Imrnunol.114:1116-25 typic and functional heterogeneity of ÷ T cells, lmmunol. Today 9: 27474. Swierkosz, J. E., Marrack, P. J., CD4 Kappler, J. W. 1979. Functional analy77 sis of T cells expressing Ia antigens. 86. Kurt-Jones, E. A., Liano, D., Hayglass, Demonstration of helper T-cell heteroK. A., Benacerraf, B., Sy, M.-S., geneity. J. Exp. Med. 150:1293-1309 Abbas, A. K. 1988. The role of antigen75. Waldman,H., Lefkovits, I., Feinstein, presenting B cells in T cell priming in A. 1976. Restrictions in the functions vivo. Studies of B cell-deficient mice. J. of single helper T cells. Immunology 31 : Immunol. 140:3773-78 353-62 87. Rock, K. L., Haber, S. I., Liano, D., 76. Tada, T., Takemori, T., Okumura,K., Benacerraf, B., Abbas, A. K. 1986. Nonaka, M., Tokuhisa, T. 1978. Two Antigen presentation by hapten-specidistinct types of helper T cells involved fic B lymphocytes. III. Analysis of the in the secondary antibody response: immunoglobulin-dependent pathway independent and synergistic effects of of antigen presentation to Interleukin ÷ Ia and Ia helper T cells. J. Exp. Med. I-dependent T lymphocytes. Eur. J. 147:446-58 Immunol. 16:1407-12 77. Janeway, C. A. Jr., Murgita, R. A. 88. Greenbaum, L. A., Horowitz, J. B., Weinbaum,F. I., Asofsky, R., Wigzell, Woods, A., Pasqualini, T., Reich, H. 1977. Evidence for an immunoE.-P., Bottomly, K. 1988. Autocrine ÷ T cells. Differential globulin-dependentantigen-specific helgrowth of CD4 per T cell. Proc. Natl. Acad. Sci. effects of IL-1 on helper and inflamUSA 74:4582-86 matory T cells. J. Immunol. 140: 155578. Bottomly, K., Mosier, D. E. 1979. Mice 60 whose B cells cannot produce the T15 89. Fernandez-Botran, R., Sanders, V. M., idiotype also lack an antigen-specific Mosmann,T. R., Uhr, J. W., Vitetta, helper T cell required for TI5 exE. S. 1988. Lymphokine-mediated pression. J. Exp. Med. 150: 1399regulation of the proliferative response 1409 of clones of TH1and TH2cells. J. Exp. 79. Thomas,M. L., Reynolds, P. J., Chain, Med. 168:543-58 A., Ben-Neriah, Y., Trowbridge, I. S. 90. Muhlradt, P. F., Opitz, H. G. 1982. 1987. B cell variant of mouseT200(LyClearance of interleukin 2 from the 5): Evidence for alternative mRNA blood of normal and T cell-depleted splicing. Proc. Natl. Acad. Sci. USA mice. Eur. J. Immunol. 12:983-85 84:5360-65 91. Kurt-Jones, E. A., Hamberg, S., 80. Saga, Y., Tung, J.-S., Shen, F.-W., Ohara, J., Paul, W. E., Abbas, A. K.

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1987. Heterogeneityof helper/inducer T lymphocytes. I. Lymphokineproduction and lymphokine responsiveness. J. Exp. Med.166:1774-87 92. Horowitz, J. B., Kaye, J., Conrad, P. J., Katz, M. E. 1986. Autocrine growthinhibition of a cloned line of helper T cells. Proc. Natl. Acad.Sci. USA 83:1886-90 93. Gajewski, T. F., Fitch, F. W. 1988. Anti-proliferative effect of IFN~, in immune regulation. I. IFN~,inhibits the proliferation of TH2but not TH1 murine HTLclones. J. Immunol.140: 4245-52 94. Parish, C. R. 1972. The relationship between humoral and cell-mediated immunity.Transplant Rev. 13:35-66 95. Katsura, Y. 1977. Cell-mediated and humoral immuneresponses in mice. III. Dynamicbalance betweendelayed type hypersensitivity and antibody response. Immunology32:227-35 96. Coulie, P. G., VanSnick, J. 1985. Enhancementof IgG anti-carrier responses by IgG2 anti-hapten antibodies in mice. J. Exp. Med.15: 79398 96a. Ptak, W., Janeway,C. A. Jr., Flood, P. M. 1988. Immunoregulatory role of Ig isotypes.-II. Activationof cells that blockinduction of contact sensitivity responses by antibodies of IgG2aand IgG2bisotypes. J. Immunol.141: 76~ 73 97. Murgita,R. A., Vas,S. I. 1972.Specific antibody-mediatedeffect on the immuneresponse. Suppression and augmentation of the primary immune responsein miceby different classes of antibodies. Immunology22:31%31 97a. Ptak, W., Flood, P. M., Janeway, C. A. Jr., Marcinkiewicz,J., Green, D. R. 1988. Immunoregulatory role of Ig isotypes. I. Inductionof contrasuppressor T cells for contact sensitivity responses by antibodies of the IgM, IgGl and IgG3isotypes. J. Immunol. 141:756-64 98. Coutclier, J. P., van der Logt, J. T. M., Heessen,F. A., Warnier, G., Van Snick, J. 1987. IgG2arestriction of murine antibodies elicited by viral infections. J. Exp. Med.165:64~69 99. Murray, H. W., Spitalny, G. L., Nathan, C. F. 1985. Activation of mouseperitoneal macrophagesin vitro andin vivoby interferon-~. J. Immunol. 134:1619 22 100. Pace, J. L., Russell, S~ W., Torres, B. A., Johnson, H. M., Gray, P. W. 1983. Recombinant mousey interferon induces the primingstep in macrophage

activation for tumorcell killing. J. Immunol. 130:201113 101. Warren, M. K., Vogel, S. N. 1985. Opposingeffects of glucocorticoids on interferon-y-induced murinemacrophage Fc receptor and Ia antigen expression. J. Immunol.134:2462-69 102. Lee, S. H., Aggarwal,B. B., Rinderknecht, E., Assisi, F., Chiu,H. 1984. Thesynergisticanti-proliferativeeffect of gamma-interferonand humanlymphotoxin. J. Immunol.133:1083-86 103. Hudak,S. A., Gollnick,S. O., Conrad, D. H., Kehry, M. R,~ t987. MurineB cell stimulatoryfactor 1 (interleukin4) increases expressionof the Fc receptor for IgE on mouseB cells. Proc. NatL Acad. Sci. USA84:4606-10 104. Zlotnik, A., Fischer, M., Roehm,N., Zipori, D. 1987.Evidencefor effects of interleukin 4 (B cell stimulatoryfactor 1) on macrophages: Enhancementof antigen presentingability of bonemarrow-derivedmacrophages.J. Immunol. 138:4275-~79 105. Sanderson,C. J., O’Garra,A., Warren, D. J., Klaus, G. G. 1986. Eosinophil differentiation factor also has B-cell growthfactor activity: proposedname interleukin 4. Proc. Natl. Acad.Sci. USA 83:437-40 106. Tite, J. P., Foellmer, H. G., Madri, J. A., Janeway,C. A. Jr. 1987.Inverse Ir gene control of the antibody and T cell proliferative responsesto human basement membranecollagen. J. Immunol. 139:2892-98 107. Ogilvie, B. M., Jones, V. E. 1969.Reaginic antibodies and helminthinfections. In Cellular and HumoralMechanismsin Anaphylaxisand Allergy, ed. H. Z. Movat. Basel/New York: R. Karger 108. Kelly, J. D., Ogilvie,B. M.1972.Intestinal mast cell and eosinophil numbers during wormexpulsion in nulliparous and lactating rats infected with Nippostrongylusbrasiliensis. Int. Arch. Allergy 43:497-509 109. Mayrhofer,G., Fisher, R. 1979. Mast cells in severelyT cell depletedrats and the responseto infestation with Nippostrongylusbrasiliensis. Immunology 37: 145 52 110. Finkelman, F. D., Katona, I. M., Urban,J. F. Jr., Holmes,J., Ohara,J., Tung, A. S., Sample, J. vG., Paul, W.E. 1988. Interleukin 4 is required to generate and sustain in vivo IgE responses.In press 111. Askenase, P. W. 1980. Immunopathologyof parasitic diseases: Involvement of basophils and mast

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cells. Springer Semin. lmmunopathol.2: 417-42 112. Locksley, R. M., Heinzel, F. P., Sadick, M. D., Holaday, B. J., Gardner, K. D. Jr. 1987. Murine cutaneous Leishmaniasis: Susceptibility correlates with differential expansion of helper T-cell subsets. Ann. Inst. Past./Immunol. 138: 744-49 113. Scott, P., Natovitz, P., Coffman,R. L., Pearce, E., Sher, A. 1988. Immuno-

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regulation of cutaneous leishmaniasis: T cell lines which transfer protective immunity or exacerbation belong to different THsubsets and respond to distinct parasite antigens. J. Immunol. Submitted 114. Sacks, D. L., Lal, S. L., Shrivastava, S. N., Blackwell, J., Neva, F. A. 1987. Ananalysis of T cell responsiveness in Indian Kalaazar. J. Immunol. 138:908-13

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Ann. Rev. lmmunol. 1989. 7:175-207 Copyright © 1989 by Annual Reviews Inc. All rights reserved

THE STRUCTURE, FUNCTION, AND MOLECULAR GENETICS OF THE 7/3 T CELL RECEPTOR David H. Raulet Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139

INTRODUCTION Distinct subclasses of functional T lymphocytesare involved in induction of antibody production by B cells (helper T lymphocytes) and cytolysis pathogen-infected host cells (cytolytic T lymphocytes, CTL).These T-cell subclasses share several properties: They are largely thymus-derived, bear the Thy-1 antigen (in the mouse), and express a clonally variable, cellsurface antigen-receptor (1-4). The receptor is noncovalently associated with a complexof several invariant polypeptides collectively called CD3 proteins. The two subclasses can be distinguished by expression of two - cells, and most other surface proteins: most helper T cells are CD4+CD8 + CTLare CD4-CD8 cells (5). Unlike B cells, T cells generally recognize fragments of protein antigens only when they are bound to major histocompatibility complex (MHC)encoded glycoproteins on the surface of host cells. Helper T cells and CTLgenerally recognize antigens boundto structurally different classes of MHC proteins, called class 2 and class 1, respectively. Recent studies have demonstratedthat recognition by most T cells of both the foreign antigenic peptide and the MHCprotein is accomplished by a single heterodimeric antigen-receptor structure, composedof disulfide-linked c~ and fl chains (6, 7). Both chains are clonally variable and are encodedby families of variable (V), diversity (D; only for the/~ chain) and joining (J) gene segments assemble by gene rearrangements during T-cell differentiation (3, 4, 8). Although helper T cells and CTLrecognize antigens associated with strut175 0732~0582/89/0410-0175502.00

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1.76

RAULET

rurally distinct MHC proteins, they utilize the same pool of ~ and/3 gone segmentsto assemble their receptors (8, 9). In t.he face of the considerable progress in. understandingthe recognition systems of functionally defined T lymphocytes, immunologists have recently been confronted with a novel T-cell subclass. Cells of this subclass differ in several respects from "conventional" T cells, but their most striking characteristic is the use of a distinct antigen-receptor-likestructure, composedof clonally variable, CD3-associated 7 and 6 chains. In common with T cells .that express ~/fl receptors (hereafter called ~/fl cells), 7/3 cells express several markers characteristic of T cells and exhibit T-cell functional activities such as cytolysis and lymphokinerelease in .vitro. Progress in defining ~/j~ T cells proceeded in a "conventional" manner after initial detection of T-cell functional activities. The properties of the cells, the structure of their antigen-receptors, and the characteristics of the corresponding gone families followed sequentially. In contrast, progress in understanding 7/6 cells is proceeding in the reverse order: the first discovery was of a gone encoding an unknownantigen-receptor-like chain (10), followed by discovery of a novel T-cell subclass bearing the chain (11). The characteristics of these cells, and the specificity of their receptors is currently under intense scrutiny. As of this writing, the biological function and specificity of 7/3 cells is unknown.7/6 cells are found in seyeral if not all vertebrate species; t.hey express clonally variable receptor mo._lecules encodedby complexgone families and exhibit various functional activiti.es similar to those of e//3 T cells--all points to be discussedlater. Theresee.ros little doubt that these cells function in vivo, perhaps to mediate heretofore unrecognized immunereactions. The purpose of this review is to assemble current knowledgeof the structure of 7/6 receptors and the corresponding genes, the phenotypic and functional properties of 7/6 cells, and the localization and ontogeny of this unique lymphocyte subclass. Murine, human, and avian systems are discussed later,, although a bias to the murinesystem is evident. This information represents most of the available pieces of the 7/6 puz.zle~ whichthe reader is invited to help solve.

HISTORICAL

OVERVIEW

In early 1984 cDNA clones encoding a T-cell receptor (TCR)subunit (the /3 chain) were firstisolated by subtractive hybridization (l 2) and differential screening (13) methods.:Thes~.meth0ds are, based onthe assumption that TCRmRNAs are prese.nt inT-cells but,not in-B cells and that the genes encoding TCRsundergo T cell-specific, clonally variable gene rearrangements. Subsequently, a candidate ~ chain cDNAclone was reported in

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mid-1984, again by subtractive hybridization cloning (10). The deduced amino acid sequence of the latter cDNAhad clear similarities to immunoglobulin and TCR-fl chains in structure and sequence; the corresponding gene was rearranged in T-cell clones; and corresponding mRNAwas detectable in cloned CTLlines. But it soon becameclear that this cDNA could not encode the u-chain, because a-chains are glycosylated on asparagine residues (1), while th~ protein sequence deduced from the cDNAcontained no consensus sites for asparagine-linked glycosylation (N-x-S/T). Bona fide a-chain cDNAs were isolated shortly thereafter (14, 15). The original candidate u chain cDNAwas renamed "~," and efforts to determine the function and structure of the ~ chain were underway. Over the next two years, the diversity, ontogeny of expression, and genomicstructure of ~ genes were investigated in considerable detail. The discovery that ~ mRNA levels were highest in the thymocyte subset that includes the least mature thymocytes (CD4-CDS-or "double-negative" thymocytes), and very low in most resting T lymphocytes, provokedspeculation that the ~ polypeptideis expressedin the progenitors of u/fl T cells and plays a role in their ontogeny (16-18). Subsequent reports demonstrating a preponderance of nonproductive ~-rearrangements in cloned ~/fl T cells raised doubts about these models and suggested that ~ genes do not function in u/fl T cells (19-25). During this period, human7 genes were first characterized (26-28), and the diversity of V~ genes in both murine and humansystems was found to be more extensive than previously thought. Altogether seven murine (2932) and seven or eight functional humanV~ genes (24, 25, 34-36) have been identified; this suggests a relatively limited repertoire of V~genes in both species. In the summerof 1986, evidence was first provided for a T-cell subset that expressed a second heterodimeric T cell receptor (11, 37). The use monoclonal antibodies reactive with all u/fl receptors on humanT cells + cells that allowed the identification of a small subset of peripheral CD3 do not express u/fl receptors. These cells, as well as certain cell lines and lymphomas, were shown to express a CD3-associated heterodimer of a ~ chain with a second unidentified chain (11, 3741). Subsequently, these findings were extended by the detection of 7-containing TCRson a subset of murine adult double-negative thymocytes (42). Expression of ?-containing heterodimeric receptors was also detected on a subset of 5-10% of murine fetal thymocytes, beginning around day 14 of ontogeny, long before u/fl receptor expression can be detected (ca. day 17, 18) (43, 44). In both humanand murine systems, evidence provided that the second chain of the new T-cell receptor was not u or/3,

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thus arguing against modelsin which ~ pairs with either the ~ or/~ chains and fueling the expectation that a fourth chain, called 6, remained to be cloned (11, 42). The derivation by several groups of ?/6 T-cell lines and hybridomas that carry out typical T-cell effector functions (cytolysis of target cells, lymphokinerelease) strengthened the notion that ~/6 cells maybe immune effector cells as opposed to ontogenic intermediates (37, 39, 40, 45-49). Other models, in which ?/6 T cells are a separate lineage that regulate the developmentof ~/fl T cells, were also proposed (42). In the summerof 1987, a surprising turn of events yielded the 6 chain gene. A study of unusual rearrangements occurring just upstream of the J~ gene segments revealed the presence of novel TCR-like C, J, and D gene segments that rearrange in double-negative thymocytes (50, 51). Subsequently, protein sequencing data and serological evidence confirmed the assignment of this novel gene as the 6 gene (52-55). In addition highly related human 6 cDNAhomologue was isolated independently by subtractive hybridization procedures (56). Around the same time, it was determined that a previously detected population of Thy-1 ÷ marrow-deriveddendritic cells resident in the skin ("Thy-1 ÷ dendritic epidermal cells" or Thy-1 ÷ DEC) express predominantly CD3-associated ~/6 receptors (57-60). Subsequent studies have demonstrated that a preponderance of T cells in the murine (61, 62) and chicken (63) intestinal epithelium are y/6 cells. These observations have provokedthe suggestion that ~/6 cells are poised as a line of defense against epidermal or gut infections and/or tumors. Recently, success has been reported in deriving several cloned 7/6 cell lines with clear specificity for allogeneic MHCor MHC-related gene products (64, 65). These reports raise the possibility that 7/6 cells normally react with foreign antigens bound to self-MHCproteins. Overall, the observations of T-cell effector activities of 7/6 cells, the peripheral locations of someof these cells, and the MHC-relatedspecificity of at least some~/6 receptors, represent suggestive evidence that ~/6 cells are a distinct subset of peripheral effector T lymphocytes with specificity for MHCor antigen-modified MHCglycoproteins. This view remains to be confirmed by direct experimentation. The most striking outstanding issues include the nature of ligands recognized by 7/6 cells, and the physiological and evolutionary necessity for a separate subset of T lymphocytes. ~ CHAINS

AND GENES

Murine Murine y genes are located on chromosome13 (67). The seven V genes and four C genes are organized as shownin Figure 1 (22, 29-32, 68). The

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179

MURINE GAMMA GENES V5

V4 V3 Jl C1

V2

VI,3

HUMAN GAMMA GENES

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n n n n/,dL.v,,

n n n n

n n,./.L_B__L/,.t

i

i

trot

t I

~

MURINE DELTA GENES

HUMAN DELTA GENES v~, v~ ~z~r~s

D6z /,. I

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DSZ ~6~ ~82 =Zl3 C6 II I I r’--I

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Fiyure l Genomicorganization of murine and human7 and 6 gene families. For simplicity the exons of the C genes are not depicted. Brackets (//) indicate gaps; the murine 7 genes have been linked by pulsed field electrophoresis (68), although the orientation of the V71.3, J73 and C73genes relative to the other genes is not known.Functional genes are listed above the lines while pseudogenes are listed below the lines. The humanVyl.5 sequence is not determined. The mapsare drawnroughly to scale. The ticks below the lines of the murine ~, and ~ genes correspond to the approximate location of EcoRI sites in the BALB/cstrain.

interspersal of V and J-C gene segmentsis reminiescent of the ,~ light chain locus and differs from most other receptor gene loci, including the human 7 genes (see below). vy GENES ANDREGIONS The seven routine V7 genes isolated thus far fall into five subfamilies based on sequence similarity (Figure 2). The VTI subfamily [according to the nomenclature of Garmanet al (30); see Table 1 for a cross-referencing of murine 7 gene nomenclatures] includes three V genes, V71.1, V71.2, and V71.3, whichare highly related at the nucleotide and amino acid levels (23, 29); V72, V~3, V74, and V75 are each quite distinct in sequence from each other and from the V7I genes (17-50% homologyat the amino acid level) (30-32). The V~ genes, like other V genes, are composed of a major exon, encoding most of the variable region chain, preceded by an exon that encodes the 5’ untranslated portion of the mRNAand the presumed

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RAULET

MOUSE -2 +i

i0

20

30

40

VYl.l MLLLRWPTFCCLWVFG LGQLEQT{LSVTRATDE~AQISCIVSL~YFS NTAIH~YRQK] V~I.2 MLLLRWFTSCCLWVFG LGQLEQTELSVTRETDENVQISCIVYLPYFS NTAIHWYRQK’ VYI.3 G LGQLEQTELSVTREQDESAQISCIVSLPYFS NTAIHWYRQK] ---PLLKVVIFLCLLTFG V?2 HGKLEQPEISISRPRDETAQISCKVFIESFR SVTIHWYRQK[ V?3 MSTSWLFLLSLTCVYG DSWISQDQLSFTRRPNKTVHISCKLSGVPLH NTIVHWYQLK< V?4 MGLLLQVFTLASLRIYSEG SSLTSPLGSYVIKRKGNTAFLKCQIKTSVQKPDAYINWYQEK V?5 MLWALALLLAFLPAGR QTSSNLEERIMSITKLEGSSAIMTCDTHRTGT YIHWYRFQ

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HUMAN vTi .2 V?l.3 V?l.8 V?3

MQWALAVLLAFLSP MRWALAVLLAFLSP MQWALAVLLAFLSP MLLALALLLAFLPP MLSLLHTSTLAVLGA MSLLEAFAFSSWA MPLVVAVIFFSLWV

AS QKSSNLEGRTKSVIRQTGSSAEITCDLAEGSNG YIHWYLH AS QKSSNLEGRTKSVTRQTGSSAEITCDLTVTNTF YIHWYLH AS QKSSNLEGRTKSVIRQTGSSAEITCDLAEGSTG YIHWYLH AS QKSSNLEGRTKSVTRPTGSSAVITCDLPVENAV YTHWYLH LCVYGAGHLEQPQISSTKTLSKTARLECVVSGIKIS ATSVYWYRE LG LGLSKVEQFQLSISTEVKKSIDIPCKISSTRFE TDVIHWYRQ FA LGQLEQPEISISRPANKSAHISWKASIQGFS SKIIHWYWQ

Figure2 Deducedamino acid sequences of murine and humanV7 genes.The nomenclatures are those of Garman et al (30) ~r the murinegenes (see Table 1) and Strauss et al (I 15) and Brenneret al (116) ~r the humangenes (an alternative nomenclature~r human7 genes has been proposed; 36). In parentheses to the right of each sequence are re~rences ~r the sequences. Gapswere inserted to align the sequences ~r comparison, and the conserved cysteine residues are indicated by asterisks. The amino-terminiof the matureproteins (+ 1) are estimated. The leader sequence of murineV72is 24 aminoacids longer than shown(30).

cleaved signal peptide of the chain. Heptamer-nonamer recombination signal sequences separated by 23 bp spacers are found just to the 3’ side of each sequencedV7 gene (29, 30). c7 CEYES aYDREC~OYS Of four murine C~ genes (Figure 3; 22, 29, 30), C73is apparently a pseudogene, at least in BALB/cmice, by virtue of a defective 5’ splice site borderingthe secondexon(29). In addition, the J73C~3gene is deleted entirely in several mousestrains, including C57BL/10 (22); it is present in the C57BL/6 and BALB/cstrains. Three of the Cy genes, C71, Cy2, and ~Cy3, are very similar in coding sequence(10, 29, 30). C?I and C~2differ by only six replacementsof 290 aminoacids, anda 5 aminoacid insertion in C71.This insertion is located just to the amino-terminalside of the cysteinc residuc used for disulfide linkage to the 6 chain, in the region connecting the disulfide-linked C domain to the plasma membrane. The C74gene differs significantly in sequencefromthe other Cy genes, with about 66%overall amino acid identity and 76%nucleotide identity in the coding regions (22). In addition, the C74 sequence contains a

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GAMMA AND DELTA TCR

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REFERENCES

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QQFEYLIYVATNYNQR PLGGKHKKIEASKDFKSSTSTLEINYLKKEDEATYYCAVWM QQFEYLIYVETNYNQR PLGGKNKKIEASKDFQTSTSTLEINYLKKEDEATYYCAVWI QGLZFLLYVLATPTHI FLDKEYKKMEASKNPSASTSILTIYSLEEEDEAIYYCSYG EPLRRIFYGSVKTYKQ DKSHSRLEIDEK DDGTFYLIINNWTSDEATYYCACWD QRLQRMLCSSSKENIV YEKDFSDERYEARTWQSDLSSVLTIHQVTEEDTGTYYCACWD R~EHLLYYNFVSSTTVVDSRFNSEKYHVYEGPDKRYKFVLRNVEES DSALYYCASW

(15) (23) (30,31) (30) (30) (32)

GKAPQRLQYYDSYNSKVVLESGVSPGKYYTYASTRNNLRLILRNLIEN DSGVYYCATWDG (34) GKAPQRLLYYDVSTARDVLESGLSPGKYYTHTPRRWSWILRLQNLIEN DSGVYYCATWDR (24,34) ,GKAPQRLLYYDSYTSSVVLESGISPGKYDTYGSTRKNLRMILRNLIENDSGVYYCATWDG (34) GKAPQRLLYYDSYNSRVVLESGISREKYHTYASTGKSLKFILENLIER DSGVYYCATWDR (34,41) GEVIQFLVSI SYDGTVRKESGIPSGKFEVDRIPETSTSTLTIHNVEKQDIATYYCALWEV (72) NQALEHLIYIVSTKSAARRSMGKTSNKVEARKNSQTLTSILTIKSVEKEDMAVYYCAAWWV (35,36) (36) NKGLEYLLHVFLTISAQ DCSGGKTKKLEVSKNAHTSTSTLKIKFLEKEDEVVYHCAC Figure 2 (Continued).

amino acid (69 bp) insertion (comparedto Cy2) immediately preceding the cysteine residue thoughtto be involved in a disulfide-linkage with the 6 chain. In BALB/c mice, the C71gene contains a single potential site for asparaginc (N)-linkcd glycosylation while the Cy2 gene contains none. In some other strains, such as C57BL/Ka and C57BL/10,the N-glycosylation site in C71is absent (66). The C74gene from C57BL/10also contains a single site for N-glycosylation. Knowledge of the potential glycosylation sites of

Table 1 Correspondencebetween the different mouseVy genenomenclatures

~ VV Vl,l VI.2 Vl.3 V2 V3 V4 V5

Haydayet al (Ref. 29) VI0.8B V10.8A V5,7

Heilig & Tonegawa (Ref. 31)

Trauneckeret al (Ref. 23)

Hucket al (Ref. 36)

V2 VI V3 V4.3 V4.1 V4.2 V4.4

V2 VI V3 V4 V6 V5 V7

V1 V2 V3 V4 V5 V6 V7

Thenomenclature usedin this reviewis fromGarman et al (30).

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182 RAULET

M

0

Ol

z

t~

0 E~

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TCR

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Cy and Vy chains has been useful in some cases in identifying the rearranged ? genes that encode particular ? polypeptides. Each of the four ~ constant regions includes a hydrophobic transmembranedomain of about 20 residues, followed by a short cytoplasmic domain of about 12 amino acids. The cytoplasmic domains of C71 and C72 are identical but differ significantly from the corresponding domain of C74. Like other TCRC genes, all C7 genes encode a lysine residue in the transmembrane domain, which is presumably buried within the membrane.It has been proposed that the lysine residues are involved in intramembrane interactions with other chains, such as those CD3components that display acidic residues within the membrane. C71, C72, and C73 are each comprised of three exons (29, 30). The first (CI) exon encodes most of the extracellular portion of the constant region, while the short CII exon encodes a stretch of variable length (see above) that separates the C domain from the plasma membrane; the CII exon also encodes the cysteine residue presumablyinvolved in a disulfide bridge with the 6 chain. The third exon encodes a short extracellular region, the hydrophobic transmembrane domain, the short intracellular domain, and the 3’ untranslated region of the mRNA. J7 GEYESF~GMEYTS Each of the four C7 genes is preceded by a single knownJ7 gene segment which is numberedaccording to the corresponding C gene (22, 23, 29). In accord with the similarities betweenC7 genes noted above, the sequences of JTl and J72 are extremely similar (identical at the amino acid level), whereas J74 differs from JT1 and J72 at 9 of 19 amino acid residues (Figure 4). Most of the differences occur at the aminoterminal end of the J segments, where they may contribute to the third complementarity determining region (CDR) of the corresponding regions. The J~/3 gene segment,like its correspondingC73gene, is defective, by virtue of two frameshift mutations in the coding regions (23). The genes are flanked on their 5’ sides by heptamer-nonamer recombination signal sequences separated by 12 bp spacers (29). REARRANGEMENT OF MURINE V’~ GENES The murine 7 gene family is often considered as clusters of VT/JTCygenes, based on the genomicorganization and the observations that most V~/rearrangements are to the Jy-C7 gene that is most proximal and in the same transcriptional orientation (Figure 1). Thus VTl.1/J74C74 is one cluster, V71.2/J72C72 is another, V7 t .3/J73C73 is a third, and the fourth cluster is V75,V72,V74,V73/J~ 1C 71. Rare instances of V7 rearrangements to more distal J7C7 genes have been reported, including V75/J74C74(32), VTI.3/J74C74(69), and V72/J74C74 (J. Segal, D. Cohen, personal communication). Table 2 summarizesthe observed rearrangements of murine ~ genes and

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RAULET Murine J.~ sequences

J~l, J~ J~4

SSGFHKVFAEGTKLIVIPS (29) GTSWV-I--K ....V---P (22)

HumanJ.~ sequences

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J~l.1 TTGWFKIFAEGTKLIVTSP (73, 73a) J~2.1 SSD-I-T--K--R ...... (73a) JTi.2 GDELGKKI-V-GP ..... I- (34,73a) J~l.3 & NYYK-L-GS--T-V-(33,72)

Murine J~ seouences J81 TDKLVFGQGTQVTVEP J82 SWD-RQMF--T-IELF---

(51) (69,76)

HumanJ~ sequences J~l TDKLIFGKGTRVTVEP J~2 SWD-RQMF--T-IKLF--J63 SWD-RQMF--T-IELF---

(55,86) (82) (82a)

JT2.3 Ftlqure4 Murineand human J7 and J6 amino acid sequences.Identities are indicated by dashes.In parentheses to the right of eachsequence are references. what is currently knownof their expression in y/6 cells in different tissue sites. Remarkably, it appears that different 7 genes are expressed preferentially in different tissues (see V~and V6usagein peripheral 7/6 cells). Human The human7 genes, located on chromosome7 (26, 28), are organized a fashion similar to that of the//chain genes and unlike that of the murine 7 genes: two neighboring JyCy gene clusters are flanked on their 5’ sides by an array of Vygenes (Figure 1; 27, 70). V~ GENES ANDREGIONS Currently 14 humanV7 genes have been identified, of which only seven or eight are potentially functional (Figure 2; 24, 25, 33-36). The functional humanV7 genes fall into four subgroups. Subgroup I consists of at least four functional genes (V1.2, V1.3, V1.4, V1.8) and four nonfunctional genes. The functional subgroup I sequences are identical at 67-87%of their amino acids. Subgroups II, III, and IV each include a single V gene (V2, V3, and V4, respectively) and differ considerably sequence from each other and subgroup IV segments (23~44%amino acid identity between the subgroups). Comparisonof the amino acid sequences to routine V7 sequences reveals similarity between humansubgroup I and murine V75 (up to 49% identity), and between the human subgroup sequence and the murine V71subfamily (up to 51%identity) and V72gene (52%identity). GENESANDREGIONS The sequences of the two human CT regions are very similar overall, although heterogeneity in the membraneproximal connector region of the C~2gene results in significant differences in the structure of the two C~regions (Figure 3). Like murine C7 genes, the humanC71 gene is composedof three exons, C7

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Table 2 V7/Cv gene rearrangement and expression in the mouse Rearrangement is rare in fetal thymus, commonin thymomas(32). Predominant7 chain of intestinal intraepithelial 3’/(5 cells (62). Rearrangementdetected in fetal thymus, frequent in adult double negative thymocytes,and in e//3 T cells (30, 31); in the latter population is often but not always nonproductively rearranged, and seldom transcribed. Protein is predominant V7of 7 chain in expressed by _adult thymic 7/6 cells (42; Figure 6) and common in splenic "~/6 cells (93). V74-J ~ 1C~1 :

Rearrangementis relatively frequent in fetal thymocytes, and infrequent in adult double negative thymocytes(30). Protein detected hybridomas of adult double negative thymocytes (69).

V?3-J71C]? 1:

Rearrangementis relatively frequent in early fetal thymocytes(30, 31) and DECcells (60, 89), but undetectable in adult double negative 3,/6 cells. Protein is predominant V~, of 7 chains of DECcells and probably in early fetal thymocytes(92, 96). Rearrangementcommonin late fetal and adult double negative cells, and in c~//~ T cells (30); in the latter populationit is often but not always nonproductively rearranged 09-23, 99). Unlike V72-JTIC~l, V1.2J72C72transcripts are detected in activated (but not resting) c~//~ T cells, particularly those of the CD8phenotype (15, 30, 74). There is evidence that 7/6 cells expressing VTI.2-J72C72chains are present in MLR activated spleen cell preparations (85, 114), and indications that an MHC-IE reactive 7/6 T cell hybridomamayexpress the V71.2-J72C72protein (L. Matis and J. Bluestone, unpublished data). Rearrangement of V71.3 to J73C73 has been rarely observed in cloned 7//~ T cells (29); because Cy3is a pseudogene,these rearrangements, if they occur in 7/3 cells, are presumably non-functional. Rearrangementof V71.3to J74C74has been detected in an adult thymic 7/~ T cell hybridoma, and appears to encode the ~ chain expressed on this cell (69). The V71.3 gene (and the J~3-C73gene) is deleted in somemousestrains (e.g. C57BL/10)(22). Rearrangementof V’~ 1.1 to Jy4C74has been observed in some~//3 T cell clones (22), and in somey/fi T cells (69). The rearrangementis detectable but present in a minor fraction of adult thymic ~/~5 cells (Figure 6). The rearrangement of V71.1 to J74C74 is the most commonC74 rearrangement observed. Protein level expression of J74C74-containing chains has been demonstratedin the case of DECcell lines (59a) a small fraction of fetal and adult thymic 7/8 cells (96), and a large fraction of splenic 7/~5 cells (86).

VV1.2-Jy2Cy2:

V71.3:

V71. I-Jy4C?4:

Sites of predominant usageof the indicated7 chain are underlined.

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corresponding to the disulfide-linked C domain (exon I), the membrane proximal connector region (exon II), and the transmembrane and cytoplasmic domains(exon III) (70). While the regions of CTl and C72 encoded by the first and third exons are virtually identical in their amino acid sequences, the membraneproximal connector regions differ markedly. First, the C72gene, but not the C71gene, includes two or three homologous tandemcopies of the CII exon (CIIa, b, and c), each of which is similar sequenceto the single CII exon of CT1(41, 70, 71, Figure 3). Becausethese tandem copies of the CII exon are spliced together in the corresponding mRNA,the size of the C~2 connector region is larger than that of C71. Due to genetic polymorphism,different individuals have two (b and c) three (a, b, and c) copies of the CII exon of C72. Secondly, while the CII exon of the C71 encodes a cysteine residue required for disulfide linkage to the 6 chain, none of the three CII exons of C72 encodes a cysteine. Therefore, this chain cannot form a covalent linkage with the 6 chain, and this accounts for the observation that manyhuman~/6 receptors are not covalently associated (71). The heterogeneity in the size and sequence the connector region of murine and human7 chains may have functional as well as structural consequences. J7 GENESEGMENTS While each murine C? gene is preceded by a single J segment, the humanC7 genes are preceded by two (C72) or three (C71) segments(Figure 1; 72, 73, 73a). All five J7 gene segments are potentially functional and rearrangements of each to V~ gene segments have been documented(compiled in 36). Twoof the J7 segments, one in each cluster, encode identical aminoacid sequences, whereasthe others differ markedly, particularly at the aminoterminal ends (Figure 4). Junctional

Diversity

of Murine and Human ~ Genes

No D elements have been identified for 7 genes in mouse or human. However,significant variability exists at manyVT-J7junctions due to the imprecision of the joining process and the addition of short stretches of nucleotides, called N regions, at the V-J junction during joining (25, 36, 73, 74). The latter process, presumably catalyzed by terminal deoxyribonucleotidyl transferase (TdT) in progenitor lymphocytes, adds nucleotides to junctions in a template independent fashion (75). Most of the published sequences are of nonfunctional 7 gene rearrangements from e//3 T cells, and there is currently only limited data concerning ~ junctional diversity in murine 7/3 cells. The sequences of four human 7 cDNAsknown to encode 7 polypeptides at the cell surface include N regions of 3 5 nucleotides (compiled in 36). In contrast several murine dendritic epidermal cell lines lack N regions (see below). Consideringthe limited repertoire

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Vyand Jy genes, and the apparent lack of D elements in y genes, junctional diversity mayaccount for muchof the overall diversity of y chains. 6

GENES

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Murine

AND CHAINS

V6 Genes

The murine C~, J~i, and De5 gene segments are located on chromosome14 between the V~ and J~ gene segments (Figure 1; 50, 51). The C6 gene ~ 75 kb upstream of the Ca gene, but only ~ 8 kb upstream of the most 5’ knownJc~ gene segments. TwoJ6 and two D6 gene segments lie on the 5’ side of C6(51, 69, 76). At present, eight murine V6 gene subfamilies have been identified (Figure 5). As assessed by Southern hybridization, several of these subfamilies appear to include only one member(V61, V62, V63, V65), while the others include two or moremembers(65, 69, 77). It is likely that more V6genes will be identified. The location of D6, J6, and C6 genes between V~ and J~ gene segments raises the possibility that a shared pool of V genes is utilized to produce both ~ chains and 6 chains. The current evidence suggests that there is indeed someoverlap in V-usage between the ~ and 6 chains. Thus, four of the eight V6 gene subfamilies (V63, V66, V67, V68) overlap with or are identical to knownVc~ subfamilies (V~6, V~7, V~4, V~I 1, respectively) (Figure 5); in fact, V63and V~6are probably identical (77). However, of the V6 subfamilies are very different than knownV~ sequences (Figure 6), including V6 genes commonlyexpressed by fetal thymocytes(V61) (76), y/g-dendritic epidermal cells (also V6I, J. Allison, personal communication) and adult thymic double negative 7/c5 cells (V65; 69, 77). That these commonlyused V6 genes have not been observed in surveys of large numbersof c~ cDNA clones indicates that they are preferentially, possibly even exclusively, used to produce 6 chains. At present the mechanismsthat account for preferential usage of certain V gene segments to produce 6 versus ~ chains are not known. The possibility that V~ and V6 genes are distinguished by flanking recombination signals is unlikely, since two sequencedV6genes, like V~genes, are flanked by recombination signal sequences separated by 23 bp (78, 80). The likelihood of rearrangement of a V gene to D6/J6 versus J~ gene segments might be influenced by its genomic location. In line with the observation that J. proximal V, gene segments rearrange preferentially to JH in pre-B cells (79), it is possible that 6 gene rearrangements, which occur earlier in ontogeny than ~ gene rearrangements (see later), might preferentially use D6-Jfi proximal V gene segments. Indeed, V~6, a "singlememberedgene family" probably identical to V63, is closer to J6 than any

Annual Reviews 18 Murine

8 RAULET V~ Sequences

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+I VSI V82 V~3 V~4 V~5

(V~6)

V~6

(V~7)

V~7 V~8

(Vd4) (V~l i)

Human V61

i0

20

30

40

5

DVYLEPVTkKTFTVVAGDPASFYCTV TGGDMKNY HMSWYKKNGTNAL ~ TQMLHQSPQSLTIQEGDEVTMSCNLST SL Y ¢ ALLWYRQGDDGSLV. QPDSMEST-EGETVHLPCSHATISGNE Y IYWYRQVPLQGPE~ AQTVSQPQKKKSVQVAESATLDCTYDT SDTN Y LLFWYKQQGGQ Vl C I TLTQSS TDQTVASGTEATLLCTYNADSPN P DLFWYRKRPDRSFQ~ AQRVTQVQPTGSSQWGEEVTLDCSYET SEYF Y CIIWYRQLFSGEMV~ AQKVTQVQSTGSSQWGE VTLHCSYET SEYF Y VILWYKQLFSGEMVI AQRVTQVQPTASSQWGEEVTLDCSYET SEYF Y RIFWYRQLFSGEMV~ AQKVIQVWSTTSRQEGEKLTLDCSYKT SQVL Y HLFWYKHLLSGEMV] QVALSEEDFL TIHCNY SASG YPTLFWYVQYPGEGPQJ ~ RG.

V~ Sequence QKVTQAQSSVSMPVRKAVTLNCLYET

SWWS

Y YIFWYKQLPSKEMI}

Figure 5 Murine and human V6 amino acid sequences. The nomenclaturefor murine V6 genes is an extension of that of Elliott et al (77). Whereapplicable, the corresponding gene subfamily is indicated. In parentheses to the right of each sequence are references for the sequences. V~7is from the DN2.3cell line (69) and V~8is the 3G8sequence in reference 65. Gaps were inserted to align the sequences for comparison, and the conserved cysteine residues are indicated by asterisks. The amino-termini of the mature proteins are estimated.

of 38 other V~ genes tested in a deletion mappingstudy (M. Kronenberg, personal communication). Since V62 is reportedly located on the same EcoRI fragment as V63(77), it is also proximal to D6/J6. In addition, the V35gene is in close proximity to D6/J3 gene segments, though it is located to the 3’ side of C6 (see below). Finally, a study of the extent of V6 gene deletion in thymocytes led to the conclusion that V61~are all relatively proximal to D6-J6 (77). Therefore, segeral of the V6 genes that are.frequently rearranged to D6-J6 in various tissues (V61, V63, V65) are proximal to D6-J6 gene segments in germline DNA,suggesting that proximity mayaccount at least partly for the preferential usage of 6 gene segments. It is unlikely, however,that proximity is a necessary distinguishing feature of V6 genes, since V66 family members are not commonlydeleted by ~ rearrangements and are therefore probably located to the 5’ side of many V~ gene segments (77). Interestingly, the V65geneis located ~ 2.5 kb to the 3’ side of C6 (Figure 1), in the opposite transcriptional orientation, and rearranges by inversion to D6/J6 gene segments (80). Despite its proximity to D6/J6 gene segments, V65is infrequently rearranged in ~/6 cells from the fetal thymus(76, 77); instead V~5-D~J~C~ rearrangements are frequent amongadult thymic y/6 cells (69, 77), accounting for 16-30%of rearranged alleles. Therefore,

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60

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90

i00

LVXKLNSNSTDGGKSNLKGKINISKN LVTLQKGGDEK SKDKITAKLD VTHGLQQNTT NSMAFLAIASDR LVILQEAYKQYNATLNRFS%rNFQ ILYRDDTSSHDADFVQGRFSVKHS LIY QTSFDTQNQRNGRYSV~Q LIY QTSFDTQNQRNSRYSVVFQ LIY QPSFDTQNQRSGRYSV-~/FQ LIR QMPSTIAIERSGRYSVVFQ LFR ASKDKEKGSSRGFEATYD LISLFYLASGTKENGRLKSAFDS

QFI LDIQKATMKDAGTYYCGSD KKMQQSSLQIQASQPSHSGTYLCGGK KS ST LILTHVSLRDAAVYHCILR KAAKSFSLEISDSQLGDAATYFCALM KANRTFHLVISPVSLEDSATYYCASG KSLKSISLVISASQPEDSGTYFCALS KSLKSISLVISASQPEDSGTYFCALS KSFKSISLVISASQPEDSGTYFCALS KSRKSISLVISTLQPDDSGKYFCALW KGTTSFHLKKASVQESDSAVYYCALS KERRYSTLHIRDAQLEDSGTYFCAAD

LIK QGSDEQNA.K

KAAKSVALTISALQLEDSAKYFCAL

SGRYSVNFK

189 Ref. (76) (76) (77) (51) (77) (77) (77) (51) (76) (69) (65)

(55,56)

Figure 5 (Cont#~ued).

least in this case, proximity alone does not lead to a high frequency of rearrangement in fetal thymocytes. Human Vc5 Genes The human6 locus, located on chromosome14, is also located between J~ and V~ gene segments (81, 82). Also similar to the murine 6 locus, three J6 and two D6 gene segments are located 5’ to C6 in germline DNA (Figure 1). As in the murine system, the humanV6 gene repertoire appears to be limited, and at least somewhatdistinct from the Vc~gene repertoire (55, 56). Only a single humanV6 sequence, V61, has been reported (Figure 5), which displays 57%amino acid identity with a humanV~ gene segment, and up to 58% amino acid identity with murine V66/V~7members. V61 is expressed by all five of the 7/6-bearing T-cell lines and leukemias examined in several reports, suggesting that it is a commonlyutilized V6 gene segment (55, 56, 78). Recently three additional humanV6 genes have been isolated (M. Krangel, personal communication). One of these genes encodes a V6 which is similar to the murine V65region, displaying 66% aminoacid identity. Interestingly, this V segmentis located to the 3’ side of the C~gene, as is murine V~5. C6 Gene In mice and humansthere is a single C6 gene (51, 81, 82). In the mouse, C6 is encoded by four exons located 75 kb 5’ to the Ca gene and about 8 kb 5’ of the most proximal J~ gene segment, J~l (76).

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RAULET

The deduced amino acid sequence of C6 includes cysteine residues separated by 51 amino acids that presumably form an intrachain disulfide linkage, followed by a connector region that carries a cysteine residue presumedto form a disulfide linkage with the ~ chain (Figure 3; 51, 55, 56). The connector region is followed by a transmembrane domain which includes a charged lysinc residue conserved in all TCRC regions at corresponding positions. The lysine residue is thought to be involved in interactions with CD3components, which carry acidic residues in their transmembrane regions. In addition, the C~ and Ca transmembrane regions each carry a second basic residue (arginine) in the transmembrane region, which may also be important for interactions with other chains. Also like Ca, the C6 intracytoplasmic tail is apparently extremely short ( _< 4 aminoacids). J~ Genes TwoJ6 gene segments, J61 and J62, have been localized 18.5 and 5 kb upstream of the murine C6 gene (69, 76). The deduced Jc~ region sequences match at only 7 of 16 residues (Figure 4). Three humanJ~ gene segments have been identified, two of which are nearly identical (Figure 4; 55, 56, 78, 82, 82a). The murine J6 sequences are very similar to the human counterparts, matchingat 13 of 16 (J61) and 18 of 19 (J62) residues. contrasts with the J7 regions, for which there is no correspondencebetween mouse and human. The J6 gene segments are flanked on their 5’ sides by heptamer-nonamerrecombination signal sequences with 13 bp spacers (69, 76, 78). D6 Genes Two D6 genes, D6I and D62, have been localized approximately 10 kb and 1.2 kb upstream of J61 in both mouse and human(76, 82a). Murine D61and D62are 11 and 16 nucleotides long, respectively, and can be read in all three reading frames. They are each flanked on both sides with heptamer-nonamerrecombination signal sequences; the 5’ signal sequence includes a 12 nucleotide spacer, while the 3’ signal sequence carries a 23 nucleotide spacer. Rearrangement

of ~ Genes

The arrangement of recombination signal sequences flanking 6 gene segments is compatible with V6-J6, V6-D6-J6, and V6-D61-D62-J6rearrangements. The latter type of rearrangement, which is unprecedented in other rearranging gene families, is commonamongadult thymic 6 rearrangements (77). V6-D62-J6 rearrangements are frequent among fetal rearrangements (76). Direct V6-J6 rearrangements may also occur,

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191

evidenced by analysis of excised circular DNAfrom thymocytes (83). Interestingly, incomplete V6-D6rearrangements are fairly commonin hybridomasof fetal thymocytes (76). In contrast, V-Drearrangements are rarely if ever observed in heavy chain or TCR-/3gene rearrangements.

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DIVERSITY OF THE 7/c~

RECEPTOR

With relatively few V genes (7-8), no D regions, and few distinct J regions (2M), 7 genes are one of the least diverse rearranging receptor gene families. The diversity of the ~ genes is also likely to be less than that of e and/~ gene families with respect to the number of V (~ 10), J (2), and D segments. In addition, neither ? nor 6 chains appear to undergo somatic hypermutations, an important mechanism for diversifying immunoglobulin V regions following antigenic stimulation (74, 76, 77, 80). Although the number of rearranging gene segments is small in both families, there is potential for considerable junctional diversity in both chains. In fact, the junctional diversity of the c5 chain is potentially enormous (77, 78). Unlike other receptor gene rearrangements, assembled genes in the adult thymus often include two D segments (V6-D61-D62J6 rearrangements). Imprecise joining can result in variable inclusion of nucleotides located at all six DNAends involved in these rearrangements, and N regions can be added at all three junctions. Therefore, although the V6 repertoire is relatively limited, the potential diversity of 6 chains may be far greater than that of/3 and c~ chains. Junctional diversity of ? chains due to imprecise joining and N region addition has also been documented (25, 36, 73, 74), although there are no D7 gene segments. Based on these considerations, it has been estimated that there could be 1017 possible ?/6 heterodimers, with almost all the diversity concentrated at the V-J junctions of both ? and 6 chains (77). As discussed below, however, ? and chain diversity maybe muchmore restricted in certain anatomical sites, such as the epidermis.

PERIPHERAL LOCATIONS OF 7/6

CELLS

In the periphery, 0.5-10%of peripheral blood T cells in the human(11, 38, 49, 84) and ~ 3%ofT cells in the murine spleen and lymph nodes (44, 85, 86) are CD4-CD8 ?/6 cells. In the chicken, the proportion of?/6 cells in peripheral blood and spleen is higher than in mammals,approaching 30% of CD3+ T cells (87, 88). Interestingly, most (~70%) chicken ?/3 + cells (88); the remainder are CD4-CD8cells in the spleen are CD4-CD8 cells, as are the ?/3 cells in chickenblood. Strikingly, ?/3 cells are the predominantT-cell type found in the murine

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epidermis and the murine and chicken intestinal epithelium. Murine Thyl + dendritic epidermal cells (DEC),the subject of extensive investigations for several years as candidate epidermal T cells, were shownrecently to be largely if not exclusively 7/6 cells (58-60, 89). 7/6-epidermal cells have not been detected in the chicken, and studies in the humanare thus far inconclusive. In the routine and chicken intestinal epithelium, 7/6 cells are also the predominant T-cell type. In both species, most intestinal + cells (61-63). The epithelial intraepithelial 7/6 cells are CD4-CD8 location of some?/6 cells has promptedthe suggestion that they function to combatepidermal and/or intestinal antigens. The findings that 7/6 cells in different peripheral locations exhibit striking differences in V7 and V6 gene usage raise the possibility that 7/6 cells in various locations carry out discrete functions (see below). ONTOGENY

OF

7/6

CELLS

7/6-bearing cells are the first TCRbearing cells to appear in ontogeny, on or before day 14 of gestation in the murine fetal thymus (43, 44, 63, 91). Six to seven percent of day 14 fetal thymocytes, bear 7/6 receptors (92). At this early stage all thymocytes are CD4CD8-, and the proportion of CD4-CD8thymocytes which bear 7/6 receptor cells, 5-10%, is maintained throughout ontogeny (4.2-44, 92); few if any CD4+ + and/or CD8 thymocytes bear 7/6 receptors. Since the absolute number of CD4-CD8thymocytes increases during ontogeny, so does the numberof 7/6-bearing thymocytes, reaching approximately 3 x 10s cells in the young adult thymus. The proportion of 7/6-bearing thymocytes, however, decreases precipitously after day 16 of gestation (to 0.2%of adult thymocytes) + thymocytes. In the parallel with the rapid increase in CD4+ and/or CD8 chicken a similar pattern of 7/6 cell ontogeny has been documented(87). 7/6-bearing splenic T cells first appear sometimebetweenbirth and four weeksof age (93). Thyl + dendritic epidermal cells, most of which are 7/6 cells, also appear between birth and three weeks of age (R. Tigelaar, personal communication).The ontogeny of 7/6 cells of the intestinal epithelium has not been reported. Thymus Dependence

of ~/~ Cells

The early appearance of 7/c~ cells in the thymus, followed later by their appearance in the spleen and epidermis, suggests that splenic and intraepithelial 7/6 cells may be thymus-derived. Direct evidence that many splenic 7/6 cells are thymus-dependentcomes from the demonstration that they are severely deficient in athymic nude mice until at least 11 weeksof age; furthermore, thymus grafting of nude mice results in near normal

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numbersof splenic ~/6 cells several weeks later (93). In the same study, however, it was shown that old (~ 24 weeks) ungrafted nude mice have detectable numbersof splenic 7/6 cells. A similar situation pertains to 7/fl T cells, which are present in small numbersin the spleens of old but not young nude mice (94). Whetherintraepithelial 7/6 cells are thymus-dependenthas been difficult to evaluate. Thyl + dendritic epidermal cells are present in reduced numbers in the skin of eight weekold nude mice, but surprisingly the cells are CD3- and are devoid of full-length TCRtranscripts (95). The obvious conclusion that epidermal 7/6 cells are thymus-dependent is confounded by the fact that nude mice mayexhibit a general epithelial defect. In fact, the Thyl + dendritic epidermal cells of athymic mice type as CD3-even after thymus-grafting (G. Stingl, unpublished data). While these results are consistent with the possibility that (some) 7/6 cells mature in the epidermis and that this process is deficient in nude skin, other explanations are equally plausible. For example, homingof.thymus-derived 7/6 cells to nude skin may be deficient. Another possible explanation is suggested by the observation that the Vy and V6 chains expressed by manyepidermal ~,/6 cells are commonlyexpressed by fetal but not adult ~/6 thymocytes (92, see below): it is possible that epidermal~/6 cells derive from an early wave of fetal thymic ~/6 cells which are not generated in the thymusgrafting experiments performed to date. While extensive additional research will be necessary to resolve this issue, the overall data suggest that manysplenic ~/6 cells, at least, are thymus-dependent. Programmed V Expression

of T/6 Cells

in the Thymus

Emergingdata indicate that during ontogeny, thymocytesexhibit a striking programmedpattern of V7 and V6 gene utilization. Most day 14-day 16 fetal thymocytes express a V73-JTIC71chain, as shownwith a monoclonal anti-V73 antibody and by Southern hybridization studies (30, 31, 92, 96). V6 usage maybe more heterogeneous at these times, but V61 is commonly expressed (76). By day 18 of gestation, V~/3-expressing thymocytes are undetectable (92, 96). Aroundday 16 of gestation, "waves"of cells appear that express distinct ~ chains, including V72-JTICT1, C74-containing chains, and possibly V74-JT1CyI chains (92, 96). In the young adult thymus, the large majority ofCD4-CD8-thymocytes express V72-J71C7I gammachains, although a minority express V74-JylCTI and C74-containing chains (Figure 6; 42, 69, 96). V6 usage by adult CD4CD8 thymocytes appears also to be largely nonoverlapping with that of fetal thymocytes, and predominantly limited to a few V6 genes, including V65 (69, 77). ProgrammedV gene usage has also been documented in pre-B cells,

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where the predominantly utilized VH-genes are proximal to the J,-gene segments (79). Similarly, common fetal Vy and Vd gene segments (Vy3, V6 1, and V63) are all relatively J-proximal (Figure I , see Murine V6 Genes, above). Models to account for programmed V-gene usage include: (a) initial preferential rearrangement of J-proximal V gene segments, with subsequent selection for cells expressing other, more distally located V gene segments (79); (h) initial preferential rearrangement of J-proximal V segments, and later replacement of the rearranged V-segments with other V segments by secondary rearrangement events (30,97,98) (two examples

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of such secondary replacement rearrangements in B cell lymphomashave recently been reported; 97, 98) and (c) sequential activation in progenitor cells of distinct subsets of ¥ genes for rearrangement. It is not clear which if any of the above mechanisms operate to program V7 and V6 usage in thymic ontogeny. In addition to distinct V gene usage, fetal and adult thymocytesdiffer in their ~ junctional sequences. Thus most fetal ~ rearrangements involve V6-D62-J6 rearrangements (76), whereas in adult CD4-CD8-thymocytes, V&D61-D62-J61rearrangements are the rule (77). Moreover, N region diversity is extensive in adult but not fetal 6 rearrangements (76, 77). LINEAGE RELATIONSHIP cz/fi CELLS

OF y/6

CELLS

AND

The weight of evidence at present, though indirect, argues persuasively that 7/~ receptor bearing cells do not differentiate to c~/fl receptor bearing cells. First, surveys of cloned e/fl-T cell lines have revealed that manylack functional rearrangements of all known7 genes (21, 99). A comparable examination of 6 rearrangements in most mature e/fi T cells is impossible since 6 genes are deleted from chromosomal DNAby Ve-Je rearrangements (51). However,the DNAdeleted by Ve-J~ rearrangements is present in thymocytes as extrachromosomal circular DNAmolecules, which can be isolated and molecularly cloned. Of more than 400 DNAclones of this type examined, all contained D62 and J61 gene segments in germline

Figure 6 Evidence that most adult thymic 7/6 cells express a ~ chain encoded by V72-JylC71 rearranged genes. 7/6 cells were enriched (to ~50-80%~,/~ cells) from C57BL/6doublenegative thymocytepreparations by short-term (~ 3d) culture in a lymphokinecocktail (42), and DNAsamples were analyzed by Southern hybridization with the indicated probes. The Cy2 probe detects C71, C72 and C~/3. Panels ADare EcoRI digests and panel E is a HINDIII digest. Lane 1 is DNAfrom 7/6 cells and lane 2 is C57BL/6liver DNA.V72-J~/1Cyl(-~Ir in panel B) and Vy1.2-Jy2C~/2(top ~ir in panel A) rearrangementsare frequent in the population. V74-JylC71(’A" in panel C) and V~I. 1-Jy4Cy4(’A" in panel E) rearrangements are detectable on the original autoradiograph, though infrequent. Vy3rearrangements are undetectable. The large majority of 7 chains in the population bind to antiserum reactive with C,/1 and C72 and are glycosylated on asparagine residues (42). Since the V,/1.2-J72C~/2 sequence lacks N-glycosylationsites, it follows that most of the 7/6 cells express a V72-J ~ 1Cy1 chain. Smaller proportions of these cells express Cy4-containing7 chains (96) and V74-J~/ICTIchains (69). Most of the Vy1.2-J~2C72rearrangements appear not to encode a y chain expressed on the cell surface. Experiment performed by D. Pardoll, A. Kruisbeek, B. J. Fowlkes, and D. Raulet.

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196 RAULI~T configuration, arguing that all or most V~-Ju rearrangements occur on chromosomes that have not previously rearranged 6 genes (A. Winoto, D. Baltimore, unpublished data). Therefore, it appears that neither rearrangements nor productive ,/ rearrangements have occurred in the precursors of most ~//3-T cells. Most likely e/// T cells and ;~/6 T cells represent distinct T-cell lineages. If 7/6 cells and e//~ cells represent separate lineages, at what stage in Tcell differentiation do they separate? Developmentalstudies suggest that in early fetal thymocytes complete rearrangements of 7 and 6 loci and partial D~-J/~ rearrangements occur nearly synchronously in the same cells (76, 100). Rearrangementsof V/~ to D/%J/~are delayed by one or two days, and ~ rearrangements are believed to occur later yet (16, 17, 100, 101). Based on these and other considerations, it was proposed that progenitor thymocytesthat initially fail to produce a functional ~/6 receptor go on to attempt complete ~ and/~ rearrangements (44). This model accounts for the findings that all c¢//~ T cells have ~ rearrangements, manyof which are nonproductive. However, the model also predicts that cells undergoing u-gene rearrangements have previously attempted 6 rearrangements, inconsistent with the aforementioned demonstration that c¢ rearrangements generally occur on chromosomes which have unrearranged Dy2 and JT1 genes. In fact, the latter finding suggests that the cells that rearrange 6 genes early cannot differentiate into c¢//~ cells. Perhaps the progenitors of ~//3 cells are a separate subset that are committed to rearrange ~ genes rather than 6 genes. Alternatively, 6 rearrangements, functional or not, may somehowprevent subsequent e rearrangements in the same cell. In either case, it appears that differentiation ofu//? cells maynot be contingent upon failed attempts to produce a 7/6 receptor. A possible mechanism whereby ~ rearrangements may prevent subsequent c¢ rearrangements on the same chromosome has been recently proposed (102, 103). A novel rearrangement event often deletes the D6, J6, and C6 gene segments in human thymocytes. The rearrangement involves heptamer-nonamer recombination signal sequences located upstream and downstreamof the D6, J6, and C6 genes. The 3’ signal sequence flanks a J-like sequence that is between C&and all known Ju gene segments. However, the upstream signal sequence, which is located 3’ of the known V~ and V6 genes, is not associated with a recognizable gene segment. It was proposed that this rearrangementeven~, is a prerequisite for subsequent c¢ gene rearrangement. Rearrangements of.V6 to D6 should delete the upstream signal sequence, thereby preventing-the deletion of the 6 locus and subsequent c¢ gene rearrangement. Further studies will be necessary to determine whether this deletion event actually plays a role in regulating e gene rearrangements.

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Vy AND Vfi y/fi CELLS

197

GENE USAGE IN PERIPHERAL

One of the most striking emerging phenomenaassociated with 7/6 cells is the selective expression of different 7 and 6 V genes in different tissues. As summarizedin Table 2, most epidermal 7/6 cells in the mouse express a receptor composed of a V73-J71C71chain and a V61-D0-J62C6chain (60, 89; D. Asarnow,J. Allison, unpublisheddata). In contrast, 7/6 cells of the intestinal epithelium commonlyexpress a Vy5-J7ICTIchain (62, 104). chains from these cells have not yet been analyzed. In the spleen, most 7/6 cells express V72-JT1C~,Ior J74C74-containingchains (86, 93). Interestingly, allogeneic mixedlymphocytereactions of splenic 7/6 cells lead to an enrichment of 7/6 cells expressing VT1.2-J~2C72 chains (85). If intraepithelial 7/3 cells differentiate in situ, their selective V7and V6 gene usage maybe determined by specific microenvironmentalsignals that influence gene rearrangements or exert a selection for cells with particular receptor structures. If intraepithelial 7/3 cells are instead thymus-derived, differential homingof particular 7/6 cells to different sites maybe operative. Thus, it has been proposedthat the earliest thymic7/6 cells, which express V73-J7ICT1chains, are programmedto hometo the epidermis (92). A later thymic "wave"of ~,/6 cells expressing V75-JT1C~lmayhomespecifically to intestinal epithelium. Most splenic 7/8 cells maybe derived from a later waveofthymic ~/~i cells, which commonlyexpress V~2-J~1C~1 and J74C~4containing chains. Selective expressionof different V segmentsin different peripheral tissues has not been observed for e//%T cells and maysuggest that the different 7//3 receptors are specialized to recognizedistinct and limited sets of ligands at these sites. It is interesting that even the junctional diversity of ~/~ receptors expressed in someperipheral sites maybe highly restricted. A striking example comes from an examination of the sequences of the expressed 6 and 7 genes of several independent Thyl + DECcloned lines (D. Asarnow, J. Allison, unpublished results). Each expresses V61-D62J62C6and V~3-J~1 C71 rearranged genes, and the junctional sequences are virtually identical. Thus, no N region nucleotides are present, and the rearrangement breakpoints are identical. The lack of junctional diversity of ~ and 6 chains expressed by manyepidermal ~,/6 cells fits with the notion that they are derived from early thymic 7/3 cells, whicharise before terminal transferase is detectable (~ day 17). In any case, the observation that many Thyl+ DECdisplay virtually no TCRdiversity suggests that the putative epidermal ligand(s) of Thyl + DECare of extremely limited diversity. A caveat is that several 7/6 Thyl + DEClines analyzed in an independent study express other V3, and V6 genes (57-59, 59a; J. Coligan,

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personal communication).Further studies will be necessary to resolve this discrepancy.

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SPECIFICITY OF 7/6 CELLS Exposition of the ligands of 3’/6 receptors is crucial for understanding the physiological role of 7/6 cells. A particularly burning issue has been whether 7/6 receptors, like ~//~ receptors, recognize MHC-associatedantigens. Several early reports demonstrated that human7/6 cell lines cause lysis of a variety of tumor target cells regardless of their MHC-antigen expression (39, 40, 45-49). A similar pattern oflysis is displayed by natural killer cells and by MHC-restrictedT-cell clones that have been cultured in high concentrations of lymphokines(105). A critical issue is whether the observed lysis by 7/6 cells involves the CD3-associated ~/6 receptor. Although some studies suggested that anti-CD3 antibodies block such target cell lysis (39, 45, 46, 48, 106), more recent studies with F(ab’)z fragments of anti-CD3 antibodies yielded the opposite conclusion (107). The blockade with intact antibody may therefore be an indirect effect involving the Fc portion of the antibodies. It appears likely that MHCindependenttumor cell lysis by these 7/(5 cell lines worksindependently of their 7/6-TCR. Morerecently, 7/6 cytotoxic cell lines reactive with allogeneic MHClinked antigens were derived from aged nude mice (64, 65; L. Matis, J. Bluestone, unpublished data). Several lines have been established; each recognizes distinct MHC-relatedantigens: a class 1 type antigen encoded in the Tla region, a conventional MHC-class1 antigen (Dk), and a conventional class 2 antigen (Ek). These results are in accord with earlier findings demonstrating enrichment of ~/6 cells in allogeneic mixedlymphocyte cultures (85). The reproducible ability to produce MHC-reactive?,/6 cell lines suggests that 7/8 receptors, like ~/3 receptors, maybe predisposed to recognize MHC-relatedproteins. Amongc~///-T cells, alloreactivity is characteristic of T cells that also react with foreign antigens associated with self-MHCproteins (108, 109). However, as of this writing no selfMHC-restricted ~,/6 cells specific for non-MHCantigens have been identified.

FUNCTIONAL ACTIVITIES

OF 7/6

CELLS

7/6 cell lines exhibit various functional capabilities similar to those of e//~ T cells. As discussed in the previous section, a variety of both humanand murine7/6 cell lines exhibit cytolytic activity, whichin somecases is specific

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for cells bearing a11ogeneic MHC proteins. Cytolytic 7/6 cell lines have been isolated from humanthymus and peripheral blood (37, 39, 40, 4549), nude mousespleen (64, 65), the murine Thyl + DECpopulation (110; W.Havran,J. Allison, unpublisheddata), and the intestinal intraepithelial T cell population (61). Interestingly, freshly isolated intestinal intraepithelial 7/6 cells are cytolytic without induction, suggesting that they maybe activated in situ (61). Production of T cell-derived lymphokinesby a variety of 7/6 cell lines has also been documented. IL-2 production in response to activation with mitogens or anti-T3 antibodies was reported for humanand murine 7/6 cell lines (37, 47, 65), murineThyl + DEClines (110; W.Havran,J. Allison, unpublished data), and T-hybridomas derived from adult murine thymic 7/6 cells (111). In the latter category, one exampleof a hybridomaproducing both IL-2 and ILo4 was documented. Furthermore, early fetal thymic y/6 cells may produce both IL-2 and IL-4 (112). An alloreactive murine 7/6 CTLline produces both GM-CSFand y-interferon (65). Although lymphokineproduction is often considered primarily a function of noncytolytic helper T cells, manyof the 7/6 cell lines examinedexhibit both cytolytic activity and lymphokineproduction. Therefore, the present data do not allow categorization of 7/6 cells into cytotoxic and helper subsets. In sum, 7/6 cell lines exhibit at least someof the typical effector functions described for e//~ T cells, suggesting that they may represent a mature subset of T cells, with a similar or overlapping range of functional activities.

OVERVIEW AND SPECULATIONS Muchearly work on y/6 cells focused on the possibility that these cells represented an immaturestage of T-cell differentiation. However,the evidence cited herein suggests that precursors of ~///cells need not express functional y or 6 chains. Moreover, 7/6 cells exhibit receptor-triggered functional activities similar to those of mature e//3-cells. Despite their "immature" phenotype (they are often CD4-CD8 cells), it is likely that ~/6 cells represent a mature functional lineage of lymphocytes. Attempts to propose a unified model of y/6-cell function are frustrated by the paradoxical properties of these cells. On the one hand, the 7/6 receptor displays enormouspotential junctional diversity, consistent with a role in antigen-recognition akin to the ~//3 receptor. On the other hand, 7/6 cells resident in specific peripheral locations, such as the epidermis, maydisplay highly restricted V gene diversity with little or no junctional

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diversity. In these locations the diversity of putative ligands recognizedby 7/6 cells are presumablyhighly restricted. Modelsof?/6 cell function based on each of these points of view will be discussed separately below, followed by an attempt to reconcile them.

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A Diverse

~/~ Receptor

As we have seen, 7/6 cells reactive with allogeneic MHCantigens can be demonstrated, raising the possibility that like the ~/fl receptor, the 7/3 receptor has a predilection for binding MHCproteins. MHCproteins function to bind antigenic peptides for recognition by the ~/[~ receptor, and self-MHC-restricted T cells are frequently reactive with allogeneicMHC proteins. It is tempting to suggest, therefore, that 7/3 cells also recognize antigenic peptides bound to MHC-proteins. The potential diversity of the 7/6-receptor is sufficient for a role in MHCrestricted recognition of diverse antigens. However,the concentration of this diversity in the third CDRregion contrasts sharply with the receptor. A recent model suggests that the third CDRof T-cell receptor chains is oriented over the bound peptide of an MHC-peptidecomplex, whereasthe first and second CDRmaybe positioned better for interactions with polymorphic MHCresidues flanking the peptide binding site (113). Extrapolated to the 7/6 receptor, the modelmaypredict a potentially large universe of antigenic peptides but a small universe of restricting elements. The finding of a 7/3 cell line reactive with a Tla-linked class 1 antigen (64, 65) has fueled speculation that the restricting elements of at least some 7/6 cells might include a subset of these MHC-likemolecules, which are relatively nonpolymorphic(69, 104). It must be emphasized however, that ~/6 cell lines that react with "conventional" MHCantigens (Dk k) and E have also been established (65; L. Matis, J. Bluestone, unpublishedresults). A Nondiverse

~/~ Receptor

7/6 cells in distinct anatomical sites express distinct V7 (and possible V6) genes. Moreover,junctional diversity of both 7 and 6 chains of epidermal 7/3 cells (at least) maybe extremely limited. These findings argue for predictable and limited set of ligands for 7/6 cells, at least in the skin. It is possible that most epidermal 7/6 cells recognize one or a few antigens (possibly associated with MHC-likeproteins) which are associated with commonskin pathogens. An alternative view is that the ligands for the receptors on intraepithelial 7/6 cells are unique se/f-antigens which are only expressed following environmental insults such as infections and/or transformation (104). In keeping with the observed MHC-reactivity some7/6 cell lines, the self-antigens maybe encoded by MHC-relatedclass 1 genes that mapin the Tla region and show tissue-specific expression.

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Reconciliation The diverse and nondiverse character of ~,/6 receptors in different anatomical sites constitutes a significant paradox which maybe resolved by recourse to an evolutionary perspective. Perhaps the ~/c5 receptor preceded the ~/fl receptor in evolution and functioned originally to survey tissues for a limited array of predictable antigens (see above). Later, evolution may have superimposed a broader, MHC-restricted antigen recognition capability on the ~/~ receptor, akin to a primitive ~//~ receptor system. Thus fetal thymic ~/fi cells, which may be restricted in diversity, may function in the former role after migration to epithelia. Later wavesof ~/fi cells mayfunction in the latter role, after migration to the secondary lymphoid organs. The two functions may thus be separated phylogenetically as well as ontogenetically. One can imagine that the pressure for yet greater diversity finally forced the separation of these two functions, resulting in the appearanceof the ~/fl receptor system. For the ~ genes the 6 genes were subserved and diversified by creation of manyJ~ gene segments and diversification of V6 genes. The fl genes mayhave evolved from ~ genes following duplication and transposition to another chromosome. The views expressed above are simply a speculative attempt to distill the patchy available information into a unified modelof y/6 cell function and evolution. While the progress to date in describing ~/~i cells and receptors is impressive, it falls short of providing confidencein any modelof 7/6 cell function. Considering our inexperience in determining functional pathways starting with a gene, this is perhaps not surprising. Wehope the experience will provide approaches and perspectives that will facilitate future such endeavors. ACKNOWLEDGMENTS

I thank the many investigators who shared with me their unpublished data; Herman Eisen, Lisa Steiner, and Alan Kormanfor reviewing the manuscript; and Chris Greco for preparing the manuscript. This work was supported by research grant CA28900from the NIH and a Cancer Research Institute/Frances L. and Edwin L. CummingsMemorial Fund Investigator Award. Literature Cited 1. Allison, J. P., Lanier, L. L. 1987. The structure, function and serology of the T cell antigen receptor complex. Ann. Rev. Immunol. 5:503-40 2. Marrack, P., Kappler, J. 1986. The antigen-specific major histocompati-

bility complex-restricted receptor on T cells. Adv. lmmunol. 38:1-30 3. Davis, M. M. 1985. Molecular genetics of T-cell receptor beta chain. Ann. Rev. Immunol. 3:537~50 4. Toyonaga, B., Mak, T. W. 1987. Genes

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V-REGION CONNECTIVITY IN T CELL REPERTOIRES P. Pereira,

A. Bandeira and A. Coutinho

Unit6 d’Immunobiologie, Institut Pasteur, 75724Paris C6dex15, France M.-A. Marcos, M. Toribio and C. Martinez-A. Centro de Biologia Molecular, Universidad Aut6nomade Madrid, 28049Madrid, Espafia INTRODUCTION Areviewof the evidenceand ideas concerningT cell repertoire (Tc Rep) selection within an immune networkis both simple, becauselittle has been done, and difficult, becauseof the waythis has beendone. Theproblemis not only conceptual(1). Ourmethodsare excellent tools for clonal analysis but certainly not appropriatefor networkstudies. If a networkis described by its structure (connectivity), dynamics,andmetadynamics (2), it is couragingto realize that wehaveno suitable techniques,evento quantitate the first of those parameters. If weexclude fromnetworkimmunology all studies that address, instead, idiotypic regulation of clonal activities, we are left with astonishinglylittle materialfor a reviewon immune networks, let alone"T cell connectivity."It is, therefore, difficult to identify among the manypaperson idiotypes (Ids), T cell reactivities, Tc Repselection, responsiveness,and tolerance, those that suggest modesof operation and selection compatiblewith a networkorganization. Westart by reviewing experimentsshowingconnectivity amongT cell receptors (TcRs) and continuewith the evidencefor interactions between TcRsand immunoglobulin (Ig) idiotypes in the selection and maintenance of Tc Reps. After briefly discussing antibody (Ab) networksand their relationships to Tc Reps, we describe a frameworkaccommodating these observations and somefindings in tolerance and immuneresponsiveness. 209 0732-0582/89/0410-0209502.00

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CONNECTIVITY AMONGT CELL RECEPTORS In a very large and diverse collection of proteins it should always be possible to find (and define) a level of connectivity typical of that set molecules in the conditions as they are. If such molecules are TcR, the interesting question relates to the frequencyof interactions with sufficient affinity to result in functional consequencesfor the T cells expressingthem, and therefore, to the importanceof such connectivity in the biology of the wholeset. Augustin and his colleagues have carried out a series of ingenious experiments demonstrating that direct TcR complementarities can be found in normal individuals (3, 4). Since alloreactive T cells to class-II major histocompatibility complex (MHC)products are abundant in normal animals, the strategy consisted in finding the respective complementarities, which would constitute some sort of "image" of the alloantigen. These authors first isolated T cell lines and hybridomasthat were directly stimulated by monoclonal antibodies (mAbs) to class II determinants and demonstrated the reaction of the mAbwith the respective TcRs. They could then showthat such T cell hybrids stimulated T cell lines and other hybrids with the same class II alloreactivity as the mAbthat had induced them. Specificity controls established the clonal nature of such interactions and thus provided direct evidence for their existence in the normal Tc Rep. Interestingly, fine specificity analysis established that, as expected, the "points of view" of antibodies and TcRthough overlapping are not identical. Furthermore, the TcR shown to be complementary to an allo-class I~reactive TcRwas itself (self) class II-reactive, that is, the twointeracting TcRscould both be described as "images" of class II molecules. Finally, although the interacting T cells were both I-A-reactive helper cells, they could productively interact in the absence of I-A but were nevertheless inhibited by the appropriate anti-I-A mAb,in its quality as anti-TcR. If suggestive and provocative, these results give no indication as to the frequency of such complementarities within the Tc Rep. Experiments by Suciu-Foca et al (5, 6) go further in this respect by showingthat normal peripheral humanT cells proliferate quite vigorously if stimulated in vitro with autologous T lymphoblasts generated in response to allogeneic classII antigens. The specificity of these autologous responses was established by secondary stimulation (primed lymphocytetest) and shownto correlate with the reactivity (and thus probably the TcR) of the stimulator, alloactivated T cells. Similarly suggestive evidence for frequencies of TcR complementarities to class II-reactive TcRs, high enoughto be detected in primary proliferative responses of normal T cells, was reported by the laboratories of Kaplan (7) and Weksler (8). Both used T cell clones

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hybrids directed to self I-A determinants as stimulator cells and argued for the abundance of such TeR complementarities to anti-self class-II TcRs. These observations were significantly expanded by the work of Quintans and colleagues (9) who showedthat auto (class II)-reaetive well as antigen-specific, self-(class II)-restricted T cell lines and clones were all capable of stimulating considerable proliferative responses in syngeneic normal T cells. As in Augustin’s experiments (2), although these responses occurred in the absence of class II-bearing cells, they could be inhibited by anti-class-II Abs with the same target specificity as the stimulator class lI-reactive T cells. Both CD4+ and CD8+ T cells were shown to participate in these autologous, putative anti-TcR responses; most interestingly, T cell populations responding to a particular TcRcould themselves stimulate second-orderreactions in other T cells, with a clear increase in degeneracy. The most documented example ofa TeR network, however, is provided by the studies on graft-vs-host (GvH) resistance conducted by Darcy Wilson and associates, which opened this area of research and constitute the best evidence for the paramountbiological significance of connectivity amongstTcRs(10-14). If pretreated with small numbersof parental strain A, CD4+ Tc, containing A anti-B (MHC)precursors, (A x B)F1 hybrid rats becomeresistant to local GvHreactions induced by large numbersof parental A T cells, but not to those induced by parental B T cells. Resist+ host T cells which display a clear ance is primarily mediated by CD8 specificity for TcRV-regions, as they suppress and kill A anti-B, but not A anti-C or anti-D T cells. Most interestingly, hosts maderesistant to A anti-B TcRs also suppress and specifically kill all other anti-B T cells prepared in other MHC-haplotypedonors. This finding, which has been interpreted to indicate a limited polymorphism and little somatic modulation among TcRs to MHC-encoded antigens in the species, has other important implications. First of all, and as seen above in other systems, direct interactions between TcRs are not MHC-restricted and thus are likely to result from complementarity between three-dimensional shapes of "unprocessed" receptor proteins. This is important in the context of the repeated observations that alloMHC-reactive TcRs "recognize" conformational determinants (e.g. 15, 16) rather than linear, processed peptide sequences, as is mostly the case for conventional protein antigens (17-20). It can then be argued that TcRs on host "anti-idiotypic" T cells are "images" of self-MHC, as demonstrated in Augustin’s example and suggested in Quintan’s experiments. If this were the case, there would be no reason to postulate a limited polymorphism for anti-MHC TcRs in the species, and Wilson’s observations wouldnot be at variance with previous descriptions of the existence

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of a polymorphicidiotypic repertoire of such receptors, as analyzedby Abs(2 l). Thus,all anti-BT cells, regardlessof their idiotypicprofile, share complementarities to B (MHC)and, therefore, to "images" of B in the form of "anti-idiotypic" TcRs.It is remarkable, nevertheless, that GvH resistance is complete and, therefore, that all "aspects" of B (MHC) are fully represented by TcR"images." This interpretation has other implications, becausethe experimentsalso showthat resistance against a very frequent reactivity (A anti-B MHC) is acquired within a very short time after injection of parental cells (D. Wilson,personalcommunication). This leaves little roomfor clonal amplification of the "anti-idiotypic" T cells andsuggestsactivation and/orrelocalization of frequent, pre-existing clones. Sucha high frequencyof TcRsthat "reproduce"the molecularprofiles of self-MHCmaybe explained in two ways. They mayarise by strong positive selection in ontogeny,implying, as arguedbefore (22), that the original modeof reaction in the immunesystem is the generation of mimicriesrather than the production of complementarities.Someevidence for this assumption has been obtained by the abundanceof "natural" serumAbs, particularly in very younganimals, that bind to anti-(self) MHC monoclonalantibodies (23). Someof these are actually allo-MHCspecific (24). Thecentral point here is that sucha selective processmust be driven by other TcRs(these being anti-self MHC) in MHC-unrestricted (but, at the origin, MHC-directed)manners.Thus, since the "points view"of MHC profiles by TcRsand antibodies do not coincide, the "copy" of self-MHC to TcRidiotypes that is seen as faithful by T cells initiating GvHreaction, can only be achieved if such T cells themselves have mediatedthe process of positive selection. Hypothesesthat postulate such basic principles for T cell selection in the thymushavepreviously been proposed (2, 25). Alternatively, if TcRsmimickingself-MHCare not positively selected, it shouldbe expectedthat the idiotypic profiles corresponding to allogenic MHC would also be present at high frequency in normalindividuals and thus wouldco-exist with high frequencies of complementary,anti-allo MHC TcRs. In other words, the Tc Rep would form a very high connectivity network. Wilson’s experiments cannot address this point, but the evidencereviewedabovewouldperhapsindicate the predominanceof self (MHC)images. Thesetwo alternatives are not mutuallyexclusive and positive selection of self-related complementaritiesand mimicriescan well be done from a high connectivitynetworkthat is "species-specific." Thestructure of such a network cannot be expected to consist of two complementarysets of TcRs(complementaritiesand mimicries of MHC) separated in two internally disconnectedhalves. As is already suggestedby Augustin’sexample

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(the anti-anti-self TcRis itself an anti-allo), a greatly interwovennetwork should apply, where complementarities to antigen should largely be outnumberedby the respective (partial) images. Such a model of Tc Reps has been extensively developed by Eichmannand colleagues (see below) with interesting general properties of suppression and stability. Evolutionarily, the stability of a germ line-encoded, highly connected TcR network has implications for other sets of genes, namely the MHC.As we argued before, to explain some characteristics of MHC polymorphisms(26, 27), this set of genes would only vary within the limits set by evolutionarily stable T cell repertoires. Apart from sporadic but recurrent examples that could be interpreted to indicate direct interactions among TcRs on helper and suppressor cells (e.g. 28-31), and from the work of K6hler (32-34), whoargued functional interactions between complementary TcRs on TH, a wealth of reports have described "cascades" and "circuits" of suppression and contrasuppression involving T cells and their clonally specific products. In manyinstances, the data have been interpreted to indicate direct V-region interactions amongTcRs (see for review, 35-38), and they contribute the notion of TcR connectivity. The discussion of these observations, however, is complicated by the intimate relationship unrevealed in those studies between the participating TcRs and Ig V-region Reps (see below). Furthermore, since TcV-region interactions were not directly shown but were deduced from experiments involving complex cell mixtures and since there are no available data on the frequencies of such T cells and interactions, we cannot use this evidence in quantitative discussions of TcR connectivity. Direct measurements in limiting dilution analysis (LDA)of normal cells and quantitative deductions on connectivity in a T cell network have been performed by Eichmann and colleagues (39-44). The basic experimental design consisted in polyclonally activating normal T cell populations with concanavalin A, letting limited numbersof activated cells expand for a week in the presence of exogeneous growth factors, testing clonal progenies for specific effector functions, and calculating clonal precursor frequencies in the starting T cell population. The approach is similar to one previously used for B lymphocytes(45), and it rests on the assumption that mitogen activation reveals an unbiased sample of the available repertoire. Since total frequencies of responding T cells were not determined, we do not knowto which fraction of all cells the results apply. These approaches, however, offer the considerable advantage of overcomingvariables in the initial activation and growth of cells (antigen concentration, accessory cell activities, etc) whichconstitute serious limitations for antigen-driven specific precursor frequency determinations.

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Furthermore, in this particular case, the system provided the opportunity of detecting interactions amongactivated T cells and thus revealed activities that wouldpass undetected with populations of resting T cells. From the shapes of the lines defined by LDAdata points in semilog plots, these authors derived several important conclusions. The first concerns the enormous degeneracy in the functional recognition of antigens by T cells: for every antigen tested (e.g. major or minor histocompatibility antigens, hapten modifications of self, heterologous erythrocytes), the frequency of specific clones approaches 1%of all normal cells. Antigen-specific effector T cells, however,could only be obtained in quantitative conditions that would fit the zero-order term of the Poisson distribution from cultures containing very low numbers of activated responder cells. Higher numbersof such clonally heterogeneous cells drastically suppressedthe generation (or detection?) of specific effector cells. These were again detected within a narrow range of even higher cell concentrations in a second (or third) straight line in the semi-log plots. Specifically primed T cell populations analyzed in the same manner, essentially show clonal frequencies with "appropriate" Poisson’s distribution at the same level as the abundant population in unprimedcells, suggesting to the authors that priming results in resistance to suppression of very frequent, specifi~ clonal precursors, rather than in their expansion. After demonstrating that established T cell clones were also suppressed by hctcrogcneous populations of activated T cells, the authors used such cloned T cells to demonstrate that antigen competes with suppression and restores functional reactivity. This observation provided the major argument for receptor-mediated mechanismsoperating in this system and, together with the demonstration that T cell populations with helper and suppressor phenotypes were both operational suppressors, led to a network model of Tc Reps. A high degeneracy of specific TcR interactions with ligands, if it ensures high specific precursor frequencies, also results in a very high degree of connectivity in Tc Reps. In a randomrepertoire, each complementary TcR to any antigenic pattern is largely (10-100 fold) outnumberedby other TcRs that interact with it with minimal affinities. Such direct TcRinteractions are postulated to be suppressive, in contrast with TcR-antigen contacts that are inductive. It follows that the normal state is dominantly suppressed and that responses can only be obtained by antigen competition with the connected TcRsat the level of high affinity cells. Activation of T cells by antigen would then commanda maturation step in the responding cell such that it would no longer be sensitive to connectivity suppression. As expected, this set of observations and interpretations met with skepticism, so much did they depart from conventional wisdom (biased Tc

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Reps to MHC,antigen-dependent clonal expansion, deletion of anti-self T cell reactivities, etc). The experimental observations, however,have been well established, and the modelhas provided a unified view of the question. Unfortunately, it has not explored the phenomenology of allo (MHC)reactivity, Ir-genes, tolerance, thymic education, and it did not include Ab V-regions in a wider view of immunenetworks. The assumption of a symmetrical networkwith "distributed" connectivity can also be criticized, since the data can as well be accommodated with the postulate of a special population of"sticky" TcRs. Furthermore, the whole of the evidence rests on the analysis of activated T cell populations and is applied to normal conditions where most lymphocytes are resting (GvHresistance, for example, which can be considered in the context of a high connectivity T cell network, requires activation of the "anti-idiotypic" T cells). Finally, it is difficult to accept that TcRinteractions with someligands (other TcRson resting T ceils) are suppressive, while being inductive with other ligands (antigen), particularly because the affinity argumentmakeslittle sense the context of a network. Current limitations in the methodsof study, together with the oddity of these approaches in comparison with "fashionable" T cell immunology, mayexplain the little attention currently given to TcRconnectivity. Unfortunately so, for it appears a major characteristic of Tc Reps and a promisingfield for direct clinical applications (see e.g. 46).

EVIDENCE SELECTION IDIOTYPE

FOR B-CELL/ANTIBODY-DEPENDENT OF T-CELL REPERTOIRES: SHARING

Up to the early 1980s, a very productive area of research employedantibodies to IgV-region determinants to probe T cell repertoires. Antibodies to idiotypes, VH-isotypes, or allotypes were repeatedly shownto identify T cell or T-cell products clonally, and to stimulate or suppress specific T cell responses. Extensive reviews (47-49) have dealt quite recently (50) with this topic, which was also addressed in very critical ways (51-53). Before the identification of the TcRproteins (54-58) and structural genes (59, 60), the dominant interpretation of those results has been the expression of Ig V~-genesby T cells. After 1983 a paradoxical development took place: manyof the most active groups deserted the field, although in the light of the new information on TcR structure, those results had extraordinarily important implications for the organization of the immune system and the selection of Tc Rep. Thus, if a minimumof credit is given to that large set of observations, "Id sharing" by structurally different

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receptors suggests powerful mechanismsdriving the somatic selection of Tc Rep on the basis of antibody idiotypes, "implying a functional idiotypic network in the most fundamental sense" (50). This would indeed appear to be a very worthwhile topic to pursue. The backlash is understandable after the dominant hypothesis (and some of the biochemical data) had been falsified, and doubts accumulatedconcerning the genetic and cellular basis of other phenomenaintimately connected with "Id sharing" (e.g. specific T cell factors, suppressorT cells, I-J). It is, nevertheless, surprising that, precisely from the momentwhen the experiments could be better controlled and interpreted, manyfewer groups continued, or started, work in this area, and there was little developing of novel experimental approaches or understanding of the phenomenology. Only a few aspects of the older work on "Id sharing" should be discussed here. First of all, howoften does it occur and, consequently, howsignificant is the phenomenonfor Tc Reps? The answer is not simple because "Idsharing" is necessarily detected by an anti-idiotypic antibody (or TcR), and it may, therefore, vary with the reagents used. This is particularly relevant when monoclonal antibodies are used to define idiotypes, and it is probably not only because polyclonal sera are "dirty" reagents that idiotypic mimicries were more often recorded with mixtures of antibodies (see 61, for discussion). The most reasonable way of addressing the question experimentally would be to use as reagents the same combiningsites (Abs or TcRs) that the system itself uses in the putative selection mimicries; in other words, to adopt the "point of view" of the systemitself, in order to understand how it works. Unfortunately, with few exceptions (62, 63) the monoclonalantibodies used as anti-idiotypic are derived after extensive immunizationsand are most likely not present in normal animals. Finding of "Id-sharing," therefore, will be a matter of chance or of extensive screening of anti-idiotype monoclonal antibodies. An operational question here is how and what to screen, since there is no good reason why"Id-sharing" should be found in T cells and B cells participating in the particular immuneresponse that the investigator has chosen to elicit. In practice, and regardless of how fundamental the phenomenonmight be for the immunesystems, it is a priori unlikely that it has evolved for the recognition of, say, TNP. In general, since T cells and B cells "see" molecular patterns in essentially different ways--short peptide sequences together with MHC,versus three dimensional shapes--"Id-sharing" may well exist, but the respective lymphocytesparticipate in immuneactivities that the investigator considers separate. Again, the solution here should perhaps be to consider the globality of the immunesystem operation and to adopt its own "point of view". "Id-sharing" should, therefore, be analyzed in concomitant immuneactivities even if these concern apparently

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distinct specificities. A clear suggestion for this is provided by the Oudin and Cazenave phenomenon: "Id-sharing" occurs among Bc/Ig molecules, all stimulated in an immuneresponse, but only someare antigen-specific Abs (64-66). In view of these limitations, we can instead ask howoften "Id-sharing" between T cells and B cells has actually been detected. The phenomenon has been described in a variety of immuneactivities concerning helper, suppressor, and cytolytic lymphocytes(and their products) responding MHCdeterminants (67-69), a number of different haptens (70-72), terial products (73, 74) and conventional protein antigens (75, (reviewedin 47-50). The list is incompleteand unlikely to be representative since with few exceptions (10, 77) failures are usually not reported, and are not aware of any systematic study addressing this point. The closest is a brief description by Martinez-A. et al on screenings of "primary", uncloned Tr~ lines, recently derived from lymph nodes immunizedagainst a variety of very little cross-reactive hapten modifications of "self" (TNP, NP, NIP, FITC, SP), with a number of monoclonal antibodies to idiotopes expressed by several TNP- or NP/NIP-binding Abs from the corresponding mousestrains. They reported that only one of seven antiidiotypic mAbsshowed inhibition of TH activity (71, 78) and immunoprecipitated a heterodimeric, clonotypic molecule from T cells (79). Incidentally, this is actually the only report on "Id-sharing" that shows biochemical evidence of anti-idiotype antibody reactivity with a T cell surface molecule bearing characteristics of TcRs(54-58). Interestingly, the context of the above considerations, these authors also reported that, if instead of conventional anti-Id antibodies they used "natural mAbs" with the same nominalanti-Id specificity, two of three tested were capable of THinhibition (62, 63). Wecould perhaps conclude as others did before (77) that "Id-sharing" between T cells and B cells participating in the response to a nominalantigen is the exception rather than the rule, at least when analyzed with conventional anti-Id mAbs. If the reagents used by the system in its operation are also used in the screenings, however, the situation might be quite different. The next question on "Id-sharing" concerns its significance. In the large diversity of Abs and TcRs it is more than likely, on simple statistical grounds, that similar shapes will be found. It is necessary, therefore, to ascertain if "Id-sharing" is the result of selection or of pure chance. Most of the described exampleshave concerned T cells and B cells with similar nominal specificities, and this has been taken to indicate "meaningful" regulation. This would only be the case, however, if the Id could not be frequently found in the responses to manyother antigens--which cannot be experimentally verified. An example of such aleatory "Id-sharing"

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between THand B cells has recently been described (80) and shown be completelyindependentof B cell/Ig-dependentselection of the T cell clonotype(81). There are no indications of the frequencythat should expectedfor this kind of cross-reactivity, but those observationscommand caution on "network interpretations" of Id-sharing, in the absence of formalevidencefor meaningfulId selection. The best argument for the conclusion that "Id-sharing" does represent selection of Tc Repsin a networkthat includes B cell/Ig is the finding that Id-expressionby T cells is controlled by IgVn-linkedgenes (71, 72, 82-88). Since TcRare not encodedby IgH-linked genes, those repeated observations strongly suggest that the Tc Reps analyzed had previously been selected by mechanismsinvolving polymorphicantibody V-regions. This argumenthas recently received a formal proof by the demonstrationthat "idiotype sharing" by TcRsdoes require the presence of B cells/Igs during Tc Repdevelopment(78, 89). Further evidence for this central point was provided by experimentsdirectly comparingIgHlinkage and B cell/Ig-dependenceof the expressionof two distinct T cell clonotypes, shared with the sameB cell idiotype, and both identified by the sameanti-Id mAb:IgH-locuscontrol ofT cell idiotype expression was foundassociated with the requirementfor the presenceof B cell/Igs along with Tc Repdevelopment, but not whenthe "shared Id" was expressed independentlyof the Bc compartment (72, 78, 80, 81). Giventhe variety of experimentalsystems in whichIgH-linked"idiotype-sharing" betweenAbs and TcRshas been documented,this process wouldappear to play a significant role in the establishmentof Tc Reps. Other examplesof mimicrybetweenantibody idiotypes and self structures (90-93) have not shownany IgH-linked polymorphismin expression. expected,these findings indicate that, given a range of alternative possibilities providedby the geneticandstructural diversity of TcRs,a coherent solution for patterns in Tc Repsemergesfrom the operation of an immune network.In other words,"Id-sharing" maywell not represent a particular advantageto the immunesystem and, therefore, maynot be selected for a given purpose. Rather, it maysimply be a consequenceof the wayit functions. EVIDENCE FOR B-CELL/ANTIBODY-DEPENDENT SELECTION OF T-CELL REPERTOIRES: IGH-RESTRICTION OF T CELLS AND T CELL-DERIVED FACTORS In the samemanneras MHC-restrictionof antigen recognition by T cells has beentaken to indicate Tc Repselection uponinteractions with MHC-

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encoded glycoproteins in T cell ontogeny, IgH-restriction of T cells and their products is suggestive of IgV-region participation in selecting functional Tc Reps. These two types of phenomena, however, are likely to have distinct structural and functional bases and to have very different impacts in Tc Reps. IgH-restriction describes the findings that T cells or their: clonally specific factors fail to interact functionally with lymphocytes from donors that carry a distinct IgH-haplotype. Originally described as limiting interactions between various types of T cells participating in suppressive activities (88), IgH-restriction was later shownto apply also to THactivities (94) and to concern V- rather than C-genes in the locus. The central point in this set of observations, constituting formal proof of B cell/Ig-dependent selection of the Tc Repsanalyzed, is the demonstration that IgH-restricted T cells require the presence of B cells/Ig for the acquisition of the self-restricted phenotype (89). Moreover,such T cells have been shownto "learn" restriction to either self (95) or allogeneic Ig Reps (96) if exposed to the appropriate environments. In contrast with MHC-restriction, peripheral T cells can be re-educated to alternative IgHrestriction patterns, in a process that takes less than two weeks(96). Because some antibody idiotype-related TH activities were shown to proceed independently of MHC-linkedcontrols, in what concerns both Irgeneeffects and restriction in target cell interactions (97, 98) the notion was developed that MHC-and IgH-encoded proteins could serve equivalent functions in the biology of two parallel sets of TH(99, 100), which would differ also in functional capabilities and surface markers (101, 102). discussed below, this division is, in our minds, unwarranted. Thus, the notion of IgH-restriction of TcRs is quite different from that of MHCrestriction. While the latter essentially concerns "presentation" of antigen fragments in the context of MHC-molecules,the former is likely related to direct complementarities between TcRs(some bearing "idiotypic" determinants of antibodies, others constituting the respective "anti-idiotypic" counterparts). Althoughit could in principle be possible, we do not know of evidence suggesting that TcRs or factors recognize antigens "in the context" of other V-regions, be they antibodies or TcRs. Even if some of the systems showing IgH-restriction are antigen-dependent, this may be due to totally different reasons. Furthermore, absence of MHC-restriction (and Ir-gene effects) in these interactions does not have to be interpreted as the property of a distinct class of T cells. Thus, MHC-restrictedT cells can readily be activated by anti-TcR or anti-CD3 antibodies in MHCunrestricted manners(57, 103). In other words, cell activation requires TcRsinteractions with threshold affinities: in somecases, that threshold is only reached if the ligand is a "peptide-MHCcomplex" (and cell interactions stabilized by accessory molecules), but the same TcRcan obviously

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directly interact with high affinity complementaryantibodies or other TcRs. The above discussion of TcRconnectivity provided several examples of T cells that interact with TcRsof other T cells in MHC-unrestricted manners. Yet, we should perhaps not define a third category of T cells that are TcR-restricted. Let us consider first, that antibody idiotype-specific T cells are likely to be: (a) both IgH-restricted and MHC-restricted, when they recognize polymorphic idiopeptides presented by MHC-molecules; (b) MHC-restricted only, whenthe V-region peptides are not polymorphic; and (c) simply IgH-restricted when they interact with three-dimensional structures on polymorphic antibody V-regions. On this non-network scheme, a second level of complication is brought in by the fact that Tc Reps are selected by B cell/Igs and, therefore, differ according to the IgHhaplotype of the mouse. T cells thus selected can also be said to be restricted by IgH-genes (although we would prefer to say "determined" or "selected"), and the expression is often used in this context. These very cells, however, can showall properties of MHC-restricted or MHC-specific recognition of targets and are also submitted to Ir-gene controls (e.g. 7 i). Provided the appropriate targets, however, these same T cells can also function in MHC-independentmanners by direct interaction with Ab or TcR V-regions. It is our impression that suppressor T cell "cascades" represent Ig-dependent selection of complementaryTcRs, while the observations on MHC-independent Id-specific TH represent Ig-dependent positive selection of TcRswith sufficient affinity to interact directly with unprocessed Ab V-regions. Given that MHC-unrestricted T cells have been very rarely detected in immuneresponses to conventional antigens (104), it is pertinent to ask whether the apparent abundance of MHC-independentT cell reactivities to TcRor AbV-regions should be given a particular significance. Clearly, if Tc Rep selection is an ongoing process throughout life, molecular patterns that are present in the "internal environment" such as Abs and TcRs have more possibilities for selecting complementaryTcRswith sufficient affinity to dispense of MHC presentation. In this case, the difference betweenV-regions and other structural protei.ns available in the organism should be explained (whenarguing for either positive or negative selection). Alternatively, it might be argued that V-regions of TcRsand even Abs are present inside the thymusand thus participate in the primordial selection, in parallel or in conjunction with MHC-proteins(25). Even if correct, however, this hypothesis cannot accommodateall available findings, particularly the B cell/Ig-dependent "education" or "re-education" of peripheral T cells transferred to adoptive hosts. A reasonable model should perhaps consider, in addition to the above possibilities, that the "preoccupation" of Tc Reps with TcRs and Abs (and MHC)may also

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partly due to germ-line gene composition but, most importantly, may simplyhave to do with V-regiondiversity. Since the numberof molecular shapes of V-regionsis several orders of magnitudelarger than that of all other proteins in the vertebrate body,there is obviouslya muchgreater chanceto find the appropriate complementaritiesamongthe former set, whatever the TcRconsidered and the requirements for "appropriate" mightbe. Again, wecomeback to the idea that the immunesystemcannot avoid existing as a networkand that manyof its properties simplyemerge fromthis organization. EVIDENCE FOR B-CELL/IMMUNOGLOBULINDEPENDENT SELECTION OF ALLO(MHC)REACTIVE T-CELL REPERTOIRES Asidefromthe IgH-linkedresponsesof T cells to anti-idiotypic antibodies and the control of suppressor T cell interactions, evidence of quite a different nature has directly implicatedB cell/Ig in the selection of Tc Reps(105-111).Sherman’sexperimentsinvolvedfine-specificity paratopic clonotypingof primaryallo-reactive eytolytic T cells in multipletests of single clones on a panel of target cells expressingdifferent mutationsof the sameclass-I antigen. Analysisof the CTLrepertoire in individual mice revealedthe existenceof"recurrent",strain-specific clonotypicreactivities expressed at high frequency which could, therefore, be used to probe influences of various loci in CTLReps. As expected, there was a marked influence of MHC in the allo-reactive repertoire, mostof whichcould not be explainedbyself-toleranceandcross-reactivities. Interestingly, neonatal mice from MHC-congenic strains expressed a far more similar Tc Rep than did adult individuals, and F1 animals co-dominantlyexpress both parental elonotypic patterns. Repertoire divergenceis, therefore, due to ongoingselection throughout life, as demonstratedby the analysis of MHC-congenic T cells obtained from double parent chimeras. Having established that such selection is MHC-dependent, Shermananalyzed the putative influence of other polymorphicnon-MHC genes and surprisingly found a major effect of IgVH-genes. Theseobservationsare interesting in several regards. First, they constitute the only evidenceto date demonstratingthat available Tc Repsin unprimedindividuals, evenif analyzedin waysthat do not involveB cells or Abreagents, are drastically selected by AbV-regions.Second,they show that B cell/Ig-dependent selection is MHC-determined and established in a time course whichsuggests the requirementfor prolongedco-existence with Abrepertoires. This is a central point as it illuminatesthe discussions

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above on "IgH-restricted" T cells (we are dealing with MHC-specific repertoires) and on TcRnetworks. Thus, the observations concernrepertoires of allo-reactive specificities andmodesof selection that cannotbe explainedby deletion. Therefore, they must represent positive selection of complementaritiesto not-self molecularprofiles. Thesedata are not compatiblewith modelsof Tc Repselection solely based on a networkof TcRs(4, 44) and imposethe inclusion of AbV-regionsin such a network, whichapparently concerns the wholeTc Rep(self- and not-self-directed reactivities). Finally, they suggesthowextensiveis the impactof B cell/Igdependentselection on Tc Repsand the interest in extendingthese studies to other aspectsof T cell reactivities, particularlyto Ir-geneeffects. EVIDENCE FOR B-CELL/IMMUNOGLOBULINDEPENDENT SELECTION OF T-CELL REPERTOIRES: B CELL-DEPRIVED ANIMALS Theevidencefor selection of T cell reactivities in a networkthat includes AbIds suggested a direct approach: to compareTc Reps ontogenically establishedin the presenceor absenceof B cell/Ig. Thus,it is possibleto producemicethat are profoundlydepleted of matureB cells andcirculating Ig by chronic treatments frombirth or embryoniclife with anti-isotypic (112) or -allotypic (113) Absto # chains. Since early studies had shown that such animalshaveconservedT cell functions (114), it waspossible compare"Id-sharing" and "IgH-restriction" of T cells in B cell/Igdeprivedmice and control littermates. Results fromthis type of experimentalsystemhavebeen re.ported by several groups, and all agree in the conclusion: T cell clonotypes expressed in B cell-deprived animals are different fromthose available in normalindividuals (78, 89). Martinez-A. and colleagues have shownthat a syngeneic mAbto the BALB/cTNPbinding Id MOPC 460 identifies a TcRclonotype on the majority of TNPBALB/c specific Tn obtained in normal animals of this strain. The mAB specifically inhibits T~proliferation and effector functions on specific targets, stimulates IL-2 production under appropriate conditions, and immunoprecipitates a clonotypic heterodimer from those TH. Although Bc-deprivedBALB/c mice produceTNP-self-specific TH,none of the antiId mAbreactivities could be reproducedwith them(78, 79). Independently,Sy et al (89, 115) and Floodet al (116), studying pressor T cells operating in DTHresponses to azobenzenearsonate(ABA) or Abresponsesto sheeperythrocytes, respectively, describedthe finding that T cells from B cell-deprived donors were both unable to produce suppressorfactors active on cells from normalanimals, and resistant to

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the activity of such factors produced by normal donors. The studies of Sy and colleagues are particularly detailed and complete, in a system previously developed by Greene and Benacerraf (reviewed in 37). ABA-specific suppressor T cells from A/J mice produce factors that react with anti-idiotypic Abs directed to the dominantAbId of the strain, and these factors are restricted in their effector activity to "acceptor" T cells from donors carrying the same IgH-haplotype (37, 115). Suppressor factors prepared from T cells of B cell-deprived donors, while competent on cells from these same animals, were no longer active on target cells from normal donors (89, 117), showingthat the repertoires of interacting T cells were altered if established in the absence of B cell/Ig. Interestingly, depletionreconstitution experiments directly support the interpretation that Tc Reps available in normal individuals are positively selected and expandedby B cell/Ig. Thus, interruption of anti-# Ab administration, which is followed by replenishment of the B cell/Ig compartment,results in acquisition of T cell reactivities that are characteristic of normal animals and absent in B cell-deprived donors (95). Moreover,complementaryexperiments to B celldeprivation, namely the transfer of peripheral, mature T cells to IgHcongenic recipients resulted in "re-education" of Tc Reps, which now contained reactivities characteristic of both the donor and the recipient (but not if the latter was anti-#-suppressed) (96). Sy’s experimentspropose another interesting conclusion on the interrelationships betweenT cell and B cell repertoires. The major anti-ABAAb Id of BALB/cis a minor Id of A/J strain Abs and is "shared" by suppressor factors of BALB/cbut not of A/J mice. Such factors prepared in BALB/cor congenic mice carrying the A/J IgH-locus (C.AL-20) are only active on cells from the strain origin. Interestingly, however, cells from anti-#-suppressed animals of either strain, are suitable targets for suppressor factors producedin the other strain, suggesting that clonal dominance(in both T cells and B cells) is established by Id network-positive selection from equivalent potential repertoires. The consequences for the T~ repertoire of autologous B cell reconstitution in anti-p-suppressed mice were also analyzed by Martinez-A. et al (118), with somewhatdifferent results. In this case, B cell-deprivation for more than 4 weeks results in a definitive loss of expression of the TH clonotype studied. This finding, together with the stability OfTHclonotype expression in mice manipulated as adults (to express either large amounts of Ab Id or none) led the authors to conclude that, once established by cell/Ig-dependent mechanisms, Tc Reps were resistant to further alterations in the B cell compartment. These observations are compatible with earlier experiments on "Id-sharing" that had been interpreted to indicate VH-geneexpression by T cells and were difficult to reconcile with the new

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interpretations of Ab-dependentselection of T cell clonotypes (119, 120). Theseconsisted in the transfer of T cell populations containing Id-positive cells from adult donors to recipients lacking expression of that Id on Abs. Whenanalyzed a long time after transfer, Id expression was still detectable on T cells. The apparent discrepancy between Sy’s and Martinez-A.’s outcomein autologousB cell reconstitution is likely to result from different rules applying to the expression of the respective Ab ld after interruption of anti-# treatment. If repertoire selection is a circular process (xc ~ B), appears to be the case in Martinez-A.’s system (see below), his results are a reflection of recursivity. Studies on T cell activities in B cell-deprived mice were actually initiated by Janewayet al (121) whoprovided evidence for altered patterns of helper effects, using adoptively transferred, carrier-primed T cells, from normalor anti-/~-suppressed donors, in conventional cooperation systems. Together with Bottomly, Janeway has also produced the original observations on the absence in anti-#-suppressed mice of a particular THspecificity found in normal animals which was indirectly defined by the idiotypic pattern of the Ab responses analyzed (122). This work integrates a series of studies conducted by Bottomly and colleagues on Id-related THcells, which provides another example of the role of B cell/Ig in the establishment of Tc Reps (97, 98, 100, 122-125). Wheninvestigating the dominanceof the T15 Id in the TH-dependent Ab responses to phosphorylcholine, Bottomly described the fact that, in contrast with carrier-primed THfrom normal donors, those obtained from animals that do not express the T15 Id were unable to help a T15-dominated Ab response, while being otherwise competent helpers for specific Ab production (123). Subsequent work showedthat this class of Id-related Tn cells, which were characterized as being simultaneously specific for whatever carrier was used in the experiments, could be obtained by priming (Ir-gene)-nonresponder animals, and these cells displayed no requirements for "linked" collaboration nor MHC-restriction in their effector activity (97, 98). Interestingly, such THpopulations can be specifically "panned" or T15-coated plates (124, 125), showingthat their receptors interact with "unprocessed" Id, and thus explaining their peculiar properties. These observations are reminiscent of those of Woodland & Cantor (126) and Eichmannand colleagues (127), and similar findings in several other systems have also been reported (102, 128-131). They have constituted a major argument for Janeway’s model of Id-restricted (MHCunrestricted) T cells, discussed above, and are interesting here for they represent positive selection of TcRs that are complementaryto an Ab Id present in relatively high concentrations in normal individuals. Indeed, cells of this kind have been found in normal, unprimed animals (130, 131)

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and are likely responsible for the stimulation of Id-positive B cells which do not bind antigen after priming with the antigen (66). Their special characteristics--as compared to "conventional" TH--maybe only apparent and result from a commondenominator: expression of TcRs that constitute somesort of "image" of the nominal antigen specific for the Id in question, a "r-type anti-Id." If this were the case, the same TcR can simultaneously be specific for any other antigen, and its interaction with Id-positive target cells would require no further "bridging" and no MHC restriction either. Anyactivation of THin vivo, even if "nonspecific" (as could be the case in genetically nonresponder mice), can be expected to generate cells with these properties. Dependingon the affinity of direct TcR-Id interaction, such "Id-related Try" will require or dispense simultaneous activation of the target B cells by other means--suchas linked, MHC-restricted cooperation with other TH--and/or concomitant activation by the antigen that originally induced them. Selective long-lasting suppression of a given Id can also be achieved by neonatal injection of specific anti-Id Abs(132) or by maternal transmission of such specificities (133-135). Such Id-suppressed animals are equally suppressed for expression of the TcRIds (72, 136). These experiments, however, even if performed in systems where "Id-sharing" was shown to be positively selected, do not have the same demonstrative value as those carried out with anti-p-suppressed mice. Thus, the absence of T cell clonotypes can as well be due to direct effects of the anti-Id Ab on target T cell. Nevertheless, since the effects of neonatal (or sometimesadult) antiId treatment are very long lasting (at least several months)it is likely that, either Id-specific active suppression is established (see e.g. 137-139), else that the absence of Id early in development leads to the positive selection (and consequent clonal dominance)of alternative (but equivalent) clonotypes normally "dominated" by the suppressed Id.

THE ANTIBODY NETWORK AND ITS T CELL-DEPENDENCE Wenow briefly discuss recent advances concerning the Ab network that will be useful for the global consideration of repertoire selection. Direct quantitation of antibody connectivity, performed in Kearney’s laboratory and by Holmberget al (140-142), has demonstrated a very high frequency of V-region interactions amongperinatal antibodies. The number of"connections" in a given set of Abs largely exceeds the number of members and reaches around 20%of all possibilities. High connectivity, however, is not a property of any diverse collection of Abs, as shownby parallel

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experiments using Abs randomly selected after polyclonal activation of adult, resting B cells. In this case, connectivity is 10-100 fold lower than that of perinatal Abs (141, 142). Furthermore, neonatal Abs are heterogeneous with respect to connectivity and seem to constitute thi:ee discrete groups, one of whichis very poorly or not at all connected(J. Stewart, F. Varela, A. Coutinho, manuscript in preparation). Interestingly, there is a clear association between connectivity and the use of VH-genesfrom the 7183 and Q 52 homologyfamilies (143), which are the first to be expressed in ontogeny due to their chromosomalposition (144). Since no somatic mutations have been detected in 12 such perinatal, high connectivity Abs (D. Holmberg,~.personal’communication), it appears that the immune system necessarily .starts as a high connectivity, germ-line encoded Ab network, the expression of which is developmentally controlled. Reactivities present in this original network stimulate B cell production by interacting mitogenically with non-Ig surface recep.tors on B cells and their precursors (93, 145; J. Kearney, personal communication). In addition, idiotypic connectivity participates in the (positive) selection of newlyarising Ab clonotypes in apparently T cell-independent manners, as elegantly shown"byVakil et al (146). At this point in development,most B cells are "naturally" activated, even in germ-free (147) and "antigen-free" animals (148). Auto-reactivity of such Abs, however, is only associated with "multispecificity," and further development is necessary, including the appearanceof peripheral T cells in sufficient numbers,before evidence for positive selection of auto-reactive B cells can be detected (see below). The interesting point to consider here is, indeed, the T cell-dependence of the establishment of Bc Reps. This topic has received little attention in the past, and very few examplesare available suggesting (or excluding) the participation of T cells,, before priming with antigens orother "artificial" manipulations. Thus, most observations can be interpreted to indicate helper or suppressor T cell participation in responses (either dealing with Ids as conventional antigens, or in Id-related mannerssuch as those discussed above), and such observations give no indication of T cell-dependent network selection of available B cell Reps. One report on the MHClinked control ofAbIds (149), however,is difficult to explain on this basis. More direct evidence might be sought by the~ analysis of B cell clonal precursor repertoires in unprimed populations, but while an early paper suggests MHC-linkedcontrol of T15 Id frequencies (150), others have provided examples of T cell-independent, IgH-controlled frequencies of other Ids (15l). Recently, Freitas and colleagues have detected very significant differences in the representation of VH-genefamilies in peripheral B cells from normal and athymic mice. Furthermore, they could show that transfer of normal T cells to nude littermates-resulted in profound

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alterations in the expressedVH-geneRep. It is unlikely that such modifications result from ongoingresponses to environmentantigens, since they are detected amongsmall, resting B cells. Theseresults provide direct evidence for T cell-dependent selection of Ab Reps (A. A. Freitas, M.-P. Lambezat, B. Rocha, manuscript in preparation). Indirect suggestionsare providedby the T cell-dependence of B cell production discovered in mice carrying the Xid defect, which whenhomozygousfor the nude mutation produce few B cells (152) and very reducedlevels of circulatingIg (152, 153). T cell influencesoperate the level of bone-marrowprecursors (154) and normallevels of B cell productionthat can be reconstitutedby matureT cell or thymusgrafts (154, 155). It is possible that an AbRepdefect is implicatedhere, since Xid mice are reported deficient in the class of perinatal B cells associatedwith the productionof high connectivityantibodies (156). Furthermore,alterations of T cell function during adult bone-marrow reconstitution of Xid mice have also been shownto result in gross modifications of B cell/Ab repertoires (157; unpublishedobservations). Otherresults concerningspecificity repertoires, can also be invokedin this context. For example,mice recovering from neonatal anti-# suppression remain negative for the expressionof a T cell-regulated idiotope (139, 158), suggestingthat the emergentBc Repis in part selected by available Tc Reps.Siskind, Weksler, and colleagues investigating the basis for the qualitatively and quantitatively reducedAbresponsesin agedmice, demonstrateda shift in Id patterns of anti-hapten Absaccompanying reduced responsiveness. Then in doubletransfer experiments,they establishedthat repertoire alterations precede immunizationand are imposedon B cells from younganimals by co-existence with T cells from old mice (159-162; M. Weksler,personal communication). Aseries of experimentson the induction of Id productionuponintravenous injection of nanogramdoses of the same Id in .the absence of antigen are also interesting here as they suggest one level of Bc Rep selection whereT cell influences could be predominant:the induction of "natural" plasma cells and serum Abs(163-167). Such Id-induced responses are T cell-dependent and MHC-(and Igh-) linked; they are quantitatively poor, as comparedwith a conventional immuneresponse, and must represent enhancementof an ongoingactivity following a rise in circulating Id, since responsivenesscorrelates with Id "recurrency,"and the doses of Id injected are muchlower than its serumlevels in normal animals. Other groups carried out similar experiments, but reading out alterations inducedby Id administrationin the Id compositionof antigeninducedresponses (134, 168, 169). Also here, enhancedId expression recorded, and the establishment of "dominance"is T cell-dependent. The

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minimal doses of Id injected and the mode of administration make it plausible that similar mechanisms continuously operate in the normal immunesystems. In support of this notion, Primi et al have reported that circulating levels of Ig molecules expressing particular V.-VK combinations are controlled by MHC-linkedgenes (170), and more recent studies have also established the MHC-controlof circulating levels of rheumatoid factors (P. Pereira, manuscript in preparation). Moreinteresting perhaps are the findings on sharing of V-region determinants between circulating Igs and TcRs. These experiments represent the converse of T cell "Id-sharing," as they have used an anti-Vfl mAb (F23.1) (171) to identify circulating antibodies carrying similar idiotopes. SuchAb specificities are positively selected by T cells into the secretory B cell compartment, but only in strains which express such TcR genes (P. Pereira et al, manuscript in preparation). If confirmed by ongoing formal genetic analysis, these studies suggest TcRVfl-linkage of expressed antibody idiotypes and thus the same interpretation as the IgH-linkage of T cell idiotypes. In both cases, the selection of mimicries (and complementarities) are likely to reflect the global operation of a networkthat includes TcRs and antibodies. The network selection of Bc Reps by T cells may well involve other molecules in the "internal environment" which are necessary components of networkconnectivity (1), and selected antibodies showself-related specificities. Evidencefor T cell-dependent selection of auto-Abreactivities has recently been obtained. Huetz et al (172) demonstrated that T cells are necessary for the positive selection--in terms of frequency and "natural" activation--of B cells producting lytic antibodies to bromelain-treated syngeneic erythrocytes. Whilepractically all clonal B cell precursors with this and other auto-Abreactivities are cycling, "naturally" activated lymphocytes in normal animals (173), the spleens of nude mice contain very few such precursors, but their positive selection could be demonstrated upon transfer of T cell populations from normal syngeneic donors (172). Furthermore, transfer of adult T cells to newbornsaccelerates the establishment of this splenic auto-reactive Bc Rep from its normal development at around 6 weeksto 3 weeks of age (F. Huetz, P. Poncet, A. Coutinho, P. Portnoi, submitted). If T cells select Bc Reps and these participate in Tc Repselection, the process is circular and recursive, Recent observations directly address this principle by demonstrating T cell-dependent selection of B cells for the "education" of Tc Reps in secondary hosts (174). The experiments investigated which B cell populations were competent in the positive selection of the T. clonotype discussed above (71, 78). They established the loss T. Id expression in animals that reconstitute the B cell compartmentfrom

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adult bone-marrow,that is, either after anti-/~ suppression for the first 4 weeksof life, or after lethal irradiation as adults with bone-marrowprotection. Positive selection of the THclonotype could be recovered, however, if bone-marrowreconstitution was supplemented with either peritoneal Ly-1 + B cells or splenic, Ly-1-, activated B cell blasts from normal, adult donors. Since equivalent B cell populations from nude mice failed to operate in T cell selection, it was possible to demonstrateT cell-dependent Bc Repselection by first transferring normalT cells to nude recipients and, a month later, determining the ability of B cell blasts from such animals to select the TH clonotype upon transfer to adult bone-marrowreconstituted recipients. These experiments not only give indications on the cellular basis of T-Bc Rep selection but have the heuristic value of adopting the "point of view" of the system. Thus, T cell-dependent Bc Rep selection was not directly analyzed on the basis of Abreactivities but simply by the ability of the Bc Rep to secondarily educate and "reproduce" the same Tc Rep that had originally selected them.

CELLULAR AND MOLECULAR MECHANISMS OPERATING IN REPERTOIRE SELECTION: NATURAL IMMUNE ACTIVITIES This review on a multitude of heterogeneous observations would gain coherence if someprinciples on the mechanisms(local rules) of lymphocyte selection could be laid down.Let us start with peripheral, positive selection of Reps. The first principle has to do with lymphocytepopulation dynamics: rates of production in the central lymphoid organs, life spans as competent cells, rates of decay, and peripheral renewal. No significant ongoing repertoire selection can indeed occur in the absence of extensive renewal rates in peripheral immunocompetentcells. These topics have been reviewed elsewhere (175), but it should be underlined that high renewal rates have previously been demonstrated in both the B cell and T cell compartments, though on distinct bases. B cells are constantly renewed, primarily by bone-marrowproduction of new specificities from uncommitted precursors (176, 177), while T cells are also extensively renewed, but mostly by peripheral division of immunocompetentlymphocytes (178, 179). At any time, most B cells in an adult mousehave just been produced in bone-marrow (and will soon disappear), while most cells are the progeny of mature lymphocytes that were stimulated in the periphery and thus have, or have had, available complementarities there. This major difference must be considered, however, with the quantitatively less notorious counterparts in the two lymphocyte compartments: the

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persistent B cell populations producedin perinatal life (156) and the recent thymic emigrants produced throughout life (180). Furthermore, special consideration should be given to early developmental periods (embryonic and neonatal). WhenT cell and B cell populations first comeinto contact, high levels of maternal antibodies are present, and peripheral lymphocyte populations do not turn-over but accumulate in numbers. These aspects are all fundamental to understanding "founder" effects in the recursive process of Rep selection. The second set of principles concerns the physiology of lymphocyte activation, because lymphocytesare produced as resting, noncycling cells, and the positive selection of clonal reactivities must rely uponfunctional activation to expansion and/or persistence of the target lymphocytes, by mechanisms that involve interactions with receptor V-regions. Most important, since lymphocyteactivation results in the expression of effector functions (antibody secretion, help, or suppression) positively selected lymphocytesare effector cells that establish the recursivity of the process by selectin# (positively or negatively) the complementarities that induced them. This "linkage" with effector functions of both partners is actually what is so special about V-region connectivity in contrast to receptor interactions with other molecules in the body (a clone stimulated by, say, transferin, has little chance, in turn, of stimulating (or suppressing) transferin production). Documentedexamples of such direct antigen-independent lymphocyte interactions are abundant: TH activation by (activated) B cells or antibodies, and B cell activation by effector TH(see above, and e.g. 181-186); suppressor and cytolytic T cell activation by antibodies or B cells, and suppressionof B cell function by effector T cells (see above, and e.g. 187-197; reviewed in 37, 198); Tr~ inactivation by suppressor cells (e.g. 29, 199) or CTL(13), and suppressor T cell activation by inducer cells (e.g. 88, 200; see 36, 37, 198 for reviews); mutual stimulation of (e.g. 29-34), and of B cells (see above, reviewed in 50). Of particular significance here are the findings that cross-linked Ab binding to TcRs results in stimulation of resting T cells (e.g. 57, reviewed in 201) and -that activated B cells are excellent "stimulator" cells for both helper and suppressor T cells (reviewed in 202;.see also 203-214). Conversely, resting B cells .are, by definition, the "targets" for THactivity (e.g. 71, 185, 186, 215, 216). The third class of principles should consider the kinds of molecular interactions in which TcRs and IgRs .may actually participate. There are at least four types of appropriate ligands for ,productive TcRinteractions with other V-regions: (a) processed lgV-region peptides on (self) molecules; (b) processed TcRV-regionpeptides on (self) MHC molecules; (c) directly complementary (MHC-independent) tridimensional shapes

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either TcRs,or (d) Ig V-regions.All these possibilities except(b) been documentedand normal immuneReps repeatedly shownto contain specificities towardssyngeneicor autologous V-regions. As originally shownby Janewayet al (217, 218), this is the case for THinteracting with processed MHC-presented peptides of IgV-regions(e.g. 72, 181-184,219221), and for unprocessedIg-V-regionsas well (e.g. 124-126, 128-131, 185, 186). Thesameapplies to suppressorTc-Ig interactions (reviewed 36, 37, 198), and to the direct (MHC-unrestricted)TcR-TcR interactions reviewedabove. In contrast, Ab"sticky ends" excel in interactions with large molecularsurfaces (222) and, therefore, with unprocessedTcRsand other Igs, although Absto peptide-MHC complexesmayactually be quite frequent (223,224; J.-P. Abastadoet al, personal communication). In essence,therefore, the molecularspecificities, the cellular mechanisms, and the population turnovers are all available in the normal immune system to provide conditions for the establishment and operation of a functional V-regionnetwork.Theaboveevidence, on the other hand, does showthat the operation of an immune networkaccounts for the selection of available lymphocyte repertoires before antigenic challenges. It follows that normalanimals should showsigns of this ongoingactivity. Pereira and colleagues have described in normal germ-free and "antigen-free" mice, before any manipulation,the existence of considerable lymphocyte activity: 10-20%of all splenic B cells, CD4+and CD8+T cells are activated, blast cells, manyof whichare in mitotic cycle (148, 225, 226). Importantly, these activated populations, in contrast with small resting lymphocytes isolated fromthe sameindividuals, are effector cells: B cells secrete IgMAbs, CD4÷Tc very efficiently "help" appropriate resting B cells into proliferation and Ig-secretion, whileCD8+ Tc directly suppress B cell responses. Becausethe quantitative levels of lymphocyte activation are quite comparablein conventionallybred and "antigen-free" individuals, this "internal activity" has beeninterpreted to indicate the operation of an immunenetwork, although no evidence has been given for the Vregion specificity of the participating lymphocytes.In turn, these observations have providedthe upper limit for the fraction of normalimmune systemwhich, at any point in time, maybe participating in a functional network. In other words, in a normal immunesystem 80-90%of all lymphocytesare not included in a network, at least in a functionally significant manner.(The above experimentsconcernexclusively splenic and peritoneal cavity lymphocytes, as "antigen-free" mice show very reduced or absent cellularity in mucosal-assoeiatedtissues and lymph nodes. In turn, together with the absenceof non-IgM isotypes, this observation showsthe very profounddeprivation of environmentalstimulation in suchmice, yet with conservedsplenic lymphocyte activity).

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A MODEL OF LOCALIZED CONNECTIVITY

LYMPHOCYTE

A simple working hypothesis was derived from the above results and considerations (227). Most immunocompetentlymphocytes produced the periphery display clonal reactivities that find no functionally productive complementarities in the molecular environment where they exist. These remain resting and decay at defined rates. In contrast, other clonal reactivities engagein functionally relevant interactions in the internal environment (with V-regions, other self "somatic" molecules or even external antigens constantly entering the body through mucosal surfaces) are activated and, by their effector functions, recruit into the set of activated lymphocytesother V-regions displaying sufficient interactive affinity (or they limit their expression, in case of CD8+ cells). Activation, on the other hand, results in positive selection and persistence of the activated clonotypes without necessarily involving extensive clonal expansion (175). This model gives rise to a series of predictions, some of which have been supported by experimental evidence and are discussed here. First, "connectivity" should be significantly higher in the "activated cell pool" than amongsmall lymphocytes; this was verified for B cells (141,142) and, indirectly, for TH(63). Connectivity, however, should not be limited to regions but should extend to other structural componentsavailable in the organism. In other words, the repertoire of "activated lymphocytes" should be biased for "self-reactivities." This has been very extensively demonstrated for B cells. While small B cell repertoires appear quite "burnettian" in all analyses of clonal precursors, "large" B cells from normal individuals contain autoreactive specificities in high frequencies (172, 173), and "natural Abs"are essentially a collection ofauto-Abs (228230). Also in the case ofT cells, recent experiments showthe preference of "large" CD4+ and CDS+lymphocytes to functionally interact with "unmodified" target (activated) B cells (A. Bandeira, P. Pereira, Coutinho, C. Martinez-A., manuscript in preparation). In contrast, small T cells are completely deprived of auto-reactivities and very enriched for allo-specificities. Finally, such "localized connectivity" wouldimply that an immunenetwork predominantly (or exclusively) operates in the compartment of activated cells, while the repertoires of small lymphocytesof normal individuals should showlittle evidence of network selection. This point has also been extensively substantiated by experiments. For example, IgH-linked, THclonotypes that are selected by B cell/Ig-dependent mechanisms are found in the activated T cell pool of normal animals, while TH "sharing" antibody idiotypes by aleatory cross-reactivities are small cells

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T CELLS ANDNETWORK 233 (63); "large" T cells from normal donors positively select auto-reactive cells, while small T cells from the same donors do not (172); B cells that positively select Tu clonotypes are "large" in normal mice, and T cells that "educate" Bc Reps for recursive THRepselection are also in the activated pool (174); the TcRVfl-linked expression of antibody idiotypes is only detected amongactivated B cells and "natural antibody," and all IgHidentical mousestrains tested contain small B cells carrying this Id (P. Pereira, C. Pefia-Rossi, S. Petersson, C. Martinez-A., A. Coutinho, manuscript in preparation). The model, therefore, makes predictions on which Ids will be (or will not be) "shared" by T cells and B cells, and on which kinds of reactivities are (or are not) the result of networkselection. This is compatible with the finding that anti-#-suppressed mice show Tc Reps that, only in some regards, are different from normal animals (e.g. 231234). A second prediction of the model is that immuneresponses to "external" antigens predominantly arise from the small lymphocyte pool, precisely because such molecular profiles are absent from the internal environment. Clonal reactivities in the "large" cell pool, even if complementaryto an immunizingantigen, will treat it as a self-molecule, and consequentlytheir "response" will merely represent a "compensation" in previously ongoing activities. Mostimportantly, because of the different levels of connectivity in either set of lymphocytes,activities in the large cell pool will alwaysbe "self-limited" and rich in "nonspecific" and degenerate components, in contrast with the high-titered specific immuneresponses induced in small lymphocyteswhich, being disconnected, are free from systemic constraints in their clonal expansion. (It is likely, however,that such clonal responses will subsequently fall under "network" control, for the very significant alterations in the composition of the molecular environment brought about by clonal amplifications.) This prediction of the modelhas been verified for B cells in two ways. The kinetics and magnitude of a specific T cell-independent immune response are muchfaster and higher if the responding clones are in the small cell pool (235); the dynamicsof "natural" antibody production, and of its compensations to perturbations in the network they form, is very different from immuneresponse dynamics, actually displaying characteristics of "chaotic" attractors (236). For T cells, however,only indirect evidence is available. Animalsthat are neonatally tolerized to semi-allogeneic cells, maintain high levels of lymphocyteactivity throughout life (237), and their activated T cell pool contains the alloreactive specificities to the tolerized haplotype, which are found amongsmall cells in normal individuals (238; P.-H. Lambert, personal communication). The reported examplesof "split" tolerance (e.g. in mice grafted with allogeneic thymic

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epithelium; 239) between in vivo behaviors and specificities revealed in vitro, can also be explained in this context. The same principles could apply to connectivity amongTcRs, a point suggested by the report of Wilson (240) that F1 animals neonatally challenged with parental T cells, cannot mount, as adults, GvH-resistance responses to the same TcRs (see above). This discussion brings us to a few points concerning tolerance (and responsiveness) in the context of this model. Clearly, if a part of normal immunesystem is organized as a high connectivity network which by "trapping" autoreactive cells limits their participation in clonal immune responses, this organization could be responsible for the establishment of some kinds of tolerance and low-responsiveness. This notion provides a satisfactory explanation for self-reactivities in the B cell compartment (241), and it is tempting to apply it to T cells as well. Thus, regardless the extent of intra-thymic clonal deletion (242-244) and/or other forms selection that limit functional responsiveness of T ceils (245), it is inescapable that someform of "tolerance" must operate in post-thymic, peripheral T cells. This modelconsiders peripheral self-tolerance as a systemic, "distributed" property, determined by the particular organization in which auto-reactive T cells are necessarily included by their normal reactivity in the molecular environmentof self. Since recursive repertoire selection is established early in ontogeny, the process predominantly applies to the antigenic universe available at that time (and thus, the ease in inducing "neonatal tolerance"). The process is strongly influenced by maternally derived molecules, but it maintains the "memoryof self" (physiological auto-reactivity) by recursivity. The model, therefore, explains the "learning" of self on the basis of normal lymphocytereactivities, an advantage to clonal models of self-tolerance which often have to invoke ad hoc postulates in this respect. Furthermore, it accommodatesin the same frameworkthe evidence reviewed here on V-region-directed T cell autoreactivities, with thc repeated observations on the existence, in normal individuals, of potentially aggressive T cell reactivities to autologous "somatic" proteins (246-251). Finally, it readily explains the finding that severe mutilations of Tc Reps, by makingit impossible to establish such a self-related network of high connectivity, may well (but paradoxically, for other interpretations) result in pathological autoimmunity(252254). Since inclusion in the network limits clonal responsiveness, somecases of genetically controlled "low responder" phenotypes could have a connectivity explanation. If the appropriate T cell specificities were brought into high connectivity because of receptor properties (such as idiotypic) other than binding to self antigen-MHC,alterations in the network struco

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ture, mayrelease those clones from connectivity and shift responder phenotypes. Such an examplehas recently been reported. In contrast with their H-2k congenics, BALB/cmice are nonresponders to low density TNP-self modifications. They predominantly "respond" to highly TNP-derivatized self with Tn that are selected by Bc/Ig-dependent mechanisms to the compartment of activated cells and to "share" an antibody idiotype (63, 71, 78). However, BALB/cmice that had their network altered immunization with anti-Id mAbs,particularly if early in ontogeny, "disconnect" TNP-self-reactive TH, show muchhigher clonal frequencies of THreacting with low TNP-self densities, and acquire a "high-responder" phenotype characteristic of H-2k mice (255; Martinez-A., unpublished). surprising (manipulation of an MHC-linked, low responder idiotype results in a "high responder" phenotype), these results can be readily explained on the basis of the IgH-linked expression of the Tn clonotype in question. Although this separation of two compartments in the normal immune system might have operational and theoretical advantages, it should be pointed out how unclear (at least in time) their frontiers must be. Moreover,while the~view of peripheral "self-tolerance" as a distributed, supraclonal property emergingfrom connectivity is central in this model, it is likely that other (clonal) properties contribute to the functional characteristics of "internally" activated lymphocytes. Werefer to a number of observations from several independent groups (256-263) describing the induction of unresponsiveness (tolerance) in mature, antigen-MHC-activated T cells by exposure to high concentrations of TcR-specific ligands, such as free peptides, peptide-MHCcomplexes on lipid vesicles or nonstimulatory presenting cells, soluble anti-TeR or anti-CD3 antibodies. Interestingly, similar unresponsiveness, which lasts for days or weeks, is induced by "appropriate" restimulation of responding T cells some hours after the responses were initiated. These are all conditions that might apply to immunocompetentT cells reacting to autologous structures. In those experiments, unresponsiveness was assessed by the inability to proliferate and, in somecases, by loss of IL-2 production,~but.effector functions such as B cell-directed help or suppression were not evaluated. It is striking that such "unresponsive" T cells maybe functionally equivalent to the "naturally" activated effector T cells isolated from the spleen of normal individuals. In spite of their excellent performance in functional assays, these cells are resistant to growth induction in vitro, express few high affinity IL-2 receptors, and produce little IL-2 (264). Even if "tolerant," therefore, such cells are perfectly competent to operate in the immunenetwork. Regulation of activated,B cell performance by specific ligand binding has been known

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for manyyears (265), and this is likely to play a determinant role in the networkdynamicsof B cells and antibodies (2). It is an exciting possibility that equivalent rules apply to T cells, as it wouldprovide new functional perspectives on lymphocyteconnectivity and further possibilities to address autoreactivity.

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CONCLUDING

REMARKS

Since the mid 1970s, immunology has been confronted with two major developments. If the progress in molecular and cell biology continues to solve many questions about the structure and function of isolated components in the immunesystem, the notion that the system owes some of its properties to a network organization has set limits to the kinds of problems that can be approached by component analysis. These two aspects of the discipline are often considered alternatives. Wronglyso. The unique goal is to understand the origin of the cognitive properties of the immunesystem: learning and memory,self-assertion, discrimination betweenprotective responsiveness and tolerance. Andif the structure of a gene and its regulatory elements cannot give us the clues for the process of learning, it is as naive to study "connectivity" without considering its structural basis, the regulation of the genes encodinginteracting molecules, or the physiology of the cells producing them. In short, for the understanding of global behaviors in a viable system, local rules describing individual componentsare as essential as the structure and dynamics of connectivity. Considering its achievements to date, it is clear that the network idea did not quite work in immunology, but the divorce of its practice from quantitation and local rules is only one of the reasons for its failure. More importantly, perhaps, has been the lack of an explicit framework with testable predictions, which introduces the qualitative step from clonal to network thinking and thus departs from some of the notions associated with the clonal selection theory. Thus, most of the so-called networkexperiments have been concerned with (idiotypic) regulation of clonal components in immuneresponses, ignoring the formal incompatibility between those two views of the immunesystemand failing to explore what the network idea can really contribute: the globality of the system’s operation (1, 2, 22, 266). Beyond the dismay to which such "pseudo-network" approaches have brought the network idea, the consequences of these confusions are very apparent. In spite of the marked contributions from molecular and cellular immunology, little progress has been achieved in auto-immune diseases, transplantation tolerance, allergy, and chronic infectious diseases. These represent obvious modifications in systemic behaviors, which cannot

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be explained and will find no solution on the basis of clonal properties. The recent tendency to study immunephysiology and normal individuals-rather than immuneresponses--will perhaps be the "road to Damascus" for modern immunology. The object of this review is a prime exampleof this state of affairs. Ever since the original observations of Simonsen,Tc Reps have never ceased to display surprising characteristics. Alloreactivity, restriction, Ir-gene effects, self-tolerance, and thymic education are all essential problemsin basic and applied immunologythat remain to be solved. Yet, we know today nearly as much about the genetics and structure of TcRs, as we know of antibodies. Even if further clues might be obtained from TcR crystals, wedo not believe that they will give us the solution to the selection of Tc Reps. First, this is because the unit-target of selection is not a molecule but a whole cell, the physiology of which involves manyother interacting molecules and ligands (as it is already understood for a number of accessory T cell surface structures and hormonereceptors). Second, this is because the process of selecting a diverse repertoire has also to do with multiple interactions that arise within that repertoire as a necessary consequenceof diversity. This simple notion that the targets and effectors of selection belong in one and same set suggests some degree of "autonomy"in the process, and it imposes the consideration of the "whole" on the determination of the "parts." Undoubtedly, the environment (MHC, self and not-self antigens) where the repertoire develops, has a very significant contribution to selection. As in other complexsystems, however, it is likely that idiotypes find their wayof being (select repertoires) within alternatives that are viable for their own composition in the "medium" where they exist. Viable repertoires are then determinant for the manners by which they evolve to alterations in the mediumor in their own composition. In short, if there is some truth in the network idea, lymphocyte repertoires are self-determined within the viability boundaries set by their genetic possibilities, the rules of lymphocytephysiology, and the molecular environment where they develop (267). It follows that the emergent "solutions" for repertoires probably cannot be understood solely by the analysis of single clones and their relationships with environmental molecules but will require the description of the others and of their interactions. ACKNOWLEDGMENTS

Wethank A. Bandella for preparation of the manuscript, our colleagues for discussions and unpublished data, and DRET(France) and CAYCYT (Spain) for support.

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182. Gleason, K., Pierce, S., K6hler, H. 1981. Generation of idiotype-specific T cell help through network perturbation. J. Exp. Med. 153:924 183. Pierce, S. K., Speck, N. A., Gleason, K., Gearhart, P. 3, K6hler, H. 1981. BALB/cT cells have the potential to recognize the TEPC15 prototype antibody and its somatic variants. J. Exp. Med. 154:1178 184. Kawahara, D. J., Miller, A., Sercarz, E. E. 1987. The induction of helper and suppressor cells with secondary antihen egg-white lysozome B hybridoma cells in the absence of antigen. Eur, J. lmmunol. 17:1101 185. Tite, J. P., Kaye, J., Saizawa, K. M., Ming, J., Katz, M. E., Smith, L. A., Janeway, C. A. Jr. 1986. Direct interactions between B and T lymphocytes bearing complementary receptors. J. Exp. Med. 163:189 186. Janeway, C. A. Jr., Broughton, B., Smith, L. A., Marion, T. N., Bottomly, K. 1987. Direct receptor: receptor interactions between T and B lymphocytes: idiotypic restriction in the antibody response to a cloned helper T cell receptor. J. Mol. Cell. lmmunol. 3:83 187. Paul, W. E., Bona, C. 1982. Regulatory idiotypes and immune networks: a hypothesis. Immunol. Today 3:230 188. L’age-Stehr, J., Teichmann, H., Gershon, R. K., Cantor, H. 1980. Stimulation of regulatory T ceil circuits by immunoglobulin-dependent structures on activated B cells. Eur. J. Immunol. 10:21 189. Abbas, A. K., Burakoff, S. J., Gefter, M. L., Greene, M, I. 1980. T lymphocyte-mediated suppression of myelomafunction in vitro. III. Regulation of antibody production in hybrid myeloma cells by T lymphocytes. J. Exp. Med. 152:969 190. Juy, D., Primi, D., Sanchez, P., Cazenave, P.-A. 1982. Idiotype regulation: Evidencefor the revolvement of IgH-C-restricted T cells in the M-460 idiotype suppressive pathway. Eur. J. lmmunol. 12:24 191. Milburn, G. L., Lynch, R. G. 1982. Immunoregulation of murine myeloma in vitro. I1. Suppression of MOPC-315 immunoglobulin secretion and synthesis by idiotype-suppressor T cells. J. Exp. Med. 155:852 192. Lynch, R. G., Millburn, G. L. 1984. Murine plasmaeytoma MOPC315as a tool for the analysis of network regulation. M315idiotopes are inducers and targets of immunoregulatory signals. See Ref. 38, p. 299

193. Kresina, T. F., Baine, Y., Nisonoff, A. 1983. Adoptivetransfer of resistance to growth of an idiotype-secreting hybridoma by T cells from idiotypically suppressed mice. J. lmmunol. 130:1478 194. Monroe, J. G., Gurish, M., Dambrauskas, J., Slaoui, M., Lowy, A., Greene, M. I. 1985. Genetic and biological characterization of a T suppressor cell induced by anti-idiotypic antibody. J. Immunol. 135:1589 195. Slaoui, M., Urbain, J., Lowy, A., Monroe,J. G., Willems, F., Benacerraf, B., Greene, M. I. 1986. Anti-idiotypic treatment of BALB/c mice induces CRib-bearing suppressor cells with altered Igh-restricted function. J. Immunol. 136:1968 196. Hausman, P. B., Sherr, D. H., Doff, M. E. 1986. Ai~ti-idiotypic B cells are required for the induction of suppressor T cells. J. Immunol. 136:48 197. Frischknecht, H., Binz, H., Wigzell, H. 1978. Induction of specific transplantation immune reactions using anti-idiotypic antibodies. J. Exp. Med. 147:500 198. Doff, M. E., Benaceraff, B. 1984. Suppressor cells and immunoregulation. Ann. Rev. Immunol. 2:127 199. Yamananchi, K., Murphy, D. B., Cantor, H., Gershon, R. K. 1981. Analysis of antigen-specific H-2 restricted cell free product(s) made I-J- Ly-2 cells (Ly-2 TsF) that suppresses Ly-2 cell-depleted spleen cell activity. Eur. J. Irnmunol. 11:913 200. Yamananchi, K., Murphy, D. B., Cantor, H., Gershon, R. K. 1981. Analysis of antigen-specific Ig restricted cell material made by I-J+ Ly-1 cells (Ly-I TsiF) that induces Ly-2+ cells to express suppressive activity. Eur. J. lmmunol. 11:905 201. Mrller, G,, ed. 1984. T cell receptors and genes. Immunol. Rev. 81:5 202. Chesnut, R. W., Grey, H. M. 1986. Antigen presentation by B ceils and its significance in T-B interactions. Adv. Immunol. 39:51 203. Frohman, M., Cowing, C. 1985. Presentation of antigen by B cells: functional dependence on radiation dose, interleukins, cellular activation, and differential glycosylation. J. Irnmunol. 134:2269 204. Lanzavecchia, A. 1985. Antigen-specific interaction between T and B cells. Nature 314:537 205. Krieger, J. I., Grammer,S. F., Grey, H. M., Chesnut, R. W. 1985. Antigen presentation by splenic B ceils: resting B cells are ineffective, whereas acti-

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T CELLS vated B cells are effective accessorycells for T cell responses. J. lmmunoL135: 2937 206. HayGlass, K. T., Naides, S. J., Scott, C. F., Benacerraf, B., Sy, M.-S. 1986. T cell developmentin B cell-deficient mice. IV. Therole of B cells as antigenpresenting cells in vivo. J. Immunol. 136:823 207. Janeway, C. A. Jr., Ron, J., Katz, M.E. 1987. The B cell is the initiating antigen-presenting cell in peripheral lymph nodes. J. Immunol. 138:1051 208. Lichtman, A. H., Tony, H.-P., Parker, D. C., Abbas, A. K. 1987. Antigenpresentation by hapten-specific B lymphocytes. IV. Comparativeability of B cells to present specific antigen and anti-immunoglobulin antibody. J. Immunol. 138: 2822. 209. Ron, Y., Isakov, N., Sprent, J. 1987. Unresponsiveness of MRL/MP-lpr/lpr mice to antigen given subcutaneously in adjuvant: partial restoration of responseafter local injection of B cells. J. Immunol. 139:400 210. Ron, Y., Sprent, J. 1987. T cell priming in vivo: a major role for B cells in presenting antigen to T cells in lymph nodes. J. Immunol. 138:2848 211. Liano, D., Abbas, A. K. 1987. Antigen presentation by hapten-specific B lymphocytes. V. Requirements for activation of antigen-presenting B cells. J. Immunol. 139:2562 212. Krieger, J., Jenis, D. M., Chesnut, R. W., Grey, H. M. 1988. Studies on the capacity of intact cells and purified Ia from different B cell sources to function in antigen presentation to T cells. J. Immunol. 140:388 213. Kurt-Jones, E. A., Liano, D., HayGlass, K. A., Benacerraf, B., Sy, M.-S., Abbas, A. K. 1988. The role of an.tig.en-p.resen.ting B cells in T cell priming ~n wvo. Studies of B celldeficient mice. J. Immunol. 140:3773 214. Gosselin, E. J., Tony, H. P., Parker, D. C. 1988. Characterization of antigen processing and presentation by resting B lymphocytes. J. Immunol. 140:1408 215. Tony, H. P., Parker, D. C. 1985. Major histocompatibility complex-restricted polyclonal B cell responses resulting from helper T cell recognition of antiimmunoglobulin presented by small B lymphocytes. J. Exp. Med. 161:223 216. Tony,H.-P., Phillips, N. E., Parker, D. C. 1985. Role of membrane immunoglobulin (Ig) crosslinking in membrane Ig-mediated, major histocompatibility-restricted T cell-B cell cooperation. J. Exp. Med. 162:1695

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217. Janeway, C. A. Jr., Sakato, N., Eisen, H. N. 1975. Recognition of immunoglobulin idiotypes by thymusderived lymphocytes. J. Itnmunol. 72: 2357 218. Sakato, N., Janeway, C. A. Jr., Eisen, H. N. 1977. Immune responses of BALB/cmice to the idiotype of T15 and other myeloma proteins of BALB/corigin: implications for an immune network and antibody multispecificity. CoMSpriny HarborSyrnp. Quant. Biol. 41:719 219. Jorgensen, T., Hannestad, K. 1982. HelperT cell recognition of the variable domains of a mouse myelomaprotein (315). Effect of the major histocompatibility complex and domain conformation. J. Exp. Med. 155:1587 220. Bogen,B., Malissen, B., Haas, W. 1986. Idiotopeospecific T cell clones that recognize syngeneic immunoglobulin fragments in the context of class II molecules. Eur. J. lmmunol. 16:1373 221. Kawahara, D. J., Marrack, P., Kappler,J. 1982. Helper T cells specific for a VHdeterminant(s) are under H-2linked Ir gene control. Fed. Proc. 41: 366 222. Alzari, P. M., Lascombe, M.-B., Poljak, R. J. 1988. Three-dimensional structure of antibodies. Ann. Rev. Immunol. 6:555 223. Kourilsky, P., Claverie, J.-M. 1986. The peptidic self model: a hypothesis on the molecular nature of the immunological self. Ann. Inst. Pasteur/ Immunol. 137D: 3 224. Kourilsky, P., Chaouat, G., Rabourdin-Combe, C., Claverie, J.-M. 1987. Working principles in the immunesystem implied by the "peptidic self" model. Proc. Natl. Acad. Sci. USA84: 3400 225. Pereira, P., Larsson, E.-L., Forni, L., Bandeira, A., Coutinho, A. 1985. Natural effector T lymphocytesin normal mice. Proc. Natl. Acad. SoL USA 82:7691 226. Bandeira, A., Coutinho, A., MartinezA., C, Pereira, P. 1988. The origin of "natural antibodies" and the internal activity in the immunesystem. Int. Rev. Immunol. 3:47 227. Coutinho, A. 1988. Tolerance to self as a global behaviour of the immune system based upon the connectivity of auto-reactive lymphocytes. In The Tolerance Workshop, ed. P. Matzinger, M. Flajnik, H.-G. Rammensee,G. Stockinger, T. Polink, L. Nicklin. 3: 54. Basel: Editions Roche 228. Guilbert, B., Dighiero, G., Avrameas,

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PEREIRA ET AL S. 1982. Naturally occurring antibodies against nine common antigens in humansera. I. Detection, isolation, and characterization. J. ImmunoL128:2779 Dighiero, G., Lymberi, P., Mazi6, J.C., Rouyre, S., Butler-Browne, G. S., Whalen, R. G., Avrameas, S. 1983. Murine hybridomas secreting natural monoclonal antibodies reacting with self antigens. J. ImmunoL131:2267 Dighiero, G., Lymberi, P., Holmberg, D, Lundkvist, I., Coutinho, A., Avrameas, S. 1985. High frequency of natural autoantibodies in normal newborn mice. J. Immunol. 134; 765 Ron, Y., De Baetselier, P., Gordon,J., Feldman, M., Segal, S. 1981. Defective induction of antigen-reactive proliferating T cells in B cell-deprived mice. Eur. J. Immunol.1 i: 964 Ron, Y., De Baetselier, P., Tzehoval, E., Gordon, J., Feldman, M., Segal, S. 1983. Defective induction of antigenreactive proliferating T cells in B celldeprived mice. II. Anti-# treatment affects the initiation and recruitment of T cells. Eur. J. Immunol. 13:167 Kim, K. J., Rollwagen, F., Asofsky, R., Lelkovits, I. 1984. The abnormal function of T cells in chronically anti#-treated mice with no mature B lymphocytes. Eur. J. Immunol. 14:476 Cerny, A., Huegin, A. W., Sutter, S., Bazin, H., Hengartner, H. H., Zinkernagel, R. 1986. Immunity to lymphocytic choriomeningitis virus in B cell-depleted mice: evidence for B cell and antibody-independent protection by memoryT cells. Eur. J. lmmunol. 16:913 Portnoi, D., Lundkvist, I., Coutinho, A. 1988. Inverse correlation between the utilization of an idiotype in specific immune responses and its representation in pre-immune "natural" antibodies. Eur. J. Immunol. 18:571 Lundkvist, I., Coutinho, A., Varela, F., Holmberg, D. 1988. Evidence for a functional network amongst natural antibodies in normal mice. Submitted Bandeira, A., Coutinho, A., Carnaud, C., Jacquemart, F., Forni, L. 1988. Transplantation tolerance correlates with high levels of lymphocyteactivity. Proc. Natl. Acad. Sci. USA. In press Luzmi,S., Merino, J., Engers, M., Izui, S., Lambert, P.-A. 1986. Autoimmunity after induction of neonatal tolerance to alloantigens: role of B cell chimerism and F~ donor B cell activation. J. ImmunoL136:4420 VonBoehmer, H., Schubiger, K. 1986. Thymocytes appear to ignore Class

I major histocompatibility antigen expressed on thymusepithelial cells. Eur. J. Immunol. 14:1048 240. Wilson, D. 1988. See Re£ 227, p. 88 241. Holmberg, D., Coutinho, A. 1985. Natural antibodies and autoimmunity. hnmunol. Today 6:356 242. Kappler, J. W., Roehm, N., Marrack, P. 1987. T cell tolerance by clonal elimination in the thymus. Cell 49:273 243. Kappler, J. W., Staerz, U., White, J., Marrack, P. C. 1988. Self-tolerance eliminates T ceils specific for Mls-modifled products of the major histocompatibility complex. Nature 332:35 244. MacDonald, H. R., Schneider, R., Lees, R. K., Howe, R. C., Acha-Orbea, H., Fester~stein, H., Zinkernagel, R. M., Hengartner, H. 1988. T-cell receptor Vfl use predicts reactivity and tolerance to Mls"-encoded antigens. Nature 332:40 244a. MacDonald, H. R., Pedrazzini, T., Schneider, R., Louis, J. A., Zinkernagel, R. M., Hengartner, H. 1988. -Intrathymic elimination of Mls~-reae tive (Vfl6+) cells during neonatal tolerance induction to Mlsa-encodedantigens. J. Exp. Med. 167:2005 245. Kisielow, P., Blfithmann, P. Staerz, U. D., Steinmetz, M., Von Boehmer, H. 1988. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4÷ 8 ÷ thymocytes. Nature 333:742 246. Ben-Nun, A., Cohen, I. R. 1982. Spontaneous remission and acquired resistance to autoimmuneencephalomyelitis (EAE) are associated with suppression of T cell reactivity: suppressed EAE effector T cells recoveredat T cell lines. J. Immunol. 128:1450 247. Ben-Nun, A., Eisenstein, S., Cohen, I. R. 1982. Experimental autoimmune encephalomyelitis (EAE)in genetically resistant rats: PVGrats resist active induction of EAEbut are susceptible to and can generate EAEeffector T cell lines, d. Immunol. 129:918 248. Naparstek, Y., Holoshitz, J., Eisenstein, S., Reshef, T., Rappaport, S., Chemke, J., Ben-Nun, A., Cohen, I. R. 1982. Effector T lymphocyte line cells migrate to the thymus and persist there. Nature 300:262 249. Maron, R., Zerubavel, R., Friedman, A., Cohen, I. R. 1983. T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmtmethyroiditis in mice. J. Irmnunol. 131:2316 250. Hooper, D. C., Taylor, R. B. 1987. Specific helper T cell reactivity against autologous erythrocytes implies that

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T CELLS AND NETWORK self tolerance need not ..depend on clonal deletion. Eur. J.ilmmunol. 17: 797 251. Hooper, D. C., Young, J. L., Elson, C. J., Taylor, R. B. 1987. Murine T cells reactive against autologous erythrocytes: evidence for in vitro and in vivo priming with mouse and rat red blood cells. Cell. Immunol.106:53 252. Sakaguchi, S., Takahashi, T., Nishizuka, Y. 1982. Study on cellular events in,postt.hymectomy autoimmune ooph, oriti~ in mice. II., Requirements of Lyt-1 c~lls in normal female mice for the prevention of oophoritis. J. Exp. Med. 156:1577 253. Taguchi, O., Takahashi, T., Seto, M., Namikawa, R., Matsuyama, M., Nishizuka, Y. 1986. Development of multiple organ-localized autoimmunediseases in nude-miceafter reconstitution of T cell function by rat fetal thymus graft. J. Exp. Med. 164:60 254. Taguchi, O., Nishizuka, Y. 1987. Self tolerance and localized autoimmunity. Mouse models of autoimmune disease that suggest tissue-specific suppressor T cells are involvedin self tolerance. J. Exp. Med. 165:146 255. Martinez-A., C., Marcos, M. A. R., Pereira, P., Marquez, C., Toribio, M. L., de la Hera, A., Cazenave, P.-A., Coutinho, A. 1987. Turning (Ir-gene) low-responders into high responders by antibody manipulation of the developing im~nunc system. Proc. Natl. Acad. Sci. USA84:3812 256. Lamb, J. R., Skidmore, B. J., Green, N., Chiller, J. M., Feldmann,M. 1983. Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J. Exp. Med. 157:1434 257. Jenkins, M. K., Schwartz, R. H. 1987. Antigen presentation by chemically modified splenocytes induces antigenspecific T cell unresponsivenessin vitro and in vivo. J. Exp. Med. 165:302 258. Quill, H., Schwartz, R. H. 1987. Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes:

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specific induction of a long-lived state of proliferative nonresponsiveness. J. Immunol. 138:3704 259. Suzuki, G., Kawase, Y., Koyasu, S., Yahara, I., Kobayashi, Y., Schwartz, R. H. 1988. Antigen-induced suppression of the proliferative response of T cell clones. J. lmmunol. 140:1359 260. Jenkins, M. K., Ashwell, J. D., Schwartz, R. H. 1988. Allogeneic nonT spleen cells restore the responsiveness of normal T cell clones stimulated with antigen and chemically modified antigen-presenting cells. J. Immunol. 140: 3324 261. Tomonari, K. 1985. T-cell receptor expressed on an autoreactive T-cell clone, clone 4. Cell. Immunol. 96: 147 262. Webb, S., Sprent, J. 1987. Downregulation of T cell responses by antibodies to the T cell receptor. J. Exp. Med. 165:584 263. Breitmeyer, J. B., Oppenheim, S. O., Daley, J. F., Levine, H. B., Schlossman, _S: F. 1987. Growthinhibition of human ’T’cells-by antibodies recognizing the T cell antigen receptor complex. J. Immunol. 138:726 264. Bandeira, A., Larsson, E.-L., Forni, L., Pereira, P., Coutinho, A. 1987. In vivo activated splenic T cells are refractory to interleukin 2 growthin vitro. Eur. J. Immunol. 17:901 265. Andersson, J., Bullock, W. W., Melchefs, F. 1974. Inhibition of mitogenic stimulation of mouse lymphocytes by anti-mouse immunoglobulin antibodies. I. Modeof action. Eur. ,;J. Immunol. 4:715 266. Jerne, N. K. 1974. Towards a network theory of the immune system. Ann. Immunol. (Inst. Pasteur) 124C: 373 267. Varela, F. J., Sanchez-Leighton, V., Coutinho, A. 1988. Adaptive strategies gleaned from immune networks. Viability theory and comparison with classifier systems. In Evolutionary and Epigenetic Order from Complex Systems: A Waddington Memorial Volume, ed. B. Goodwin, P. Saunders. Edinburgh: Edinburgh Univ. Press. In press

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THE IMMUNE SYSTEM OF XENO P US Louis Du Pasquier,* Martin F. Flajnik**

Joseph Schwager* and

* Basel Institute for Immunology, Grenzacherstrasse487, CH-4005 Basel, Switzerland

INTRODUCTION Comparativestudies of the immunesystem require that for each key systematicposition at least one animalmodelis investigated in depth. For anuranamphibians,modernrepresentatives of the first vertebrates that achievedthe transition to terrestrial life, this modelis the clawed-toad or SouthAfricanfrog (genusXenopus).Since these frogs are easy to maintain and breed in captivity and are commerciallyavailable, and since they present developmentaland genetic advantagescomparedto other amphibians, they becamethe modelof choice for manyinvestigators. In 1988, after about 20 years of work, weare heading toward a comprehensive view of the Xenopusimmunesystem, thanks to use of a cross-fire of methodologies, rangingfromclassical graft rejection to geneanalysis. This reviewfirst presentsa descriptionof the structural elementsof the Xenopus immunesystem: the lymphoid system, the major histocompatibility complex,and the immunoglobulins. Three functional issues will be then considered:immune responses, tolerance, and antibodydiversity duringontogeny.Finally, a section will be devotedto the impactof polyploidy on the Xenopusimmunesystem. THE LYMPHOID LYMPHOCYTES

ORGANS

AND THE

The Thymus (Table 1) Thethymusarises throughan invagination of the dorsal epitheliumof the second pharyngeal poucharound day 3 after fertilization (stage 40 of **Present address: Department of Immunologyand Microbiology, University of Miami, Miami, Florida, 33101.

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Annual Reviews IMMUNE SYSTEM OF XENOPUS

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Nieuwkoop& Faber) (1-4). It is colonized during the following days cells from the dorsal lateral plate and ventral mesodermthrough the head mesenchyme(5-9). These large lymphoblasts proliferate in situ. One day later, the epitheliumbegins expressing class-II moleculesbut not the classical MHC class-I molecules (10). By day 6-8 the cortex-medulla architecture becomesvisible (11). The cortex contains mainly proliferating lymphocytesintricated in the digitations of the epithelial cells. The medullacontains epithelial cells with cytoplasmic granules and rough endoplasmic reticulum. Unlike the case in mammals,the cortex and medulla are separated by a distinct cellular barrier (12, 13). This area is rich in blood vessels and IgMproducing plasma cells (14). Macrophagesare present from early stages on. After metamorphosis a new type of macrophage presumed by some to be a nurse cell equivalent appears (2). Within the cortex large dendritic cells resembling those described in Rana(15) have been found in Xenopus(16). Myeloidcells, mucouscells and cysts are occasionally seen in the medulla (2). The thymusreaches its peak larval size (about 1-2 x 106 lymphocytes) at stage 58 (3, 17, 18). It involutes during metamorphicclimax (17-19) during which more macrophagesare seen in the shrinking cortex. At this time the thymus is translocated towards the tympanum(3). A second histogenesis follows, with the appearance of myoepithelial cells, aminecontaining cells, and larger cysts (2). The size of the organ reaches about 1-3 x 107 cells about 2-3 months after metamorphosis, and then it undergoes a regression at the time of sexual maturity, when it becomes more and more embeddedin fatty tissue (17, 19). A cell surface glycosylated membraneprotein of 120 kd has been identified with a monoclonal antibody XT-1 on a subpopulation of Xenopus T cells (20-23). Thymectomyseverely impairs allograft and mixed leucocyte reactions (MLRs)(24~26), proliferative responses to classical T-cell mitogens 27), and antibody responses against T-dependent antigens (25, 28). It does not affect response to B-cell mitogens (29), nor antibody response thymus-independentantigens (30, 31), nor to xenografts (32). IgY is absent in the serum of early thymectomized (Tx) animals, whereas IgM is more abundant and IgX not affected (33-35). Late thymectomy(after 8 days) does not impair allo-responses but still interferes with the antibody response to T-dependent antigens, as if someregulatory cells had not yet left the thymus(36). This, together with the stepwise effects ofthymectomy on MLRand phytohemagglutinin (PHA) responsiveness, suggests sequential maturation of the T-cell function (37), The thymus both generates and contains T helper cells (38), although thymic T cells are relatively poor helpers and poor graft-vs-host effectors. ThymicT cells obtained by nylon wool filtration can suppress ongoing T-B

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collaboration in vitro, althoughno quantitative titration of this suppressive activity was ever possible for unknownreasons (38). However, thymic dependent suppression or direct thymus cell suppression is a recurrent finding also under allogeneic conditions (39-43). Thymocytesfrom adults but not from larvae can produce the equivalent of IL-2 after allogenic or mitogen stimulation. Larval thymocytes will respond to mitogens when exogenous IL-2 is added (44). The thymus of Xenopus also contains IgMplasma cells or B cells ready to switch to IgY synthesis (14, 38, 45). In all these experimentscare must be taken of the specificity and affinity of the antibodies. Cross-reactive IgMantibodies can give apparently normal titers in Tx animals although the T-dependent compartmentis indeed affected (46). The Spleen

(Table

1)

The spleen appears about 12-14 days after fertilization as a mesenchymal thickening in the mesogastrium. The mature spleen has delineated regions of red (hematopoietic) and white (lymphopoietic) pulp (1, 47). The nodules of white pulp, with their central arteriola surrounded by lymphocytes, are lined by a boundarylayer of cells. Scattered lymphocytesmainly of the T lineage are present in the perifollicular area (2, 32, 48-50). The larval spleen is not hematopoietic but is richer in B cells than that in the adult. During metamorphosis the spleen cell number reaches a plateau (0.51 x 106), or may even drop. Afterward the organ grows steadily until it contains about 4 x 107 lymphocytesin 300 g adults (17). Large mitotically active dendritic cells are located in the white pulp and can extend pseudopods toward T cells. They are not classical macrophages, and they remain after thymectomy(51). Unlike the case in other species, Xenopusspleen is the only organ to accumulate and retain antigen for several weeksin a specific zone near the periphery of the white pulp (1, 52). Since Tx animals do not showthis type of antigen maintenance it has been proposed that it was due to antigenantibody complexes(53). Particular and soluble antigens have a different distribution (1). During an ongoing immuneresponse, proliferation cells in both the red and the white pulp can be observed in some but not all cases (1, 54). The spleen is a source of antibody forming cells (55). Most of its B cells produce IgM and a very few produce IgY or IgX. Lipopolysaccharideinduced proliferation of Xenopus B cells is poor and may be due to a contaminant of LPSpreparation (56). Lipopolysaccharide (LPS) itself, doses comparable to those used in mammaliansystems, induces Ig synthesis (E. Hsu, T. Leanderson, personal communication;34). Spleen cells

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respond well to PHAand Con A (57). Pokeweedmitogen (PWA)and antiIg reagents are potent stimulators of proliferation and differentiation (58, 59); phorbol myristateacetate (PMA)induces 80%of splenic leukocytes to synthesize DNA(60). Spleen cells respond muchearlier in ontogeny mitogens than do thymocytes (61). Adult spleens from day-10 thymectomizedanimals do contain IL-2 producing and IL-2 responding cells (44). Splenocytes from Tx animals that have rejected an allograft display a "nonspecific" MLRagainst any MHChaplotype (62). Spleen contains also specific alloreactive T cells capable of MLR and of differentiation into cytotoxic lymphocytes. Helper cells are more abundant or more efficient in the spleen than in the thymus (38). Splenectomy has no major effect immuneresponses (63).

Other Sources of LymphoidCells In the anterior part of the tadpole the ventral and dorsal cavity bodies occupythe central part of the pharynx (11, 64). They are depleted of their lymphocytes after thymectomy(1, 48) and disappear at metamorphosis. Lymphoidnodules are present in the adult but not the tadpole intestine. They are rich in B cells. Numerousplasma cells producing IgM and IgX, but not IgY, are also visible in the mucosa(65). In the liver, lymphopoiesis is associated with the lymphomyeloidperipheral layer (11, 66) whichpersists throughout the life of Xenopus,but not in other anuran species where it disappears at metamorphosis. The liver traps circulating particles by meansof Kupffer-like cells (67) which are class-II positi, ve (10). Free mononuclearliver cells can be stimulated PHAor by the purified protein derivative of tuberculin, but not by Con

A(68). In the kidney, lymphocytes, mainly B cells, accumulate along the whole mesonephros, between the renal tubules (1). Injected antigen has been traced to intertubular areas (67, 69). Mesenteries and gills contain random accumulations of lymphomyeloid cells, and no homologyof lymph glands or jugular bodies found in other anura can be seen in Xenopus(1). Peritoneal cells produce factors with l-like activity (70). The bone marrow of Xenopus develops after metamorphosis. From functional studies with mitogen or from cytological observation it may not be lymphopoietic(68, 71; discussed in 1). Blood contains B and T cells and monocytes: the experiments that led to the demonstration ofT- and B-cell collaboration and the MHCrestriction of this phenomenonin Xenopus were done mainly with peripheral blood cells (72, 73). After immunizationthe numberof plasma cells is only two times lower than in the spleen.

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Embryonic Origin of Lymphoid Stem Cells Twomesodermalstem cell compartmentsexist in the embryo:the ventral bloodisland (VBI)and the dorsal lateral plate mesoderm (5, 74-76). cells associated with early hemopoiesisare derived fromthe VBI.Studies with the X. borealis marker(77) provedthat the thymusrudimentat stage 43 is colonizedonly by cells derived fromthe VBI(78), whereasin adult life, similar experimentswith chimerashave shownthat the thymuswas essentially populatedby cells fromthe dorsal lateral plate (79). Thetwo compartmentsare in fact not completely independent from each other (80). It remainsdifficult to assert the type and function of VBI-derived cells becausethey appear to be a transient population. Morerecently it has been suggestedthat "accessorycells" and thymocytescould arise from a bipotential precursor that diverges into these two lineages after the colonizationof the epithelial thymicrudiment(81, reviewin 82). THE MAJOR HISTOCOMPATIBILITY

COMPLEX

Theexistence of a majorhistocompatibility complexwasoriginally proven by family studies in whichthe cosegregationof classical mammalian MHC functional or serological markers were analyzed. Thus, MLR (83) acute gra.ft rejection occurred, and somecell surface antigens detected with alloantisera segregatedtogether (84); this providedevidencefor a complex called XLAby analogy with HLA. Structural

Aspects (Table 1)

The abovementionedantisera and anti-humanclass-II DRflxenoantisera were used to characterize the XenopusMHC molecules(85, 87). Class I-like molecules have been immunoprecipitatedfrom 125I cell surface labeled leukocytes and erythrocytes. The heavychain has a mol wt ranging from 40-44 kd dependingon the allele examined,of which3 kd consist in N-linked carbohydrate. The light chain, presumablythe homologueof f12 microglobulin, had a mol wt of 13 kd and no N-linked carbohydrates. The family studies confirmedthat the heavy chains are encoded by XLAgenes. Size and charge polymorphismwas obvious from the twodimensional (2d) gel electrophoresisanalysis of 7 alleles (85). Class-II antigens were identified with alloantisera recognizing an approximately 30-kd molecule on the surface of lymphocytesin MHCtypedfamilies (86). Moreover the IgGfraction of a rabbit antihumanclassII, fl chain-inhibited, bidirectional MLR recognizedthe samemoleculesas the alloantisera, and immunoprecipitated the Xenopusclass-II molecules. Theyare composedof two 30- to 35-kd integral membrane glycoproteins.

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The ~ chains have some N-terminal sequence homology with mammalian ~ chains. Unlike most mammalianclass-II molecules, the deglycosylated fl chains are larger and more acidic than the ~ chains. Each XLAhaplotype seems to carry two ~ and up to 5 fl genes. Both class-II chains are polymorphic.In family studies the 2d gel patterns precisely matched MLRdata. However, for some outbred animals that were attributed a MLRJ haplotype (a poor stimulator in most MLR), the 2d gel pattern failed to display the J pattern. Clearly MLR underestimates the true level of polymorphismin a Xenopus population (87). Until now the following haplotypes of Xenopus MHChave been described a, b, c, d, f, g, r, j, k (88-90). Frequencyoff and r are similar (3.3%) whereas g is rare (0.8%). Linkage disequilibrium has been found between class and class II in a population that had otherwise reached equilibrium (91). A polymorphism based on isoelectric focusing (IEF) pattern has been exploited to study the linkage of the complement component C4 (92) MHCin two strains (J and K) of Xenopus. In a backcross of progeny of 25 individuals there was correlation between MHCtype (defined by MLR and acute graft rejection) and the C4 type. However,the C4 IEF type of one individual did not correlate, perhaps indicating a high frequency of crossover (90). C3 is not MHC-linked;therefore the possible date of separation of C3 and C4 predates the emergenceof amphibians in evolution (93). One Xenopus erythrocyte glycoprotein of 38-43 kd displays some of the hallmarks of chicken BGmolecules. It is only expressed on red cells, does not bind f12 microglobulin, and appears to be MHClinked. It is polymorphic,by size and reactivity with alloantisera, but unlike class I, it has the same V8 protease pattern. Under nonreducing conditions, homoor heterodimers are formed as with BGmolecules (94). It remains determine whether its linkage to MHCis physical or genetic. Functional

Aspects

Cytotoxic T lymphocytes (CTL) can be generated in Xenopus although it is not possible to generate killers after a primary MLR.Anin vivo priming is required, followed by MLR.The CTL specifically recognize MHClinked target molecules on lymphoblasts (95). In vitro assays for T and B collaboration have been set up using T and B separated by nylon wool, from either carrier or hapten primed animals of various MHCtypes (72, 73). Antibodies of isotype IgY, which are highly thymus dependent, and high affinity IgMantibodies (73), could detected only in combinations where T- and B-cells shared at least one MHChaplotype. IgM antibodies of low affinity were produced normally in all cultures, regardless of the MHC type of T- and B-cells. To further determine the homology of MHCfunction in Xenopus,

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IMMUNE SYSTEM OF XENOPUS

259

several types of experiments were done in which T cells matured in the "wrong" MHCenvironment. Antibody production to a thymus-dependent and graft rejection was examined. Reconstitutions of early Tx Xenopus have been attempted using either genetically undefined, partially defined, MHCtyped, or isogenic thymus donors (96, 97). The best system remains that of LG hybrids (98) where truly isogenic and MHCpredetermined replacements can be done. Tx tadpoles were later reconstituted with larval irradiated thymus of all possible XLAtypes. Under those circumstances one tested whether T cells that differentiated in an allogeneic thymuswere able to collaborate with B cells. Muchless IgY was produced in fully MHC mismatched chimaeras than in chimaeras where one MHChaplotype was matched or in control. IgM antibody production was the same in all groups. To better address the issue of education, the chimaera had to be done. earlier, before the thymus was colonized. In such chimaeras (8), made by sectioning the embryos just behind the gill anlagen, the thymus epithelium is derived from the head, and all the lymphocytes are hematopoietic cells from the body. The results were similar to those of the larval chimaera but even less pronounced (89). MHC fully mismatchedchimaeras made IgY antibodies with low kinetics or in low amounts depending on the MHChaplotypes involved. In summary,although it was not absolute, there was a thymic selection of the T-cell repertoire in Xenopus. Early attempts to generate CTLagainst minor histocompatibility antigens have failed (95). This maynot be surprising since the generation CTLagainst MHC was already difficult. Recently T-cell lines generated to both major and minor histocompatibility antigens have been obtained with growth factors from mitogenstimulated cultures (99). These lines will be useful to study in vitro the MHC restricted recognition of such antigens. Experiments done with chimaeras in vivo have suggested that responses to at least some minor antigens are MHCrestricted. LG15(a/c) and LG6 (a/c) strains have the same MHC genotypes a/c and differ only by minor H antigens. LG3(b/d) differs from these strains at the level of the two MHChaplotypes and rejects a/c skin acutely in 21-25 days. LG3can be made tolerant to LG15(a/c) tissue by grafting an eye anlage during embryoniclife (within 24 hours after fertilization). Such an animal, which b/d lymphocyteshave matured in a b/d thymus, is not able to reject either an LG6or LGI5 graft. Presumably the minor antigens of LG6, to be recognizcd by T cells, have to be recognized in a MHC-restrictedfashion by the host lymphocytes. LG15(a/c head)/LG3 (b/d body) chimaeras which b/d lymphocytes have matured in an a/c thymus, on the other hand, reject LG6(a/c) grafts as rapidly as normal LG15, but tolerate LG15 grafts. This was the first evidence for a positive selection of the T-cell clones in the thymus(89).

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THE IMMUNOGLOBULIN THEIR GENES

MOLECULES

AND

Heavy Chain Isotypes (Table 1) (reviewed in 100) IgM is the most abundant isotype (2 mg/ml) (101). Its heavychain is heavily glycosylated. IgMexists in a slightly different size in outbred Xenopus (102). All the anti-IgM monoclonal antibodies precipitated from serum a thick diffuse band at mol wt 72,000-73,000 which can be resolved into two distinct bands at mol wt 61,700 and 60,000, whenB cells are treated with tunicamycin (102). Polymeric IgM associated with J chain and forms hexamers (103, 104). The full sequence of an IgM heavy chain has been determined from a cDNAclone (105). It has four domains. The CH3and CH4domains are the most conserved (47% and 42%identities, respectively) whereas CH1 and CH2 show little homology with mammalian IgM (31% and 32%). This # chain is encoded by a four-exon germline gene. The Xenopus~t gene has much larger introns than do those of mammals (106). A putative switch region of about 3 kb in the intron between J, and C/~ consists in multiple repeats of 150 bases rich in AAGCTCAGCT elements (107).

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IgM

IgY The Xenopus analog of IgG has been called IgY because its mol wt, most likely due to four constant region domains, is greater than IgG (Table 1) (100). Glycosylated and nonglycosylated forms are present in equimolar amounts in the IgY of all normal serum samples (present at 0.7 mg/ml), as if during biosynthesis only one chain in each dimer could bear the carbohydrate (102). IgY preferentially associates with the heaviest light chain. IgX The existence of a third Ig isotype had been suspected after the immunoprecipitation of what was considered as a second # (58). In fact IgX is an antigenetically distinct isotype but is polymeric like IgM. Its heavy chain (mol wt 63,000) is more glycosylated than/~ (34). ISOTYPE DISTRIBUTION The three isotypes differ by their V8 peptide digest, by several antigenic epitopes, and by their tissue distribution. Splenic IgM+ B cells range from 20%(with monoclonal antibodies) (57, 108) to (with xenoanti-Ig sera) (109). Gut epithelium, liver hematopoietic layers, and thymusmedulla are rich in IgMplasma cells (14). IgY and IgX positive cells are muchrarer in the spleen but manyIgX p~asmacells are found in the gut. All B cells positive for IgY hlso produce IgM (102). The thymus does not contain surface IgM positive cells in large numbers (110) originally described (111).

Annual Reviews IMMUNE SYSTEM OF XENOPUS 261

The Light Chain Three categories of light chains from 25-29 kd have been detected. Two of them, recognizedby monoclonalantibodies, are antigenically distinct (34, 102). L chains from anti-DNP antibodies have been partially sequencedand are related to both VKand V2 sequences (112). A cDNA clone for the constant region of one of the light chain has beenisolated and showscharacters in common with both ~ and 2 (113). Annu. Rev. Immunol. 1989.7:251-275. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

The Variable Region of Heavy Chains The aminoacid heterogeneity of V, peptides is not pronounced,since sequencecould be determinedfor the first frameworkeven in nonimmune samples(112, 114). Thediversity of the variable regions has beenestimated by IEF (llS), maturation of the immuneresponse (ll6), and idiotype analysis (112). All studies revealed a restricted heterogeneitywhich, addition to the inheritance of IEF spectrotypesand idiotypes in isogenic frogs (55, 112), suggested that somatic mutationplayed a minorrole Xenopus(117). TheIg genecluster (Figure 1) and the rearrangementshavebeenstudied to see whetherthe lowheterogeneitywasdueto a special geneorganization. The sequenceof the Vnpart of a completeIgMcDNA clone (105), or the sequence of a genomicVHclone obtained by cross-hybridization (llS), showedgreat conservationof the prototypic V/~structure. Mammalian and Xenopussequences showthe classical frameworksand complementarity determiningregions (CDR).The percentage of identical nucleotides for the entire VHis about 65%.As in most other species the genomicVH element is preceded by a conserved octamer and a TATA box 5’ to the split leader (Figure 1). Someputative DHand the JH elementsare highly conserved because up to 15 of 16 aminoacids of CDR3and framework4 are identical in human,Xenopusand shark. Oneputative D. sequenceis ?

?

?

7

(VHN) (VIlli) n (VHI)f~

(VHI)n

DH?

JH

",,,,,,, 2~

S~t

~

Cp.

C x

Cu

, =l,~, -"- ,,"¯~: ||-"| 1

234

12

34

1 2 3 4

7997 AGTCAAAT TATA

Figure 1 Genomicorganization of Xenopusimmunoglobulingene segments. 7 and 9: heptamersandnonamers.23 and 12, length of spacers in bp. S/t: putative switch region, C#, Cx, Co:genomicconstant regions genes. C/~, representedto scale, correspondto a fragment of 4.2 kb. A single VHelementis representedwith its octamer,TATA box and split leader. Thestructure of the D,uelementis hypothetic.

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identical (one gap of 2 bp necessary) to one of the shark DHelements (119). To this date, three different VHfamilies have been identified, based on the absence of cross-hybridization under stringent conditions. Southern blot analysis suggests that there are at least 60 VHgenes per haploid locus (119). This numberis large and in principle could generate more antibodies than are detected by IEF. It is possible that not all of them are used. The incidence of pseudogenes in the Vu elements so far analyzed is < 20%. Yet each V~/family seems to have a distinct set of putative regulatory sequences 5’ to the coding region. Within each family, sharing of entire CDRbetween one gene and the other has been noticed. Thus, such a pool of Vuelements mayindeed generate a smaller repertoire than that deduced from the number of its members(120). The cDNAsequences, and the recombination signals and spacers of VH and Jn (Figure 1) suggest the existence of Dnelements. As in other species, presumably the same element can be used in different frames. Genomic Xenopus Dn have not been isolated. Data from cDNAclones suggest that they encode 1 to 11 residues (119). Seven JH have been described. All have the appropriate recombination signal sequences and 22 bp spacers (119). Southern blots with B-cell DNAvisualized the rearrangements in Xenopus. There are multiple rearrangements presumably on both alleles (119). Theseevents result in allelic exclusion (108). The expression of these genes is illustrated in Figure 2 wheretwo typical immuneresponses are represented. Antibody responses are slower in Xenopus than in mammals;the cell division time for the temperature at which they live is about 24 hr. At the beginning only IgMis produced; then it is produced in conjunction with IgY (100). Incomplete switch has been reported, since many lymphocytes keep on producing IgM and IgY antibodies simultaneously (121). A modest affinity maturation of IgM has been noticed (Figure 1) (115). PFCnumbers, kinetics, titers, and spectrotypes are very similar between isogenic individuals (28, 55, 122). A second injection of antigen generated a stronger secondary response (memory). The affinity of the antibodies is immediately that of specific antibodies (115). The contribution of IgX to the immuneresponse unknown. THE

ONTOGENY

Tolerance

OF

THE

IMMUNE

SYSTEM

Induction

Based on transplantation of pituitary, Triplett’s experiments (123) suggested that an early hypophysectomized Hyla would recognize its own pituitary, grownon a transient host, as foreign. In Xenopusclones, better

Annual Reviews IMMUNE SYSTEM OF XENOPUS

10o~

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100_

100.

263

10-8

10_

Time(weeks after immunization) Figure 2 Antibody responses in Xenopus. Left, plaque forming cell (PFC) response after an injection of sheep red blood cells. The upper and lower curves give the range of the response in a group of four outbred individuals. Right, anti-DNP response. Left Y axis: inactivation constant of DNP-T4bacteriophage. (,~, primary ~ secondary responses.) Right Yaxis: relative affinity estimation. Inhibitor concentration in inhibition of modifiedT4 bacteriophage inactivation assays measuringrelative affinities of antibodies to the DNP(black symbols) and the cross-reacting TNPligand (open symbols) in a primary (Q, ©) or secondary (I, []) response.

experiments contradicted Triplett’s results, A Xenopus grown without its eyes, thyroid, or pituitary will always recognize these isogenic organs as self. Thus, the processes of tolerance generation are not restricted to one phase of the ontogeny (124, 125). Tadpoles of Xenopus develop their allorecognition capacities around stage 49 (126). Whethera tadpole will reject or tolerate an allograft depends on a sensitive balance of parameters. As a rule (review in 132) minor nonMHCdifferences do not promote graft rejection in tadpoles. However, exceptions have been reported (e.g. HDfamily siblings; Ref. 125), and all possible thresholds of graft rejections have been recorded (129-135). Maintenance of tolerance depends on the continuous presence of the tolerizing graft (136). Somehave found a higher frequency of tolerance to MHC-disparate skin graft during metamorphosis (130, 134). Others, working under different genetic conditions, have found that the ability of tadpoles to reject grafts emerges gradually during ontogeny (128, 129). Metamorphosis,nevertheless, represents a crucial period (Figure 3; 127) separating two rather different immunesystems. Although tadpoles

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ET AL

LARVALSTAGES

PROMETAMORPHOSIS METAMORPHOSIS

ADULT AGEIN DAYS

0

10

20

30

14191,5~5’1 1~25~1814 1155

36 45

40

50

60

75

90 105

I I Ill 16364t 661I /,, I ,, I ,l I 5611517 58160611’ 59 62 65 STAGES

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CIRCULATION BEGINS - HATCHING

~

PRE8-CELLS,N LIVERT

I

I I PREB-CELLSIN LIVER~" B-CELLSIN LIVER(1)ANDSPLEEN(2)

1 I 1 +2

IgM IN SERUM IgY IN SERUM TADPOLE REPERTOIRE "~’~,

ADULT REPERTOIRE FULLT-CELL HELP

L

[~y

THYMUS INVADEDBY STEMCELLS ~" THYMECTOMY AFFECTSALLOGRAFT RESPONSE AND MLR

MECTOMYAFFECTS ANTIBODYRESPONSE I Tx~TOL.

I

? I

? ’~= i INCOMPLETE

MLR ALLOGRAFT

IMPAIREDMLR REACTIVITY IMLR RESPONSES Ii~,~ ~ ALLO 9¯ ~. TRANSFER OF I SUPPRESSION I

MHCCLASS][ ONTHYMUS EPITHELIUM(1)AND 8-CELLS(2) MHCCLASS][ ONTHYMOCYTES AND T-CELLS SKINCLASS If + CELLS

I

MHCLINKEDAg ON THYMUS EPITHELIUM CLASSICAL MHCCLASS]~ RBCAG~ [___

Ilx103

¯ 3X104

3x105

9x108 1.5xlo s 9x105

lx108 2x106

lx107 I

THYMICLYMPHOCYTES NUMBERS

RELATIVETETRAIODOTHYRONIN CONCENTRATION Figure

3 The ontogeny

of the immune response

in )Senopus

(modified

from Ref.

135).

Abbreviations: Tx, thymectomy; Tol, tolerance. Skin class II÷cells: appearance of class II positive cells in the skin. RBC AG red blood cell antigen linked to MHC. Relative tetraiodothyronin concentration: visualization of level of the T4 hormone. For real values consult specific

chapters in Ref. 168.

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IMMUNESYSTEMOF XENOPUS 265 express ~z and fl class-II chains identical to those of adults, their tissue distribution is different. Only 50-70%of tadpoles splenic lymphocytes including the B cells are class-II positive, whereasall adult splenic lymphocytes are positive (10). As determined by immunoprecipitation and immunofluorescence, the classical polymorphic membrane-boundclass-I molecules are not expressed before metamorphosis (137-139). The MLR is present during larval and adult life but is impaired at metamorphosis. The larval and adult immunesystems differ to the point that syngeneic larval anti-adult MLRcan be detected (136). It is not knownwhether class-I molecules are the stimulators for this MLR.MHC class-I and adult globin chains follow the same pattern of expression. Within l0 days after climax there is a large influx of erythroblasts producingadult hemoglobulin and class-I molecules 038). Lymphocytes from metamorphosing animals can suppress the response of a young adult to minor H disparate grafts (140). Moreover, cells from larval animals made tolerant to adult skin can inhibit the rejection of semiallogenic graft (133). Moreover, thymectomyat stage 56 decreases the number of tolerance cases in subsequent grafting (141), suggesting that suppression is an important mechanismfor tolerance induction and maintenance. The question of clonal deletion has never really been addressed. Further insight into tolerance came from thymectomies and reconstitution, and from embryonic chimaera experiments. Implantation of MHC-disparatethymusin late larval life of Tx hosts restores the response to MHCdisparate grafts but induces tolerance to skin from the thymus donor (142, 143). However, spleens of reconstituted animals can display MLRtowards the donor stimulator. This situation is analogous to the cases of split tolerance noticed in Tx animals reconstituted in adult life (144) and in head/body chimeras (89). In animals grafted at metamorphosis, tolerance to skin with retained proliferative responses has also been noticed when the animals are challenged in vitro by donor cells. It has been suggested that the cytotoxic effector part of the alloresponse is inhibited (145). These results concur with the observation that tolerated grafts are invaded by host-derived, class-II positive cells (10) and that certain chimaeras are able, upon challenge with head-type red cells, to synthesize antithymus MHC"auto" antibodies (146). So clearly, tolerant animals are not simply unable to recognize the tolerated antigens. Antibody

Responses

The maturation of B cells starts in the liver, at stage 45, where the first pre-B cells (surface IgM- cytoplasmic Ig +, L chain-) and then B cells can be detected (147), followed by the appearance of Ig in the serum (148)

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(Figure 3). There seem to be two wavesof pre-B cell production (149), this would account well for the detection of the two different antibody repertoires (139). The stability of the larval and the adult repertoires suggests that the rearrangements mayoccur only during these two waves. The ability of Xenopus tadpoles to recognize foreign antigens appears around stage 51-52 (148, 150, 151, 153). Tadpoles, probably limited in their T-cell function, produce IgMantibodies and have difficulties switching to IgY, which can be improvedby injection of adult T cells (152). Tx animals, reconstituted with larval thymus, show a normal repertoire (148, 149). "Bridges" exist between the larval and the adult immunesystems, since memoryis transferred through this period, at least in the case of T- and B-cell memoryfor an anti-DNP.KLHresponse (116) and to some extent for graft responses (153). The repertoire abruptly shifts from tadpole to adult at metamorphosis (E. Hsu, unpublished). With respect to the perimetamorphic period, the thymus at stage 57/58--when its cells bind less lectin than in larval or adult life--seems to lose its suppressionactivity on antibody responses (154). THE EFFECT OF POLYPLOIDY IMMUNE SYSTEM

ON THE

In principle the polyploid forms of this genus could express more than one MHCallele. Studies were performed using MLRas a MHCmarker in Xenopus of various ploidy (Table 1, 155). Diploid expression of MLR loci was seen in all species except X. ruwenzoriensis, apparently a recent polyploid. Thus, the need to becometolerant to manyself-MHCantigens appears to have been solved by the silencing of all but one diploid set of loci (156). In contrast to the natural polyploid, all constitutive MHC and other histocompatibility loci were expressed in laboratorymadepolyploids. This suggests that time is involved in the "silencing" phenomenon. With respect to immunogloblin production, allopolyploid species with three genomesof one species and one genomeof the other express specific Ig proportionally to the number of chromosomes.However, a lymphocyte never expresses more than one of its immunoglobulin constant-region Ig genes. This suggests that if a stochastic model for allelic exclusion is correct, the frequency of multiple successful rearrangements has to be very low. The resulting huge waste of lymphocyte precursors would be incompatible with the development of the Xenopus immunesystem characterized by its small numberof lymphocytes(108). Allopolyploidy could

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267

a source of extra V genes which the individual might express. Indeed, in contrast to its effects on MHC,polyploidy is accompaniedby an increase in antibody diversity (157) (Table 1). Lab-madeaneuploid Xenopus (158) had their anti-DNP repertoire affected by the presence or the absence of a given chromosomein the genome (159).

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CONCLUSION 1. The Xenopus lymphoid system is less complex than in mammals. A smaller numberof lymphocytes and an absence of lymph nodes are associated with a restricted antibody repertoire. This restriction maybe explained by the fact that in spite of an Ig-gene organization similar to that of mammals,the pool of usable VHelements may be smaller, and there do not seem to be somatic mutations. 2. The Xenopus larval immunesystem functions without the classical MHC class-I molecules expressed on cell surface. MHC serves as a marker of adulthood at the time of metamorphosis, in the hematopoietic lineage. The larval immunesystem differs from the adult, and it is not yet clear whichdifferences are due to class-I expression, class-II distribution, endocrine regulations, and cytokine production. 3. The use of thymectomy, embryonic chimaeras, and the natural situation at metamorphosis have deepened our knowledge about tolerance, stressing the role of regulatory interactions versus clonal deletion. 4. Evolutionary studies, madepossible by the polyploidy of someXenopus species, have revealed the trend for the expression of a single MHC and the strong pressure for allelic exclusion at the Ig locus level. For further development, many methods (160) and strains are now available in different species (161), not to mention more revolutionary tools such as interspecies somatic cell hybrids (162) or nuclear transplantation (163) and transgenic Xenopus(164). If nuclear transplantation has failed so far to provide an ideal tool for single cell genetics, it nevertheless has resulted in the production of tadpoles from single lymphocyte nuclei (165). It could be interesting to use it again in conjunction with embryonictransplants and to complementfuture transgenic frog studies. No lymphoid cell lines are available, but chemically induced tumors may provide this precious tool (166, 167). In the future, one can envision that endocrine regulations (154, 168), and the elucidation of the gene organization, and roles of membersof the Ig supergene family and complementwill be areas of predilection where the peculiar genetic, phylogenetic, and ontogenetic characteristics of Xenopuswill be exploited further.

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ACKNOWLEDGMENTS We thank Ms. S..Kirschbaum The Basel Institute’for F. Hoffman-La

for typing Immunology

Roche & Co. Ltd.,

Basel,

the manuscript. was founded and is

supported

by

Switzerland.

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IMMUNE SYSTEM OF XENOPUS bian ontogeny. Immunogeneties 3:38191 117. Du Pasquier, L. 1982. Antibody diversity in lower vertebrates--why is it so restricted? Nature 296:311-13 118. Yamawaki-Kataoka, Y., Honjo, T. 1987. Nucleotide sequences of variable region segments of the immunoglobulin heavy chain of Xenopuslaevis. Nucleic Acids Res. 15:5888 119. Schwager, J., Grossberger, D., Du Pasquier, L. 1988. Organization and rearrangement of immunoglobulin M genes in the amphibian Xenopus. EMBOJ. 7:2409-15 120. Du Pasquier, L., Schwager, J. 1987. Diversity of VHgenes and Vnfamilies in Xenopus. Basel Inst. Immunol. Ann. Rep. 1987:32 121. Hadji-Azimi, I., Parrinello, N., Perrenot, N. 1978. The simultaneous production of two classes of cytoplasmic immunoglobulins by single cells in Xenopuslaevis. Cell. Immunol.39: 31624 122. Du Pasquier, L., Wabl, M. R. 1976. Antibody diversity studied in amphibians. In The Generation of Antibody Diversity: A New Look, ed. A. J. Cunningham, pp. 151-64. London: Academic 123. Triplett, E. 1962. On the mechanismof immunologicself recognition. J. Immunol. 89:505-10 124. Rollins-Smith, L. A., Cohen, N. 1982. Self-pituitary grafts are not rejected by frogs deprived of their pituitary anlagen as embryos. Nature 299: 82021 125. Rollins-Smith, L. A., Cohen, N. 1983. The Triplett phenomenon revisited: self tolerance is not confined to the early developmentalperiod. Transplant. Proc. 15:871-74 126. Horton, J. D. 1969. Ontogeny of the immuneresponses to skin allografts in relation to lymphoid organ development in the amphibian Xenopus laevis Daudin. J. Exp. Zool. 170:449-66 127. Cohen, N., Dimarzo, S., RollinsSmith, L., Barlow, E., VanderschmidtParsons, S. 1985. The ontogeny of allo tolerance and self-tolerance in larval Xenopus laevis. In Metamorphosis, ed. M. Balls, M. Bownes, pp. 388-419. Oxford: Clarendon 128. Obara, N., Kawahara, H., Katagiri, C. 1983. Response to skin grafts exchanged amongsiblings of larval and adult gynogenetic diploids in Xenopus laevis. Transplantation 36:91-95 129. Du Pasquier, L, Chardonnens, X. 1973. Induction of skin allograft toler-

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ance during metamorphosis of the toad Xenopus laevis: a possible model for studying generation of self-tolerance to histocompafibility antigens. Eur. J. lmmunol. 3:569-73 130. Du Pasquier, L., Chardonnens, X. 1975. Genetic aspects of the tolerance to allografts induced at metamorphosis in the toad Xenopus laevis, lmmuno9enetics 2:431-40 131. Barlow, E. H., Dimarzo, S. J., Cohen, N. 1981. Prolonged survival of major histocompatibility complex disparate skin allografts transplanted to the metamorphosingfrog, Xenopus laevis. Transplantaion 32:51-57 132. Dimarzo, S. J., Cohen, N. 1982. Immunogeneticaspects of in vivo allotolerance induction during the ontogeny of Xenopus laevis. Immunogenetics 16:103-16 133. Nakamura, T., Marno, M., Tochinai, S., Katagiri, C. 1987. Tolerance induced by grafting semi-allogeneic adult skin to larval Xenopus laevis: possible involvement of specific suppressor cell activity, Differentiation 35: 108-14 134. Bernardini, N., Chardonnens, X., Simon, D. 1969. Drveloppement aprrs la m6tamorphose des comp~tences immunologiques envers les homogreffes cutan+es chez Xenopuslaevis (Daudin). C. R. Acad. Sci. Paris 269D: 1011 14 135. Flajnik, M. F., Hsu, E., Kaufman, J. F., Du Pasquier, L. 1987. Changes in the immune system during lnetamorphosis of Xenopus. Immunol. Today 8:58-64 136. Kaye, C., Schermer, J. A., Tompkins, R. 1983. Tolerance maintenance~de~ pends on persistence of the tolerizing antigen: evidence from transplantatiOn’ . on Xenopus laevis. Dev. Comp. lmmunol. 7:497-506 M. F., Kaufman, J. F., 137. Flajnik, Hsu, E., Manes, M., Parisot, R., Du Pasquier, L. 1986. Major histocompatibility complex-encoded class 1 molecules are absent in immunologically competent Xenopus. before metamorphosis. J. Immunol.’q37:38"91-99 138. Flajnik, M. F., DuPasquier, L. 1988. MHCclass I antigens as surface markers of adult erythrocytes during the metamorphosis of Xenopus. Devel. Biol. 128:198-206 139. Du Pasquier, L., Blomberg, B., Bernard, C. C. A. 1979. Ontogeny of immunity in amphibians: changes in antibody repertoire and appearance of adult MHCantigens J. Immunol. 9:900~5in Xenopus. Eur.

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140. Du Pasquier, L., Bernard, C. C. A. 1980. Active suppression of allogeneic histocompatibility reactions during metamorphosis of the clawed toad Xenopus. Differentiation 16:1-7 141. Barlow, E. H., Cohen, N. 1983. The thymus dependency of transplantation allotolerance in the metamorphosing frog, Xenopus laevis. Transplantation 35:612-19 142. Horton, S. D., Horton, T. 1975. Development of transplantation immunity and restoration experiments in the thymectomized amphibian. Am. Zool. 15:73 143. Nagata, S., Cohen, N. 1984. Induction ofT cell differentiation in early thymectomized Xenopus by grafting adult thymuses from either MHC-matched or from partially or totally MHCmismatched donors. Thymus 6: 89103 144. Arnall, J. C., Horton, J. D. 1986. Impaired rejection of minor histocompatibility antigen-disparate skin grafts and acquisition of tolerance to thymus donor antigens in allothymus implanted, thymectomized Xenopus. Transplantation 41:766-76 145. Arnall, J. C., Horton, J. D. 1987. In vivo studies on allotolerance perimetamorphically induced in control and thymectomized Xenopus. Immunology 62:315-19 146. Flajnik, M. F., Du Pasquier, L. 1986. Anti-MHCauto antibodies in Xenopus head-body chimeras. Basel Inst. Ann. Rep. 1986:96-97 147. Hadji-Azimi, 1., Schwager, J., Thiebaud, C. 1982. B lymphocyte differentiation in Xenopuslaevis larvae. Devel. Biol. 90:253-58 148. Hsu, E., Du Pasquier, L. 1984. Ontogeny of the immunesystem in Xenopus. I. Larval immuneresponse. Differentiation 28:109 15 149. Hadji-Azimi, 1., Coosemans,V., Canieatti, C. 1988. B lymphocyte population in Xenopuslaevis. Submitted 150. Kidder, G. M., Ruben, L. N., Stevens, J. M. 1973. Cytodynamics and ontogeny of the immune response of Xenopus laevis against sheep erythrocytes. J. Embryol. Exp. Morph.29: 7385 151. Hsu, E., Du Pasquier, L. 1984. Ontogeny of the immune system in Xenopus. II. Antibody repertoire differences between larvae and adults. Differentiation 28:116 22 152. Jurd, R. D., Luther-Davies, S. M., Stevenson, G, T. 1975. Humoral antibodies to soluble antigens in larvae

of Xenopus laevis. Comp. Biochem. Physiol. 50B: 65-70 153. Manning, M. J., A1Johari, G. M. 1985. Immunological memory and metamorphosis. In Metamorphosis, ed. M. Balls, M. Bownes,pp. 420-39. Oxford: Clarendon 154. Ruben, L. N., Clothier, R. H., Jones, S. E., Bonyhadi, M. L. 1985. The effect of metamorphosis on the regulation of humoral immunity in Xenopus laevis, the South African clawed toad. In Metamorphosis, ed. M. Balls, M. Bownes, pp. 360-87. Oxford: Clarendon 155. Kobel, H. R., Du Pasquier, L. 1986. Genetics of polyploid Xenopus. Trends Genet. 2:310-15 156. Du Pasquier, L., Miggiano, V. C., Kobel, H. R., Fischberg, M. 1977. The genetic control of histocompatibility reactions in natural and laboratorymade polyploid individuals of the clawed toad Xenopus. Immunogenetics 5:12941 157. Du Pasquier, L., Blomberg, B. B. 1982. The expression of antibody diversity in natural and laboratory-made polyploid individuals of the clawed toad Xenopus. Immunogenetics 15:25140 158. Kobel, H. R., Du Pasquier, L. 1979. Hyperdiploid species hybrids for gene mapping in Xenopus. Nature 279: 15758 159. Du Pasquier, L., Kobel, H. R. 1979. Histocompatibility antigens and immunoglobulins genes in the clawed toad: expression and linkage studies in recombinant and hyperdiploid Xenopus hybrids. Immunogenetics 8: 299310 160. DuPasquier, L., Flajnik, M. F., Guiet, C., Hsu, E, 1985. Methods used to study the immune system of Xenopus (Amphibia, Anura). In Immunological MethodsIII, ed. I. Lefkovits, B. Pernis, pp. 425-65. NewYork: Academic 161. Afifi, A., Picard, J. J., Querinjean, P. 1985. A partially histocompatible family of Xenopus borealis. Lab. Amin. Sci. 35:139~J,1 162. Hengartner, H., Du Pasquier, L. 1981. Somatic cell hybrids from frog lymphocytes and mousemyelomacells. Science 212:I034~35 163. Du Pasquier, L., Wabl, M. R. 1977. Transplantation of nuclei from lymphocytes of adult frogs into enucleated eggs: special focus on technical parameters. Differentiation 8:9-19 164. Etkin, L. D. 1986. Gene expression in transgenic Xenopus laevis. Prog. Clin. BioL Res. 217A: I1 16

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165. Wabl, M. R., Brun, R., Du Pasquier, treated Xenopuslaevis. In Proc. 1st Int. L. 1975. Lymphocytes of the toad Colloquium on Patholoyy of Reptiles Xenopus laevis have the gene set for and Amphibians, ed. C. Vago, G. Matz, promoting tadpole development. Scipp. 163-72. Angers: Univ. Angers ence 190:1310-12 168. Balls, M., Clothier, R. H., Rowles, 166. Balls, M., Clothier, R. H., Knowles, J. M., Kiteley, N. A., Bennett, G. W. K. R. 1981. The phylogeny of tumour 1985. TRHdistribution, levels and sigimmunity. Dev. Comp. Immunol. 5 nificance during the development of (Suppl.) 1:37-48 Xenopus laevis. In Metamorphosis,ed. 167. Balls, M., Clothier, R. H., Knowles, M. Balls, M. Bownes, pp. 260-70. K. R. 1983. Tumourincidence in NMU Oxford: Clarendon

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Ann. Rev. lmmunol. 1989. 7:277-307 Copyright © 1989 by Annual Reviews Inc. All rights reserved

MOLECULAR GENETICS OF CHRONIC GRANULOMATOUS DISEASE Stuart

H. Orkin

Department of Hematology, Children’s Hospital, Boston, Massachusetts 02115, and Department of Pediatrics, Harvard Medical School, and HowardHughes Medical Institute INTRODUCTION Chronic granulomatous disease (CGD)is an uncommoninherited disorder in which phagocytic cells (neutrophils, monocytes, macrophages, and eosinophils) fail to produce antimicrobial oxidants (1, 2). Affected individuals have traditionally suffered from recurrent and often lifethreatening bacterial and fungal infections. In fact, upon its recognition as a clinical entity nearly 30 years ago, the disorder was termed "fatal granulomatousdisease" (3), which attests to the severity and progressive nature of the infections often witnessed in CGD patients. Although it is a rare condition, CGDhas been the focal point for research efforts to define the biochemical events that participate in the cellular production of the major oxidant, superoxide anion. The underlying theme has been that identification of the specific defect(s) in the pathway to superoxide generation that characterize the disease would provide an understanding of the normal biochemistry of this important host defense system and, perhaps, suggest new approaches to treatment of the inherited condition or modulation of tissue oxidant damagein inflammatory states. While the physiologic deficiencies of phagocytes from CGDpatients have been recognized for nearly two decades (4), it is only of late that some components of the superoxide-generating system of phagocytes have been fractionated and, more recently, shownto be the primarily affected gene products in CGD.Althoughdefinitive biochemical and genetic solutions to some long-standing questions posed by CGDhave recently been attained, 277 07324)582/89/04104)277502.00

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278 ORKIN fundamental problems remain to be addressed nowwith new tools and perspectives. In this review I summarizethe biochemistryand moleculargenetics of CGDwith particular emphasison those aspects that suggest whichavenues might be pursued in further efforts to understand howthe superoxidegenerating system of phagocytesis assembledand controlled, and howit functions. First, I review the cellular biochemistry of superoxidegeneration, its abnormalities in CGD,and the evident heterogeneity of the condition. Withthis as a background,I then reviewrecent genetic and molecularfindings that haveled to the characterization of specific components, sorted out primary defects from secondary consequences on cellular metabolism,and begunto suggest howparts of the system may be regulated. Finally, I suggest wherenewapproachesthat couple conventional biochemical maneuvers with molecular techniques maybe applied to understand further howthe superoxide generating system of phagocytesis assembledand howit functions. THE CELLULAR BIOCHEMISTRY OF THE RESPIRATORY BURST AND CGD Whenphagocytesencounter bacteria, or other appropriate particulate or soluble stimuli, they undergoa profound metabolic transformation in whichtheir consumptionof oxygenincreases rapidly; soon thereafter, large amounts of superoxide (O~) and hydrogen peroxide (H2Oz) producedand liberated into the surroundingmedium.This process, known as the "respiratory burst," functions to generate the powerfuloxidants that constitute an importantlimb of our antimicrobialhost defense(1, 2). Thegeneration of superoxidereflects the end-productof activation of an otherwise dormantmembrane-bound enzymatic system that catalyzes the one-electron reduction of oxygento O2. In CGDthe failure to produce superoxidecould, in principle, be the consequence of a defect in activation or at a subsequentstep. Theenzymethat catalyzes the electron transfer is generally referred to as the NADPH-oxidase, to reflect its apparentpreference for NADPH, over NADH, as a cofactor. A brief description of various aspects of the superoxide-generatingsystemprovides a framework in whichto consider the componentsthat mightbe defective in CGD. Activation Manydifferent stimuli can activate the respiratory burst. At least two pathwaysof activation appearto exist (5). One,whichis thoughtto involve an interaction with guaninenucleotide-binding(G-) proteins, can be trig-

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gered by binding of the chemotactic peptide N-fmet-leu-phe to its receptor on the cell surface. Alternatively, stimulation by a soluble mediator, phorbol myristate acetate (PMA),is believed to occur via activation of protein kinase C and subsequent phosphorylation of components of the oxidase. Although considerable work now focuses on the relationship of specific G-proteins to the respiratory burst of phagocytes, at present it is not possible to relate functional abnormalities in CGDto any particular G-protein species.

Protein Phosphorylation Upon stimulation of neutrophils with PMA,some dozen or so proteins, from 11-80 kd in size, exhibit changes in phosphorylation, the majority of which show increased labeling (6-11). Establishing that altered phosphorylation of any of these proteins is intrinsically related to the activation pathwayof the superoxide-generating system has been difficult. However, as described below, several polypeptides in the 44-48 kd size class appear to be reasonable candidates for critical components.

MembraneDepolarization One of the earliest recognized events accompanyingactivation is a depolarization of the neutrophil plasma membrane(12). Largely because depolarization is not observed upon stimulation of neutrophils obtained from CGDpatients, it was hypothesized that defective activation of the superoxide-generating system, rather than a defect in the oxidase itself, might account for the physiologic abnormalities in the disease. More recent findings, however, mitigate against this conclusion. For one, the respiratory burst in normal neutrophils can occur even in the presence of agents that inhibit this membrane depolarization (13, 14). In Vitro Activation One of the more promising developments in neutrophil research has been the development of in vitro assays for activation of the NADPH oxidase (15-19). Nearly simultaneously, several laboratories independently reported that arachidonic acid and somedetergents can activate the oxidase activity in cell-free preparations. The dormant, but activatable, oxidase is located in the plasma membranefraction and requires a cytosolic componentfor activation. The nature of this cytosolic factor is poorly understood. Preliminary data suggest that it is a large protein (~ 240 kd) that may dissociate to ~40 kd subunits (17, 20). It does not appear synonymouswith protein kinase C. Induction of differentiation in cultured promyelocytic leukemia HL60cells is accompanied by the appearance of this cytosolic factor activity (21, 22). Whetherthe cytosolic cofactor is

Annual Reviews 280 ’ ORKIN single protein species or a complexof similarly sized (,,~ 40 kd) subunits currently under investigation in several laboratories.

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The Respiratory

Bi~rst

Oxidase

Despite considerable efforts, the enzymatic machinery that performs the one-electron reduction of oxygen to O~ has been one of the components of the superoxide-generating system most refractory to definitive biochemical characterization. Several laboratories have attempted purification of the O7 forming enzymefrom the particulate fraction of neutrophils (1, 23, 24). In some purified preparations major polypeptides 66, 48, and 32 kd were seen under denaturing conditions (24). However, no clear-cut evidence exists, as yet, that these polypeptides are integral components of this enzyme system. Affinity-labelling of a 66-kd polypeptide by NADPH-analogsprovides some tentative supportive evidence for the potential involvementof the similarly sized chain seen in the oxidase preparations (25). Until rigorous purifications of the oxidase are reported and confirmed by several laboratories, it is not possible to discuss the biochemistry of this enzymatic complexin greater detail. Electron

Carriers

and the Neutrophil

Cytochrome

b

Like manyaspects of the cellular biochemistry of the respiratory burst, there has been considerable controversy in the literature regarding the electron carriers involved in catalysis by the oxidase. Althougha detailed discussion of the various possibilities is beyondthe scope of this review, it is sufficient to mentionthat at least three groups--a quinone (ubiquinone50) (26), a flavin (27), and a berne--have been considered as potential carriers. Evidence in favor of involvement of ubiquinone-50 generally has been considered weak (28). An FADrequirement for O7 production particulate preparations in vitro has provided strong support for flavin participation (29). Compellingdata indicates that heine, the prosthetic group for a phagocyte-specific protein, designated cytochrome b55a or b-24~, is involved in superoxide-generation. Although characterization of this protein has proved formidable and controversial, recent data have not only demonstrated its existence but established its structure and critical role in the oxidas~system. The first descriptions of the phagocyte-specific cytochrome appeared in the Japanese literature in the mid-1960s. These reports presented data on horse and rabbit neutrophils (30-32). It was not until more than 10 years later that the observations of Segal & Jones brought this unusual cytochrome to the fore (33, 34). This b-type cytochromehas been observed only in neutrophils, monocytes, macrophages, and eosinophils

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in humans. During in vitro differentiation of HL60cells and monocytic U937cells the abundance of the cytochrome (as determined by spectral measurements) increases. In normal neutrophils the cytochrome b is particularly abundant, roughly 100 pmol per mgof protein (35). The midpoint potential, the point at which it is balanced between oxidation and reduction, is the lowest for this cytochrome (-245 mV)of any knownb-type cytochrome. This property has suggested that it maybe the terminal electron donor in a short electron transfer chain and maydirectly reduce oxygen to superoxide (36, 37). Since it displays an absorption band at 558 nm, the cytochrome has alternatively been designated cytochrome b558. Hereafter in this review, we refer to this protein as the neutrophil cytochrome b. Absence of the cytochrome spectrum in the majority of patients with CGD(see below) first led Segal and his associates (35) to propose that the cytochrome was important to normal superoxide generation and that abnormalities of the cytochromewere, in fact, likely to reflect primarydefects in this disorder. Purification of the neutrophil cytochromeb has been a formidable task, accomplishedonly recently. Initial estimates of its size ranged from 11127 kd (38~t 1). The disparate sizes reported were in part the consequences of proteolysis, anomalousmigration of some proteins on acrylamide gels, and difficulties involved in distinguishing relevant polypeptides from contaminating species. Even before purification of the cytochrome was attained, Harper and colleagues recognized that the cytochrome was, in fact, a glycoprotein, on the basis of its staining with periodic acid, lectin binding, and reduction in apparent size following treatment with endoglycosidase F (42). Deglycosylated cytochrome appeared to migrate as 50-55 kd species. Within the past two years the laboratories of Segal & Jesaitis independently achieved purification of the neutrophil cytochromeb (43, 44). Surprisingly, the protein was found to be composedof two subunits, a 90kd glycosylated heavy chain (corresponding to the glycosylated cytochrome first noted by Harper et al; 42), and a 22-kd nonglycosylated chain. These subunits are tightly associated. Dissociation is achieved only upon harsh treatment (heat and SDS), conditions under which the heme spectrum is lost. Because of this close association of the componentsof the cytochrome b heterodimer, it has not been possible to determine by biochemical criteria whether the heme prosthetic group is primarily associated with one or the other subunit. Immunoprecipitation and hydrodynamicstudies have suggested that the subunits are associated as heavylight chain heterodimers (45). The structures of the cytochromeb subunits are described in greater detail below.

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INHERITANCE OF CGD

AND GENETIC

HETEROGENEITY

Fromthe earliest descriptions of the disorder it wasapparent that the majorityof affected families display X-linkedtransmission(1). In carrier females, mosaicismof neutrophil function, as revealed by a histochemical stain for oxidase activity (the NBTtest) (46), can be demonstrated, sistent with the Lyonhypothesisof randominactivation of one X-chromosomein each cell (47). DNAlinkage data and the cloning of the gene involved in the X-linked form of CGD have provided definitive evidence, as described below. In the overwhelmingmajority of patients with Xlinked CGD(X-CGD)the spectrum of the neutrophil cytochrome b absent, although rare patients with partial, or even complete, spectral activity have been described. X-CGD in which the spectral activity is absent is generally designated X-, whereasthe variety with cytochrome +. spectrumis X Autosomalrecessive inheritance of CGDis nowwell established and accounts for perhaps 25-35%of affected families (48, and see 1). The occurrenceof CGD in femalesfirst led to the discoveryof this formof the disorder. Themajority of autosomallyinherited CGDis of the cytochrome b positive variety, A+ (49, 50, 51); although a less common subtype in which the cytochromespectrum is absent (A-) (18, 52) is also documented. Althoughbiochemicaland genetic criteria can nowbe used quite effectively to classify formsof CGD,as presentedin moredetail below,elegant demonstrationof distinct subtypes of the disorder wasprovided several years ago by complementationanalysis in somatic cell hybrids derived from monocytesof affected individuals (52, 53). Hybridsformedbetween X- and A+ monocytesexhibit functional complementation, as assayed by the NBTtest, whereasX cells of one patient cannot complementXcells of other individuals. Furthermore,two forms of autosomaldisease could be distinguished, as X- and A cells were also seen to complement each other. However,a more prolonged period of culture was required before functional reconstitution was apparent. The complementationof X- and A- monocyteswas interpreted as evidence that two genes, one of X-chromosome origin and the other located on an autosome,are necessary for ultimate expression of the neutrophil cytochromeb. This conclusion can nowbe refined in light of morerecent biochemicaland moleculardata, as discussed below. In addition to the broad subtypesnoted above, patients with "variant" forms of disease havebeen described. Thesepatients generally havepresented with a milder clinical course, usually evident frompresentationat

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a later age, and often with a NADPH oxidase that exhibits kinetic alterations in cell preparations (50, 51, 54, 55). Neutrophils of these patients have generally produced small but measurable amounts of superoxide uponactivation, in contrast to those of"classical" patients that produce no superoxide. "Variant" patients maybe either of the X-linked or autosomal variety. The existence of "variant" CGDpatients underscores the clinical heterogeneity of the disorder. Although other explanations have been entertained in the past, it is increasingly apparent that someof these "variant" patients probably have gene defects that are not as severe in a functional sense as those found in more "classical" patients. Again, additional evidence in favor of this view is presented below.

BIOCHEMICAL DEFECTS IN CGD: CANDIDATES FOR THE PRIMARY GENE PRODUCTS A myriad of biochemical abnormalities have been described in neutrophils from CGDpatients. In a complex cellular system, such as that which generates superoxide, it is difficult to establish conclusively the role of specific constituents without suitable cell-free reconstitution assays and/or information contributed by genetic approaches. The problems inherent in dissecting the componentsof the oxidase system and in proving the functional relevance of any purified (or semi-purified) species, and the disparate abnormalities reported for CGDphagocytes, conspired to render conclusions regarding any one finding controversial and often confusing. Clearly the most prominent candidate for the primary protein product affected in CGDhas been the neutrophil cytochrome b, first shown by Segal and coworkers(35, 56) to be absent at the spectral level in the vast majority of X-CGD patients. Difficulties in achieving purification of the cytochrome (38-41), questions regarding its kinetics, and the apparent absence of heme from enriched preparations of putative respiratory burst oxidase (24) contributed to doubts as to whether the spectral abnormalities in the disorder reflected a primary or secondary defect. The more recent purification of the neutrophil cytochrome b by Segal & Jesaitis and their associates provided evidence that both the 90-kd glycosylated and 22-kd nonglycosylated subunits were lacking at the protein level in X-CGDphagocytes (43, 44). While this finding may have strengthened the association of the cytochrome b with the X-CGDphenotype, quantitative deficiency of two subunits, not apparently related in a precursor-product manner, made it impossible to conclude on biochemical grounds alone where the genetic defect(s) in X-CGDresided. Although the defects might be located within the gene(s) for either (or both) subunits (the latter possibility being extremely unlikely), an equally plausible

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ORKIN

hypothesis might involve deficiency or abnormality of an unrelated protein, whoseintegrity is necessary for proper assembly or stability of the cytochrome b heterodimer. The description of other biochemical defects in CGDphagocytes, either of the X-linked or autosomal variety, presented alternative candidate molecules in virtually all disease classes. Since activation of the dormant NADPH oxidase is a critical step in superoxide generation, manyinvestigators have focused on the possible role of protein phosphorylation in this process and potential disturbances in CGD.Particular attention has been paid to a cluster ofpolypeptides, 44~48kd in size, that becomerapidly phosphorylated upon stimulation of neutrophils with phorbol esters or during activation in cell-free preparations. Segal and associates first reported failure of phosphorylation of a 4448 kd protein in autosomal recessive, but not X-linked, CGDpatients (11, 57, 58). Others, using two-dimensional rather than single dimension acrylamide gel analysis, have described changes in both X- and autosomal CGDneutrophils, but more limited alterations in the X-linked variety (7). Finally, others have failed to observe alterations in either disease class (59). Nonetheless, the emergingconsensus is that changes in protein phosphorylation accompanyactivation and that alterations in such events do occur in CGDneutrophils. However, again, it has not been established whether the observed phosphorylation disturbances in CGDreflect primary defects or secondary consequences of other underlying quantitative or qualitative protein abnormalities. In the case of X-CGD,based on the evidence to be reviewed below, we must conclude that any alterations in phosphorylation of 44-48 kd polypeptide are secondary phenomena. In light of this, we must reserve judgment as to whether the apparent failure of phosphorylation of a 44-48 kd polypeptide in autosomal CGD reflects deficiency or a structural defect of the substrate (the 44 kd polypeptide) or merely indicates a more proximal defect that ultimately results in a change in phosphorylation. Although available data suggest that protein kinase C itself is not abnormalin autosomal CGD,this conclusion also must be considered tentative in light of the increasing complexity of protein kinase C isoforms that have been described (60). Howthese 4448 kd polypeptides are related (if at all) to the cytosolic factor activity defined in cell-free, reconstitution assays is uncertain. Reconstitution assays using purified 44-48 kd protein species have not yet been reported. These could serve to more strongly associate specific protein deficiencies and functional deficits. Recently a protein species of apparent size 40-47 kd that is rapidly phosphorylated by protein kinase C in activated platelets h/is been characterized by cDNAcloning (61). As this protein is also expressed

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promyelocytic HL60cells that are induced to differentiate with retinoic acid, we maywonderwhether it, or a related species, is in actuality one of the neutrophil componentsthat,iszrapidly phosphorylated upon activation. A direct analysis of these issues is nowpossible. Fromthe different isoelectric points observed for the platelet and neutrophil species, however, it is likely that they are not closely related (J. Curnutte, personal communication). Given its postulated role in superoxide-gene~ation,the respiratory burst oxidase (or flavoprotein) has also been considered as the primarily affected product in CGD(62). However, in view of the incomplete biochemical characterization of this enzyme(or enzymecomplex), no investigations protein abnormalities in CGDneutrophils have been reported. In sum, from biochemical data alone the primary protein defects in CGDhave been difficult to assign with confidence. In the X-linked variety of disease, the neutrophil cytochromeb, as proposed by Segal and associates (35), constituted an excellent candidate, although recognition that two subunits associate to form the cytochromedid not immediatelyclarify the situation from a genetic perspective. In the major autosomal form of the disease, the 44-48 kd phosphorylated polypeptides have emerged as candidates for the primarily affected proteins; however, the complexity of the system makesalternative models~plausible.

MOLECULAR APPROACH TO THE X-CGD LOCUS In light of the problems inherent in defining the .critical proteins of a complex cellular system without availability of each purified component and a suitable in vitro reconstitution assay, a molecular genetic approach to unraveling the basis of CGDin its various forms has fundamental advantages. While such an approach has led to specific, definitive conclusions regarding some of the polypeptides described above in their relation to CGD,it has also provided reagents that mayprove useful in addressing someof the manyquestions nowevident in the normal cellular biochemistry of the superoxide-generating system. Chromosomal Location of the Gene for X-CGD As with manyfindings relating to the primary ~basis, of CGD,the initial assignment of the locus for X-CGDto the distal portion of the short arm of the X-chromosomefrom limited family segregation data has been revised with the introduction of newtechniques for analysis. Studies in the pre-DNAlinkage era reported evidence for linkage of CGDto the blood group antigen Xg, a distal Xp marker (situated in Xp22.3-Xpter) (63). addition, the reported association of CGDwith the absence of Kell-related

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286 ORKIN antigen Kxas well as its linkage to Xg(64, 65) seemedto strengthenthis assignment.Absence of the Kxantigen on red cells results in acanthocytosis and a mild hemolytic anemia, designated the McLeodphenotype. Althoughinitial reports of CGD frequently described association with the McLcodphenotype (63, 66), possibly suggesting a commonmembrane defectin red andwhitebloodcells, it is nowevidentthat only rare patients haveboth phenotypes(67) [a point discussedbelow]. By 1984 it had becomeapparent that the assignment of the X-CGD locus to distal Xpwasinconsistent with cytogeneticdata derived at first from two rare cases studied by Franckeand her associates. In the first instance, a womanwith mild mental retardation and heterozygosity for CGDwas found to be heterozygous for a deletion within chromosomal bandXp21(68). Asecond patient [B.B.] whowasafflicted simultaneously with Duchenne muscular dystrophy (DMD),CGD,McLeodsyndrome, and retinitis pigmentosahad an interstitial deletion of Xp21involving perhaps 5 million basepairs of DNA(3-5 megabases)(69-71). Both these cases, taken together with another patient [N.F.), whohad DMD, McLeod syndrome,and CGDin association with an interstitial deletion of Xp21(72), suggested a more proximal location of X-CGD gene than that suggestedby linkage to Xg. In as muchas complex,but unseen, cytogenetic rearrangementsmight be invokedto explain the apparent Xp21location of the gene in the face of earlier evidencefor linkage to Xg, a formalgeneticlinkage analysis was performedin typical X-CGD families (72), using a collection of cloned probes that recognizerestriction fragmentlength polymorphisms (RFLPs) along Xp. DNA probes within Xp21(designated p754 and PERT84), but not probes moredistal or Xgitself, were found to be linked to the CGD locus (72). Thecalculated Lodscore for linkage to p754was 3.7, which indicates nearly a 10,000: 1 chancethat the X-CGD locus is nearbyrather than elsewherein the genome.In addition, these linkage data, combined with DNAmappingdata of Kunkel & Monacoobtained in DMD patients (73), indicated that the X-CGD locus resided proximal(or centromeric) to the DMD gene (72). Onthe basis of cytogenetic and DNA linkage data therefore, the X-CGD locus was reassigned to Xp21. In addition, no evidence has been obtained that suggests the existence of more than a single locus on the X-chromosome. Identification of the Gene on the X-Chromosome That Is Defective in CGD The assignment of the X-CGD gene to Xp21 within the BBdeletion narroweddownthe area in which the gene resided to about 0.1%of the human genome, roughly 5 megabases, at a minimum.Because major

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questions existed regarding the nature of the protein primarily affected in X-CGDand because adequate biochemical reagents were lacking for any of the candidate proteins, a direct approach to identification of an RNA transcript or DNAsegment of the relevant gene was attempted (74). pool or radioactive cDNA,derived from reverse transcription of mRNA from differentiated HL60cells, was enriched for granulocytic sequences by hybridization subtraction with RNAfrom the B-cell line of a patient NF with an Xp21 deletion. The enriched cDNAwas used directly as a probe in Southern blot hybridization to screen a collection of genomic bacteriophage clones derived from Xp21 which had been isolated by Kunkel and coworkers (75, 76) in their search for the DMDlocus. Two independently isolated, but overlapping, bacteriophage clones reacted with the pooled cDNAprobe and thereby identified a region of Xp21 transcribed in differentiated HL60cells (74). With the portion of the bacteriophage clones that hybridized as a probe, the specific mRNA present in these phagocytecells was identified. The RNAtranscript detected in Northern blot analysis is 4.5 kb in length and is expressed specifically in differentiated HL60cells, normal neutrophils, and monocyte/macrophages, and at a lower level in EBVtransformed B lymphocytes(74). Undifferentiated HL60cells contain little of this mRNA.Cells of nonphagocytic lineages are devoid of the mRNA. Strong evidence in support of the relevance of this RNAtranscript to XCGDwas provided by Northern blot analysis of monocyte RNAisolated from patients with X-CGD. In the first four classical (i.e. cytochromebnegative) X-CGD patients so examined, three revealed markedly deficient mRNA(74). Formal genetic criteria more firmly established the RNAtranscript as that derived from the X-CGDlocus. In the only mRNA-positivepatient amongthe first four studied, a relatively small interstitial deletion (74) (of about 1 kb) removed the coding capacity for the C-terminal 41-amino acids of the protein predicted from the cDNAsequence (S. H. Orkin, unpublished data). That this deletion was entirely contained within the transcribed region of the gene and removed a segment of the predicted protein constitutes a genetic proof that the putative X-CGD transcript is from the relevant locus. Additional biochemical evidence noted below provides confirmation of this conclusion. The Predicted

X-CGD Protein

The complete X-CGDcDNA is 4.27 kb in length and contains a single open reading frame encoding 571 amino acids (including the initiation methionine) (calculated 65-kd polypeptide), which is displayed in Figure 1. The initiator codon in the first published report was

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Annual Reviews 288 ORKIN

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not correctly identified due to a DNAsequencing error in the 5’-untranslated region (77). Of particular note and unknown significance, if any, is the short length of the 5’-untranslated region of the X-CGDmRNA (12 nucleotides). What conclusions could be drawn from the predicted sequence of the X-CGD protein? Unfortunately, surprisingly few. As shown in Figure 2, the open-reading frame predicts five potential N-glycosylation sites and at least three substantial hydrophobic domains. Together, these features suggested that the encoded protein was a membraneglycoprotein. Searches of the GenBankand Protein Database failed to reveal significant similarity to other knownproteins or genes. In particular, it has not been possible to discern any similarity to knowncytochrome species or to identify a potential heme-binding motif. On the basis of the computer searches it could only be speculated initially that the X-CGDgene encoded a membrane glycoprotein that might be involved in the assembly of the cytochromeb but was unlikely to be the cytochromeitself.

THE PRODUCT OF THE X-CGD LOCUS AND THE NEUTROPHIL CYTOCHROME B: RELATIONSHIP, STRUCTURE AND EXPRESSION Identification of the X-CGDProtein as the 90-kd Subunit of the Cytochromeb Twoindependent approaches, however, established that the predicted XCGDprotein is synonymouswith the larger subunit of the cytochrome b. First, antisera raised either to a synthetic peptide directed to 20 amino acids near the C-terminusof the predicted protein or to a fl-galactosidase fusion protein reacted with a 90-kd glycoprotein in normal neutrophils, as well as the larger subunit of purified cytochromeb (77). The antisera also reacted with the deglycosylated (roughly 55-kd) form of the large subunit.

5’

N N-glgcosyletion

Sites

Hgdrophobic Domains ¯

ORF e ee e

~

C e

( PolyT-TTTATT

¯

0.5 kb 2 Structureof X-COD cDNA and predictedprotein. Theopenreadingframe(ORF) is shown bythe hatchedbox. FixTure

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290 ORKIN Second,directly determinedN-terminalaminoacid sequencesof the 90kd chain matchedthe sequence predicted from the corrected cDNA sequence(78). In the vast majorityof X-CGD patients studiedto date, the 90-kdsubunitis virtually absentin Western blot analysis of neutrophils (77).

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Structure of the 22-kd Subunit of the Neutrophil Cytochrome b The 90-kd X-chromosome encodedsubunit is tightly associated with a nonglycosylated 22-kdpolypeptide(43, 44). Theprimarystructureof the 22-kdchain has morerecently been determinedby cloning of its cDNA via immunoscreening of a 2gt 11 library (79). DirectN-terminalaminoacid sequencingconfirmedthe authenticityof the cDNA.Thepredictedprimary translationproduct,depictedin Figure3, contains195 aminoacids; the

GCAGTGTCCCAGCCGGGTTCGTGTCGCCATGGGGCAGATCGAGTGGGCCATGTGGGCC~ ~etGlyGlnlleGluTrpAl~e~TrpAl~As CGAGCAGGCGCTGGCGTCCGGCCTGATCCTCATCACCGGGGGCATCGTGGCCACAGCTGG nGluGlnAlaLeuAl~SerGlyLeuIleLeuIleThrGlyGlyIleV~iAl~ThrAlaGl GCGCTTCACCCAGTGGTACTTTGGTGCCTACTCCATTGTGGCGGGCGTGTTTGTGTGCCT yArgPheThrGlnTrpTyrPheGlyAlaTyrSerIleValAlaGlyValPheValCysLe GCTGGAGTACCCCCGGGGGAAGAGGAAGAAGGGCTCCACCATGGAGCGCTGGGGACAGAA uLeuGluTyrProArgGlyLysArgLysLysGlySerThr~etGluAr~TrpGlyGlnLy GCACATGACCGCCGTGGTGAAGCTGTTCGGGCCCTTTACCAGGAATTACTATGTTCGGGC sHisMetThrAlaVaiValLysLeuPheGlyProPheThrArgAsnTyrTyrValArgA1 CGTCCTGCATCTCCTGCTCTCGGTGCCCGCCG~CTTCCTGCTGGCCACCATCCTT~GGAC aValLeuHisLeuLeuLeuSerValProAlaGlyPheLeuLeuAlaThrlleLeuGlyTh CGCCTGCCTGGCCATTGCGAGCGGCATCTACCTACTGGCGGCTGTGCGTGGCGAGCAGTG rAl~CysLeuAl~IleAl~SerGlyIleTyrLeuLeuAl~Al~V~lArgGlyGluGlnTr GACGCCCATCGAGCCCAAGCCCCGGGAGCGGCCGCAGATCGGA~GCACCATCAAGCAGCC pThrProlleGluProLysProArgGluArgProGlnlleGlyGlyThrlleLysGlnPr GCCCAGCAACCCCCCGCCGCGGCCCCCGGCCGAGGCCCGCAAGAAGCCCAGCGAGGAGGA oProSerAs~ProProProArgProProAl~GluAl~ArgLysLysProSerGluGluG1 GGCTGCGGCGGCGGCGGGGGGACCCCCGGGAGGTCCCCAGGTCAACCCCATCCCGGTGAC uAlaAl~Al~AlaAlaGlyGlyProProGlyGlyProGlnValAsnProIleProYalTh CGACGAGGTCGTGTGACCTCGCCCCGGACCTGCCCTCCCACCAGGTGCACCCACCTGCAA rAspGluValValEnd TAAACGCAGCGAAGGCCGGGAAAAAAA Figure3Deducedproteinsequenceofthecytochrome b 22 kd subunit

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sequence is particularly notable for a proline-rich C-terminus (27%of the 63 C-terminal residues). Three or four hydrophobic regions appear to precede the proline-rich tail. Computeranalysis has revealed no overt similarities to other proteins, including a variety of cytochromespecies. Althougha short stretch of similarity (29% identity over a 31-amino acid region) to polypeptide I of mitochrondrial cytochrome c oxidase was identified, its significance is unknown. Therefore, the phagocyte cytochrome b appears to be a unique cytochromespecies in that it is not overtly related to previously studied cytochromes. In light of this circumstance, the location of the hemeprosthetic group(s) in the cytochrome b heterodimer remains unknown. Howthe neutrophil cytochrome b is positioned in the plasma membrane and specific granules is currently poorly understood. On the basis of a cluster of potential N-glycosylation sites near its N-terminus, it has been proposed that an external domainof the 90-kd glycoprotein is likely to exist (80). Consistent with this notion, a monoclonalantibody to the 90kd subunit reacts with the external surface of neutrophils (81). In addition, endoglycosidase F cleaves N-linked carbohydrates from the cytochrome at an extracellular site (82). Preliminary data also suggest that the extreme C-terminus of the 90-kd subunit may lie on the cytoplasmic face of the plasma membrane(82). In as muchas deletion of the C-terminal 41-amino acids of the 90-kd polypeptide in an X-CGD patient leads to characteristic findings of the disease (74) [i.e. absence of both protein subunits], it likely that the C-terminus plays an important role in either cytochromeb assembly or interaction with the 22-kd subunit. Preliminary cell-free assay findings have also been interpreted as consistent with an important functional role for the C-terminusof the 90-kd protein (82). Reyulation Subunits."

of mRNAs Encodin# the Cytochrome Implications.for Assembly

b

As noted above, the mRNA transcript for the 90-kd subunit is expressed in a highly lineage-specific manner (74). In neutrophils, monocyte/ macrophages, and presumably eosinophils, the mRNAis particularly abundant, perhaps accounting for 0.1% or more of cellular mRNA. In addition, the mRNA is inducible during in vitro differentiation of cultured HL60cells along either a granulocytic or monocytic pathway. A low level of mRNA is detectable in normal EBV-transformed B-cells, which are reported to have low levels of NADPH-oxidase activity (83, 84). Although the pattern of expression of the 90-kd subunit mRNA mirrors the distribution of neutrophil cytochromeb and oxidase activity in various cell types, that of the 22-kd subunit does not. Surprisingly, the 22-kd subunit mRNA, which is derived from an autosomal locus (M. C. Dinauer,

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292

ORKIN

S. H. Orkin, unpublisheddata), is present in a wide variety of cell lines in abundance comparable to that in phagocytic cells. However, although the mRNA is constitutively expressed, non-phagocytic cells contain very little if any stable 22-kd light chain polypeptide. These observations seem most compatible with a model in which the 22-kd subunit may be unstable intracellularly in the absence of the 90-kd glycosylated chain. Overall, the appearance of the cytochromeb heterodimer is temporally correlated with expression of the 90-kd subunit mRNA.Although the rationale for this regulatory scheme, rather than coordinated expression of the individual subunits, is obscure, it implies that the 90-kd glycoprotein may serve a critical role in directing the 22-kd polypeptide into the functional membrane complex (79). Since neither cytochrome b subunit has a cleavable N-terminal signal peptide, targeting to the membranecompartment must involve internal signal sequences. A likely possibility is that the 22-kd subunit is anchored into the membranecomplex via its tight interaction with the 90-kd chain. Modulation of Expression mRNA by Interferon-~

of the 90-kd Subunit

Macrophage functions can be augmented by treatment with the lymphokine interferon-~, the major constituent of macrophage-activating factor (85). Specifically, treated macrophagesacquire the capacity to generate ir~creased amountsof superoxide and to kill microbes more efficiently (8689). Biochemicalcorrelates of this phenomenonof macrophageactivation are largely unknown. Recently it has been observed that recombinant interferon-~ induces the level of 90-kd subunit mRNA in normal cultured monocytes, neutrophils, and promonocytic leukemic cell lines, such as THP-1and U937 (90). In cultured promonocytie THP-1cells the level induction is approximately 10-fold. The abundance of mRNA encoding the 22-kd subunit is virtually unchangedupon interferon-~ treatment; this is consistent with its independent regulation. Nuclear run-on eperiments suggest that transcriptional regulation plays a major role in the induction of the 90-kd subunit mRNA (90). Although interferon-7 undoubtedly has diverse effects on macrophageconstituents, it is reasonable to infer that induction of the 90-kd subunit mRNA may contribute to increased availability of functional cytochromeb and ultimately to augmentedsuperoxide production, mRNA in the 90-kd subunit is the first defined componentof the superoxide-generating system regulated in a physiologically appropriate mannerduring macrophageactivation induced by interferon-~,. Recently Cassatella et al (91) have confirmed the induction of 90-kd subunit mRNA by interferon-7 in cultured HL60, U937, and ML3cells, and they have shown that the induction is insensitive to inhibition of

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OF CGD

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protein synthesis. Therefore, it is likely that modification of pre-existing nuclear factors mediate the transcriptional increase in 90-kd subunit mRNA expression. Tumornecrosis factor (TNF) also induces an increase in specific mRNA in cultured HL60,U937, and ML3cells (91). In association with increases in 90-kd subunit mRNA upon interferon- 7 or TNF treatment, the amount of spectrally determined cytochrome b increased (91, 92).

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Structure of the X-CGDGene Although the detailed physical organization of the X-CGDgene has not been reported, several general features can be briefly summarized. The transcribed region (i.e. that represented by the cDNA)encompasses approximately 25-30 kb of genomic DNAisolated in several overlapping bacteriophage clones. The last 41 amino acids of the encoded protein and the 2.5 kb T-untranslated region of the mRNA are included in the last exon. The remaining 12 exons are more conventional in size. As noted above, the 5’-untranslated region is unusually short. The cis-acting DNA sequencesthat mediate lineage-specific expression of the gene or its responsiveness to interferon- 7 have yet to be identified. Consensus sequences for interferon-responsive elements (93-96) are not evident in genomic sequences (S. Orkin, unpublished data). No restriction fragment length polymorphismsof the gene or its immediate flanking regions have been identified in a search with more than 40 different enzymesusing several genomic and cDNAfragments as probes (S. C. Goff, S. H. Orkin, unpublished data). Therefore, the locus appears to be highly conserved.

THE MOLECULAR BASIS

OF CGD

On the basis of the data reviewed herein it is nowpossible to begin to discuss the various forms of CGDin molecular genetic terms.

X-CGD(X-, X- Variants,

and +)

The commonest X-linked form of the disorder, designated X- [for Xlinked, cyt b-negative], is caused by mutations in the gene encoding the 90-kd cytochrome b subunit. At present, there is no evidence from DNA linkage data or from analysis of patient material with the cloned cDNA that morethan a simple Xplocus, that within Xp21,exists. Th¢,,d~fe.c, ts ~in the gene are heterogeneous. Deletion of the entire gene has,been, observed in rare patients with complex phenotypes, such as BB .and NF who~had CGD, DMD,McLeodsyndrome, with or without retinitis pigmentosa, and others that presented with CGDin association with McLeodphenotype alone (see below) (69, 72, 74). ,Partial :gene deletion that predicts

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synthesis of a truncated protein has also been described (74). In the majority of X-CGD patients, however, the gene encoding the 90-kd subunit appears grossly normal by conventional Southern blot analysis (74). these instances, mRNA is often, but not always (97), markedly deficient in phagocytes, presumably due to mutations that alter RNAtranscription, mRNA processing, and/or mRNA stability. In the vast majority of patients both cytochromeb protein subunits are absent in phagocytes (43, 44, 79). Variant X-CGDpatients have been described who have either residual detectable superoxide-generation (so-called variant X- disease) or linked disease in which the cytochrome spectrum is present at normal or (X+) (50, 55, 98, 99). Althoughmore studies need to be executed, patients of the former subgroup often appear to have exceedingly low, but detectable, levels of 90-kd subunit mRNA (100). These patients are formally analogous to patients with hemoglobinopathies termed/~+-thalassemia in which a reduced amount of normal/%globin mRNAis produced (101). Individuals with the X+ phenotype will most likely be found to have single amino acid substitutions in the cytochrome b heavy chain that interfere with its electron transfer function, but not with heterodimer assembly, homebinding, or stability. Additional investigation is needed, however,to provide direct evidence for these suppositions. A--CGD Although they are very rare, patients with autosomally inherited cytochrome b-negative CGD(A) have been identified (52, 102). By Western blot X -patients analysis, these patients have a protein phenotype that is indistinguishable from the X--patients: both the 90-kd and 22-kd subunits are lacking in phagocytes (M. C. Dinauer, J. Curnutte, S. H. Orkin, unpublished data). By virtue of its autosomal location and the intimate association of the two subunits of the cytochromeitself, the gene encoding the 22-kd subunit is the likely site of mutations leading to A--CGD. Preliminary data (M. C. Dinauer, S. H. Orkin, unpublished) are consistent with this hypothesis, in that we have studied one A--CGDpatient whose monocytes lack 22-kd mRNA,but not mRNA for the 90-kd chain. Alternatively, A -CGDcould result from mutations in regulatory proteins that primarily affect expression of the 90-kd subunit gene in trans. In such instances, one would anticipate normal levels of 22-kd mRNA and deficiency of 90-kd mRNA in the face of autosomally inherited disease. Currently, the molecular basis of autosomal cytochrome b-- positive disease (A+) is not formally established. Polypeptides of 44-48 kd that are rapidly phosphorylated upon neutrophil activation are potential candidates for the proteins primarily affected. Evidence is already emerging

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that autosomal, cytochrome b-positive CGDmaybe a heterogeneous entity. Specifically, cell-free complementation of cytosolicfractions of two different A÷-CGD patients has been reported in preliminary form (103). Purification and cloningofcDNAs for the 44-48kd species will be required to characterizethe relevant proteins. X-CGD in Association with McLeod Syndrome The McLeodSyndromeis rarely seen in association with Duchennemuscular dystrophy (DMD)and/or CGD.Whensuch complex phenotypes are evidentin affectedindividuals,it is inferredthat deletionof the relevant Xp21loci is the molecularbasis. Withthe development of specific probes for the DMD and X-CGD loci and also for anonymoussequences distributed throughout other regions of Xp21, DNAdeletions have been readily established by Southernblot analysis (69, 73, 74). Examination a series of McLeodpatients (with and without DMD or CGD)with specific DNA probes has led to construction of a deletion mapof Xp21 (displayedin Figure4) in whichthe McLeod locus is deducedto lie between the DMD and CGDloci, but within approximately 500 kb of the latter (73). The existence of McLeodpatients whodo not have DMD or CGD or evident DNA deletions demonstratesthat a distinct McLeod locus must be present in Xp21. In two patients with X-CGD and McLeodsyndrome the CGDlocus is completelydeleted (73, 104). It is inferred that the McLeodlocus is also removedby a chromosomal deletion in these individuals. Theprecise nature of the McLeod locus protein product is uncertain, althougha 37-kdprotein (the Kxantigen) is a candidate(73). The molecular bases of various forms of CGDare summarizedin Table i. OTC m

COD l’Ic Leod mmmm~m

OTC

CGD

CX5.7

DI"ID III 754 p84

p87

GENE LOCI L1

cen BBdeletion

I

I

I

OHdeletion (CGD,HcLeod)

Figure 4 Organization of the Xp21region and the relative position of gene loci. The centrometic(cen) andtelomeric(tel) directions are indicated. Thetop line and solid boxes indicate genetic loci. OTC= ornithine transcarbamylaselocus. In the middle, probesfor specific loci and anonymous sequencesare shownwith downward arrows. Thepositions of deletions in two patients BB(Ref. 69) andOM(Ref. 73, 104) are depicted.

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Clinical genetic subtype

Cytochromeb (spectrumor protein)

X : classical "’variant"

Absent Very reduced

+ X

Normalor reduced

A

Absentor greatly reduced

÷ A

Normal

X-CGD in association Absent with McLeod syndrome ( + DMD)

Primarygene defect 90 kd cyt b subunit 90 kd cyt b subunit (heterogenous:transcription, RNAprocessing, RNA translatability) presumed90 kd cyt b subunit (presumedmissense) probably22 kd cyt b subunit (likely to be heterogenous:? possible existenceof regulatorydefects in trans to genefor 90 kd subunit) ?? 44M8kd polypeptide(s) cytosolicfactor(s) deletion of 90 kd cyt b subunit gene

CLINICAL IMPLICATIONS OF IDENTIFICATION OF THE X-CGD GENE Diagnosis The reduction of NBT(46) by neutrophils or related assays of superoxide production are generally sufficient to diagnose CGDand the carrier state for X-CGDin females. As with many other genetic disorders, availability of molecular reagents could greatly facilitate prenatal diagnosis of XCGD.This would obviate the use of fetal blood sampling (105) for prenatal diagnosis. Either RFLPs or detection of specific gene defects in fetal DNA obtained by chorion villus biopsy or animocentesis could provide the means to a definitive diagnosis for families at risk (106). Where a complete or partial gene deletion underlies X-CGD, prenatal diagnosis can be applied quite simply using Southern blot analysis alone. In the family at risk for CGDdue to the interstitial deletion that removed the terminal 41amino acids of the 90-kd subunit, DNAanalysis has been employed to demonstrate that a subsequent male fetus was unaffected (S. Orkin, J. Curnutte, unpublished data). Unfortunately, however, most families at risk for X-CGDdo not have DNAabnormalities that are detectable in this manner (74). In addition, RFLPs within or flanking the X-CGDgene have not been identified (S. C. Gott, S. H. Orkin, unpublished data). In that X-CGDis essentially an

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X-linked lethal disorder in genetic terms (until recently whentreatment has improved), one would expect extensive heterogeneity in the specific mutations leading to gene dysfunction, in part based on the hypothesis of Haldane. With new methods for detection of point mutations in genes, coupled with convenient techniques for in vitro amplification of specific gene segments(107), it is reasonable to anticipate that prenatal diagnosis may be accomplished in some instances by identification of a specific family-based mutation. Treatment

of

CGD

The use of chronic antibiotic prophylaxis in the past decade has led to a marked decrease in the incidence of life-threatening infections seen in patients with CGD(108). An improved understanding of the critical components of the NADPH-oxidasesystem and development of molecular reagents raise newpossible strategies for consideration. PHARMACOLOGIC MANAGEMENT-INTERFERON-)Y The recognition that the lymphokine interferon-~ augments superoxide production from phagocytic (25, 109, 110) cells and induces mRNA encoding the 90-kd cytochrome b subunit has led to an evaluation of this agent in various forms of CGD (90, 91). Initially, phagocytic cells harvested from patients were examined for their response to interferon- 7 in vitro (100). In several patients with classical X-CGD,characterized by absent cytochrome b spectrum and superoxide production, no in vitro improvement in NBTreduction in either neutrophils or macrophageswas noted. In patients with "variant" X-CGD,in which baseline superoxide production was measurable (1-10% or normal), interferon-~ treatment has led to relatively consistent increases in NBTreduction and superoxide production. The results obtained with phagocytes of two brothers were particularly informative, as treatment was associated with a restoration of superoxide production to 30-50%of normal (100). Accompanyingthis improved function was induction mRNA for the 90-kd subunit from nearly undetectable levels to about 35% of normal. In light of promising in vitro findings preliminary studies have nowbeen performed in CGDpatients on the effects of interferon-~ administered in vivo. In a Boston study (111) the "variant" X-CGDpatients (who responded in vitro) and a classical X-CGDpatient (who did not) were given subcutaneous injections of interferon-~ at 0.1 mg/mgon two consecutivc days. Dramatic responses were noted in the two brothers with "variant" disease who displayed a partial correction in vitro. In vivo treatment led to restoration of superoxide production to normal levels within’3-5 days. Surprisingly, normal function persisted for nearly one

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298 monthdespite subcutaneousinjection of interferon-~ limited to the first two days of the trial. In associationwith increasedsuperoxideproduction, in vitro bacterial killing of Staph. aureusreturned towardnormal,cytochromeb spectral measurementsimprovedfrom less than 10%to approximately 20-30%of normal, and the 90-kd subunit (as detected by Western blot analysis) increased in abundance,while still remainingonly a small fraction of normal. Improvements in superoxideproductonwere seen after in vivo treatment of another patient with "variant" disease, and also with a "classical" XCGD patient whohad failed to display an in vitro response(111). Although the augmentationin superoxide production evident following treatment of these patients was muchmore modestthan that seen in the mildly affected brothers, detectable superoxideproduction, cytochrome b spectra, and improvedbacterial killing were demonstrated.Giventhese findings wecan expect that phagocytes of these patients in vivo wouldbe more efficient at disposingof microorganisms than in their baseline state. Preliminary findings from Sechler and colleagues (112) surveying a larger numberof patients, including both X-CGD and A+ subtypes, generally confirmedthe responsiveness of somepatients to interferon- 7 administration. Of particular interest is that, whereasthe phagocytesof only 2520%of X-CGD patients produced more superoxide following lymphokine treatmentin vitro, cells of nearly all A+ patients appearedto respond. Fromthe early clinical experience with interferon-7 in CGD,several points shouldbe addressed.First and foremost,the agent appears to offer promiseas a pharmacolc/gicagent in the management of selected patients with the disorder. Considerableexperience with interferon-~ in cancer chemotherapy trials suggests that it is well tolerated in the dosagesused in the CGD trials. Second,the heterogeneityin response that is already evidentin the limited trials is likely to reflect underlyinggeneticheterogeneity (111, 112). Specifically, amongX-CGD patients we might expect those whohave some residual cytochromeb synthesis in their baseline state (although vastly reduced from normal) to benefit most, particularly if underlying mutations merely impededquantitatively the production of otherwise normal gene product, such as in the case of fl+-thalassemia. Amongpatients with molecular defects that prevent expressionof any cytochromeb (such as partial gene deletions or translation termination mutations), no response might be anticipated. Third, the augmentationof superoxide production in responders has exceeded the increase in cytochromeb measuredspectrally or by Western blot analysis (111), suggesting that very small amountsof cytochromeb are required to permitsubstantial oxidasefunction. This is formallyanalogous to other enzymesystems wherea residual level of activity maylead to

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appreciable conversion of substrate to product. Wemayinfer, then, that the neutrophil cytochrome b is functionally rate-limiting in X-CGD. Fourth, although interferon-~ can induce cytochrome b heavy-chain expression (90, 91), it is quite likely that other important components the oxidase system maybe induced or altered in such a way as to function moreefficiently (11 l, 112). In this regard it is noteworthythat cells of +o CGDpatients, in the experience of Sechler and coworkers (112), have rather consistently responded to interferon-y in vitro. This observation suggests that the gene product deficient in this subtype of CGDmayalso be induced by interferon treatment. Finally, the potential contribution of nonoxidative mechanismsof microbial killing induced by interferon-y may be substantial and mayeventually be found to play a role in any clinical efficacy established for interferon-~ in CGD(113). To imply at this stage that the improved function of phagocytes of treated patients (apart from improved superoxide production) is due solely to induction of cytochrome b expression is not warranted. Genetic management--somatic 9ene therapy Since X-CGDis a disorder of marrow-derivedcells that is nowknownto be due to genetic defects in the gene encoding the 90-kd subunit of the neutrophil cytochrome b, transfer of an expressible copy of its mRNA into pluripotent hematopoietic stem cells, in principle, wouldconstitute definitive therapy (114). Because bone marrow transplantation where applied to X-CGDhas resulted in functional correction (115), somatic genetic therapy has a strong rational basis. In view of the substantial restoration of superoxide production associated with only miniscule amounts of cytochrome b protein, correction of the disease by somatic gene transfer would not necessitate complete correction of protein levels. An approach under investigation is the use of recombinant retroviruses (114) to transfer expressible cDNA for the 90-kd subunit into hematopoietic stem cells and their progenitors. Althoughthis approach has considerable promise, unexpected difficulties encountered in the expression of sequences transferred into primary hematopoietic stem cells and in their efficient infection, as well as the lack of immortalized,deficient phagocyticcells to test for functional reconstitution in culture, makethe practical application of this technology uncertain for the forseeable future (114). The availability of molecular reagents for the X-CGD locus, combined with site-specific gene disruption techniques applicable in murine embryonicstem cells (116-118), mayultimately lead to production of a mouse model for X-CGD.Such an experimental animal wouldgreatly facilitate studies of reconstitution by gene transfer and also provide cultured lines for in vitro cell biologic investigation of cytochrome b assembly and targeting.

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NEW DIRECTIONS AND REMAININGQUESTIONS While it has provided clearcut resolution of several long-standing issues in the field of phagocyticcell biology and suggested newpotential strategies for treatment of CGD,research in the past two years has raised a wealth of questions. AlthoughCGDin its various forms is rare, lessons learned about critical proteins in the superoxide-generating system will undoubtedly lead to general insights into the normal biology of phagocytic cells and their role in host defense. Whathave we learned so far and where are the new challenges? First, biochemical and genetic studies have converged to provide compelling evidence for a critical role for the neutrophil cytochrome b in the pathway of superoxide generation. Although its role may have been considered controversial by someinvestigators until quite recently, little evidence can be mustered now to exclude its participation in superoxide production. This important conclusion supports the insightful research of Segal and his colleagues (35). Nevertheless, even though theprimary amino acid sequences of the two subunits of the cytochrome are now complete from cloned cDNAs(74, 77, 79), manyaspects pertinent to its structure and regulation remain unresolved. The topologic organization of the subunits, the domainsinvolved in their interaction, the identity of the axial ligands of the prosthetic group(s) in the heterodimer, and the structural basis for participation of the cytochrome b in production of superoxide are unknown. Second, the responsiveness of expression of the 90 kD subunit mRNA to differentiation and to interferon-v treatment suggests that it plays an important role in synthesis of the functional membranecomplex and is a critical determinant in different states of macrophageactivation. The manner in which the assembly of the cytochromeb is regulated, a process that is likely to be important in establishing the functional membranecomplex, needs direct analysis. Combinedbiochemical and genetic approaches, perhaps using gene transfer of the subunit cDNAsinto heterologous cells, will be required to address these issues. A better understanding of the organization and modulation of the oxidase may suggest new types of anti-inflammatory agents that may modify damagingeffects of superoxide generation, yet preserve antimicrobial activity. Third, the availability of molecular reagents for both cytochrome b subunits nowpermits direct assessment of the mutations that underlie the X-, X+, and A- forms of CGD. Studies of X-CGDshould clarify the relationship between "variant" disease and residual cytochrome b production and between interferon-v responsiveness and genetic pathology. Identification of presumedpoint mutations that lead,,tcr:X%disease

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mayindicate specific aminoacids that play a role in the electron transfer function of the cytochromeb heterodimer. Studies of X- and A- disease mayprovide insights into the regions of the subunits that specify their tight interaction. Identification of instructive mutationsmaylead the way for directedsite-specific mutationsof the subunitsin the future in an effort to correlate protein structure andfunction. Fourth, studies of the cis-DNAsequences and trans-nuclear protein factors that specify lineage and temporal expression of the X-CGD gene and its inducibility by interferon-y maycontribute morebroadly to an understanding of regulated expression in hematopoietic cells and the molecular basis of interferonaction. It is likely that lineage-specificnuclear factors participate in the regulation of expression of the X-CGD gene. An understanding of their nature maysuggest more generally howthe expressionof genes is coordinatedduring myelomonocytic differentiation. Fifth, recent data on treatment of patients with interferon-~/ suggests that future understandingof various forms of CGDand the regulation of specific components of the oxidase mayprovidethe logical basis for more effective management in the future. In this respect CGD represents one of the fewdisorders in whichbasic studies of genetics haveled to the design of new forms of medical management. Manyoutstanding issues remain untouched.Elucidation of the primary basis of A+-CGD, presumedto be related in somemannerto the rapidly phosphorylated4448kd polypeptidesof neutrophils, will open up investigation of another limb of the NADPH-oxidase system. The structure and role of the elusive "respiratory burst oxidase"merit attention if the various components implicatedby in vitro reconstitution assays are to find their place in the pathway.Althoughin vitro reconstitution assays are being developedto assess the activity of various cytosolic and membrance fractions and purified proteins, progressin understandingthe interactions of specific geneproductswouldbe greatly facilitated if a suitable heterologouscell systemwereestablished. OncecDNAs for a minimalbattery of essential proteins are available, onemightenvisionattemptsto transfer the superoxide-generating system into nonphagocytic cells. If this ambitiousgoal wereaccomplished,the regulatory effects and interactions of the various componentscould be addressed in a direct, systematic manner. ACKNOWLEDGMENTS

The work in the author’s laboratory was supported by a grant from the National Institute of Health and general support from the HowardHughes MedicalInstitute. I amgrateful to numerousindividuals for the developmentof research on CGDin mylaboratory; these include LouKunkel,

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Tony Monaco, Bob Baehner, Brigitte Royer-Pokora, Mary Dinauer, Peter Newburger, Alan Ezekowitz, and A1 Jesaitis. I also very much appreciate Marie Fennell for her work in preparation of the manuscript. The author is an Investigator of the HHMI.

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Monoclonal antibody 7D5 raised to cytochrome b558 of human neutrophils: Immunocytochemical detection of the antigen in peripheral phagocytes of normal subject, patients with chronic granulomatous disease, and their carrier mothers. Blood 69: 140408 82. Rotrosen, D., Hunoi, H., Tiffany, H. L., Albert, J., Maloy,W. L., Gallin, J. I., Malech, H. L. 1988. A cytoplasmic carboxyterminal domain of the transmembranous large subunit of neutrophil cytochromeb is involved in activation of the respiratory burst. Clin. Res. 36: 582A 83. Volkman,D. J., Buescher, E. S., Gallin, J. I., Fauci, A. S. 1984. B cell lines as models for inherited phagocytic diseases: Abnormalsuperoxide generation in chronic granulomatous disease and giant granules in Chediak-Higashi syndrome. J. Immunol. 133:3006-3009 84. Pick, E., Gadba, R. 1988. Certain lymphoid cells contain the membraneassociated component of the phagocyte-specific NADPH oxidase. J. Immunol. 140:1611-17 85. Nathan, C. F., Tsunawaki, S. 1986. Secretion of toxic oxygen products by macrophages: Regulatory cytokines and their effects on the oxidase. Ciba Found. Symp. 118:211-30 86. Berton, G., Zeni, L., Cassatella, M. A. 1986. Gammainterferon is able to enhance the oxidative metabolism of humanneutrophils. Biochem. Biophys. Res. Commun. 138:1276-82 87. Cassatella, M. A., Cappeffi, R., Delia Bianca, V. 1988. Interferon gamma activates human neutrophil oxygen metabolism and exocytosis. Immunology 63:499-506 88. Nathan, D. G., Kaplan, G., Levis, W. 1986. Local and systemic effects of intradermal recombinant interferon gammain patients with leptomatous leprosy. N. Engl. J. Med. 315:6 89. Nathan, C. F., Murray, H. W., Wieve, M. E. 1983. Identification of interferon gammaas well as the lymphokine that activates humanmacrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670~89 90. Newburger,P. E., Ezekowitz, R. A. B., Whitney, C., Wright, J., Orkin, S. H. 1988. Induction of phagocyte tyrochrome b heavy chain gene expression by interferon gamma.Proc. Natl. Aead. Sci. USA 85:5215 19 91. Cassatella, M. A., Hartman, L., Perussia, B., Trinchieri, G. 1988. Tumor necrosis factor and immuneinterferon

synergistically induce cytochrome b245 heavy chain gene expression and NADPHoxidase in human leukemic myeloid cells. Submitted 92. Andrew, P. W., Robertson, A. K., Lowrie, D. B., Cross, A. R., Jones, O. T. 1987. Induction of synthesis of components of the hydrogen peroxidegenerating oxidase during activation of the human monocytic cell line U937 by interferon-gamma. Biochem. J. 248:281-83 93. Reich, N., Evans, B., Levy, D., Fahey, D., Knight, E. Jr., Darnell, J. E. Jr. 1987. Interferon-induced transcription of a gene encoding a 15-kd protein depends on an upstream enhancer element. Proc. Natl. Acad. Sci. USA 84:6394-98 94. Friedman, R. L., Stark, G. R. 1985. Alpha-interferon-induced transcription of HLAand metallothionein genes containing homologous upstream sequences. Nature 314:637-39 95. Korber, B., Mermod, N., Hood, L., Stroynowski, I. 1988. Regulation of gene expression by interferons: control of H-2 promoter responses. Science 239:1302q5 96. Zinn, K., Maniatis, T. 1986. Detection of factors that interact with the human beta-interferon regulatory region in vivo by DNAseI footprinting. Cell 45: 611-18 97. Lomax, K. J., Burch-Whitman, C., Tiffany, H. L., Gallin, J. I., Malech,H. L. 1988. Analysis of chronic granulomatous disease kindreds reveals distinct genetic lesions affecting the same gene product. Clin. Res. 36: 413A 98. Seger, R. A., Tiefenauer, L., Matsunaga, T., Wildfeuer, A., Newburger, P. E. 1983. Chronic granulomatous disease due to granulocytes with abnormal NADPH oxidase activity and deficient cytochrome-b. Blood 61: 423-28 99. Lew, P. D., Southwick, F. S., Stossel, T. P., Whitin, J. C., Simons,E., Cohen, H. J. 1981. A variant of chronic granulomatous disease: Deficient oxidative metabolism due to a low-affinity NADPH oxidase. N. En#l. J. Med. 305: 1329-33 100. Ezekowitz, R. A. B., Orkin, S. H., Newburger, P. E. 1987. Recombinantinterferon gamma augments phagocyte superoxide production and X-chronic granulomatous disease gene expression in X-linked variant chronic granulomatous disease. J. Clin. Invest. 80: 1009-16 101. Orkin, S. H. 1987. Disorders of hemoglobin synthesis: The Thalassemias.

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MOLECULAR GENETICS OF CGD In The Molecular Basis of Blood Diseases, ed. G. Stamatoyannopoulos, A. Neinhuis, P., Majerus,1: 106-26. Philadelphia: Saunders 102. Ohno,Y., Buescher,E. S., Roberts,R., Metcalf, J. A., Gallin, J. I. 1986. Reevaluationof cytochromeb and flavin adeninedinucleotidein neutrophils from patients with chronic granulomatousdisease and description of a family with probable autosomalrecessive inheritance of cytochromeb deficiency. Blood67:1132-38 103. Nunoi, H., Gallin, J. 1., Malech, H. L. 1988. Different defects in cytosolic factors maybe responsible for autosomalforms of chronic granulomatousdisease (CGD).Clin. Res. 415A 104. Frey, D., Machler, M., Seger, R., Schmid, W., Orkin, S. H. 1988. Genedeletion in a patient with chronic granulomatousdisease and McLeod syndrome: Fine mapping of the Xk genelocus. Blood 71:252-55 105. Newburger, P. E., Cohen, H. J., Rothchild,S. B., Hobbins,J. C., Malawista, S. E., Mahoney, M.J. 1979.Prenatal diagnosis of chronic granulomatousdisease. N. Engl. J. Med.300: 178-81 106. Orkin, S. H. 1984. Prenatal diagnosis of hemoglobin disorders of DNA analysis. Blood63:249-53 107. Saiki, R. K., Scharf, S., Faloona,F., Mullis, K. B., Horn, G. T., Erlich, H. A., Arnheim,N. 1985. Enzymatic amplification of ~-globin genomic sequencesand restriction site analysis for diagnosisof sickle cell anemia.Science 230:1350-54 108. Forrest, C. B., Forehand,J. R., Axtell, R. A., Roberts, R. L., Johnston, R. B. 1988.Clinical features and current managementof chronic granulomatousdisease. See Ref. 5, 2:253-66 109. Babior, B. M., Peters, W.A. 1981.The O2-producing enzymeof humanneutrophils: Further properties. J. Biol. Chem. 256:2321-23

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110. Babior, B. M., Curnutte,J. T., McMurrich, B. J. 1976.Theparticulate superoxide-forming system from human neutrophils: Properties of the system and further evidence supporting its participation in the respiratory bursts. J. Clin. Invest. 58:989-96 111. Ezekowitz,R. A. B., Dinauer, M. C., Jaffe, H. S., Orkin, S. H., Newburger, P. E. 1988. Partial correction of the phagocytedefect in patients with Xlinked chronic granulomatousdisease by subcutaneousinterferon gamma.N. En#l. J. Med.319:146-51 112. Sechler, J. M. G., Malech, M. L., White,C. J., Gallin, J. I. 1988.Recombinant human interferon-y reconstitutes defectivephagocyefunction in patients with chronic granulomatous diseaseof childhood.Proc.Natl. Acad. Sci. USA85’. 4874-78 113. Lehrer, R. I., Ganz,T., Selsted, M.E. 1988. Oxygen-independent bactericidal system. Mechanismsand disorders. Hematol./Oncol.Clin. N. Am.2: 159269 114. Williams, D. A., Orkin, S. H. 1986. Somaticgene therapy: current status andfuture prospects.J. Clin.Invest. 77: 1053-56 115. Rappeport, J. M., Newburger,P. E., Goldblum, R. M., Goldman, A. S., Nathan, D. G., Parkman, R. 1982. Allogeneic bone marrowtransplantation for chronic granulomatousdisease. J. Pediatr. 101:952-55 116. Jackson, I. J. 1987. Thereal reverse genetics: targeted mutagenesisin the mouse.TrendsGenet. 3:119-20 117. Robertson,E. J., Bradley,A., Kuehn, M., Evans, M. 1986. Germ-linetransmissionof genes introduced into cultured pluripotentcells byretroviral vectors. Nature323:445-48 118. Thomas,K. R., Fogler, K. R., Capecchi, M.R. 1986.Highfrequencyis targeting of genesto specific sites in the mammaliangenome.Cell 44:419-28

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Ann.Rev. Immunol.1989. 7.’309-37 Copyright© 1989by AnnualReviewsInc. All rights reserved

CELL BIOLOGY OF CYTOTOXIC AND HELPER T CELL FUNCTIONS"Immunofluorescence MicroscopicStudies of Single Cells and Cell Couples AbrahamKupfer* and S. J. Singer Department of Biology, University of California at San Diego, La Jolla, California 92093

INTRODUCTION This review deals with certain important features of the cell biology of natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and helper (Th) cells. Thesefeatures appearedoriginally in studies of the cell biology of cells other than immunological cells, and their relevance to immune cell interactions was then appreciated and explored. This brief review is therefore meant to be selective rather than exhaustive, and it focuses primarily on problemsthat have been intensively studied in our laboratory over the past decade. At the outset, it should be emphasized that we concentrate on investigations of single (generally cloned) T cells and cell couples, rather than of cell populations. Recent reviews have appeared that deal with other aspects of the cell biology of T cells and their interactions (1-7). The entr+e to this work stemmedfrom our investigations of eukaryotic cell motility; these have been separately reviewed (8). These studies are considered briefly first, since they not only introduce the ideas that have played a prominent part in our immunological work, but they also demon-

*Present Address: National Jewish Center for Immunologyand Respiratory Medicine, Denver, Colorado 80206.

309 07320582/89/0410-0309502.00

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strate the generality of someof the phenomenaobserved for understanding cellular interactions in immunology.

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THE DIRECTED MIGRATION OF CELLS Whena eukaryotic cell undergoes directed migration in response to a polarized signal, it is generally agreed that its leading edge is propelled forward in the direction of the signal by intracellular forces mediated by the actin cytoskeleton. In addition, however,the cell’s normalrecycling of its surface membranebecomes polarized, so that new membranemass is continually inserted at the leading edge of the cell while other membrane mass is internalized rearwards of the leading edge (8). This polarized recycling of membranemass appears to be essential for directed migration to occur (9), but whether the essential feature is the insertion of new membranemass per se, or the directed secretion of adhesive or other soluble proteins at the leading edge that accompanies such membrane insertion, or both, is not clear. What mechanism determines that membrane recycling becomes polarized? The answer we found (10, 11) is that the Golgi apparatus (GA)inside the cell undergoesa reorientation to face the region of the cell surface that is to becomethe leading edge, within minutes after receipt of a polarized signal to move. The GAis the organelle that generates the membraneboundvesicles containing secretory componentsthat go on to fuse with the plasma membrane. The vesicular membranemass is thereby introduced into the cell membraneand the secretory contents of the vesicles are simultaneously released to the cell exterior (12). In most eukaryotic cells the GAis a compactset of stacked vesicular structures usually present to one side of the cell nucleus. Reorientation of the GAin a particular direction apparently ensures that vesicular traffic derived from the GAis delivered to that region of the plasma membraneapposed to the GA(9, 13). Howthe GAreorientation is achieved is not yet clear, but microtubules probably play an important role. Interphase metazoancells usually contain a single organelle, the microtubule-organizing center (MTOC) that acts the nucleation center for the polymerization of all of the microtubules in the cell (14). Double immunofluorescence observations of the MTOC and the GAin individual cells of widely different types indicate that labeling for the MTOC and GAis always found to be superimposed, and a reorientation of the GAis always accompaniedby the same reorientation of the MTOC (8). This codistribution and coordinated reorientation of the two organelles probably reflects somephysical linkages between them, but this is not established. Oneplausible possibility is that the process that signals

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the cell to move may somehowinduce a microtubule-mediated torque on the MTOC which reorients the MTOC along with the GAthat is physically linked to it.

CYTOTOXICITY

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NK Interactions Midwaythrough these studies of cell motility in our laboratory, Dr. Gunther Dennert informed us of his recent investigations on the cloning of mouseNKcells and the evidence that an NKcell lysed its bound target cell by secreting cytotoxic componentsto it (15). It occurred to us that, like cell motility, the NKcell : target cell interaction mightbe an instance of a polarized signal (this time from the boundtarget to the NKcell) inducing a polarized secretion (from the NKto the target). The prediction was that in couples formed between cloned NKand target cells, the MTOC/GA inside the NKcell would be found oriented to face the boundtarget. This was then shownto be the case (16). Similar results were found with human NKcells (17, 18).

CTLInteractions In the NK: target cell system, the nature of the surface molecules whose transcellular bonding is responsible for the cell:cell interaction is unknown,and the cytotoxicity has limited specificity. A similar cytotoxic phenomenonoccurs when CTLinteract specifically with allogeneic target cells; in this system, however, it is knownthat the specific binding of a clonotypic T cell receptor (TcR) on the surface of the CTLto a class-I MHCmolecule on the surface of the target cell initiates the cytotoxic process. Wetherefore next examined individual cell couples formed between cloned CTLand their targets. MXO¢/~h REORIEYa’ha’ION With allospecific CTL:target cell couples we found by immunofluorescence observations that, in a way similar to the NKcell couples, the MTOC/GA inside the CTLwas oriented to face its boundallospecific target (19). Earlier electron microscopic studies with CTLpopulations provided evidence that the GAinside the CTLbecomes reoriented to face the contact region with the target (20). Geiger et al (21) had also reported that in CTL: target couples the MTOC within the CTL was oriented towardthe area of cell : cell contact. Theysuggested that the target cell binds to an already polarized CTLat a cell surface region apposed to the MTOC.The cumulative evidence cited below, however, strongly indicates that this interpretation is incorrect; rather, the CTL binds to the target cell at randomregions of the twocell surfaces, following

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which the MTOC inside the CTLis rapidly reoriented to face the cell :cell contact region. Onedemonstration of this involves the effects of Ca+2 on the properties of the allospecific CTL: target cell couples. It had been shown(22-24) that such couples could form in the presence of Mg÷2 and in the absence of Ca+2, but that cell killing would no~ occur unless Ca+2 was restored. We found (25) that CTL: target cell couples formed in Mg+2-containing÷2free media exhibited random orientation of the MTOC/GA inside the CTL (Figure 1D); the addition of +2 tothese couples, however, led to n early all couples having the MTOC inside the CTLfacing the cell : cell contact site (Figure 1A). Theseresults strongly suggest that cell : cell binding occurs at cell surface regions unrelated to the position of the MTOC/GA inside the CTL, and that a subsequent Ca+Z-mediatedpolarized signal induces the MTOC/GA reorientation inside the CTLto face the cell : cell contact region. That the binding of specific target cells induces a rapid Ca2+ influx into the CTLhas been demonstrated (26). Another demonstration involved multiple cell conjugates, with several specific target cells attached simultaneously to a single CTL.It is known that in such cases, the targets are lysed sequentially and not simultaneously (27-29). Our results (25) were entirely consistent with the conclusion the MTOC/GA in the CTLfirst orients to face one of the bound target cells which then proceeds to lyse, with its ghost remaining attached to the CTL; the CTLMTOC/GA then reorients to face the second bound target to be lysed; and so on until all the target cells are destroyed. Again,cytolysis appears to be coupled to a signalled reorientation of the MTOC/GA. In addition to the reorientation of the MTOC/GA, cinematographic observations by Nomarskioptics indicated that a rapid redistribution of cytoplasmic granules occurs inside CTLto face toward the boundallospecific target cell and that the fusion of these granules with the CTLmembraneat that region begins within minutes after specific cell: cell contact (30). In these cases of NKand CTLcell couples, the polarization of the MTOC/GA that always accompanies a cytolytic interaction is unidirectional; it occurs inside the effector cell but not the target. A question that then arose was whether such organelle reorientation is a specific characteristic of cytolytic cells that is not shared by non-T-cell targets, or whether instead it reflects the engagementof specific receptor molecules on the effector cells. This question was answered by studies carried out with cell couples formed between two CTL, of the types a anti-b and b anti-c. In such mixedCTLpopulations, it was knownthat only the b antic CTLis lysed (31). Correspondingly, we found (32) that with a two system that showed unidirectional killing, the MTOC/GA in the effector CTL, but not that in its bound target CTL, was reoriented to face the

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Figure 1 The effect of extracellular Cai2 on the orientation of the MTOC and the distribution of talin inside CTL : target cell couples. The allogeneic cloned CTL 18C3 (H2bantiH2d) were mixed with an equal number of the target cells P815 (H2d) in the absence ( A X ) or presence (D-F) of Mg+2/EGTA,as in (25). The cells were plated out, fixed, and double immunofluorescently labeled with affinity purified guinea pig anti-tubulin (A,D) and affinitypurified rabbit anti-talin antibodies (B,E) followed by the appropriate fluorescent-conjugated secondary antibodies. The Nomarski pictures of the same cells that are immunolabeled in A,B, and in D,E are shown in C and F, respectively. The thick arrows point to the cell : cell contact area, and the thin arrows to the position of the MTOC in the CTL. Note that in cell couples formed in the presence of extracellular Ca+*(A-C), the MTOC is oriented to face, and talin is concentrated at, the contact site, but that in the absence of extracellular Ca+Z, the MTOC (D) is not oriented to the contact area but talin (E) is still concentrated at this site. The MTOCs in the target cells in both cases faced away from the contact area. The bar in C represents 10 ,urn (A. Kupfer, S . J. Singer, unpublished).

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cell:cell contact region. These results indicate that it is the polarized engagementof the TcRon the bound effector CTLthat is required for the reorientation of the MTOC/GA in that cell, whereas the simultaneous engagement of the class-I MHCmolecules on the target cell does not reorient the MTOC/GA inside the target. In all of the experimentsso far described, and in additional ones reported in the original publications, a perfect correlation was found between the reorientation of the MTOC/GA inside the effector cell and the productive lysis of its bound target cell. This suggests that the MTOC/GA reorien¯ tation is an obligatory accompaniment of the lytic activation of the effector cell. In this connection, it is of interest that certain combinationsof allogeneic CTLand target cells do not require Ca+2 in the mediumfor the targets to be lysed; yet even with such CTL:target cell couples in the ¯ absence of Ca+2, the MTOC/GA reorientation is’observed inside the boundCTL(33). In other studies of cloned CTL: CTLcouples (34), killing was never observed without a reorientation of the MTOC/GA, although in one case with a cloned CTLtarget resistant to lysis, the MTOC/GA reorientation occurred in the effector CTLof the couple, but the target was not lysed. These results support the proposal that the MTOC/GA reorientation in the effector cell is a necessary but not sufficient condition to produce lysis of a boundtarget. CYTOSKELETAL REORGANIZATION In the case of the two CTLsystem a anti-b and b anti-c, where only the b anti-c CTLwas lysed (see, however, 35-37), we showedthat part of the explanation for the unidirectionality of killing is that there is a unidirectional polarization of the MTOC/GA in the boundeffector CTL. The function served by this organelle reorientation is presumablyto direct secretion of cytotoxic componentsfrom the effector to the target where the two cells are bound together (see below). As there is no indication that the two cell surfaces at the cell: cell contact are rendered discontinuous, such putative cytotoxic componentswould be expected to be secreted into the confined intercellular spaces in the region of cell:cell contact. Therefore, even if secretion was unidirectional, both the effector and target CTLmembranes would presumably be equally exposed to the action of the secreted cytotoxic components.Whythen was the effector CTLnot as susceptible to killing as the target CTL? One explanation that occurred to us is that a unidirectional cytoskeletal rearrangement under the membraneof the bound effector cell might occur which somehowprotected the membraneagainst the cytolytic process. Whateverthe merits of this~explanation in retrospect (see below), it led to examine by immunofluorescencemicroscopy the distribution of several important cytoskeletal proteins in couples formed with CTL. These cyto-

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skeletal proteins included ~-actinin (38), vinculin (39, 40), talin (41), 200 kd protein (42), and fimbrin (43). They were chosen for investigation because in a wide variety of different cell types each of these proteins has been proposed to play a role in the linkages of actin microfilaments to one another or to the cell membrane(44). Few studies of these proteins have been made in lymphocytes. In a related connection, there are reports that actin is concentrated inside CTL(45) and NKcells (46) at the sites contact with target cells, but as the targets were not themselves CTL, whether such actin concentration is unidirectional was not clear. In the cell couples formed between two CTLof the type a anti-b and b anti-c, we showed(32) that talin, but not any of the other four cytoskeletal proteins examined, became concentrated under the effector CTLmembrane where it was in contact with the specific target CTL.However,all five proteins remaineduniformly distributed in the boundtarget. The talin redistribution is therefore specific and unidirectional. This talin effect was observed generally inside the CTLin specific CTL-target cell couples whateverthe type of lysable target cell (Figure 1B,E), as well as inside cells boundto lysable targets (32). Interestingly, the redistribution of talin was not dependent upon the presence of Ca+: in the medium(Figure IB,E). +2 The reorientation of the MTOCwhich is dependent on Ca (Figure IA,D), is therefore not tightly coupled to the talin redistribution. The properties of the talin molecule and its interactions are discussed further below. At this stage in our studies of cytotoxic cell interactions, we had discovered two rapid, readily detectable intracellular events in cytolytic effectot cells that invariably accompaniedtheir binding to specific target cells: a remarkable reorientation of the MTOC/GA complex to face the bound target; and a unidirectional redistribution of the cytoskeletal protein talin under the contacting CTLmembrane. In the case of CTL, these effects must be the consequence of the appropriate engagementof the clonotypic TcRmolecules on its surface. Becausesecretion (of lymphokines)by helper T cells was knownto be involved in T cell help, and because the TcR molecule on CTLis closely similar to the TcRon helper T cells (cf 47), then becameinterested in whether,in cell : cell interactions involving helper T cells and antigen-presenting B cells, similar intracellular reorganization phenomena occurred. T CELL HELP: INTRACELLULAR REORGANIZATIONS Cytotoxicity is extremely important physiologically, serving to protect the organism against viral and some bacterial diseases, and it is perhaps

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involved in immunesurveillance against cancer (48). However,the cell : cell interaction is a dead end: the target cell is lysed. In the case of Thcells and antigen-presenting cells (APC), the interactions have been implicated the proliferation and ultimate differentiation of B-cell APCinto antibodysecreting ptasmacytes, as well as in the proliferation of, and lymphokine secretion by, the Th cells. The helper T cell system therefore involves cell differentiation rather than cell killing, and as such maybe related mechanistically to other important binary cell:cell interactions in developmental biology. Cell couples of CTLand their allogeneic target cells can be produced and isolated quite easily (49), but the direct binary interaction of syngeneic Th and APChas been muchmore difficult to investigate. This has had to await the cloning of specific Th cells, or the production of hybridomasof specific Thcells (50). In our initial studies in collaboration with S. L. Swain and C. A. Janeway, Jr. (51, 52), cloned mouseTh cell lines were used, and mouse cell hybridomas or lymphomaspulsed with very large concentrations of appropriate antigens served as APC. This allowed us to produce and isolate specific Th : APCcell couples, as well as control nonspecific couples, for immunofluorescencestudies by the same methods that we had earlier used with NKand CTLsystems. The cloned Th cell lines used included D10.G4.1(referred to as D10) and D8 specific for the egg white antigens conalbumin(Con) and ovalbumin(Ova), respectively, in the context of in addition, some experiments were carried out with the T-T hybridoma 2H.10.H1 with a helper response specific for pigeon cytochrome c (Cyt) and Iak. As APC,the B-cell hybridomasLK(Ia d, Iak), LB(Ia d, Iab), BCL1 (Ia d) and CH12Oak) (52) were employed. The Th cells and antigen-pulsed APCwere mixedin equal numbers, the mixtures lightly pelleted, incubated for 10 rain, and then resuspended for plating. They were then fixed and immunofluorescently labeled. Under these conditions, cell couples were formed with both specific and nonspecific combinations of Th and APC. The specific couples could not readily be distinguished from the nonspecific by Nomarski optics. However, in every specific Th:APC combination examined, the Th of most (80-95%) of the cell couples exhibited MTOC/GA oriented to face the bound APC, whereas in the case of nonspecific couples, either presenting a nonspecific antigen or the wrong Ia on the APC, the Th MTOC/GA was randomly oriented. Further studies of such couples showed that talin, but not ~-actinin or vinculin, was concentrated under the Th membranein contact with specific APC, but not with nonspecific APC(52). As predicted, therefore, these results were closely similar to those we had earlier obtained with CTL:target cell couples.

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These two objective criteria--MTOC/GAreorientation and talin redistribution in the boundTh cell--therefore served to establish that a specific binary interaction of Th and APChad occurred. Although prior binding experiments with cell populations (53) provided compelling evidence for specific interactions, these studies together with the cell:cell binding studies of Sanders et al (54) represent the most direct evidence for a binary interaction. In the system consisting of D10Th cells and antigen-pulsed LKcells, evidence was then obtained from chloroquine inhibition and other experiments (52) that antigen processing by the APCwas required in order produce the MTOC/GA reorientation in the Th cell. It was also shown that, as observed with the cytotoxic cell couples, the MTOC/GA reorientation in the Th required extracelllar Ca÷2, whereasthe talin redistribution did not (52). In our studies ofTh : APCcouples up to this point, the B-cell hybridomas and lymphomasthat were used for antigen presentation, lacked the appropriate surface IgM,and therefore were not specific for the antigens recognized by the cloned Th cells we employed. Antigen presentation by such cells required that they be pulsed with very large doses of the antigen, of the order of 500 #g/ml. To achieve conditions that were more nearly physiological, we took advantage of the production of an antigen-specific B-cell hybridoma by Hozumi and colleagues (55). They had stably transfected A20 B hybridoma cells (Ia ~) with the rearranged genomic DNA for the H and L chains of a surface IgMwith binding specificity for the 2,4,6-trinitrophenyl (TNP)hapten. These transfected cells are referred as A20-HL.In our experiments (A. Kupfer, D. R. Wegmann,S. J. Singer, in preparation) the A20-HLcells were pulsed with 2,4-dinitrophenyl (DNP)-modified ovalbumin (DNP-Ova), the DNP and TNP haptens cross-reacting extensively. The DNP-Ovawas used at varying concentrations below I/~g/ml (contrast this with the 500 #g/ml of antigen used with nonspecific APC). Within this concentration interval, the DNP-Ova was endocytosed and processed by the cells via the surface anti-TNP IgM, and peptide fragments of Ova were presented. These APCwere then used to form specific Th : APCcouples, using as Th the cloned cell line D20.36 (referred to as D36) with specificity for the peptide sequence 323-339 Ova in the context of la d (D. R. Wegmann, in preparation). These couples were then examined, as in the previous studies, by double immunofluorescence for their MTOC orientations and talin distributions, as well as for LFA-1on the T-cell surface, as functions of the DNP-Ovaconcentration used. Onaliquots of similar cell mixtures, the activation of the population of D36 Th cells by the APCto undergo proliferation was measured by 3H-thymidine incorporation. The results for MTOC and talin

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are discussedin this section andthe LFA-l-datain a later section. At the highest concentrations of DNP-Ova used (0.2 #g/ml), the D36cells were fully activated, and most of the D36: APCcouples showedreorientation of the MTOC/GA inside the Thcell, as well as a collection of talin in the region of the D36cell membrane that was in contact with the APC.With decreasing concentrations of DNP-Ova,below 0.1 #g/ml, there was a progressivedecreasein the fraction of D36cells that wereactivated, and this wasparalleled by a decreasein the fraction of cell couplesexhibiting a reorientation of the MTOC/GA. At DNP-Ova concentrations less than 0.02/~g/ml, little cell activation or MTOC/GA reorientation was found, althoughtalin wasstill collectedin the cell : cell contactregion. Theseresults provide further strong evidencethat a strict correlation exists betweenthe rapid reorientation of the MTOC/GA inside the effector T cells, andthe activationof these cells, inducedby the specific interaction with their congenercells. In addition, they confirmthat the reorientation of the MTOC/GA and redistribution of talin are not tightly coupled. ON THE FUNCTIONS OF INTRACELLULAR REORGANIZATIONS IN EFFECTOR T CELLS Theevidence presented to this point indicates that closely similar and rather massive rearrangements of organelles and the cytoskeleton invariably occur inside NK,CTL,or Th cells whenthey are specifically boundto their congenercells. Whyare these changesso critically associated with two such different effector cell functions as cytotoxicity and T cell help7This questionis addressedin the next two sections. The Possible Functions Served by Reorientation of the MTOC/GA As mentionedearlier, the MTOC/GA reorientation could serve to polarize and direct secretion and insertion of membrane massto the region of the surfaceof an effector cell that is in contactwith its specific congenercell. In the case of motile cells (13), the directed insertion of newmembrane massinto the leading edgeof the cell, oppositeto the perinuclear GA,has beendirectly demonstrated.In the case of cytotoxic cells, proposalshave been madethat one or morecytotoxic componentsare secreted from the effector cell to its boundtarget (cf 4); such componentshave included perforins, possessing complement-likemembrane permeability properties; proteasespresent in cytotoxic cell granulesalong with perforins; and tumor necrosisfactors. Though eachis capableof causingcell lysis in vitro, none of these components,has been definitively shownto be the "magicbullet" of physiological NKand CTLactivities. Furthermore,other lytic mech-

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anisms than secretory ones are not ruled out (56). Nevertheless, the obligatory and specific reorientation of the MTOC/GA that occurs upon stimulating these cells is strongly consistent with a secretory process for cytotoxicity, although, of course, it does not establish it. In the case of Th cell function, a number of soluble T cell-derived lymphokines can serve as B-cell growth and differentiation factors in vitro, apparently depending on the Th subset (57). The T cell is also known to express IL-2 receptors on its surface upon activation (58). The reorientation of the MTOC/GA inside the Th cell ensures that the secretion of these growth and differentiation factors, via secretory vesicles derived from the GA, is specifically directed towards the bound APC. It is of great significance that, by this means, only the specific APC would be stimulated by the soluble, non-antigen-specific secreted factors derived from the Th cell that was bound to it. Similarly, newly expressed membrane molecules, such as TL-2 receptors, would be inserted directly from GA-derived vesicles into the Th membrane where it is in contact with the APC. This would provide confined access of these receptors to their soluble ligands that were also present in the intercellular space. A preliininay experiment that is suggestive of such a directed stimulation of APC in Th : APC couples is shown in Figure 2 (A. Kupfer, S. L. Swain, S. J. Singer, unpublished experiments). The in vivo B-cell tumor line CH12

Figure 2 A demonstration of a possible polarized induction of B-cell proliferation by direct contact with cloned Th cells. Cells of the in vivo B lymphoma CHI2 were pulsed overnight in vitro with Con (500 pg/ml) and mixed with D10 cells at a 2 : 1 ratio. By 10 hr later, the cells were plated out, fixed, and double immunofluorescently labeled with rabbit anti-tubulin antibodies (A) to display the MTOCs, and goat anti-mouse Ig antibodies (B) to identify the CHI2 cells in the aggregates. Panel (C) shows the Nomarski picture of the same aggregate of cells. Note that one D10 cell is bound to two CH12 cells, one of which is larger than the other, and is undergoing mitosis (see small arrows pointing to the asters). Note also that the MTOC in the D10 cell (large arrow) is facing the dividing CH 12 cell. The bar in C represents 10 ,urn (A. Kupfer, S. L. Swain, and S. J . Singer, unpublished).

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Oak), which does not normally grow or divide in in vitro culture, was used as APC.After pulsing the CH12cells with 500/~g/ml Con, D10 cells were mixed at a 2:1 ratio with the APC. Compared to 1:1 mixtures, this resulted in an increased production of individual cell clusters containing two specific APCbound to one DI0 cell (Figure 2C). Whensuch cell mixtures were processed 10 hr after their formation and incubation at 37°C, clusters were occasionally observed in which simultaneously one large and one small CH12APCwere attached to the same DI0 cell; the MTOC/GA in the DI0 cell was oriented to face the larger APC(Figure 2A, lower arrow); and the larger APCwas in the process of cell division, exhibiting two aster-like foci of microtubule concentration (Figure 2A, upper arrows). Further studies of this system need to be carried out, but these preliminary results are consistent with the following suggestions: that the cluster remained stable during the 10 hr after its formation; that the MTOC/GA in the DI0 Th cell became reoriented to face one of the two originally identical small APC;that thereafter proliferative signals were transmitted from the D 10 to this APCwhich caused it to undergo enlargement and cell division, while the other bound APCwas not stimulated. The Possible

Functions

Served by Talin Redistributions

The unidirectional redistribution of talin under the membraneof the effector T cell where it contacted the specific congener cell was first observed with CTL: target cell couples (32). The notion then was that the talin redistribution might reflect a mechanismto protect the effector CTL from its own cytotoxic secretions. However, when it became clear that a similar talin redistribution occurred in Th cells bound to specific APC, more general functions were indicated. Several possible additional functions can be suggested, including: (a) the further stabilization of specific cell : cell adhesions, to allow prolongedperiods for the cellular interactions within individual couples as maybe required for the secretion of possibly late-acting T cell-derived B-cell growth differentiation factors, such as IL5 (59); (b) the expedition of the localized fusion of GA-derivedsecretory vesicles with the effector T-cell membrane at the region of cell : cell contact; and (c) the participation of talin in the recruitment of LFA-1into the cell : cell contact region (see below) to promoteintercellular adhesion. have no direct evidence, however,bearing on these or other possible talinmediated functions. T CELL

HELP:

SURFACE

PHENOMENA

After concentrating on intracellular events in effector T cells accompanying their interactions with congener cells, we next turned to investigate

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phenomenaat the T-cell surface. This transition has had some drastic sociobiological consequences, particularly with respect to the population density of cellular immunologists involved. An appropriate metaphor for leaving the interior of the T cell for its surface is that it was like going from central Wyomingin the dead of winter to Coney Island on Labor Day. Finding and making appropriate reference to all of the relevant contributions to T cell surface-properties is about equivalent to naming all the ConeyIslanders wearing bikinis. Webegin with some general remarks about membrane phenomena. In polarizedspecific cell : cell interactions, suchas are characteristic of effector T cell:congener cell couples, everything that happens is initiated at the two cell surfaces. Oneor more types of integral protein receptor molecules in the surface membrane of one cell initially bind to their respective ligand molecules in the other. The early productive consequencesof this transcellular binding include: (a) the induction of one or more polarized signals that ultimately affect the intracellular states of one or both cell partners; and (b) the development of a cell:cell adhesion of some appropriate stability. To discuss how these consequences mayarise, a few aspects of membranedynamics first need to be introduced. Mutual

Cappin9

It is important to realize that the transcellular binding of two integral membraneproteins can often lead to their mutual redistribution in their respective fluid membranes. This phenomenonwas originally pointed out by Singer (60) and has been experimentally (61, 62) and theoretically investigated. Let us suppose that we have two cells P and Q with monomeric cell surface molecules A and a, respectively, that can form a transcellular bond (Figure 3A). Supposethat the intrinsic reaction A + a ~ A has a first-order reverse rate constant kr. If the membraneconcentrations of A and a are sufficiently large, and kr is sufficiently small, the formation of one or a small number of transcellular A-a bonds can maintain a localized cell : cell contact long enough(Figure 3B) for the diffusion in the membraneof other A and a molecules into that contact to form additional A-a bonds and thereby to stabilize the area of cell:cell contact (Figure 3C). Because A and a molecules jointly becomecollected into the contact area, the phenomenonis referred to as "mutual capping," by analogy to the well-knownprocess of antibody-induced capping of manycell surface molecules. It seems likely that mutual capping phenomenaprovide the molecular basis for the formation of cell:cell adhesions in general. This proposal predicts that the cell surface area exhibiting mutual cap formation should alwayscoincide precisely with the area of morphologicalcell : cell contact.

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Figure3 Aschematicrepresentationof mutualcappingof a single receptor-ligandpair. Underappropriateconditions,the formationof oneor a fewtranscellularreceptor-ligand bonds(B) results in the diffusionalrecruitmentandbindingof other pairs (C) to make stablecell : cell contact.In the process,the local morphology of the cell surfacesmaychange (C). Reproduced withpermissionfrom(64).

Weshowbelow that this is the case (see also Figure 6 in 60). In addition, however, several other factors may comeinto play. If extensive areas of two interacting cells are "zippered" together by a mutual capping process, the local morphologyof the two cell surfaces may change in a concerted fashion to accommodate the making of the maximumpermissible number of A -a bonds at equilibrium. This maylead to a local flattening together of the two originally curved cell surfaces (as depicted schematically in Figure 3C; cf Figure 5 in 17), or to an extensive interdigitation of villous projections of the two cell surfaces at their contact regions (cf Figure in 17). Furthermore, cytoskeletal-membrane attachments, which often accompanythe antibody-induced capping of membraneintegral proteins, may also be induced during mutual capping processes (see Figure 1B,E and below), Such cytoskeletal involvements mayaffect the morphologyof cell:cell contacts, as well as the stability of the cell:cell adhesion by restricting the diffusibility or turnover of membranecomponents. The situation can be more complicated than that indicated in Figure 3

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if cell P expresses independentsurface molecules .4, B .... , that can form transcellular bondsto moleculesa, b ..... respectively on cell Q. Depending on the conditions, "mutual co-capping" of ,4-a, B-b, etc, pairs may occur into the samecell : cell contact region (64). If the properties of‘4 and B molecules are not independent of one another, still other complexities can arise (next section).

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Syn-capping There are several possible types of noncovalent interactions of different integral protein molecules ‘4 and B in a membrane. The two monomer molecules mayhave a strong affinity for one another and form a molecular complexin the membrane.In such instances, the antibody-induced capping of A results in the capping of B along with A, and vice versa with antibodies to B. This is referred to as co-capping. This is the case, for example,with the TcR and the T3 complex in T-cell membranes(65). There are other instances, however, where the antibody-induced capping of ‘4 results in the collection of B with the A caps, but the capping of B does not affect the uniform distribution of A in the membrane(66). One explanation for such cases is that when an appropriately induced aggregation of A molecules into small clusters occurs, such clusters of A have a higher affinity for B molecules than does monomericA. This is not necessarily reciprocal, however; clusters of B need not have an increased affinity for A. Alternatively, certain signals maymodifyA so that it acquires an affinity for B, whereas unmodified A and B molecules do not interact. Such apparently nonreciprocal intramembranous interactions, which have been encountered, for example, in the association of viral glycoproteins and other integral proteins with class-I MHC molecules (66, 67), have been called syncapping (66), to distinguish them from co-capping. The evidence cited below suggests that, under appropriate conditions, certain assessory molecules in T-cell membranesmayundergo syn-capping with the T-cell receptor. The Dynamics of the T-Cell

Receptor

in Th ." APC Couples

Our experimental studies of cell surface phenomenahave been largely confined to Th cell interactions. Th cells bear on their surfaces clonotypic TcRwith specificity for a complexligand on the APC;this ligand consists of a fragment of the antigen molecule bound to a class-II MHC molecule (Ag/class-II MHC).With cell couples formed between D10 cells (specific for Con/Ia k) and LKcells pulsed with Con, immunolabeling of the TcR on fixed couples showed that in about 70%of the couples that exhibited a redistribution of talin, the TcRwas significantly (but not completely) concentrated into the cell : cell contact region (68). The area of TcRconcentration corresponded exactly to the morphological cell:cell contact

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region as viewed by Nomarski optics. In nonspecific couples that were morphologically similar to the specific couples, no significant TcRredistribution was observed. These results therefore correspond to the prediction of a mutual capping of the TcRon the Th cell with its Ag/class-II MHCligand on the APC. (As the specific ligand on the APCcould not be discriminated from free class-II MHCby immunolabeling, its distribution on the cell couples could not be determined.)

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The Dynamics

of

CD4 in Th’APC Couples

In addition to the TcR, several accessory molecules (including CD4, LFA1, CD2) which are integral membraneproteins of the T cell appear to play important roles in most Th : APCinteractions, because monoclonal antibodies (MAb)directed to these molecules often inhibit T-cell activation (cf 69). With most CTL: target cell interactions, CD8is involved rather than CD4. The amino acid sequences, and putative structures in the membrane, of CD4and CD8are known(70). CD4appears in addition to + T lymphocytes the receptor for HIVattachment to, and infection of, CD4 (71). The precise mechanismsby which the accessory molecules participate in the interaction of effector T cells and their congenercells are not clear. Because CD4is closely associated with class-II MHC-restriction, and CD8 with class-I MHC-restriction, it has been suggested (cf 72) that the CD4 molecule forms a transcellular bond to a monomorphicdeterminant on class-II MHCmolecules, and similarly for CD8with class-I MHC.Evidence for such a transcellular interaction between CD4and class-II MHC has been presented (73). On the other hand, other evidence has suggested that CD4interacts with the TcR, or the TcR/T3 complex, in Th cell membranes(74-80). Furthermore, it is not clear what functions would served by either the putative transcellular binding of CD4to class-II MHC, or the suggested intracellular binding of CD4to TcR. In cell couples formed with D10Th cells and Con-pulsed LKcells, it was found (68) that CD4was concentrated, along with the TcR, on the Th cell membranewhere it was in contact with the APC. On the other hand, in nonspecific cell couples formed by the same D10 and LK cells (the latter, however, presenting the nonspecific antigen Ova instead of Con) CD4was not concentrated in the cell : cell contact region. If in the specific couple, CD4had been clustered into the contact region by a simple process of mutual capping with class-II MHCmolecules on the APC, then the CD4clustering would have been expected to occur equally in the nonspecific couple, whose CD4and class-II MHCwere identical to the specific case. The results rather suggest that the concentration of CD4in the contact region of spec.ific Th : APCcouples is somehowdependent on the specific engagementof the TcR.

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The Dynamics of TcR and CD4on Th Cells Treated with Monoclonal Abs to TcR The clustering of TcR and CD4into the contact regions of specific but not nonspecific Th:APCcouples raises several questions: (a) Is such TcRclustering critical in order to generate one or more signals that are transmitted into the Th cell; (b) What is the molecular mechanism responsible for CD4co-clustering with the TcRinto the contact region; and (c) Whatfunctions maybe served by such CD4clustering? To examine these questions, we took advantage of the findings of Janeway and colleagues (81, 82) whoraised a battery of MAbagainst the TcRon D10cells and madea detailed study of their properties and effects on D10cells. All anti-TcR MAbdid activate D10cells ifa cross-linking secondary antibody was additionally employed (81). However, one particular MAb,3D3, directed against a clonotypic determinant on the D10 TcR, activated D10 cells by itself (82), without requiring a secondaryantibody. If TcRclustering on the Th cell were critical for activation, then uniquely amonganti-TcR MAb, 3D3 added alone should induce TcR clustering (capping) on D10 cells. This turned out to be the case (83). Wenext asked whether the 3D3induced clustering of the D10 TcR had any effect on CD4, and found by double immunofluorescence experiments that CD4was co-clustered with the TcR (83), although CD4and TcR molecules are normally independent of one another in Th cell membranes.J. M. Rojo, K. M. Saizawa, and C. A. Janeway, Jr. (personal communication) have obtained similar immunofluorescence results, and a numberof other types of experiments have also suggested an interaction of CD4with the TcR under particular conditions (74-80). Our interpretation of these results with both MAbsand APCsis as follows: (a) The strong correlation between specific clustering of the TcRand Th cell activation, as observed upon stimulation of Th both with soluble antibodies and with specific APC,suggests that appropriate TcRclustering may be required to generate one or more signals for activation of the cell. Manyother types of receptor-mediated cellular stimulations are also thought to require receptor clustering induced by binding of the specific ligand (cf 84-86). That this might be true of T-cell stimulation, therefore, is not unprecedented. TcRclustering has also been proposed to be critical for the activation of antigen-specific B-cell differentiation by antigen-bridged Th cells (86) and also to induce exocytosis from CTL

(88). (b) This clustering of the TcR may be promoted by a conformational change in the TcR, or the TcR-T3 complex, that is proposed generally

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to accompany TcR binding to its specific Ag/class-II MHCligand on an APC, or to certain clonotypic MAbsuch as 3D3 (82). In the case of Th:APC couples, the clustering of the TcR is further promoted by a mutual capping process of TcR with its Ag/class-II MHC (Figure 3). (c) This ligand-induced TcR conformational change and promotion TcRclustering results in the syn-capping of CD4with the TcR-T3clusters. That syn-capping is involved is indicated by the facts that anti-TcR MAbs directed to either clonotypic or nonclonotypic determinants on the D10 TcR, when allowed to cap only upon addition of secondary antibodies, produce no significant degree of co-clustering of CD4with the TcRcaps; and capping of CD4on D10 cells produces no co-clustering of the TcR (68, 83). (d) If CD4molecules have only a weakaffinity for class-II MHC(73), the CD4that is co-clustered by syn-capping with the TcRinto the specific Th:APCcell:cell contact region may now be concentrated enough to bind to class-II MHCmolecules on the APC. Such transcellular binding wouldcontribute to cell : cell adhesion, and maybe required under certain conditions to transmit another signal between the two cells (78, 89). Such conjectures, therefore, provide a basis to explain how CD4molecules mayexercise a class-II MHC-mediated critical function in helper T cell interactions. Parallel considerations mayapply to CD8function in cytotoxic T cell interactions (90, 91). The Dynamics

of LFA-1 in Th ."

APC Couples

LFA-1 is a monomorphic member of the superfamily of membrane proteins called integrins (92, 93). The molecule of each memberof this family consists of two chains, ~ and /~. Someof the integrin molecules have binding sites on the externally exposed domains of their c~ chains for componentsof the extracellular matrix, such as fibronectin (93). On the cytoplasmic surface of the membrane,integrin molecules are associated with the actin microfilaments of the cytoskeleton (94, 95). In cells such fibroblasts, integrin molecules therefore serve to mediate transmembrane linkages betweenthe extracellular matrix and the cytoskeleton. In lymphocytes, whichgenerally lack an extensive extracellular matrix, there is strong evidence that LFA-1molecules are involved in cell:cell adhesions or in attachments to solid substrata (5). Furthermore, in cell:cell adhesion, there is evidence (5) that LFA-Ion the T cell maybind transcellularly a ligand on the other cell, called I-CAM-1. Wediscuss next our recent studies (A. Kupfer, D. R. Wegmann,S. J. Singer, in preparation) on immunofluorescencemicroscopy of LFA-I distribution on the Th cells in cell couples formed between DNP-Ova-pulsed

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A20-HLcells, as APC,and the cloned Th cell line D36, introduced in an earlier section. Lacking an appropriate antibody specific for the TcR on D36 cells, we did not simultaneously immunolabel the TcR in these experiments. LFA-1, and talin, were found to becomeconcentrated into the specific cell:cell contact regions over a wide range of concentrations of added DNP-Ova,including concentrations lower than those required to activate the Th cells or to induce a reorientation of its MTOC/GA. Neither LFA-1nor talin was collected, however,into the cell :cell contact regions if no DNP-Ovawas presented. Therefore, the specific engagement of the TcRon the Th cells was required to induce the clustering of LFA-1/talin into the cell : cell contact region, as well as to induce the reorientation of the MTOC of the Th cell. However, clustering of LFA-1and reorientation of the MTOC were not tightly coupled. On the other hand, over the entire range of concentrations of DNP-Ova,LFA-1and talin were always found to be co-distributed into the contact region, and to about the same extent, as judged by the immunofluorescentintensity. Other unpublished studies showedthat LFA-1is also clustered, along with talin, into the cell:cell contact regions of other specific Th:APCcouples, and of productive CTL-target cell and NK-target cell couples, but not in the corresponding nonspecific couples. The co-clustering of LFA-1/talin in these couples is +2. independent of extracellular Ca Clustering of LFA-1into the contact regions of specific effector T cell: congener cell couples, as with CD4, is a striking and unusual result, because LFA-1and TcR molecules are not normally associated in T-cell membranes. This clustering is clearly a reflection of the importance of LFA-I in these cellular interactions. However,it does not appear that LFA-1is involved in generating a simple cell:cell adhesion; the fact that LFA-1does not cluster into nonspecific cell:cell contacts argues against an adhesion resulting from a simple mutual capping of LFA-1 with its putative transcellular ligand I-CAM-1.This is because the LFA-1and ICAM-1 were identical in manyof the specific and nonspecific couples (as with DNP-Ovapresented or not presented, respectively, on the A20-HL cells). On the other hand, we have no evidence that LFA-1is syn-capped with TcR clusters, as we have inferred happens with CD4. Whenthe antiTcR MAb3D3 alone was used to cluster the TcR on D10 Th cells and to induce a co-clustering (syn-capping) of CD4with the TcR clusters (82) (discussed above), LFA-1was generally found to remain uniformly distributed on the D10cell surface. At present, we can only speculate about the mechanismfor the clustering of LFA-Iin specific T cell: congener cell couples, but for reasons discussed next, it maybe related to an induced interaction between the integral membraneprotein LFA-1and the cytoskeletal protein talin in the T cell.

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The Association

of Talin

and LFA-1

Amonga large number of cytoskeletal proteins talin uniquely becomes concentrated underneath effector T cell membraneswhere they contact their specific congener cells; this fact deserves an explanation. Talin is a 215-kd protein originally isolated from smooth muscle (41); talin or its isoforms, however,is present in a wide range of different cell types (44). At sites where cells form specialized regions of adhesion to other cells (such as the dense plaques that interconnect smooth muscle cells), or to substratum (such as the focal adhesions that attach cultured fibroblasts to a substratum), talin is also present (44) along with several other cytoskeletal proteins. These are specialized regions where actin microfilaments are knownto be attached to the membrane. In the cell membranesat these same sites, one or another memberof the integrin family of integral proteins (which includes LFA-1)is also often found (93). The possibility therefore arises that talin and LFA-1might interact with one another wherethe latter molecule protrudes from the cytoplasmic surface of a T-cell membrane.In vitro studies (96) have provided evidence for an interaction between talin and chicken smooth muscle integrin, but it appeared to be quite weak. In order to investigate this question in vivo, the antibody-induced capping of LFA-I was carried out on D36 Th cells. By double immunofluorescence we examined whether talin became collected with the LFA-1 caps, as wouldbe expected if the two molecules were associated (A. Kupfer, P. Burn, S. J. Singer, in preparation). Similar experiments have been published on possible integrin/talin associations in chicken lymphocytes (97). Whenthis experiment was carried out on the D36cells, talin was found to remain uniformly distributed on cells that exhibited LFA-1caps. However,if the cells were treated with phorbol myristoyl acetate (PMA), an activator of protein kinase C (98), just prior to the antibody-induced capping of LFA-1,talin was then found to be precisely co-distributed with the LFA-1caps. This redistribution of talin did not occur upon capping membraneproteins unrelated to LFA-I, such as CD4on D36 cells, in the presence of PMA. These experiments indicate that in mouseT cells, LFA-1and talin are not normally associated with one another; when, and only when, an appropriate signal is received by the cell do the two proteins become, directly or indirectly, linked. That PMA delivers such a signal suggests that protein kinase C activation is involved, and that phosphorylation of one or more proteins mediates the LFA-l/talin association, but this has not been established. These findings provide someinsight into the results previously discussed concerning the coordinate concentration of talin under the membrane,and

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LFA-1within the membrane,of an effector T cell where it is in contact with a specific congener cell. The transcellular engagementof the TcRon CTLor on Th cells by a specific ligand on the target cell or APC,respectively, maylead to one or more signals being conveyed into the effector T cell, one consequence of which is the association of talin with LFA1. What factors then induce the recruitment of LFA-1/talin into the contact region are not clear, except that syn-capping of LFA-1with the TcR, which was invoked in the case of CD4, does not appear to be involved. It is not ruled out, however, that the signal that causes LFA-1to become associated with talin also alters the LFA-1molecule so that its affinity for its putative transcellular ligand (I-CAM-I)increases enough to induce their mutual capping into the specific cell:cell contact region. This signal-induced mutual capping of LFA-I and I-CAM-1could make a major and crucial contribution to the overall specific cell : cell adhesion, and thereby account for the important role played by LFA-1 in T-cell functions (5). Because a closely similar redistribution of LFA-1/talin as well as the reorientation of the MTOC/GA accompanies productive NK: target cell binding, the unknownreceptor on NKcells must have signal-induction properties closely similar to those of the TcR of CTLand Th cells as described above.

CONCLUSIONS AND SUGGESTIONS Upto this point we have discussed our experiments largely in the sequence in which they were carried out, in order to indicate the logic behind what was done. In this section, however, the results are summarizedin the sequenceof their probable occurrenceuponinitiation of a specific cell : cell interaction. This requires that cell surface phenomenon be considered first, before their intracellular sequellae. Mostof our studies involving the cell surface were carried out with Th : APCinteractions; it is likely, however, that closely parallel cell surface effects accompany CTLand NKcell interactions with their specific targets. A general commentis that membranedynamics play a key role in the cell : cell interactions studied, being critically involved in the formation of specific cell: cell adhesions and most probably also in the transmission of signals into cells. The global diffusion of integral proteins in lymphocyte membranes, a phenomenonfirst recognized by immunologists nearly 20 years ago, allows these proteins to become clustered and to interact specifically with other molecules within or between cell surfaces. The occurrence of mutual capping and syn-capping phenomena needs to be appreciated, because they provide plausible mechanismsto explain a number of experimental facts that have been difficult to understand.

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At Least Two Distinct Signals are Transmitted T Cell in Th : APC Interactions

to the

In the experiments described above (A. Kupfer, D. R. Wegmann,S. J. Singer, in preparation) with cell couples formed between D36Th cells and Ova peptide-presenting A20-HLcells as APC,clear evidence was obtained for two separable signals transmitted to the Th cell, one at very low doses of the presenting antigen, the other at higher doses. At low doses we observed a specific coordinate redistribution of talin under the membrane, and of LFA-1in the membrane,on the Th cell where it was bound to the APC.Only at higher antigen doses, however, were both a reorientation of the MTOC/GA and activation to proliferate also conferred on the Th cell, along with the redistribution of talin/LFA-1. Fromthese and other results discussed and cited above, we propose the following plausible scenario of membraneevents, although many of the details must still be considered speculative. At low antigen doses, only a limited number of Ag/class-II MHCligands are presented on the A20-HL APC,and these engage an equivalently small number of the TcRmolecules on the D36 Th cell. The low concentration of Ag/class-II MHCligand molecules mayor maynot be sufficient to induce a limited degree of mutual capping with TcR. The small degree of TcR : Ag/class-II MHC binding is sufficient, however,to transmit a first signal (possibly involving a ligandinduced conformational change in the TcR) into the D36 cell; this PMAlike signal induces talin to become linked to LFA-1 and must also be responsible for their joint and massive collection into the contact region forming between the two cells. For example, if the first signal, although involving only a small numberof TcRmolecules, caused an alteration of most LFA-1molecules so that they could now effectively bind not only intracellularly to talin but also transcellularly to their ligands (I-CAM-l) on the APC, the mutual capping of LFA-1 and I-CAM-1 could provide the main source of a specific cell : cell adhesion. No mutual capping of LFA-1and I-CAM-1would occur in the nonspecific case, because no first signal via a small numberof ligand-bound TcRwouldarise. In this scheme, therefore, LFA-I would not contribute to nonspecific adhesion. Such a crucial involvement of LFA-1in the formation of signal-induced specific adhesion could explain whyLFA-1is required for productive T cell interactions (5). This first signal wouldbe independent of extracellular +2 (cf Figure 1). However,this first signal alone is insufficient to activate the Th cell to proliferate or to induce its MTOC/GA to reorient. These phenomena require at least another specific signal, which is only realized at larger antigen doses, and which is Ca÷2 dependent. As more antigen is presented by the APC, a larger number of TcR molecules is engaged, above a

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threshold numberin order for the following effects to occur extensively. The proposed conformational change induced in the TcR by ligand binding, it is suggested, promotesTcRclustering in the Th cell membrane. The TcR would then undergo an extensive mutual capping with its Ag/class-II MHCligand into the contact regions between the two cells, where LFA-1had already been mutually capped via the first signal. These clustering events are considered to be crucial for transmission of a second signal into the Th cell, perhaps functioning directly or indirectly to open a Ca+2 channel in the Th membrane. The CD4that was syn-capped with the TcR would thereby also be collected into the contact region, and perhaps in that concentrated location, CD4could then, and only then, form transcellular bonds to a domain of the class-II MHC molecule. Such transcellular CD4: class-II MHC bonds might be required for still another signal to be transmitted between the Th and its bound APC;this could explain the importance of CD4in manyTh : APCinteractions (and correspondingly CDgin many CTLinteractions). The proposed mutual cocapping of TcR : Ag/class-II MHCand of CD4: class-II MHCpairs into the cell : cell contact region wouldcontribute additionally to the cell : cell adhesion that was primarily due to the first-signal-induced mutual capping of LFA-1 and I-CAM-1. Oneor both of these additional specific signals might then be responsible for inducing the reorientation of the MTOC/GA as well as for turning on a whole cascade of processes leading to activation and proliferation of Th cells. The Reorientation

of the

MTOC/GA

All the evidencepoints to the rapid polarization of the MTOC/GA inside the effector T cell as a prerequisite to the subsequent killing (CTL) stimulation (Th) of the congener cell boundto it. In general, the functions served by such a reorientation wouldbe to direct both the insertion of new membranemass into the T-cell surface and the exocytosis of secretory componentsfrom the T cell within the confined intercellular space generated after cell : cell adhesion.In the case of CTL: target cell interactions, secretion of one or more cytotoxic componentsinto the intercellular space is an attractive modelfor cell lysis, but the unidirectional resistance of the CTLto such toxic componentshas yet to be satisfactorily explained. Talin accumulation under the contacting effector cell membrane,as well as all the integral protein componentscollected into that membraneregion~ may play a role in such resistance. In the case of Th : APCcouples, the directed secretion from the Th cell of a numberof B-cell growth and differentiation factors (57, 99-101) into the intercellular space could act to stimulate only the APCboundto the Th. If the cell : cell adhesion was sufficiently stable and prolonged, sequential delivery of such secretory factors, or the sequen-

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tially stimulated appearance of receptors for these factors on the APC, could allow several stages in the differentiation pathwayof the B cell to occur during a single Th : APCencounter. The potentialities presented by a directed insertion of new membrane mass derived from the GAinto the effector T cell membraneat the cell contact region should not be ignored. Newly synthesized or recycled lymphokinereceptors, or other functionally important integral proteins, or proton pumpspresent in GA-derivedvesicle membranes(8), if inserted into the confined cell : cell contact area, could have important consequences for the cellular interaction. ACKNOWLEDGMENTS

Weare grateful to our collaborators in several of the cellular immunological studies discussed in this review: Drs. G. Dennert, C. A. Janeway, Jr., S. L. Swain, and D. R. Wegmann.The outstanding technical assistance of Mrs. Hannah Kupfer, and the invaluable help and advice of Mrs. Margie Adams and Dr. Anne Dutton, are acknowledged. Weare also particularly indebted to Dr. N. Hozumifor the generous gift of the A20H1cell line, and to Drs. M. Bevan, K. Osami, and J. Kayefor gifts of cell lines and reagents. Our work reported here was supported in part by N.I.H. grants AI-23764 to Abraham Kupfer, and AI-06659 and GM15971to S. J. Singer. S. J. Singer is an AmericanCancer Society Research Professor. NOTEADDEDIN PROOF In connection with the possibility that was raised on p. 331 that the engagement of CD4might invoke a separate signal into the APC-bound Th cell, it has recently been shown (102, 103) that CD4molecules (and CD8on CTL)are physically complexedto the lymphocyte-specific protein ~ck. tyrosine kinase, p56 Literature Cited 1. Adkins, B., Mfiller, C., Okada, C. Y., Reichert, R. A., Weissmann, I. L., Spangrude, G. J. 1987. Early events in T-cell maturation. Ann. Rev. Immunol. 5:325-65 2. Boehmer, von H. 1988. The developmental biology of T lymphocytes. Ann. Rev. Immunol. 6:309-26 3. Sitkovsky, M. V. 1988. Mechanistic, functional and immunopharmacological implications of biochemical studies of antigen receptor-triggered cytolytic T-lymphocytes activation, lmmunol. Rev. 103:127-60 4. Young; Y. D.-E., Liu, C.-C., Persechini. P. M.. Cohn. Z. A. 1988. Per-

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CELL BIOLOGY OF CELL COUPLES 96. Horwitz, A., Duggan, K., Buck, C., Beckerle, M. C., Burridge, K. 1986. Interaction of plasma membranefibronectin receptor with talin--a transmembrane linkage. Nature 320: 53133 97. Burn, P., Kupfer, A., Singer, S. J. 1988. Dynamicmembrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorhol ester treatment of peripheral blood lymphocytes. Proc. Natl. Acad. Sci. USA 85:497-501 98. Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science 233:305-12 99. DeFranco, A. L. 1987. Molecular aspects of B-lymphocyteactivation. Ann. Rev. Cell Biol. 3:143-78 100, Sideras, P., Noma,T., Honjo, T. 1988.

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Structure and function of interleukins 4 and 5. Immunol. Rev. 102:189-212 101. Cambier, J. C., Ransom, J. T. 1987. Molecular mechanisms of transmembrane signalling in B lymphocytes.Ann. Rev. Immunol. 5:175-99 J. M., 102. Rudd, C. E., Trevillyan, Dasgupta, J. D., Wong,L. L., Schlossman, S. F. 1988. The CD~, receptor is complexedin detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc. Natl. Acad. Sci. USA 85:5190-94 103. Veillette, A., Bookman,M. A., Horak, E. M., Bolen, J. B. 1988. The CD4and CD8T cell surface antigens are associated with the internal ~ck. membranetyrosine-protein kinase p56 Cell 55:3018

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Annual Reviews www.annualreviews.org/aronline ,,Inn. Rev. ImmunoL1989. 7:339-69 Copyright © 1989 by Annual Reviews Inc. All rights reserved

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THE LEUKOCYTE COMMON ANTIGEN FAMILY Matthew

L. Thomas

Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110 INTRODUCTION The leukocyte-commonantigen (L-CA) family is a group of high molecular weight glycoproteins uniquely expressed on the surface of all leukocytes and their hemopoietic progenitors (1-14). Membersof this family differ both protein sequence and carbohydrate structures and are expressed by leukocyte populations in specific patterns (15-27). An example of the differential expression is shownfor the rat L-CAfamily in Figure 1 (19). Thymocytesexpress the lowest apparent molecular weight form of 180 kd; B lymphocytes express the highest form, 220 kd; and T lymphocytes express multiple forms. Differences also exist between T-cell subsets (Figure 1C); CD8T cells (Tc/~) express the higher molecular weight forms more abundantly than do CD4T cells (TH). The cell-type-specific patterns of expression are conserved throughout mammalianevolution (1, 2, 9-11, 19, 28), and there appear to be similar patterns of expression in chicken lymphocytes(29). L-CAis referred to in the literature by different names, including T200 (30), B220 for the B cell form (12), the mouseallotypic marker Lyo5 (31) and more recently CD45(32). L-CAis the most accurate descriptive nameand is used for the purpose of this review. The L-CAfamily is a major cell surface componentof lymphocytes and carries muchof the carbohydrate of these cells. It has been estimated that 10%of the lymphocyte surface is occupied by one or more L-CAmembers (33). Because of this abundance, L-CAwas easily detected on SDS-polyacrylamide gels of lymphocytemembranes(36-39) (Figure 1A). It was initially characterized as the major specificity of antilymphocytesera (34) and as an allotypic marker (31, 35). The primary protein structure has been determined from the analysis of cDNAclones, and this information, 339 0732-0582/89/04104?339502.00

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(A) (B)"THYMOCYTES TOTAL APPARENT r PASSED T CELLS .3 Mrx10 / IELUTED I[B [ELLS

200 150 100 -

TOTALT

(c)

i rTc/~

50IBO" Fi#ure1 SDS-PAGE of NaB[3H]4 labeled L-CA purified by monoclonal antibodyaffinity chromatography fromrat lymphoid cell surfaces. A. Thetracks showtotal cell extract (TOTAL), the unretainedmaterialpassedthroughthe affinity column(PASSED), and materialeluted fromthe column(ELUTED). B. Comparison of affinity purified L-CA from thymoeytes, T cells andB cells. C. Comparison of affinity purifiedL-CA fromtotal T cells, CD4T cells (TH)andCD8T cells (Tc/s). Reproduced fromRef. (19) withpermission.

along with studies on the genomicorganization, has delineated the molecular basis for the L-CAfamily (40-48). The function of L-CAhas been enigma, but studies with antibodies to L-CAhave implicated this family in lymphocyteactivation (49-74). This review examinesthese observations, and the discussion is divided into four sections. The first section describes the genomieorganization and the molecular basis of the family. The second section details the glycoprotein structure, and the third section examines the differential expression of family members.In the last section, functional studies are correlated with family memberexpression and the glycoprotein structure. I use these observations to argue that the carbohydrate structures on L-CAare of functional importance and may well be involved in

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determining cell-cell interactions. Theseinteractions will likely result in transmembrane signaling to the cytoplasmic domain.

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GENOMIC

STRUCTURE

The L-CAgene maps to chromosome1 in both mice (75) and humans0q32; 44) and is part of a syntenic region between humansand mice (76). This syntenic group has several genes of immunologicalrelevance. Besides the L-CAgene, the genes for Factor H and C4 binding protein map to this region for both species (76-78). Additionally, in mice this region contains the genes for the IgG Fc receptor, and the genes responsible for the GLD lymphoproliferative disorder and MLSantigens (76, 79). In humans, the genes encoding the complement receptors CR1and CR2as well as decay accelerating factor and membranecofactor protein map to this region (76,79-82). The conserved linkage group appears to be approximately centimorgans in length (76). The significance of the conserved linkage group is not yet understood. The mouse L-CAgene is composed of 34 exons encoding a protein of 1291 amino acids (Figure 2) (N. A. Johnson, M. L. Thomas,unpublished). Transcription appears to be initiated at either of two exons, la or lb, that encode most of the 5’ untranslated region, although the precise start site has not yet been mapped(47). These two exons are not used in a cell-typespecific fashion and it is not clear whethertheir differential use results in any functional difference in the mRNA.Exon 2 encodes the remainder of the 5’ untranslated region and all of the leader sequence. Betweenexons 2 and 3 is an intron of approximately 50 kb (47). Exons 3-33 are located within a 60 kb stretch of DNA,and therefore, the mouse L-CAgene is approximately 110 kb in length. Exons 3-15 encode amino acid residues 1-537 of the external domain; exon 16 encodes the membranespanning region residues 538-574; and exons 17-32 encode residues 576-1178 of the cytoplasmic domain. Exon33 is the largest exon and encodes the remaining

17 19 20 21 1alb2

leader

79

11

34

s 618\1ol 12

I

I I O-linked Cys1

Figure 2 Genomic structure

16 18~ 122 1314

I Cys2

15]

~1

II

11

I repeat1 transmembrane

for the mouse L-CA gene.

25

28 30 31

232,128

Exons that

~71~, ~13z33

~I

I repeat2

encode possible

domains are grouped and labeled below the line. The size of each exon is not to scale. data is from Refs (46, 47) and our unpublished observations.

subThe

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portion of the cytoplasmic domain, residues 1179-1268and the 3’ untranslated region of 1.1 kb.

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From One Gene, Multiple Messayes The L-CAfamily is generated by alternative splicing of three exons. Exons 4, 5, and 6 are used differentially to generate potentially eight different mRNAs (46-48). Six of the eight possible transcripts have been isolated as cDNAsfrom three different species: humans, mice, and rats (42, 4448). 13 lymphocytesuse all three exons resulting in the largest molecular weight form thymocytes splice between exon 3 and 7. Multiple transcripts occur in peripheral T lymphocyte populations (27, 42-48). Other than coding region

mRNA

7 1) L-CA456

cDNA h,m,r

~

Expression gp Mr (kD) B Thy T4 230-240 +

7 2) L-CA4s 7 3) L-CA4s

210-220

+

+

210-220

?

?

210-220

+

+

190-200

?

?

190-200

+

+

190-200

+

+

7 4) L-CAs6 7 5) 4 L-CA 3

6) 5 L-CA

~ 3

7

5

h,r 6

7

7) L-CAe 3 8) L-CA37

7 170-180

+

+

+

Figure 3 Schematic diagram of the differential exon usage for L-CA mRNA.Exons 3-7 are indicated by open rectangles. The exons used within each form are indicated by the LCAsubscript and the splicing events by the V. The forms that have been cloned are indicated in the cDNAcolumn. The species from which each form was isolated are indicated by: h; human;m: mouse; r: rat; and no indicates that no cDNAshave been isolated for that form. The label gp Mrindicates the approximate molecular weights of the glycoprotein for each type of L-CA.The expression columnindicates the possible expression patterns for different L-CAmembers.Definitive proof for the expression patterns within the T cells subsets is not available and the ? indicates only a possible expression based on SDS-PAGE. Data is from Refs (44-48).

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thymocytesand B cells, the exact use of the variable exons in leukocyte populations is not known.Decipheringwhenin normal cell development each of the various mRNAs is expressedis an importantissue that remains to be resolved.

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Regulation of Alternative

Splicing

Manyexamples exist of differential exonsplicing, but little is knownabout the regulation of these events. Cell-type-specificalternative exonusage, however,tends to be the exceptionrather than the rule (83). Splicing mammalianpre-mRNA involves the use of small nuclear ribonucleoprotein particles (snRNPs) (reviewedin 84, 85). It is likely that snRNPs, or associatedfactors, are involvedin regulating the differential splicing events, andthe mostlikely sites of interaction wouldbe 5’ to the 3’ splice site. A comparisonof humanand mousesequences 5’ and 3’ to exons 49 is shownin Figure 4A. There are stretches of significant homologyin the sequence5’ to the 3" splice site of the differentially splicedexons4-6. Of particular note, is a similar nanomersequencewhichis found 5’ to exons 4-6 in both humansand mice (Figure 4B) and which contains conserved TGAT sequence. The importance of these conserved sequences in the regulation of the differential splicing remainsto be determined. In summary,L-CAis transcribed from a single gene, and the family is generatedby the alternative splicing of three exons,4, 5, and6, to generate potentially eight mRNAs. Thepatterns of expression are controlled in a cell type-specificfashion. L-CA GLYCOPROTEIN: CONSIDERATIONS

STRUCTURAL

The completeprimary sequence for human,mouse,and rat L-CAhas been determinedfrom the analysis of cDNA clones (Figure 5) (40, 42, 44-48). The glycoprotein consists of an amino-terminal,external domainthat is (depending upon the family member)391-552 amino acids in length, membrane spanningregion of 22 aminoacids and a very large cytoplasmic domainof 705 aminoacids. It is apparent from the consensus sequence (Figure 5) that the cytoplasmicdomainis highly conservedbetweenspecies, 85%identical residues over 705 aminoacids, while the external domainis muchless so, 35%over 538 aminoacids. The disparity in conservation betweenthe external and internal domainsindicates that the structural requirements,presumably in interacting with other molecules,are different. The External Domain Theprimarysequencehas yielded insights into the architecture of the LCAmolecule. Biochemicalanalysis combinedwith protein sequence data

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0

0

0

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345

divides the external domain into at least three subdomains: an O-linked region and two separate cysteine rich regions. The rat thymocyteform (LCA37; Figure 3) is the only memberthat has been extensively studied at the glycoprotein level (3, 40, 45, 86). It is heavily glycosylated (25% weight carbohydrate; 86) with 14 potential N-linked carbohydrate sites, all but one of which are glycosylated (45). Other L-CAfamily members contain between 1 t and 18 N-linked carbohydrate sites.

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SEQUENCE ENCODEDBY THE VARIABLE EXONS The

immediate mouse aminoterminal sequence, encoded by exons 3-8, and the intron/exon boundaries are shown in Figure 6. The sequence is rich in serines and threonines (34%). There is 14%proline content and no cysteines, indicating a random structure, a sequence characteristic of O-linked carbohydrate sites (87). Therefore, the sequence encoded by exons 3-8 are potential sites for Olinked carbohydrate attachment, and the variable use of exons 4, 5, and 6 changes the numberof potential site~. For the rat thymocyteform, it has been demonstrated that all the O-linked carbohydrates are found within the first 32 amino acids (45). (This corresponds to sequence encoded mouseexons 3, 7, and 8; Figures 5 and 6.) O-LINKED CARBOHYDRATE STRUCTURES The use of the variable exons in a cell type-specific mannerimplies that the sequence encoded by these exons will be of functional consequence.There are two reasons for believing that it is the carbohydratesstructures that are of importance. First, the protein sequencesencodedby exons 3-8 are very similar, rich in serines, threonines, and prolines and are characteristic of O-linked glycosylation sites. Second, a comparison of the human, mouse, and rat protein sequence for this region (Figure 5) shows that the overall homology is low, only 40%. This wouldindicate that it is not the precise protein structure that is of importance. Little is knownabout the types of carbohydrate structures found in this region, with one exception. Lefrancois & Bevan described two monoclonal antibodies, CT1 and CT2, that inhibit cytolysis ofcytotoxic T lymphocytes (CTLs) (88). The CT1and CT2antigens are expressed on few cells types; amongleukocytes, only fetal thymocytes, intestinal intraepithelial CD8T lymphocytes and CTLsexpress these antigens (89-91). These antibodies

Figure 4 A. Comparisonof humanand mouse 3’ and 5’ intron sequences adjacent to exons 4-9. The variable exons are exons 4~6. Sequencesof significant homologyare overlined for humanand underlined for mouse. The amino acids encoded by the exons are given in the single letter code. B. Acomparison of a nanomersequence located 5’ to the 3’ splice site, Humansequence data is from Ref, (48) and the mouse sequence is from Refs (46, 47) our unpublished data.

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Annual Reviews 346 ~r~or~as

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Annual Reviews 348

THOMAS T--3--T

4

GQT~ PT~ P S~ DELS~,~ENALLLPQ~, DP LPARTTE~S TPP s, i s~ ERGNGS~S~ ETTYH49

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PGVLS, ,TLLPHLS, PQPDS, Q,TPS, AGGADT, QT.FS..SQADNP ,TLT. eA~GGG~, DPP 99 T6__ T-GT~G ~.R’~VPGT 1 P/kD T.~kFPVD"I’P $ L/~RN S S/S,k,.S P ~B~ SNVS T ~ ~ SS ~S ~z19 --7 -T-- 8 --T LT,T,LT,PS,T,LGL~S, ,TDPP S,,T,TIAT, T,,TKQT. CA 1’79 Figured The mouseamino-terminal sequenceencodedby exons3-8. The slartof the malure protein is indicaled by ~ andthesequence encoded by eache×onis indicated by the

lines abovethe seqnence. Adot is beloweachserineandthreonineresidue,andpotentialNlinedcarbohydrates sites are overlined.Thesequence is fromRefs(42, 46).

recognize an O-linked car.bohydrate structure found predominantly on the L-CAof these cell types (92). Although the inhibitory effects of the CT1/CT2antibodies on CTLs are not necessarily through L-CA, since there are two other cell-surface glycoproteins that express the antigen. The carbohydrate structure recognized by CT1and CT2 is unusual, involving N-acetylgalactosamine in a ill,4 linkage to galactose and sialic acid in an c~2,3 linkage (GalNAcfll~,(NeuAce2,3)Gal)(93). Since all O-linked carbohydrate structures appear to be found at the amino-terminal end of L-CA,this region most likely contains the CT1/CT2epitopes. It may, however,be located at multiple sites within the O-linked region. Another example of a monoclonal antibody that recognizes a restricted carbohydrate epitope on L-CAis the NK-9antibody (84). The antigen expressed by virtually all humanT cells and NKcells. Whilelittle is known about the structure of this epitope, it is of muchinterest to determine whether it is another unusual O-linked carbohydrate structure. CYSTEINE RICHREGION The exterior domain is approximately 540 amino acids in length for the largest molecular weight form (L-CA456;Figure 3), the amino-terminal serine/threonine rich region is 177 amino acids in length, and therefore there are approximately 360 amino acids between the O-linked region and the membranespanning region. The homology between species for this region is 33%over 352 residues. This level of homologyis unusually low for the same molecule amongdifferent species and is more similar to that found amongmembersof a supergene family, such as the immunoglobulinfamily (95). This supports the argument that the carbohydrates in this region are of functional importance, and indeed this region contains manyN-linked carbohydrate sites. However,similar to membersof the immunoglobulinfamily, there are key conserved residues such as the cysteines, tyrosines, prolines, and tryptophans (Figure 5),

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and therefore the exterior domain of human, mouse, and rat L-CAmay have a conserved three-dimensional structure. The region carboxy-terminal to the O-linked subdomain contains 16 cysteine residues in humanand rat L-CAand 18 in mouse, and it is divided into two subdomains. All the cysteine residues are presumably disulfide bonded. However,the position of the disulfide bonds is unknown,and the only evidence to show disulfide linkage is the separation upon reduction of two 50-kd tryptic fragments of the exterior domain(40). The positions of the cysteine residues, with the exception of the two extra cysteine residues of mouse, are conserved (Figure 5) and are clustered into two groups, of eight cysteines each. Thefirst eight cysteines are within a stretch of approximately 100 amino acids (ca. 180-280; Figure 5), and the next eight (disregarding the two nonconserved cysteines in mouse) are within the following 220 aminoacids (ca. 280-500). The potential N-linked carbohydrate groups follow this pattern. One third of the potential sites are found in the O-linked region, one third in the first cysteine-rich region, and the last one third in the second cysteine region. After the first cysteine of the second cluster (humanresidue 319; Figure 5) there is a short stretch of about 20 aminoacids that is very divergent betweenthe three species. This region contains the two extra cysteines in mouse L-CA. The exterior domain of rat L-CAcan be isolated as a 100kd fragment (40). Further digestion with trypsin cleaves in this region lysinc residue 289, yielding two fragments of 50 kd each that are disulfide bonded(40). This is consistent with the theory that this stretch of 20 amino acids is a loop structure linking the two cysteine clusters. In summary, amino acid sequence and protein chemistry data suggest that the exterior domainis divided into at least three subdomains: an Olinked region, which varies between the different forms, and two cysteine clusters, one of approximately 100 amino acids and the other of 220 amino acids. Cytoplasmic

Domain

The cytoplasmic domain (see note added in proof) is remarkable its size, 705 aminoacids. It is the largest reported to date, corresponding to an approximate molecular weight of 83 kd. An internal duplication of about 300 aminoacids (Figure 7) indicates that the cytoplasmic domain consists of at least two subdomains. In marked contrast to the exterior domain, comparison of the human, mouse, and rat sequence shows that the cytoplasmic domain is highly conserved (Figure 4). The level of homology is 85% over 705 amino acids. Furthermore, if conservative amino acid substitutions are accounted for, the level rises to 95%.The large size of the cytoplasmic domainsuggests that it will interact

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350 THOMAS

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with cytoplasmic components,and this is likely to be important in transmembranesignaling. The L-CAcytoplasmic domain in mice is known to be phosphorylated at serine residues (96). The mouse thymomacell line, BW5147,L-CA constitutively phosphorylated, and two-dimensional tryptic peptide maps indicate that there are several sites ofphosphorylation,all at serine residues (96). In recent studies by Autero & Gahmberg,L-CAof peripheral human T cells becamephosphorylated after treating cells with tumor promoting phorbol esters (97). This would be through the protein kinase C pathway, and L-CAis a knownsubstrate for this enzyme(98). There are multiple potential sites for phosphorylation by protein kinase C in the cytoplasmic domain. Most notable of these is the conserved lys-lys-arg-ser sequence immediately past the membrane spanning region. This sequence is in relation to the membranesimilar to that of the protein kinase C phosphorylation sites of the IL-2 receptor and the epidermal growth factor receptor (98). There are other potential phosphorylation sites of interest: a proline/ serine rich region at approximateposition 900 (914 for humans,Figure 4) and a glutamic acid/aspartic acid/serine rich region at approximate position 950 (964 for humans; Figure 4). These are potential sites for glycogen synthase kinase 3 and 5, respectively (99). The glutamic acid/ aspartic acid region is of particular interest because if the serines at this site are phosphorylated, this area of the molecule becomes extremely acidic. Whetherany or all of these sites are phosphorylated in a physiological situation and how this relates to L-CAfunction remain to be determined. L-CAhas been reported to associate with other molecules; however, only one has been characterized. Bourguignon and her colleagues have recently presented evidence that L-CAcan associate with the cytoskeletal protein fodrin (100, 101), and this interaction wouldbe through the cytoplasmic domain. It is knownthat L-CA will form caps on lymphocytes whencoupled with antibodies and that other surface glycoproteins will cocap (102). This phenomenonmaybe due to a cytoskeletal association with L-CA.The physiological significance is of course uncertain, but perhaps this is an indication that L-CAmaybe involved in somefunction involving the cytoskeleton such as cell motility or membraneorganization. L-CA, therefore, has a large, highly conserved cytoplasmic domain which may interact with cytoskeletal components. Cytoplasmic interactions of L-CAare likely to be important in signal transduction. Electron Microscopy Rat thymocyte L-CA, as viewed by electron microscopy after low-angle shadowing, consists of a rod-like structure of 18 nmand a globular head

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352

THOMAS

of 12 nm(103). The electron micrographs ofWoollett et al (103) are shown in Figure 8. The rod-like structure is the external domain(Figure 8C). This is demonstrated by the isolation and electron microscopy of a 100-kd tryptic fragment that contains the external portion of the molecule and the membranespanning region but very little of the cytoplasmic domain. It is apparent that the globular domain is missing. WhenL-CAis viewed in the absence of detergent, multimers are formed clustered around the head group with the tail structure pointing outwards (Figure 8B). Remarkably, as seen by electron microscopy, multimers of L-CAexist in the presence of detergents. This suggests that L-CAexists as a multimer on the cell surface. There are several lines of evidence to support this conclusion. Multimers of L-CAexist in gel filtration chromatographyin the presence of detergents, suggesting that the hydrophobic interactions of L-CAare strong (103). Second, L-CAimmunoprecipitated from cells treated with a reducible cross-linking reagent are completely cross-linked to one another (61, 96). Upon reduction of the cross-link, only L-CA molecules are found; no other molecules are released. This indicates that only and all L-CAmolecules are within the distance of the cross-linking reagent to one another. Finally, it should be noted that there is high homology in the membranespanning region of the L-CAmolecule between humans, mice, and rats (Figure 5), and this maybe important in determining multimer associations. The L-CAglycoprotein consists of an amino-terminal, heavily glycosylated exterior domain, composedof at least three subdomains; a membrane spanning region; and a large cytoplasmic domain, composed of at least two subdomains. L-CAfamily membersdiffer by the size of the Olinked carbohydrate region in the exterior domain.

L-CA FAMILY MEMBER EXPRESSION Understanding the pattern of expression for each of the L-CAfamily membersis a critical question that needs to be resolved. In cells where only one form is expressed, as in B cells, the pattern is clear. However,in cells, such as T cells, wherea single cell expresses multiple forms and the pattern changes in differentiation and activation, elucidating the pattern of expression is difficult, although some inference can be made through studies with monoclonal antibodies. There are two types of antibodies to L-CA:antibodies that recognize commonepitopes (epitopes found on all members) and antibodies restricted epitopes (epitopes found on some but not all members;CD45R, 32). Antibodies to restricted epitopes discussed in this review are listed in Table 1. Restricted epitopes can be generated by either of two means:

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Annual Reviews 354 THOMAS

I1~.~

I1~1AA

~A

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sequences encoded by the variable exons (including novel epitopes created by the joining of two exons) or posttranslational modifications, such as glycosylation. Whilethe sites of the restricted antigenic epitopes have been dctcrmincd for only a fcw monoclonal antibodies, manyof the restricted epitopes have been shownto be protein in nature (19, 112, 114). However, there are somecarbohydrate epitopes such as the CT1carbohydrate antigen described above (93). Three monoclonalantibodies that recognize restricted epitopes and have been useful in examining L-CAfamily memberexpression are 2H4, OX22 and UCHLI(104, 109, 115). The antigenic epitope recognized by the monoclonal antibody 2H4 was localized to the sequence encoded by humanvariable exon 4 (or junctions with exon 4) by in vitro translation (106). The antigenic determinent of the monoclonal antibody MRC 22 was localized by isolating and sequencing from rat L-CA,a tryptic peptide containing the antigenic site (45). The partial sequence of the OX22 antigenic peptide is Gly-Ala-Asp-Thr-Gln-Xaa-Leu-Ser-Ser-Gln-AlaAsp-Leu-, which corresponds to a sequence encoded by variable exon 5 (Figure 4). However,since the complete sequence of the tryptic peptide not known, the tryptic sequence could extend into sequence encoded by variable exon 6 or, depending upon the family member, constant exon 7 (45). The UCHL1antibody recognizes a restricted site on human L-CA (109). This antibody only recognizes the low molecular weight form of CA. Therefore, this interesting epitope is most likely contained in the sequence encoded by the junction of exon 3 with 7 (110). While these and other antibodies have been useful in examining L-CAfamily member expression, they do not, with the exception of UCHLI,determine which family memberis expressed. For example, cells that stain with 2H4 are positive for the expression of exon 4, but whether exon 4 is expressed in conjunction with other variable exons cannot be determined. Macropha#es and Granulocytes Most human macrophages and granulocytes express one to two forms of L-CAand bind UCHL1,indicating that at least the low molecular weight form is expressed (61, 108, 116). However, some macrophages bind the 2H4antibody and, in certain circumstances, mayexpress higher molecular weight forms (104). It should be noted that while L-CAis an abundant cell surface glycoprotein on tymphocytes, the expression is lower on macrophages and granulocytes (117). However, stimulation of granulocytes rapidly increases the amountof L-CAexpressed from intracellular stores (118). It will be int~esting to determine whether the increased level expression is also followed by a concomitant change in the family members expressed.

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T~xorvtAs

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Variable

Exon Expression

in Lymphoid Populations

B L¥~PHOCV’rES All B cells express the highest molecular weight form of L-CAand, therefore, use all three variable exons. The appearance of the high molecular weight form occurs very early in B-cell differentiation, probably preceding immunoglobulin rearrangement (119). Whether there are changes in L-CAfamily memberexpression when B cells are activated to becomeantibody secreting cells is less clear. The forms expressed by virally transformed B cells, plasmacytomas,and leukemic cell lines are not always the high molecular weight form (L-CA456)(119, 120), yet it is clear whether this reflects a physiological situation. Mouseplasma cells and germinal centers are positive with the monoclonal antibody RA3-2C2 (111), and rat memoryB cells are OX-22positive (115). However, RA3-2C2and OX-22recognize restricted epitope found on several different forms of L-CA,and therefore whether these cells express the highest molecular weight form, L-CA456, cannot be determined with the use of these antibodies. Differences in the carbohydrate structures between L-CAon T cells and B ceils has been well documentedby differential lectin binding (20, 22, 24), analysis of carbohydrate structures, and reactivity with antibodies directed against carbohydrate determinants (20~26). The differences in carbohydrate structures between L-CAon T and B cells is mainly due to the O-linked carbohydrate structures. For example, soybean lectin, which recognizes terminal N-acetyl galactosamine residues, and to a lesser extent galactose, binds selectively to B lymphocytes through O-linked structures on L-CA(22). The carbohydrate structures on B-cell L-CAchange upon activation (121). The addition of IL-2 to LPS/dextran sulfate-stimulated mouse cells causes a five- to fifteen-fold increase in the numberof peanut agglutinin and soybean lectin sites on L-CA.The total level of L-CAon these cells stays relatively constant and, therefore, these changes are through desialation or the addition of sugar moities. The increase in the number of peanut agglutinin sites on L-CAof B cells is of particular interest. Mouse B lymphocytes from germinal centers of lymph nodes and Peyer’s patches are in various stages of activation and can be selectively labeled with peanut agglutinin (122). It is likely that the specific labeling of mouse germinal centers with peanut agglutinin is through the L-CAcarbohydrate structures on these cells. T LYMPHOCYTES Amongleukocytes, T cells express the most intriguing, yet most difficult, L-CApatterns to interpret. There are two reasons for this. First, individual T cells express more than one form and, second, it is not possible to distinguish between some forms by SDS-PAGE.For

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example, the form that uses exons 4 and 5--L-CAas--will be a similar molecular weight to the form that uses exons 5 and 6, L-CA56.It is clear, however, that there is a developmentally regulated expression of L-CA family membersin differentiation from prothymocytes to peripheral T-cell subsets. Immature thymocytes express the lowest molecular weight form, L-CA37. As thymocytes mature, higher molecular weight forms of LCAare expressed (112, 115). This is shown by the small percentage thymocytes that express higher molecular weight forms, the observation that thymocytes expressing higher molecular weight forms are located primarily in the medulla (112, 115, 118) and that the thymocytesexpressing higher molecular weight forms are positive for either CD4or CD8(112). The forms of L-CA expressed by mature thymocytes, although similar when visualized by SDS-PAGE, are not identical to the forms exprcsscd by lymphnode T cells. This is shownby differences in restricted epitopes (112). Likewise, the L-CAforms expressed by CD4and CD8T cells are similar when examined by SDS-PAGE(see Figure 1) (19, 112); however, react differently with antibodies to restricted L-CAepitopes. For example, the 14.8 antibody to mouseL-CAreacts with all CD8T cells but not with CD4T cells (112, 113). The OX-22antibody to rat L-CAreacts with all CD8T cells but with only two thirds of the CD4T cells (115). The 2H4antibody has a similar distribution profile for humanT cells (104). Therefore, while these antibodies are not recognizing equivalent epitopes, they do show that the L-CAfamily membersexpressed by CD4cells are not identical to those expressed by CD8cells. This observation can be extended to the clonal level; CD4T-cell clones are dissimilar to CD8Tcell clones (27, 46). Both CD4and CD8T cells change in their expression of L-CA forms upon activation; however, activated CD4T cells tend to express lower molecular weight forms of L-CAwhile activated CD8T cells tend to express higher molecular weight forms. Recent studies have shown that humanand rat CD4T-cell populations can be divided into functional subsets on the basis of antibodies that bind to restricted L-CAepitopes. For example, the OX-22phenotype subdivides the rat CD4T-cell population (115, 123). The OX-22÷, CD4T cells mediate graft-vs-host reactions, suppress antibody synthesis in animals undergoing these reactions, respond well in both mixed lymphocyte reactions and Concanavalin A (Con A) stimulated cultures, and produce IL2 on activation (115, 123). The OX-22-, CD4T-cell population provides help for B cells in an antigen specific driven assay. The 2H4, WR16, and UCHLIantibodies separate human CD4T cells into homologous populations (71,104, 107, 109, 116). The 2H4÷, CD4T cells induce suppression of IgG secretion in a pokeweedmitogen (PWM)driven B cell response, and they proliferate well to Con A, while the 2H4-,

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CD4T-cell populations respond poorly to mitogens and provide helper signals for PWM-inducedIgG synthesis (104). The WR16antibody seems to divide the CD4T cells into similar populations (7 l, 107). The UCHLI antibody recognizes the reciprocal population to the 2H4antibody. That is, the CD4T cells that are 2H4- are UCHLl+ and have the corresponding functional phenotype (109, 116). Interestingly, the 2H4 antibody recognizes an epitope in exon 4 and immunoprecipitates two higher molecular weight forms of L-CAfrom CD4T cells (105), while the UCHL1 antibody appears to recognize the sequence encoded by the join of exon 3 with exon 7 and recognizes the low molecular weight form (110). Therefore, the 2H4+, CD4T cells express the higher molecular weight forms of L-CA while the UCHL1 +, CD4T cells express the lower molecular weight forms, and this correlates exon usage with functional differences in CD4T-cell subsets. There are manyantibodies to mouserestricted L-CAepitopes, although none have been described that will functionally split the mouseCD4T cell population. However, mouseCD4T-cell clones can be separated into two groups based on their lymphokine production (124, 125). The two groups appear to be remarkably similar to the rat and human CD4T-cell subpopulations separated on the basis of L-CAphenotype. Most mouse CD4 T-cell clones express the lowest molecular weight form of L-CA(L-CA37) regardless of their lymphokineproduction, although there are exceptions (C. T. Weaver, M. L. Thomas, unpublished). Therefore, mouse CD4 cell clones express L-CAforms similar to the rat OX-22-, CD4T cells and the human2H4-, CD4T cells, even though some mouse T-cell clones + or 2H4+, CD4T cells. have a functional phenotype similar to the OX-22 Clearly, the forms of L-CAexpressed by T cells change upon activation. +, CD4T cells and the rat OX-22+, CD4T cells Both the human WRI6 becomenegative for these epitopes upon activation (7 l, 123). Unstimulated mouseCD4T cells, similar to rat CD4T cells, express multiple forms of L-CA.It is likely, therefore, that mouseCD4T-cell clones express the low molecular weight form of L-CAsince they are continuously being activated by in vitro maintenance and that as for humansand rats, there exist for mice in vivo, functional subsets of CD4T cells. Whether the two CD4 subpopulations represent separate or linked lineages is disputed (126128, 128a). However,. this problem is difficult to solve based on L-CA phenotype, since the forms of L-CAexpressed change upon both differentiation and activation. The use of monoclonal antibodies to restricted human L-CAepitopes in immunohistochemicalanalysis reveals interesting patterns of reactivity (108). One antibody, PD7, that immunoprecipitates 220-, 205-, and 190kd forms, reacts with most lymphocytes but fails to react to a subset of

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lymph node T cells and sinusoidal macrophages. In the thymus, PD7stains many medullary thymocytes but fewer cortical thymocytes. The RP1/11 and UCHL1antibodies immunoprecipitate single forms of 220- and 180kd, respectively, and react with a smaller number of leukocytes. RP1/11 reacts with a subpopulation of T cells in the paracortical zone of both tonsils and lymph nodes and strongly with B cells in germinal centers and mantel zones. A small number of medullary thymocytes react with RP 1/11 and in this regard are similar to the reactivity of OX-22in the rat (115). UCHL1 reacts with a small number ofT cells in both the germinal centers and paracortical regions of lymph nodes. In the thymus, UCHL1 reacts with all but a few medullary thymocytes. CT1/CT2 carbohydrate antigens are localized to CD4, CD8thymocytes in the subcapsular region of the thymus of the newbornmouse(89). The differential staining patterns antibodies to restricted L-CAepitopes mayreflect the importance of variable exon usage by leukocytes in tissues. In summary,L-CAfamily memberexpression is precisely controlled in leukocyte differentiation and activation: This suggests that each L-CA family memberwill have distinct interactions. As variable exon usage results in changing the numberof potential O-linked carbohydrate sites and since there are differences between L-CAcarbohydrate structures between leukocyte populations, the distinct interactions for each L-CA family memberwill most likely be mediated through carbohydrate structures. This maybe important for cellular location and for interactions betweenleukocytes and other cells in tissues.

FUNCTIONAL ASPECTS The use of antibodies to disrupt cellular function has provided evidence that L-CAis involved in early lymphocyte activation and is important in transmitting signals across the membrane.These results have been intriguing, and will be even more interesting whenthe precise interactions of L-CAwith other molecules are defined.

B Lymphocytes Antibodies to both commonand restricted epitopes of humanL-CAwill inhibit B-cell proliferation induced by anti-IgM and T cell-replacing factors (65). This inhibition is more effective on small resting B cells and appears to interfere with an early stage of activation as the antibody needs to be addedwithin the first 24 hours of culture. The addition of Ly-5 monoclonal antibodies to Mishell-Dutton cultures inhibits the generation of plaqueforming cells to T cell~lependent antigens, but not T cell-independent antigens (59). Ly-5 will also inhibit IgG responses, but not IgMresponses

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or proliferation, whenB cells are inducedwith lipopolysaccharide(63). This effect is dueto a decreasein the number of cells that switchto become IgG-secretingcells rather than to a decreasein clonal expansionof IgGsecreting cells (129). Theseresults implicateL-CAin B-cell activation and differentiation. However,since the antibodies recognizecommon epitopes, these experimentsleave unanswered the question of whetherthe antibodies are inhibiting an L-CAfunction uniqueto B cells. Natural Killer Cells NKcells expressseveral different tbrmsof L-CAand are similar to T cells in this regard (61, 94). Antisera to the mouseL-CAallotypic marker,Ly5, is a potent inhibitor to NKcell cytolysis, as are somemonoclonal antibody to common L-CAepitopes (49-51, 53). Carbohydratestructures havebeen implicated in mouseNK-cellcytolysis, and it is of interest, therefore, that the poly-N-acetyllactosaminestructures on NKcell L-CA are involvedin target binding and that binding is required for cytolysis (72). Purified L-CAincorporated into liposomes can inhibit conjugate formationbetweenNKcells and target cells. However,no inhibition occurs if the liposomesare treated with Ly-5 or endo-/~-galactosidase. These experimentsare importantin that they are the first demonstrationlinking L-CAfunction with the carbohydrate structures and in coupling L-CA functionwith cell-cell interactions. The monoclonalantibody 13.1 to humanL-CAinhibits NKcell cytolysis. Theinhibition appears to occur at a stage post NKcell binding to target cell (55-57). Whilethe 13.1 antibody recognizes an L-CAepitope on all peripheral blood lymphocytes,the epitope is different from other commonepitopes and maps more distal to the membrane(32, 61). The nature of the 13.1 epitope, whetherit be protein or carbohydrate,has not been determined. Together, the data suggest that L-CAcarbohydrate is importantin binding to target cells and that L-CAis importantin mediating post-binding events, perhapsin the proper positioning of membranes andintracellular signaling. T Lymphocytes L-CAhas been implicated in CTLcytolysis. The mouseallotypic sera Ly5 can inhibit the generation and effector function of CTLsin a mixed lymphocytereaction (54, 60), and at least one monoclonalantibody to commonL-CAdeterminant appears to inhibit CTLfunction. However, not all investigatorsfind this inhibition(50), andothersfind that antibodies to commonL-CAepitope do not inhibit CTLcytolysis (58, 130). The variation betweenexperimentalresults maybe due to differences between

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L-CAepitopes, and it is seen in the effects of antibodies to L-CAon lectin stimulation discussed below. Antibodies to L-CAwill also inhibit NKlike activity of CTLswhen they are stimulated with growth factors (64). It is possible that whenantigen specificity is lost, the interactions of molecules such as L-CAmay become more critical. The correlation betweenfunctional differences in T-cell subsets with the differential expression of L-CAfamily memberssuggests that L-CAis important in mediating these functions. Further evidence is provided by the observation that the 2H4 antibody, which subdivides the humanCD4 T-cell population (discussed above), will also modulate function (66, 73). 2H4÷, CD4T cells mediate the induction of suppression. Whenthese cells are treated with 2H4 antibody, they no longer are able to induce suppression (66). The antibody, however, when included during the culture period, enhances suppression (73). Althoughit is not clear whythe antibody has opposite effects under different conditions, the point is that LCAis involved in activating T cells in this system, perhaps in mediating cell-cell interactions. This is not inconsistent with the effects seen in an NKcell system. Recent observations have shown that the ratio of T-cell subsets, as defined by the expression of various restricted L-CAepitopes, changes in diseases such as multiple sclerosis (131,132), multiple myeloma(133) rheumatoid arthritis (134). Lymphnode T cells from mice with recessive lymphoproliferative disorder, either lpr or yld, aberrantly express the high molecular weight form of L-CA (135). These mice have an abnormal proliferation of T cells resulting in a 50-fold increase in lymph node weight. Whether the forms of L-CAexpressed by T cells contribute to the manifestation or are a result of the disease is an intriguing question that remains to be determined. The WR16antibody, which identifies the same subset as the 2H4 antibody, inhibits pokeweed mitogen-induced proliferation (71), again suggesting an effect in intracellular signaling and T-cell activation. This observation is supported by data that show that antibodies to commonLCAepitopes can inhibit phytohemagglutinin (PHA)-induced proliferation (69). Whenperipheral blood monocytesare cultured with suboptimal doses of PHAin the presence of antibody to a restricted L-CAepitope, the proliferative response is augmentedwith a concomitant increase in the IL2 receptor expression (62). This has also been found with mouse spleen cells (74). Later in the response, the antibody inhibits proliferation (62). This suggests that L-CAis involved in triggering the PHA-inducedmitogenic response, but depending upon the system employed and the antibody used, proliferation is either inhibited or augmented.This provides evidence

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that the carbohydrate structures on L-CAfunction by interacti..ng with lectins, and these:interactions are important for cellular signaling. Further support for-L,GAin T-cell transmembranesignaling is provided by the observations that’antibodies to commonhumanL-CAepitopes can function to replace the signals given by monocytesin triggering lymphocyte activation with anti-CD3 sepharose (68), and antibodies to commonmouse L-CAepitopes can inhibit proliferation induced with anti-Thy-1 antibodies

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(52). The critical feature in the above experiments is that antibodies to L-CA inhibit activation and proliferation of lymphocytesand that the effects are dependent upon the antibodies used. This implies that the cause of inhibition is not steric hindrance~0f antibodies binding to an abundant surface glycoprotein. In somecases, such as in NKcell-target cell bifiding and in modulation of lectin-induced responses, the carbohydrate structures are clearly important in L-CAfunction. Finally, it should be noted that while the differential use of the variable exons implies distinct interactions for each L-CAfamily member, leukocyte populations that express the same protein forms of L-CAmayhave distinct L-CAinteractions due to different glycosylation patterns..Thenext step forward is defining the interactions for each L-CAfamily member.

CONCLUSION I have presented several lines of evidence to argue that L-CAfunctions by interacting through its various carbohydrate structures with other cell surfaces, and this then results in transmembrane signaling to the cytoplasmic domain, The interactions..of the L-CAare likely to be important in lymphocyteactivation. The data can be summarizedas follows: (a) There is precise differential use of three exons within leukocyte populations; (b) the variable exons encode potential O-linked carbohydrate sites; (c) there is a lack of protein sequence conservation betweenspecies for the external portion of the molecule; (d) the glycosylation patterns of L-CAvary between leukocytes; (e) antibodies to L-CAmodulate lymphocyte activation and proliferation; and (f) the large cytoplasmic domainis highly conserved betweenspecies. A critical question that needs to be resolved is what other molecules interact with L-CA. ACKNOWLEDGMENTS

I thank Paul Allen, Nancy Johnson, Jeff Milbrandt, and Emil Unanuefor careful reading of the manuscript and critical comments;NancyJohnson, Chris Meyers, and Jeanette Pingle for the genomic data in Figures 2 and

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4; Matt Haffner for preparing the figures; and Alan Williams for the ph6tographs in Figures 1 and 8 and for permission to reproduce them. M. L~ Thomas is recipient of an Established Investigator Award from the American Heart Association.

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NOTE ADDED IN PROOF

Two,recent reports have identified molecules with significant homologyto the cytoplasmic domain of L-CA. Charbonneauet al (136) have isolated several different protein tyrosine phosphotases. Sequenceanalysis of one of these, PTP 1B, shows homology to each of the L-CA cytoplasmic subdomains. Interestingly, the homology between the subdomains is approximately the same as to PTP 1B with the areas of highest homology (Figure 7) also being the area of highest homologyto PTP1B. This suggests that the cytoplasmic domain of L-CAwill have tyrosine phosphotase activity. Streuli et al (137) have isolated cDNAs for a cell surface molecule in which the external domain has homologyto the external domainof NCAMand the cytoplasmic domain has homology to the cytoplasmic domain of L-CA. It is intriguing to speculate that L-CAbelongs to a family of tyrosine phosphotases in which the interactions of the external domain regulate the activity of tyrosine phosphotase domains. This, of course, maybe important in the regulation of cell growth. Literature Cited 1. Trowbridge, I. S. 1978. Interspecies spleen-myeloma hybrid producing monoclonal antibodies against mouse lymphocytesurface glycoprotein, T200. J. Exp. Med. 148:313-23 2. Standring, R., McMaster, W. R., Sunderland, C. A., Williams, A. F. 1978. The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigens. Eur. J. Immunol. 8: 83239 3. Sunderland, C. A., McMaster, W. R., Williams, A. F. 1979. Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes. Eur. J. Immunol. 9:155-59 4. Michaelson, J., Scheid, M., Boyse, E. A. 1979. Biochemicalfeatures of Ly5 alloantigen. Irnmunogenetics 9: 19397 5. Scheid, M. P., Triglia, D. 1979. Further description of the Ly-5 system. Immunogenetics 9:423-33 6. Hoessli, D. C., Vassalli, P. 1980. High molecular weight surface glycoproteins

of murine lymphocytes. J. Immunol. 125:1758-63 7. Dunlap, B., Mixter, P. F., Koller, B., Watson, A., Widmer, M. B., Bach, F. H. 1980. Molecular relationships between large membrane proteins (LMP) expressed on T and B lymphocytes. J. Immunol. 125:1829-31 8. Triglia, D. 1980. Expression of Ly-5 on yolk sac and fetal liver cells of the mouse. Immuno#enetics 11:303-7 9. Omary, M. B., Trowbridge, I. S., Battifora, H. A. 1980. Humanhomologue of murine T200glycoprotein. J. Exp. Med. 152:842-52 10. Andersson, L. C., Karhi, K. K., Gahmberg, C. G., Rodt, H. 1980. Molecularidentification of T cell-specific antigens on humanT lymphocytes and thymocytes. Eur. J. Immunol. 10: 359-62 1 I. Dalchau, R., Kirkley, J., Fabre, J. W. 1980. Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat. Eur. J. Immunol. 10:737-44

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12. Coffman, g. L., Weissman,I. L. 1981. B220: a B cell-specific memberof the T200 glycoprotein family. Nature 289: 681-83 13. Dalchau, R., Fabre, J. W. 1981. Identification with a monoelonalantibody of a predominantly B lymphocyte-specific determinant of the human leukocyte commonantigen. J. Exp. Med. 153: 753~55 14. Tung, J. S., Scheid, M. P., Pierotti, M. A., Hammerling, U., Boyse, E. A. 1981. Structural features and selective expression of three Ly-5+ cellsurface molecules. Immunogenetics 14: 101~5 15. Watson, A., Dunlap, B., Bach, F. H. 1981. The biosynthesis of Ly-5 in T and B cells. J. Immunol. 127:38~12 16. Sarmiento, M., Loken, M. R., Trowbridge, I. S., Coffman,R. L., Fitch, F. W. 1982. High molecular weight lymphocytesurface proteins are structurally related and are expressed on different cell populations at different times during lymphocyte maturation and differentiation. J. Immunol. 128: 1676-84 17. Tung, J.-S., Scheid, M. P., Palladino, M. A. 1983. Different forms of Ly-5 within the T-cell lineage. Immunogenetics 17:649-54 18. Tung, J.-S., Deere, M. C., Boyse, E. A. 1984. Evidence that Lyo5 product ofT and B cells differ in protein structure. Immunogenetics 19:149-54 19. Woollett, G. R., Barclay, A. N., Puklavec, M., Williams, A. F. 1985. Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes. Eur. J. Immunol. 15:168-73 20. Axelsson, B., Kimura, A., Hammarstrom, S., Wigzell, H., Nilsson, K., Mellstedt, H. 1978. Helix pomatia A hemagglutinin: selectivity of binding to lymphocyte surface glycoproteins on T cells and certain B cells. Eur. J. lmmunol. 8:757~54 21. Childs, R. A., Feizi, T. 1981. Differences in carbohydrate moieties of high molecular weight glycoproteins of human lymphocytes of T and B origins revealed by monoclonal autoantibodies with antMand anti-i specificities. Biochem. Biophys. Res. Commun. 102:1158 64 22. Brown,W. R. A., Williams, A. F. 1982. Lymphocytecell surface glycoproteins which bind to soybean and peanut lectins. Immunology46:713 26 23. Morishima, Y., Ogata, S.-I., Collins, N. H., Dupont. B., Lloyd. K. O. 1982.

Carbohydrate differences in human high molecular weight antigens of Band T-cell lines. Immunogenetics 15: 529 35 24. De Petris, S., Takacs, B. 1983. Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA). Eur. J. lmmunol. 13:831M0 25. Childs, R. A., Dalchau, R., Scudder, P., Hounsell, E. F., Fabre, J. W., Feizi, T. 1983. Evidence for the occurrence of O-glycosidically linked oligosaccharides of poly-N-acetyllactosamine type on the human leucocyte common antigen. Biochem. Biophys. Res. Commun. 110:424-31 26. Ewald, S. J., Refling, P. H. 1985. Analysis of structural diff6rences between Ly-5 molecules of T- and Bcells. Mol. Immunol. 22:581-88 27. Lefrancois, L., Thomas,M. L., Bevan, M. J., Trowbridge,I. S. 1986. Different classes of T lymphocyteshave different mRNAsfor the leukocyte-common antigen, T200. J. Exp. Med. 163: 133% 42 28. Maddox, J. F., Mackay, C. R., Brandon, R. 1985. The sheep analogue of leucocyte commonantigen (LCA). Immunology 55:347-53 29. Houssaint, E., Tobin, S., Cihak, J., Losch, U. 1987. A chicken leukocyte commonantigen: biochemical characterization and ontogenetic study. Eur. J. Immunol. 17:287-90 30. Trowbridge, I. A., Mazauskas, C. 1976. Immunological properties of murine thymus-dependent lymphocyte surface glycoproteins. Eur. J. Immunol. 6: 55762 31. Komuro,K., Itakura, K., Boyse, E. A., John, M. 1975. Ly-5: a new T-lymphocyte antigen system. Immunogenetics1: 452-56 32. Cobbold, S., Hale, G., Waldmann,H. 1987. Non-lineage, LFA-I family, and leucocyte commonantigens: newly and previously defined clusters. In Leucocyte Typing HI, ed. A. J. McMichaelet al, pp. 788-803. Oxford: Oxford Univ. Press 33. Williams, A. F., Barclay, A. N. 1985. Glycoprotein antigens of the lymphocyte surthce and their purification by antibody affinity chromatography. In Handbook of Experimental Immunology, ed. D. M. Weir, L. A. Herzenberg, pp. 22.1-22.24. Oxford: Blackwell Sci. 34. Fabre, J. W., Williams, A. F. 1977. Quantitative serological analysis of a rabbit anti-rat lymphocyte serum and

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splicing. Proc. Natl. Acad. Sci. USA preliminary biochemical characteriza84:5360q53 tion of the major antigen recognized. Transplantation 23:34~59 47. Saga, Y., Tung, J.-S., Shen, F.-W., 35. Lubaroff, D. M. 1973. Analloantigenic Boyse, E. A. 1987. Alternative use of marker on rat thymus and thymus5’ exons in the specification of Ly-5 derived cells. Transpl. Proc. 1:115 isoforms distinguishing hematopoietic cell lineages. Proc. Natl. Acad. Sci. 18 36. Trowbridge, I. S., Ralph, P., Bevan, USA 84:5364-68 M. J. 1975. Differences in the surface 48. Streuli, M., Hall, L. R., Saga, Y., proteins of mouseB and T cells. Proc. Schlossman, S. F., Saito, H. 1987. Natl. Acad. Sci. USA 72:157-61 Differential usage of three exons generates at least five different mRNAs 37. Trowbridge, I. S., Hyman, R., Mazauskas, C. 1976. Surface molecules encoding human leukocyte common of cultured humanlymphoid cells. J. antigens. J. Exp. Med. 166:1548-66 49. Kasai, M., Leclerc, J. C., Shen, F.-W., lmmunol. 6:777-82 Cantor, H. 1979. Identification of Ly 5 38. Andersson, L. C., Wasastjerna, C., Gahmberg, C. G. 1976. Different suron the surface of "natural killer" cells face glycoprotein patterns on human in normal and athymic inbred mouse T-, B- and leukemic-lymphocytes. Int. strains. Immunogenetics 8:153-59 J. Cancer 17:40-46 50. Minato, N., Reid, L., Cantor, H., Lengyel, P., Bloom, B. R. 1980. Mode 39. Gahmberg, C. G., Hayry, P., Andersof regulation of natural killer cell son, L. C. 1976. Characterization of surface glycoproteins of mouse lymactivity by interferon. J. Exp. Med.152: phoid cells. J. Cell Biol. 68:642-53 124-37 40. Thomas, M. L., Barclay, A. N., 51. Seaman, W. E., Talal, N., Herzenberg, Gagnon,J., Williams, A. F. 1985. EviL. A., Herzenberg, L. A., Ledbetter, dence from cDNAclones that the J. A. 1981. Surface antigens on mouse rat leukocyte-common antigen (T200) natural killer cells: use of monoclonal spans the lipid bilayer and contains a antibodies to inhibit or to enrich cytocytoplasmic domain of 80,000 Mr. Cell toxic activity. J. Immunol. 127:982-86 41:83-93 52. Maino, V. C., Norcross, M. A., 41. Shen, F.-W., Saga, Y., Litman, G., Perkins, M. S., Smith, R. T. 1981. Freeman, G., Tung, J.-S., Cantor, H., Mechanism of the Thy-l-mediated T Boyse, E. A. 1985. Cloning of Ly-5 cell activation: roles of Fc receptors, cDNA.Proc. Natl. Acad. Sci. USA 82: T200, Ia, and H-2 glycoproteins in 7360q53 accessory cell function. J. lmmunol. 42. Saga, Y., Tung, J.-S., Shen, F.-W., 126:1829-36 Boyse, E. A. 1986. Sequences of Ly-5 53. Brooks, C. G., Kuribayashi, K., Sale, cDNA:isoform-related diversity of LyG. E., Henney, C. S. 1982. Charac5 mRNA.Proc. Natl. Acad. Sci. USA terization of five cloned murine cell 83: 6940-44, and correction (1987) 84: lines showing high cytolytic activity against YAC-Icells. J. Immunol. 128: 1991 43. Raschke, W. C. 1987. Cloned murine 2326-35 T200 (Ly-5) cDNAreveals multiple 54. Nakayama,E. 1982. Blocking ofeffectranscripts within B- and T-lymphocyte tor cell cytotoxicity and T-cell prolineages. Proc. Natl. Acad. Sci. USA liferation by Lyt antisera, lmmunol. 84:161-65 Rev. 68:117-34 44. Ralph, S. J., Thomas,M. L., Morton, 55. Targen, S. R., Newman, W. 1983. C. C., Trowbridge, I. S. 1987. StrucDefinition of a "trigger" stage in the tural variants of humanT200 glycoNKcytolytic reaction sequence by a protein (leukocyte-common antigen). monoclonal antibody to the glycoprotein T-200. J. Immunol. 131:114~ EMBOJ. 6:1251-57 45. Barclay, A. N., Jackson, D. I., Willis, 53 A. C., Williams, A. F. 1987. Lympho56. Fast, L. D., Beatty, P., Hansen, J. A., cyte specific heterogeneity in the rat Newman,W. 1983. T cell nature and leucocyte commonantigen (T200) heterogeneity of recognition structures due to differences in polypeptide of humannatural killer (NK)cells. sequences near the NHz-terminus. lmmunol. 131:2404-10 EMBOJ. 6:1259-64 57. Newman,W., Fast, L. D., Rose, L. M. 46. Thomas,M. L., Reynolds, P. J., Chain, 1983. Blockade of NKcell lysis is a A., Ben-Neriah, Y., Trowbridge, I. S. property ofmonoclonal antibodies that 1987. B-cell variant of mouseT200(Lybind to distinct regions of T-200. J. 5): Evidence for alternative mRNA Immunol. 131: 1742-47

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366 THOMAS 58. Harp, J. A, Ewald, S. J. 1983. Modulation of in vicro immuneresponses by monoclonal antibody to T200 antigen. Cell. Immunol. 81:71-80 59. Yakura, H., Shen, F.-W., Bourcet, E., Boyse, E. A, 1983. Onthe function of Ly-5 in the regulation of antigen-driven B cell differentiation. Comparisonand contrast with Lyb-2. J. Exp. Med. 157: 1077-88 60, Harp, J. A., Davis, B. S., Ewald, S. J. 1984. Inhibition of T cell responses to alloantigens and polyclonal mitogens by Ly-5 antisera. J. lmrnunol. 133: 1015 61. Newman, W., Targen, S. R., Fast, L. D. 1984. Immunobiological and immunochemical aspects of the T-200 family of glycoproteins. Mol. Immunol. 21:1113 21 62. Ledbetter, J. A., Rose, L. M., Spooner, C. E., Beatty, P. G., Martin, P. J., Clark, E. A. 1985. Antibodies to commonleukocyte antigen p220 influence human T cell proliferation by modifying IL 2 receptor expression. J. Immunol. 135:1819 25 63. Yakura, H., Kawabata, I,, Shen, F.-W., Katagiri, M. 1986. Selective inhibition of lipopolysaccharide-induced polyclonal IgG response by monoclonal Ly-5 antibody. J. Immunol. 136: 2729-33 64. Brooks, C. G., Holscher, M. 1987. Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes, J. Immunol. 138:1331 37 65. Mittler, R. S., Greenfield, R. S., Shacter, B. Z., Richard, N. F., Hoffman, M. K. 1987. Antibodies to the common leukocyte antigen (T200) inhibit an early phase in the activation of resting humanB cells. J. Immunol. 138:3159 66 66. Takeuchi, T., Schlossman, S. F., Morimoto, C. 1987. The 2H4 molecule but not the T3-receptor complexis involved in suppressor inducer signals in the AMLRsystem. Cell. Immunol. 107: 107 I~1 67. Takeuchi, T., Rudd, C. E., Schlossman, S. F., Morimoto, C. 1987. Induction of suppression following autologous mixed lymphocytereaction: role of a novel 2H4 antigen. Eur. J. Immunol. 17:97-103 68. Matorell, J., Vilella, R., Borche, L., Rojo, I., Vives, J. 1987. A secondsignal for T cell mitogenesis provided by monoclonal antibodies CD45(T200). Eur. J. Immunol. 17:1447 51 69. Bernabeu, C., Carrera, A. C., De Landazuri, M. O., Sanchez-Madrid, F.

1987. Interaction between the CD45 antigen and phytohemagglutinin. Inhibitory effect on the lectin-induced T cell proliferation by anti-CD45 monoclonal antibody. Eur. J. Immunol. 17:1461-66 70. Small, R. M., Walden, S. M., Ewald, S. J. 1987. Effects of Ly-5 antibodies on antibody-dependent cell-mediated cytotoxicity (ADCC). Immunology 60:15965 71. Moore, K., Nesbitt, A. M. 1987. Func+ T lymtional heterogeneity of CD4 phocytes: two subpopulations with counteracting immunoregulatory functions identified with the monoclonal antibodies WR16 and WR19. Immunology 61:159-65 72. Gilbert, C. W., Zaroukain, M. H., Esselman, W. J. 1988. Poly-N-acet.yllactosamine structures on murme cell surface T200glycoprotein participate in natural killer cell binding to YAC-1targets. J. Imrnunol. 140: 282l 28 73. Morimoto, C., Matsuyama, T., Rudd, C. E., Forsgren, A., Letvin, N. L., Schlossman, S. F. 1988. Role of the 2H4molecule in the activation of suppressor inducer function. Eur. J. ImrnunoL 18:731-37 74. Marvel, J., Mayer, A. 1988. CD45R gives immunofluorescence and transduces signals on mouseT cells. Eur. J. Immunol. 18:825-28 75. Shen, F.-W., Tung, J.-S., Boyse, E. A. 1986. Further definition of the Ly-5system. Immunogenetics 24:14(:~49 76. Seldin, M.F., Morse, H. C. III, Reeves, J. P., Scribner, C. L., LeBoeuf,R. C., Steinberg, A. D. 1988. Genetic analysis of autoimmune gld mice I. Identification of a restriction fragmentlength polymorphismclosely linked to the gld mutation within a conserved linkage group. J. Exp. Med. 167:688-93 77. de Cordoba, S. R., Lublin, D. M., Rubinstein, P., Atkinson, J. P. 1985. Human genes for three complement components that regulate the activation of C3 are tightly linked. J. Exp. Med. 161:1189-95 78. D’Eustachio, P., Kristensen, T., Wetsel, R. A., Riblet, R., Taylor, B. A., Tack, B. F. 1986. Chromosomallocation of the genes encoding complement components C5 and factor H in the mouse. J. lmmunol. 137:3990-95 79. O’Brien, S. J., ed. 1987. Genetic Maps, A compilation of linkage and restriction mapsof genetically studied organisms. Volume 4. New York: Cold Spring Harbor Lab.

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LEUKOCYTE-COMMON ANTIGEN 80. Weis, J. Y. H., Morton, C. C., Bruns, G. A. P., Weis, J. J., Klickstein, L. B., Wong, W. W., Fearon, D. T. 1987. A complement receptor locus: genes encoding C3b/C4b receptor and C3d/ Epstein-Barr virus receptor map to 1q32. J. Immunol. 138:312-15 81. Lublin, D. M., Lemons,R. S., Le Beau, M. M., Holers, V. M., Tykocinski, M. L., Medof, M. E., Atkinson, J. P. 1987. The gene encoding decay-accelerating factor (DAF)is located in the complement-regulatory locus on the long arm of chromosome 1. J. Exp. Med. 165:1731 36 82. Lublin, D. M., Liszewski, M. K., Post, T. W., Arce, M. A., Le Beau, M. M., Rebentisch, M. B., Lemons, R. S., Seya, T., Atkinson, J. P. 1988. Molecular cloning and chromosomal localization of human membranecofactor protein (MCP). J. Exp. Med. 168: 18194 83. Left, S. E., Rosenfeld, M. G. 1986. Complextranscriptional units: diversity in gene expression by alternative RNAprocessing. Ann. Rev. Biochem. 55:1091-1117 84. Sharp, P. A. 1987. Splicing of messenger RNAprecursors. Science 235: 766-71 85. Maniatis, T., Reed, R. 1987. The role of small nuclear ribonucleoprotein particles in pre-mRNAsplicing. Nature 325:673-78 86. Brown, W. R. A., Barclay, A. N., Sunderland, C. A., Williams, A. F. 1981. Identification of a glycophorinlike moleculesat the cell surface of rat thymocytes. Nature 289:456-60 87. Davis, G. C., Elhammer, A., Russell, D. W., Schneider, W. J., Kornfeld, S., Brown, M. S., Goldstein, J. L. 1986. Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblast. J. Biol. Chem. 261:2828 38 88. Lefrancois, L., Bevan, M. J. 1985. Functional modifications of cytotoxic T-lymphocyte T200 glycoprotein recognized by monoclonal antibodies. Nature 314:449-51 89. Lefrancois, L. 1987. Expression of carbohydrate differentiation antigens during ontogeny of the murine thymus. J. Immunol. 139:2220-29 90. Lefrancois, L. 1987. Carbohydrate differentiation antigens of murine T cells: expression on intestinal lymphocytes and intestinal epithelium. J. lmmunol. 138:3375-84 91. Lefrancois, L., Bevan, M. J. 1984.

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Novel antigenic determinants of the T200 glycoprotein expressed preferentially by activated cytotoxic T lymphocytes. J. Immunol. 135:374-83 92. Lefrancois, L., Puddington, L., Machamer, C. E., Bevan, M. J. 1985. Acquisition of cytotoxic T lymphocytespecific carbohydrate differentiation antigens. J. Exp. Med. 162:1275-93 93. Conzelmann, A., Lefrancois, L. 1988. Monoclonal antibodies specific for T cell-associated carbohydrate determinants react with humanblood group antigens Cad and Sda. J. Exp. Med. 167:119-31 94. Nieminen, P., Saksela, E. 1986. NK-9, a distinct sialylated antigen of the T200 family. Eur. J. Immunol. 16:513-18 95. Williams, A. F., Barclay, A. N. 1988. The immunoglobulin superfamily-domains for cell surface recognition. Ann. Rev. Immunol. 6:381-405 96. Omary, M. B., Trowbridge, I. S. 1980. Disposition ofT200 glycoprotein in the plasma membrane of a murine lymphomacell line. J. Biol. Chem. 255: 1662-69 97. Autero, M., Gahmberg, C. G. 1987. Phorbol diesters increase the phosphorylation of the leukocyte common antigen CD45in humanT cells. Eur. J. Immunol. 17:1503-6 98. Shackelford, D. A., Trowbridge, I. S. 1986. Identification of lymphocyteintegral membraneproteins as substrates for protein kinase C. J. BioL Chem. 261:8334~41 99. Hemmings,B. A., Aitken, A., Cohen, P., Rymond, M., Hofmann, F. 1982. Phosphorylation of the type-ll regulatory subunit of cyclic-AMP dependent protein kinase by glycogen synthase kinase 3 and glycogen synthase kinase 5. Eur. J. Biochem. 127:473-81 100. Bourguignon, L. Y. W., Suchard, S. J., Nagpal, M. D., Glenny, J. R. 1985. A T-lymphoma transmembrane glycoprotein (gp 180) is linked to the cytoskeletal protein, fodrin. J. Cell Biol. 101:477-87 101. Suchard, S. J., Bourguignon, L. Y. W. 1987. Further characterization ofa fodrin-containing transmembrane complex from mouse T-lymphoma cells. Biochim. Biophys. Acta 896:35-46 102. Bourguignon, L. Y. W., Hyman, R., Trowbridge,I., Singer, S. J. 1978. Participation of histocompatibility antigens in capping of molecularly independent cell surface components by their specific antibodies. Proc. Nail. Acad. Sci. USA 75:2406-10 103. Woollett, G. R., Williams, A. F.,

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368 THOMAS Shotton, O. M. 1985. Visualisation by low-angle shadowing of the leucocytecommonantigen. A major cell surface glycoprotein of lymphocytes. EMBO J. 4:2827-30 104. Morimoto, C., Letvin, N. L., Distaso, J. A., Aldrich, W. R., Schlossman, S. F. 1985. The isolation and characterization of the human suppressor inducer T cell subset. J. Immunol.134: 1508-15 105. Rudd, C. E., Morimoto, C., Wong, L. L., Schlossman,S. F. 1987. The subdivision of the T4 (CD4) subset on the basis of the differential expressionof LC/T200 antigens. J. Exp. Med. 166: 1758-73 106. Streuli, M., Matsuyama, T., Morimoto, C., Schlossman, S. F., Saito, H. 1987. Identification of the sequence required for expression of the 2H4 epitope on the humanleukocyte commonantigens. J. Exp~ Med. 166: 156772 107. Moore,K., Nesbitt, A. M. 1986. Identi+ supfication and isolation of OKT4 pressor cells with monoclonalantibody WRI6. Immunology 58:659~64 108. Pulido, R., Cebrian, M., Acevedo, A., de Landazuri, M. O., Sanchez-Madrid, F. 1988. Comparative biochemical and tissue distribution study of four distinct CD45antigen specificities. J. Immunol. 140:3851-57 109. Smith, S. H., Brown,M. H., Rowe, D., Callard, R. E., Beverley, P. C. L. 1986. Functional subsets of human helperinducer cells defined by a new monoclonal antibody, UCHL1.Immunology 58:63-70 110. Terry, L. A., Brown, M. H., Beverley, P. C. L. 1988. The monoclonal antibody, UCHL1,recognizes a 180,000 MWcomponent of the humanleucocytecommonantigen, CD45. Immunology 64:331-36 111. Coffman, R. L. 1982. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B cell development, lmmunol. Rev. 69:5 23 112. Lefrancois, L., Goodman, T. 1987. Developmental sequence of T200 antigen modifications in murine T cells. J. Immunol. 139:3718-24 113. Scheid, M. P., Landreth, K. S., Tung, J.-S., Kincade, P. W. 1982. Preferential but nonexclusive expression of macromolecular antigens on B-lineage cells. Immunol. Rev. 69:143-59 114. Dalchau, R., Flanagan, B. F., Fabre, J. W. 1986. Structural implications of the location and stability to proteolytic enzymes of immunodominant deter-

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minants of the human leukocyte common molecule. Eur. J. Immunol. 16: 993-99 Spickett, G. P., Brandon, M. R., Mason, D. W., Williams, A. F., Woollett, G. R. 1983. MRCOX-22, a monoclonal antibody that labels a newsubset of T |ymphocytes and reacts with the high molecular weight form of the leukocyte-common antigen. J. Exp. Med. 158:795-810 Akbar, A. N., Terry, L., Timms, A., Beverley, P. C. L., Janossy, G. 1988. Loss of CD45R and Gain of UCHL1 reactivity is a feature of PrimedT cells. J. lmmunol. 140:2171-78 Shah, V. O., Civin, C. I., Loken, M. R. 1988. Flow cytometric analysis of human bone marrow. IV. Differential quantitative expression of T-200 commonleukocyte antigen during normal hemopoiesis. J. lmmunol. 140: 1861-67 Lacal, P., Pulido, R., Sanchez-Madrid, F., Mollinedo, F. 1988. Intracellular location of T200 and Mol glycoproreins in humanneutrophils. J. Biol. Chem. 90:9946-51 Kincade, P. W. 1987. Experimental models for understanding B lymphocyte formation. Adv. lmmunol. 41: 181267 Raschke, W. C. 1980. Transformation by Abelson murine leukemia virus: properties of the transformed cells. Cold Spring HarborSyrup. Quant. Biol. 44:1187-94 Cook, R. G., Landolfi, N. F., Mehta, V., Leone, J., Hoyland, D. 1987. Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes. J. lmmunol. 139:991 97 Butcher, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R., Weissman, I. L. 1982. Surface phenotype of Peyer’s patch germinal center cells: implications for the role of germinal centers in B cell differentiation. J. Immunol. 129:2698-2707 Arthur, R. P.~ Mason,D. 1986. T cells that help B cell responses to soluble antigens are distinguishable from those producing interleukin 2 on mitogenic or allogeneic stimulation. J. Exp. Med. 163:774-86 Mosmann,T. R., Coffman, R. L. 1987. Two types of mouse T helper T-cell clone. Implication for immune regulation. Immunol. Today 8:233~7 Janeway, C. A. Jr., Carding, S., Jones, B., Murray, J., Portoles, P., Rasmussen, R., Rojo, J., Saizawa, K., West, ÷ T cells: J., Bottomly, K. 1988. CD4

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LEUKOCYTE-COMMON ANTIGEN specificity and function, lmmunol.Rev. 101:39-80 126. Sanders, M. E., Makgoba, M. W., Sharrow, S. O., Stephany, D., Springer, T. A., Young, H. A., Shaw, S. 1988. Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-I) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN-~, production. J. Immunol. 140:1401-7 127. Serra, H. M., Krowka,J. F., Ledbetter, J. A., Pilarski, L. M. 1988. Loss of CD45R(Lp220) represents a postthymic T cell differentiation event. J. lmmunol. 140:1435-41 128. Sanders, M. E., Makgoba, M. W., Shaw, S. 1988. Humannaive and memory T cells: reinterpretation of helperinducer and suppressor-inducer subsets. ImmunoLToday 9:195 99 128a. Mason, D. W. 1988. Subpopulations of T lymphocytes. Immunol. Lett. 14: 169-70 129. Yakura, H., Kawabata, I., Ashida, T., Katagiri, M. 1988. Differential regulation by Ly-5 and Lyb-2 of lgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4). J. Immunol. 141:875-80 130. Davignon, D., Martz, E., Reynolds, T., Kurzinger, K., Springer, T. A. 1981. Lymphocytefunction-associated antigen I (LFA-1): a surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing. Proc. Natl. Acad. Sci. USA 78:4535-39 131. Rose, L. M., Ginsberg, A. H., Rothstein. T. L.. Ledbetter, J. A.,

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Clark, E. A. 1985. Selective loss of a subset of T helper cells in active multiple sclerosis. Proc. Natl. Acad. Sci. USA 82:7379-93 132. Sobel, R. A., Hailer, D. A., Castro, E. E., Morimoto, C., Weiner, H. L. 1988. The 2H4 (CD45R) antigen selectively decreased in multiple sclerosis lesions. J. lmmunol. 140:2210-14 133. Serra, H. M., Mant, M. J., Ruether, B. A., Ledbetter, J. A., Pilarski, L. M. 1988. Selective loss of CD4+ CD45R+T cells in peripheral blood of multiple myelomapatients. J. Clin. Immunol. 8:259-65 134. llowite, N. T., Wedgewood, R. J., Rose, L. M., Clark, E. A., Lindgren, C. G., Ochs, H. D. 1987. Impaired in vivo and in vitro antibody responses to bacteriophage (I)X 174 in juvenile rheumatoid arthritis. J. Rheumatol. 14: 957~3 135. Tung, J.-S., Saga, Y., Boyse, E. A. 1987. The incongruous Ly-5 phenotype of Ipr/lpr and gld/gld T cells. Immuno,qenetics 25:126-29 136. Charbonneau, H., Tonks, N. K., Walsh, K. A., Fisher, E. H. 1988. The leukocyte commonantigen (CD45): putative receptor-linked protein tyrosine phosphotase. Proc. Natl. Acad. Sci. USA 85:7182-86 137. Streuli, M., Krueger, N. X., Hall, L. R., Schlossman, S. F., Saito, H. 1988. A new member of the immunoglobulin superfamily that has a cytoplasmic region homologous to the leukocyte commonantigen. J. Exp. Med. 168: 1553-62

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Ann. Rev. Immunol. 1989. 7:371-405 Copyright © 1989 by Annual Reviews Inc. All rights reserved

T CELL RECEPTORS IN MURINE AUTOIMMUNE DISEASES Hans Acha-Orbea,

L. Steinman,

and H. O. McDevitt

Departments of Medical Microbiology/Immunology and Pediatrics/Neurology, Stanford University, Stanford, California 94305 INTRODUCTION A critical site in the regulation of the immuneresponse is the formation of the trimolecular complexbetween T cell receptor (TCR), class-II molecules of the major histocompatibility complex (MHC),and antigen. Susceptibility for manyautoimmunediseases is associated with genes in the class-II region of the major histocompatibility complex(MHC)(1, 1 a). The gene products of this region are expressed on antigen presenting cells (APC), and their function is to bind fragments of proteins (peptides) present them to T cell receptors, primarily on CD4+ T cells (2-5). These class-II antigens are very polymorphic (6, 7). While any given class-II molecule can bind a wide variety of peptides, different class-II molecules showdistinct broad specificity patterns. This is reflected in the variation between alleles of MHC molecules in their capacity to present different peptides to the immunesystem (8-10). After successful activation of the T cell, the other functions of the immunesystem are initiated whichinclude production of lymphokines (factors influencing other cells of the immune system), activation of antibody production, and maturation and proliferation of cells (11). TCRsrecognize peptides primarily in the context of the appropriate "self" MHCmolecules. This corecognition is termed MHC-restriction (12). The repertoire of TCRsis generated by a "random" somatic recombination event (13-15). In the normal individual a fine balance is maintained to allow the immunesystem to distinguish between "self" and "nonself." Because the MHC molecules cannot make this distinction (16), 371 0732-0582/89/0410~371502.00

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372

ACHA-ORBEA,

STEINMAN & McDEVITT

the T cell provides some of the regulatory mechanismsthat prevent selfdestruction. Such mechanismsare associated with tolerance induction. Both positive selection for MHC restriction and negative selection against self-reactive T cells upon maturation in the thymus have been suggested as possible mechanisms(17-19). Yet another control might be suppression of self-destructive T cells in the periphery (20, 21). Furthermore, the expression of class-II molecules on only a fraction of an individual’s cells maylimit the frequency of T cells that encounter self plus class-II MHC. Whenthis balance of self-tolerance is perturbed, several additional mechanisms may contribute to the autoimmuneresponse: Aberrant expression of class-II antigens on cells normally devoid of class-II antigen (which then present self-peptides against which tolerance had not been induced), inappropriate lymphokine production, or induction of an immune response against a microbe which cross-reacts with "self" components. This may then induce a cascade of reactions that culminate in an autoimmunedisease. The aim of specific immuneintervention in autoimmuneconditions is to prevent or reverse such self-destruction. Experimental approaches have focused on addition or removal of specific lymphokinesand administration of anti-class I! or anti-CD4 monoclonal antibodies. Recent efforts have been directed at the depletion of T-cell subsets expressing particular TCR variable genes. This review includes a short introduction on the structure and function of the TCRand a brief review of a particularly clear model of T cellmediated murine autoimmunedisease, experimental allergic encephalomyelitis (EAE). A summaryon various approaches for immuneintervention emphasizes the role of anti-TCR antibodies in treatment of autoimmunedisease. The review concentrates on the role of CD4+T cells in autoimmunedisease. Although it is well knownthat this T-cell subset is not responsible for all the steps observed in pathogenesis, these cells play an important role in most of the autoimmunediseases analyzed. T CELL

RECEPTOR

One of the most striking features of the immunesystem is its ability to mount a highly specific immuneresponse toward an indefinitely large numberof foreign antigens. This specificity is mediated by two types of receptor molecules, the TCRexpressed on the cell surface ofT lymphocytes (22-24) and the immunoglobulins (antibodies) expressed on B lymphocytes, and which can be secreted upon activation. These two antigen recognition systems are functionally connected. Most B lymphocytes require help from T lymphocytes for activation (25).

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TCR IN AUTOIMMUNE DISEASE

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Antibodies and TCRsperform their tasks using different strategies: The immunoglobulinsusually have a high affinity with dissociation constants in the range of 10-s_ 10-14 (26) and tend to recognize the three-dimensional (conformational) structures of antigens. TCRs,in contrast, generally not recognize native antigen alone but recognize for the most part linear peptides, associated with the highly polymorphic MHCmolecules, and they require accessory molecules such as CD2, CD4, CD8, and LFA-1to enhancetheir ability to bind strongly enoughfor activation (27-29). These antigenic peptides are degraded componentsof proteins processed by the antigen-presenting cells and presented on the cell surface in association with MHCmolecules. Only the MHCmolecules present during the maturation of the T cells are recognized as restriction elements (12). These peptides apparently bind to a single site on the MHCmolecule (30). This observation is supported by the crystallographic structure of a functionally similar class-I MHCmolecule in which a noncovalently associated molecule was found embeddedbetween two s-helices. Most of the polymorphic residues pointed into this grove (3 I, 32). A structure similar to class I has been proposed for class II molecules (33). Generation

of Diversity

Both the B and T cell antigen recognition systems use the same basic mechanismto generate vast diversity. These molecules require a highly diverse antigen-binding region and a constant anchor region. The constant region mediates functions such as complementfixation and B-cell activation for antibodies (34), while the constant region anchor of the TCR is involved in T-cell activation uponantigen contact (35). In the germline, pools of variable (V), junction (J), diversity (D), and constant (C) gene segments are encoded. Unique receptor structures are generated by somatic recombination of these gene segments (13-15, 36). In the TCR 20-30 Vr, 12 Jr, 2 Dr, an estimated 100 V~ and 50-100 J~-gene-segments are encoded in the germline. For the ~-chain the functional molecules are composed of VJCand for the fl-chain of VDJC-segments. Diversity is achieved by the joining of these elements in all the distinct permutations possible and is diagrammaticallyshownin Figure 1. On each clone of T cells only one type of TCRheterodimer is expressed. This is achieved by allelic exclusion (14, 37). In addition to these somatic recombination events, further heterogeneity can be introduced by the following mechanisms: JUNCTIONAL DIVERSITYThe mechanism which joins the different elements is not precise. Nucleotides at either side of the VrDr and DaJr or the V,J~-junctions can be deleted upon recombination. In immunoglobulins deletion of nucleotides have been observed in the J-region only.

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V

V

rC~ 3. germ[l~.

O1

I1II I I I Ill I II I II II Hill I~

J 1.1-17 C D 2 d 2,1-2.7 C V Illllll-I

,

--V

! IlJllll

J 2,4-7C v II II I II IIIII

V

D J

IlaHI !_/

I

C

H TM CY

TCR ~ OrOlein

Figure 1 Germline organization

and rearrangement

of TCR c~ and/~ chains.

N-REGION DIVERSITY In these junctional regions random sequences of nucleotides can be inserted. This mechanismis found in both the ce- and the fl-chain of the TCRbut only in the heavy chain of immunoglobulin molecules. O REGION The D region can be translated in all three reading frames in TCR/?-chains, but in the immunoglobulin heavy chain usually only one of the three possible reading frames is found. sOMATIC MUTATIONHigh frequencies

of point mutations are found in antibody molecules. In TCRsno convincing evidence for such a mechanism had been found in sequence analysis of manydifferent TCR-molecules. J-REGIONELEMENTS The TCRlocus encodes more J-region elements than either immunoglobulin locus (50-100 J~, 12 J~ in the TCR,vs 4 in the heavy and 4 in each light chain of immunoglobulins). In immunoglobulinvariable region sequences, three clear hypervariable

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IN AUTOIMMUNE DISEASE

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regions were defined that were found to interact with antigen; these were termed complementarity determining regions CD1, CD2, and CD3(3840). The third coincides with the VJ-junctional region. In TCRmolecules the whole V region seems to be hypervariable, although the major peak of hypervariability is found at the same location as CD3in antibody molecules (41, 91). Similarities in amino acid sequence between TCRand antibody molecules have been found especially in the V- and J-regions. Manyof the amino acids important for immunoglobulin heavy and light chain pairing are conserved (41). Recognition

of Peptides

by the TCR

T cells recognize antigens as peptide fragments associated with MHC molecules. Antigen processing can be mimickedby proteolytic cleavage of proteins or by addition of short synthetic peptides (4, 42). The shortest peptides that can initiate an immuneresponse are 7-9 amino acids long, but usually longer peptides elicit better responses (27). The length peptides bound to MHCantigens in vivo is unknown. Whenit became possible to measure the binding of peptides to solubilized MHCmolecules it becameclear that different MHC molecules select different peptides to be presented to the TCR(43, 44). This is due to the polymorphism MHC molecules. Nevertheless, there is not a complete correlation between binding or particular peptides and initiation of an immuneresponse (44). Using peptides with single amino acid substitutions it was possible to define amino acid residues that interact with MHCmolecules, the TCR, or possibly both (45, 46). Competitionexperiments with different peptides showed that, with the peptide combinations tested, probably only one major peptide binding site exists on the class-II molecule. This agrees with crystallographic structure analysis of a class-I molecule(31, 32). In relating the observations from the competition experiments to a structural model, two possibilities have been suggested: (a) there is only one binding site the MHC molecule, or (b) peptide binding to the MHC induces an allosteric modification in the protein which precludes binding of a second peptide. In addition, structural motifs are present in those peptides which bind MHCmolecules (4749). In several experimental systems, it has been observed that certain mouse strains can elicit an immuneresponse to peptides but not to the proteins containing these peptide sequences (5053). In these instances, nonresponsivenessto particular protein is not due to the inability of the synthetic peptide to bind the MHC molecule or holes in the repertoire of the TCR.One possible interpretation is that naturally proccsscd peptidcs arc bigger than the synthetic peptides used in these studies and that these longer peptides cannot bind strongly enough to certain MHCmolecules or cannot be recognized by TCR(52). Other

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explanations are the presence of distinct suppressor epitopes within the protein (53) or deletion of the epitope by antigen processing. A peptide must bind to a MHC molecule to elicit a T-cell response (43, 44). Analysis of T-cell epitopes in different antigens revealed that MHC disparate mousestrains often recognize different peptides of the same protein. Even small epitopes on antigens can elicit a heterogeneousimmune response as determinedby peptide fine specificity for cytochromes(28), repressor (55), staphylococcal nuclease (54), myoglobin(27, 56), simplex glycoprotein (57), ovalbumin (45), and lysozyme (53, 58-60). showthe heterogeneity of the response in one mousestrain, the following example is given: Betweenamino acids 111-118 there are at least three sperm whale myoglobinepitopes seen in the context of I-Ed, whenanalyzed on peptides with single aminoacid differences, peptides of different length, or allo cross-reactivity patterns. Betweenresidues 108 and 117 at least three epitopes could be mappedin the context of I-Ad when analyzed with peptides of different length (27, 56). In experimentally induced autoimmunediseases, antigens involved in pathogenesis are easily defined, and several have been characterized: 1. Arthritis in rat and mouse:Native type-II collagen in collagen-induced arthritis and mycobacterial heat shock protein in adjuvant arthritis (61, 62). 2. Experimental allergic thyroiditis (EAT)in mouse:Thyroglobulin(63). 3. Experimental allergic myasthenia gravis (EAMG):Acetylcholine receptor (AChR)(64). 4. Experimental allergic encephalomyelitis (EAE) in mouse and rat: Myelin basic protein (MBP)and proteolipid protein (PLP) (65-67). It is muchmore difficult to find the initiating antigen in spontaneous autoimmunediseases. Finding a structure in the target organ that can elicit an autoimmuneresponse upon injection in adjuvant does not prove it is the autoantigen involved in spontaneous pathogenesis. Nevertheless, target antigens have been defined in humanrheumatoid arthritis (type-II collagen) (68) and myasthenia gravis (acetylcholine receptor) (64). In the AChRpeptides p195-212 and p257-269 have been partially characterized as epitopes in myasthenia gravis patients of the HLA-DR5and HLA-DR3,DQw2MHChaplotypes, respectively (69). TCR Structure-Function Since it has been possible to obtain cDNAsequences of TCRs, attempts have been made to correlate the complex antigen/MHCrecognition with TCRstructural components. These studies have sought to correlate the TCRcDNAsequences from T-cell clones or hybridomas with patterns of

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TCR IN AUTOIMMUNEDISEASE

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fine specificity and cross-reactivity. It is clear that the TCRis the sole structure on the T-cell surface that determinesthe fine specificity in recognition, but in most cases accessory molecules are needed to stabilize or initiate the interaction (70-72). The results obtained in a variety of antigen recognition systems are summarizedin Table 1. T-cell clones reactive to pigeon cytochromec provide the best analyzed antigen-recognition system. It has been selected because of its manypatterns of allo-cross-reactivity, different fine specificities towardspecies variants of cytochromemolecules, and various allo restriction patterns (28). Nevertheless, a limited heterogeneity of TCR-elementshas been detected in this response (73-77). The majority of analyzed T-cell clones utilize memberof the V~I 1 family. Most of the differences in TCRgene-segment usage correlated with the different fine specificity patterns. In T-cell hybridomas with a specific allo-cross-reactivity, selection of TCRelements was observed (76). At least three out of the four V and J elements were found shared between the T-cell clones which exhibited a specific allocross-reactivity pattern. Differences in the VJ~junction can changepeptide Table 1 TCRelements used preferentially

Antigen k’b bCyt.cI-E

Responder mouse strain BI0.A

in antigen/MHCrecognition

Most frequently used aTCR~elements V~ll (100%), J~84 (57%)

bCyt.c I-E k’sorS B10.A

V~I 1 (40%), J~84 (60%)

MBPpl-9

PL

V~4 (100%), J~TA31

MBPpl-ll

B10.PL

Most frequently used TCR~ elements a V~3 (100%), J~l.2 (78%), J~2.5 (22%) Val (71%), Jal.2 (58%), Ja2.1 (43%) Va8.2 (>78%), Ja2.7 (50%)

(75%) V,2.3 (58%), J~39

Va8.2 (79%), Ja2.7 (79%)

(100%) V~4.2 (42%) MBPp89-100 aMls + I-E Arsonate

SJL -SJL, SWR, C57L, C57Br a I-Aa, I-Ak, L

C57BL10

bml2

None None V~3 (80%), J~TA20’ (40%) None

Val3 (21%), Ja2.2 (21%) Val7 (100%) cV~8.1, V~6 cV~17 Va2 (40%), Ja2.5 (40%) None

~Forreferencessee text. ThemostfrequentVand J regionsare listed withthe percentof clonesanalyzed expressingthis element. ~Pigeoncytochrome c peptide 81-104-specificT cell clones. Allorestriction on the indicated class II antigens. ~Themajorityof T cell clones expressingVal7are specific for I-E+antigen;the majorityof Va8.1or Va6expressingT cell clones react with Mls". Thefrequencyof T cell clonesreacting with these antigen/ MHC complexes is unknown.

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fine specificity, and T-cell clones distinct in one aminoacid in the N-region of the//-chain and in the usage of a different V~-chainshowdifferences in allo-cross-reactivity (73-75). Comparisonof TCRsequences with specificity for cytochrome c and restriction to I-Ek and b revealed that one amino acid, asparagine at position 100, of the TCRfl-junctional region is found in the majority of T-cell clones. In those T-cell clones restricted to I-Ek and s, another amino acid, aspartic acid, is found at the same position in the majority of TCRsequences (77). All the T-cell clones with restriction to I-Ek and b express V~3 and one of two J~-regions, and a memberof the V~I 1-family with one out of three J~ regions. Similar patterns are found in the T-cell clones restricted to I-Ek and s. The length of the VaJa-junctionalregion seemsto be critical. In contrast, the junctional region of the e-chain showsneither specifically selected amino acids nor specific length. In the response to arsonate (a hapten that covalently binds to free amino groups) four out of five T cells utilize V~3, independent of the MHC restriction element used. After 4 weeks, in vitro culture of Ars-reactive Tcell lines V~3 was enriched. Transfer of the e-chain from an arsonatespecific T cell clone to cells that utilized the samerestriction element but exhibited different antigen reactivity, resulted in specificity for arsonate and the patterns observed in the recipient T-cell clone (78). In contrast to pigeon cytochrome e and arsonate reactive TCR, the response to modified APCs with the hapten N-iodoacetyl-sulfonicnaphtyl-ethylene-diamine (AED), responses show greater heterogeneity TCRusage. Twoout of four T-cell clones use the same J~ and J~ elements, two other ones use a memberof the same V, family (79). Even greater heterogeneity was found for beef-insulin and leukocytic choriomeningitis virus (LCMV)responses (80~82). In all three systems, the exact antigen structures have not been identified, and the greater diversity in TCRusage mayreflect the heterogeneity of peptides recognized. In the response to AED,a hapten reacting with free sulfhydril groups on the APCsurface, the TCRrecognition could be dependent on peptide sequences in addition to the hapten structure. dSperm whale myoglobin-specific T-cell clones in mice expressing I-A and I-Ed showa correlation between the restriction element and usage of Va8(83). Ten out of eleven I-E~ restricted T-cell clones utilized V~8. With alloreactive T cells different results were obtained in different systems. In short-term culture in vitro, two- to four-fold enrichment for specific V regions was observed. Analysis of a large number of T-cell hybridomas in C57BL/10anti-bm 12 responses did not reveal any significant increases in specific TCRelements (84-86; E. Palmer, personal communication).

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Strong selection for TCRelements has been found in specific antigen/ MHCrecognition systems, but use of these elements was not confined to any one antigen or MHCrestriction pattern. In addition analysis of TCRsof T cells with manydifferent specificities and restriction elements has shownthat there is no correlation in the usage of specific TCRelements and the recognition of different classes of MHCmolecules (88, 89). For example, the same V~ and Va regions were found in a class I-restricted cytotoxic T cell and a class II-restricted helper T cell hybridoma.The cells did not showcross-reactivities (89). In addition a T-cell clone specific for sperm whale myoglobinpeptide in association with I-Ed used the same V~, Jt~ and a memberof the same V~ family as an I-Au restricted myelin basic protein (MBP)-specific T-cell clone (A. M. Livingstone, personal communication; 90). The primary difference between TCRsequences of those two clones was found in the D region, although the J,-region usage in the sperm whale myoglobin-specific T-cell clones has not yet been determined. Despite the considerable sequence similarity, these T-cell clones showno cross-reactions (90). In antibody molecules three hypervariable regions in both the heavy and the light chain are found. These hypervariable regions are called complementarity determining regions and are designated CD1, CD2, and CD3(3840). The third coincides with the V J-junction. In TCRmolecules the VJ-junction represents the only significant hypervariable region (91). Assuming that TCRand antibodies have a similar three-dimensional structure, a model has been proposed in which the third hypervariable region in TCRs, which shows the greatest variability, interacts with antigen, whereas the others interact primarily with MHCmolecules (41). The selection of a particular amino acid in the N-region of cytochromespecific T-cell clones with a defined allorestriction pattern agrees with such a model(77). Clonal deletion, selection of mutants, cell fusion and transfer experiments implicated the r-chain more often in MHCrecognition than the a-chain (18, 19, 71, 73). Careful analysis of the results showthat the structure of the r-chain is important for MHCrecognition but does not by itself determine specificity for a particular MHC epitope. In transfer experiments,/?-chain genes were introduced in cells which had a similar antigen specificity but a different MHC restriction pattern. After transfection of the r-chain both patterns were observed. Because both T cells expressed a similar a-chain, a contribution of the a-chain to MHC recognition could not be excluded (71). Whenthe thymomacell line BW5147 is used as a fusion partner to generate T-cell hybridomas, the resulting cells are specific for the primary antigen and often are cross-reactive for allo MHCantigens. Because BW5147contains functional TCRc~ and fl chains, and variants of BW5147 which lack expressed ~ and/3 chains do

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not showthis pattern of cross-reactivity (92), it is possible to conclude tentatively that the ~ chain contributes significantly to antigen and allo MHC recognition in the T cell. A strong correlation between V~17-usageand I-E recognition has been demonstrated (18, 19). In addition Val7 does not recognize I-E per because macrophagesor transfected fibroblasts which express high levels of I-E on the surface cannot stimulate T cell hybridomasspecific for I-E, whereasI-E on the surface of a B cell can (93). The TCR V regions show hypervariability throughout the entire sequence with the exception of conserved amino acids which are probably involved in chain-chain pairing (41). It could be argued that different parts of the V regions can take part in antigen/MHCrecognition. This would explain whycertain V regions such as V~8 are found in manyantigen/MHC recognition systems. Furthermore, the apparent requirement for accessory molecules in the formation of a stable T cell-target cell interaction may suggest a requirement for a conformational change before a stable interaction can occur. It is possible to conclude from the studies with cytochromec that more than one region of the TCRis selected in a specific recognition pattern, that several elements are selected in concert. It appears that a specific three-dimensional structure of TCR,in a response to a single antigenic epitope in association with MHCmolecules, is required, not merely a particular element. It is possible that a single element plays a major role in recognition of the target antigen or MHC;however, the complete combining site is formed by contributions from all the variable parts of the TCR.A more careful analysis on T cells with identical fine specificity should help to clarify thi~ issue. Clonal Deletion The class-II molecules determine which peptides will be presented to the TCR.Because it has been shown that APCscan present self-antigens in vivo (16), additional mechanisms must operate to protect an organism from self-destruction. Goodevidence for one of several possible mechanisms has recently been presented. With the availability of monoclonal antibodies specific for certain TCRV~regions, analysis of expression levels of various TCRV genes in different mouse strains became possible. Surprising results were obtained: It was found that in I-E-expressing mouse strains and FI hybrids between I-E+ and I-E- mice, the majority of V~17and V~ 11-expressing T cells were detectable in thethymus, but absent from the peripheral T-cell population (18, 19; E. Palmer, personal communication). This was not due to TCRrecognition of cells expressing native I-E molecules but was correlated with antigen presented by I-E

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TCR IN AUTOIMMUNEDISEASE

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molecules (93). In another series of experiments, it was shown that mouse strains expressing at least one copy of Mlsa (new nomenclature system: Mls-la), a strong mixed lymphocyte stimulatory determinant, both V~6and V~8.1expressing T cells were depleted in the periphery (94, 95). As seen with Val7, levels of expression in the thymus were normal, and furthermore Va6-positive cells were found in the cortex but not in the medulla of the thymus (H. Hengartner, personal communication). From the strikingly similar results in these two systems, it is possible to conclude that under some circumstances particular V~l-bearing T cells are depleted in the thymus. It is possible that clonal deletion does not alwaysresult in such complete removal of V regions in the periphery but that in other antigen-recognition systems, certain ~-heterodimeric structures will be eliminated. Becauseof the lower frequency of specific heterodimeric TCRs, these instances are muchharder to detect.

TCR IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS Characteristics of EAE Experimental allergic encephalomyelitis (EAE) is an induced autoimmune disease of the central nervous system (65) and mimics in manyrespects the humandisease of multiple sclerosis (MS) (96-98). The disease can induced in manyspecies including mice and rats and, accidentally, in humans(through contamination of a vaccine preparation with spinal cord material). The disease is characterized by the acute onset of paralysis. Perivascular infiltration by mononuclearcells in the CNSis observed in both models. Demyelination is found in mice (99-102, 108, 116). An inflammation similar to a delayed type hypersensitivity (DTH)reaction is found in the lesions. Onlya small proportionof the participating cells is actually specific for the target antigen(s). In rats, irradiation of recipient rats before transfer of disease with activated spleen cells reveals no such DTH-likereaction, even though paralysis is observed (105). If sublethal doses of antigens are administered, rats recover several days after the onset of clinical signs. After one weekrats often showone relapse and thereafter are protected for the rest of their lives fromfurther induction of EAE(102, 103, 106). Mice develop acute, chronic and chronic-relapsing forms of the disease, depending on the methodof induction (65, 96, 97, 107-109). The disease is more difficult to induce in mice than in other species. Coinjection of pertussis vaccine is usually required for induction of disease (107-109).

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EAE is clearly mediated by CD4÷ T lymphocytes. This was shown originally in adoptive transfer studies with lymph node cells (110), cells (111-113), selected T-cell subsets (114), and later by isolation encephalitogenic T-cell lines or clones (115, 116). One of the genes mediating susceptibility is localized in the MHC classII region (50a). The best analyzed encephalitogenic protein is myelin basic protein (MBP),but other encephalitogenic antigens are found in the brain (65-67). Using synthetic peptides it was possible to map the epitopes accurately without the problems with contamination involved in puriu) fication of proteolytic peptide fragments. In the PL mousestrains (H-2 two encephalitogenic peptides in MBPhave been characterized: The MBPpeptide p35-47 and the acetylated N-terminal nonapeptide MBPpl-9 (MBPis naturally acetylated at its N-terminal in vivo) (50, 117, 118). N-terminal nonapeptide is also encephalitogenic in the B10.PL mouse strain. The MBPp35-47 peptide has not been tested in B10.PL. In the SJL mouse(H-2s) at least three overlapping encephalitogenic peptides at positions MBPp96-109, p89-100, and p92-101 have been identified (119121). In the Lewis rat the major encephalitogenic peptide is MBPp68-88, and another unmappedepitope lies outside this region (12~124). Using autologous MBPas an immunogenin rats results in about 50%of hybridomasdirected against either epitope. It has been observed that induction of EAEis easier with xenogenic MBP.The encephalitogenic T-cell clones elicited against these xeno-determinants also react with self-determinants. Therefore it was argued that suppression of self-reactive immuneresponses is responsible for this effect (124). The

PL-Mouse

To analyze the heterogeneity of TCRin MBP-specific T-cell clones from PL or (PL x SJL)F1 mice, a panel of 21 MBP-pl-9-specific T-cell clones was analyzed for expression of TCRscontaining V~8with the monoclonal anti-Va8 antibodies KJ-16 and F23.1 (125, 126). These T-cell clones were derived from individual mice primed and stimulated with different forms of antigens, or if pairs of clones were taken from the same cloning experiment, their TCRcomposition was distinct by anti-V~8 antibody reactivity. At least 78%of the PL and (PL x SJL) derived MBPpl-9-specific T-cell clones were Va8+. This represents a minimumestimate because more than one clone per experiment was taken only whenit was distinguishable with the Ve8 marker (127). In most mouse strains V~8 is the predominant TCRV region expressed on 10-25%of peripheral T cells. Therefore it was important to see whether the increased frequency of V~8 expressed by MBPp 1-9 reactive T cells represents the situation in vivo or whether it is a cloning artifact. To

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÷T + cells were sorted into Va8 address this issue, MBPpl-11-primed, CD4 and Va8- cells and then directly stimulated in vitro. Becausevery little selection occurs under these conditions, the response of the V~8+ but not + T cells to MBPpl-11 reflects the finding with the T-cell the Va8-, CD4 clones. In the response to a second encephalitogenic MBP-peptide, MBP p35-47, the majority of clones are Va8-, and in the same experiment the ÷ T-cell population (90). bulk of the response was found in the Va8-, CD4 To further characterize the heterogeneity of TCRin MBP-p1-9-specific, I-Au restricted T-cell clones, cDNA clones of TCR~- and r-chains of eight representative, independent T-cell clones were sequenced. The summary of these experiments is shownin Table 2. T-cell clones from PL mice with this fine specificity pattern use a very limited set of V regions for both chains. All utilize the same V,-chain and as mentioned above over 78% the same Va-chain. There seems to be no stringent usage of specific Ja and junctional Da regions. Amongthe eight analyzed T-cell clones, four different VaJa and three different J~ patterns were observed, but the repertoire is very limited considering all the available possibilities. The predominant pattern was found in 50%of T-cell clones for the J~-chain and in r 75% for the J,-chain. As shown in Figure 2 the region in the VaJ junction revealed a unique amino acid sequence for every VaJ/~ combination (90). Analysis of fine specificity differences with MBP-pl-9peptides with single amino acid substitutions surprisingly revealed very similar dose response curves in all the T-cell clones. The only difference betweenclones that share only the V~ region was lack of reactivity with one very weakly stimulatory peptide (90). TwoT-cell clones that shared the amino acid sequence for both TCR Table 2 TCRgene elements used by MBP-p1-9-specific, I-Au restricted T cell clones, derived from PL mice T cell clone PJB-20 PJPR-2.2 PJPR-6.2 F1-21 P JR-25 PJB-18 PJPR-7.5 Fl-12

Origin

Va

PL PL PL (PL x SJL)F1 PL PL PL (PL x SJL)F1

8.2 8.2 8.2 8.2 8.2 8.2 8.2 4

Ja 2.7 2.7 2.7 2.7 2.3 2.3 2.5 2.5

V~

J~

aP JR-25 P JR-25 P JR-25 PJR-25 P JR-25 PJR-25 PJR-25 PJR-25

TA31 TA31 TA31 TA31 TA31 TA31 TT11 FI-12

Encephalitogenicity

ND + +

~ V~PJR-25 is a memberof the V,4 family with 95%homologyto V,TA65.J,FI-I 2 has 94%homology u. in nucleotidesequenceto J,5CC7(90). All the clonesare restricted to I-A

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ACHA-ORBEA,STEINMAN~ McDEVITT

~ I : GGGACAGGGGGC GROUP

CLONE

V~

~ 2 : GGGACTGGGGGGGC

Junctionalregion TGC TCC TAT ...

P~-20

ASG 8.2 GCC AGC GGT G

GLGE ~ CTG ~G GAG

Annu. Rev. Immunol. 1989.7:371-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

A s G

S 5 G

A S G D 8.2 GeeAAGC$GGTGGATDG ~’~% PJR-25 PJB-18

P~R-2.2 P~-20

A

~ AC-C AC,C

P~-25 P~-25

TGC C~LR G CALR ~ G~ CALR

$

~ G

CTG AG

~

~

GG

AG

~ P~R-7.5

4 FI-12

~-25

P~-25

GG CALS

CALS T~ GCT ~G AG CAL$ T~ S~ ~G AG

D T AT A ER T ~G A~

d~

AGT GCA ... A G GCAA "’" GT GCA ...

~

0

CAA GAC Cc-,r._,TX,~;._.C~ AAC CAA

CALR

2P~-25~-25~T~GAG

G

ACG T~S CT TCC G

8.2 GCC AGC GGT GAT G

A s

-4 F~-~

A

Y TAT ...

A Y

d~ 2.3

J~ 25

d~"=~’"

PNYGNE CC ~C TAT G~ ~T ~G ANYGNE CC ~C ~AT GGA ~T GAG PNYGNE

0 ~ T~

ANYGNE ~CTATG~T~G PNYGNE

J=T~J

NGGSG AT ~G ~C A~ GGC NTDK ~r Ace ~C ~

J~11

J u ~

Figure 2 TCRnucleotide and anaino acid sequences of MBP1-9 specific T cell clones. In Figure 2A the TCRanucleotide and aminoacid sequencesof the junctional region are shown. The groupswere defined by the different VJcombinations.In Figure 2B the c~-chain sequences are shown. Reproducedwith permission from Cell.

chains differed in their ability to transfer disease. Thereforethe TCR on a CD4÷T cell does not by itself determine encephalitogenicity. Other factors such as production of lymphokinesand homingpatterns in vivo could determineencephalitogenicity.

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Based on these findings it was possible to analyze whether treatment of autoimmunedisease was possible with anti-V~-specific monoclonal antibodies. The monoclonal antibodies used efficiently deplete V~-expressing T cells in vivo. As shownin Figure 3, treatment with anti-V~8 results in remission of an ongoing autoimmunedisease which was induced with an encephalitogenic Va8-expressing T clone (90). This clearly demonstrates that the autoimmuneresponse is not just triggered by the encephalitogenic T cell and then is self perpetuating; rather, removal of the critical T cell blocks an ongoing disease and the health of the experimental animal is regained. In addition, EAEinduced with MBPpl-ll peptide could be prevented with anti-V~8 treatment (see Table 3), and ongoing disease induced with whole MBPcould significantly reverted (see Table 4) (90). This is remarkable because least.two encephalitogenic peptides mapto this protein in the PL mouse. Although it is not knownwhich of the two epitopes is immunodominant in vivo, the MBPpeptide p35-47 elicits mostly Va8 T cells. The remission of MBPinduced disease with anti-V~8 implicates a dominantrole for these cells in EAE. The BIO.PL

Mouse

The B10.PL mouse strain differs from the PL mouse strain in that the genes outside the MHCare derived from B10. In this strain MBPpl-9

3

0

10

20

30

40

Daysafter injection o! F23.1 Fit~ure 3 Reversal of T cell clone-induced disease with the monoclonal anti-V8 antibody F23.1. (PL x SJL)F1 mice were injected with the encephalitogenic T cell clone P Jr-25, 5 × 106 cells intraveneously (i.v.). After paralysis was apparent the mice were randomizedand treated with monoclonalantibody F23.1 or as a negative control an isotype matched antibody $5.2 twice on day one and three after paralysis was observed. Severity scale: 3, paraplegia; 2, paraparesis; 1, limp tail; 0 normal. Darkboxes represent the control experimentwith antibody $5.2, open boxes F23.1-treatment. Reproduced with permission from Cell.

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Table3 Preventionof MBP-peptide p 1-11 inducedEAE withV/38-specific monoclonal antibodyF23.1. Monoclonal ~ antibody

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F23.1 $5.2

Incidence of bdisease

Mean onset of clinical disease (day)

1/19 9/20

20 15

~(PL × SJL)FI mice were immunizedon day 0 with MBPpl11 in completeFreund’sadjuvant and pertussis vaccine. They received500~g of the purified antibodieson days - 1, ~ and 9. Antibody$5.2 represents an isotype matchedcontrol antibody whichdoes not bind to mouseT cells. bNumber of animalssick/numberof total animals Reproducedwith permissionfrom Cell.

also is encephalitogenic, MBPp35-47 has not been tested. In a study analyzing the TCRheterogeneity of MBPp 1-9-specific T-cell hybridomas, similarities to as well as differences from the above results described for PL mice were obtained (128). The majority of MBPpl-9-specific T-cell clones utilized V~8(79%)and J~2.7. (This region is called either J~2.6 J~2.7 by various authors, depending on whether the sixth J region of the second cluster, a pseudogene, is counted or not). The remaining hybridomasutilize Vfl3 and J~2.2. Fifty-eight percent of the clones use V~2.3; the rest use V,4.2. Both types of T-cell clones rearrange the same J~ gene segment, J~39 (128). In a way similar to the above results, different V~J~ rearrangements use strikingly different junctional regions, whereas rearrangements including the same V and J regions show a conservation

Table 4 Reversal of guinea pig MBPinduced disease with Vfl8 specific monoclonalantibody F23.1. Number of mice with clinical symptoms72 hours after treatment Treatment F23.1 KJ23a

Number of mice with clinical symptoms14 days after treatment

None

Mild

Severe

None

Mild

Severe

Deaths

12 1

5 12

2 9

14 9

3 2

1 7

1 4

aDiseasewasinducedas described in Table3 but with guinea pig MBP.Treatmentwas started 24 hours after the first clinical signs of EAEweredetected. Theexperimentwasdone in a doubleblind fashion. The micereceived 200#g of the antibodies i,p, Monoclonal antibodyF23,1depletes the Va8positive T cells and KJ23adepletesthe Vgl7 positive T cells in vivo, Bothantibodiesdeplete similar amountsof T cells in the (PLx SJL)F1miceused in these experiments.Reproduced with permissionfromCell.

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of aminoacid sequencesimilarity. Contraryto the results with PLmice, all the ~-chain junctional sequencesof the V~2.3-expressingclones were identical at the nucleotidelevel, and both, V~2.3and V=4.2,use the same J, region. In addition, the most predominant 3-chains in both mouse strains use the sameV andJ elementsbut different D-regions. Howcan these differences be explained? 1. The major differences between the PL and B10.PLmousestrains are the genes outside the MHC. Thevariation in the results betweenthese twostrains mayreflect strain-to-strain variations in V-regionsequences. In support of this, the V,4-sequencefromthe PLmicerepresents a new memberof the V~4-familywhichhas not yet been shownto be present on the B10background.A similar situation could be found for the V~2.3sequence.If there are strain-related differences in the V gene segments,critical residues could be altered, and this wouldexplain why PLmice use only one V-regionin the response to the MBP 1-9 peptide. 2. T cells expressing the V=2.3-sequence maybe clonally deleted due to peptides present in the PLbut not the B 10 background. 3. T-cell cloningcould select for specific T-cell populations.Becausethe T-cell clones analyzed comefromdifferent cloning experiments, such a selection uponcloning probably wouldreflect differential growth requirementsof different types of clones. SuchT-cell clones of the two groupswouldprobablyalso have a different function in vivo. u in hy4. The V~2.3 chain could have a higher affinity for MBP/I-A bridomasthan in T cell clones due to higher levels of accessorymolecules or the contribution of the BWTCRelementsin binding. Further experimentsare neededto clarify this issue. The SJL Mouse SJL mice are not susceptible to induction of EAEwith the N-terminal MBP-peptides. In this mousestrain, a peptide in the C-terminalregion of the MBP protein is responsible for encephalitogenicity(50). It has been shownthat at least two encephalitogenic peptides are located between position p89-101(120). About50%of the T-cell clones recognizing MBP p89-101in association with I-As expressVal7and require proline at position 101 to be stimulated. The T-cell clones reactive to MBP 89-100do not require position 101 of the peptide for recognition (120). A third encephalitogenicpeptide, MBP p96-109,has beenidentified in this region of MBP(121). Althoughthe TCRusage of the Val7- T cell clones has not yet been + T cell clones is blockedby the characterized, disease inducedwith Va17 Val 7 specific monoclonal antibodyK J-23. In contrast, anti-Val7 does not

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block disease induced with MBPp89-101or intact MBP(120). Until the TCRusage of the Val7-negativepopulationis analyzedin moredetail, it is not clear whether the SJL as well as the PLand B10.PLmice use a limited set of TCRelements. The response in SJL mice is clearly more complexthan in either PL or BI0.PL mice and maybe similar to the responsepattern seen with the modelpeptides. EAE in Rats In the Lewisrat one of the major encephalitogenic epitopes is located between positions MBPp68-88 (122-124). About 50%of hybridomas raised against rat MBPrecognize a different undefined epitope (124). Independent groups determined the sequences of TCRsfrom a total of three encephalitogenicT-cell clones. Bothfound that the V-regionsused by these MBPp68-88-reactiveT cell clones were strikingly homologous to the mouseV~8.2region (80 and 93 %in aminoacid sequence).Furthermore the TCRof one T-cell clone sequencedclones used a V~2like sequence (70%homology)(129; E. Heber-Katz,submitted). Analysis of Southern blot andNorthernblot data revealed that these V regions are found in the majority of the MBP specific T-cell clones analyzed.Becauseof the usage of different restriction elementsin mouseand rat and the recognition of different non-cross-reactivepeptides, these results cannot be explained. Thesefindings in the rat modelof EAEstrongly underscorethe importance of TCRV-regionsequencesto disease. An anti-TCR monoclonal antibody raised against a MBPp68-88specific T-cell clone, detecting less than 1%of peripheral blood cells, decreased the intensity of or prevented MBP-induced autoimmune disease in 67%of the experimental Lewisrats (130). Dueto the short course the disease in rats, reversal of ongoingdiseasehas not beenreported. Summary of EAE Similar to describedmodelproteins, there are multiplediscrete epitopes on the autoantigen MBP,associated with different MHC restriction elements. However,in two of three systemsanalyzedthus far, the encephalitogenic epitope(s) elicited a T-cell responsewith very little heterogeneity.In the MBP pl-9 response, no majordifferences in fine specificity towarda series of peptides with single aminoacid substitutions could be detected among the T-cell clones analyzed. Surprisingly, in both Lewisrat and H-2u mousestrains, whichrecognize different peptides and different MHC molecules, very similar TCRVregion elementswereused. Differencesbetweenthe two H-2u mousestrains, PLand B10.PL,were observedin the usage of one or two V,-regions, in

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the V~J~-junctional diversity, and the heterogeneity of the J~-regions. Tcell clones with different TCRcomposition possess nearly identical fine specificity patterns, when tested on MBP-peptideswith single amino acid substitutions or cross-reactivities to allo MHC antigens. This suggests that the overall structure of the TCRmolecule is highly selected for and is not simply correlated with the primary sequence of someof its structural components. This raises the possibility of obtaining more specific antibodies that wouldallow depletion of specific idiotypes and lead to highly specific immuneintervention. Treatment of ongoing autoimmune disease with anti-TCR monoclonal antibodies clearly showed that removal of the responsible autoimmuneT cell is sufficient to reverse a clinical condition. Theseantibodies, however, recognize about 15%of all peripheral T cells, and these results should be extended with more specific reagents. EAEcould theoretically recur since in mice treated with anti-V~8, encephalitogenic T lymphocytesexist which are V~8-negative and recognize the encephalitogenic epitope MBPpl-9. Of MBPpl-9-specific T-cell clones, 5-20% are V~8-negative. Moreover V~8-negative T cells recognizing other encephalitogenic epitopes such as MBPp35-47 exist in the PL mouse. Yet in the mice given anti-V~8 antibodies, EAE is reduced even when the immunogen is whole MBP, an autoantigen with at least two discrete encephalitogenic epitopes, including MBPpl-9 and p35-47 (see Tables 3 and 4). In SJL mice, a group of nested epitopes can induce encephalomyelitis. About 50%of the T-cell clones share V~17, and these T-cell clones are specific for one particular peptide, MBPp89-101. Depletion of V~17positive T cells in vivo does not influence susceptibility to peptide-induced disease. The complexity of TCRused in this response remains to be determined. Overall the response of SJL mice is more similar to the above described examples of model peptides like myoglobin or lysozyme. From experiments in different strains of mice it appears that the MHC alleles and not TCRrepertoire determine whichepitope is encephalitogenic. SJL (/-A s) and SWR(I-A q) mice have deleted about 50%of their TCRV~regions, including Ve8, but express an additional Ve gene element, Ve17 (18, 131). B10.T(6R) mice (I-A q) and A.SWmice (1-As) express Ve8 but not V~17. The peptide MBPp89-101 is encephalitogenic in all four strains of mice. These observations indicate that, in mice sharing MHC genes, the T-cell repertoire does not influence susceptibility towards N- or C-terminal MBP-peptides. The bias toward susceptibility to the N-terminal MBP,fragments in the (PL x SJL)Fl-mice is therefore not due to depletion V~17-positive T cells but could be explained either by low levels of expression (50, 132) and/or dominance of the epitopes where Vo8expression is involved.

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OTHER INDICATIONS FOR LIMITED HETEROGENEITY OF TCR IN AUTOIMMUNE DISEASES

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Collagen-InducedArthritis Native type-II collagen can be used to induce arthritis in mice (61). Mice expressing H-2q or H-2r are prone to disease (133). Of the H-2q expressing strains of mice, SWRis unique in that the disease cannot be induced (134); this suggests that other genes outside the MHC influence susceptibility. One of the characteristics of the SWRmouse strains is a deletion of about 50%of the TCRV~ gene elements (131). Genetic backcrosses with C57BL/6mice have shown that (C57BL/6 × SWR)F1mice arc susceptible, and the majority of the F1 × C57BL/6 or F1 × SWRbackcross mice which are susceptible had inherited one or two copies of the TCR~genes from the C57BL/6 parent (134). The two diseased mice which did not express V~8(one of the deleted V~-regions in SWRmice) inherited part the C57BL/6TCR~genes only, including Va6 as determined by restriction fragment length polymorphism (RFLP) at the V~ locus between the SWR and C57BL/6mice (135). In crosses between M1 s"-positive and -negative mice, the V~6expressing T cells are climinated during maturation in the thymus (95). In agreement with the above results, crosses between SWR (M l sa-negative) and CBA(Mlsa-positive) showed reduced incidence disease whereas crosses with M1 sa-negative mousestrains such as C3Hor C57BL/6resulted in susceptibility (136). These results implicate specific TCRgenes in the susceptibility to collagen-induced arthritis. Alternative explanations for these observations are possible however. One of the problems not addressed in the study is that SWRmice express normal amounts of mRNAfor Va6 in lymphatic organs (131). Whether the RFLPpattern represents coding sequence differences between the two alleles is not known. Because autoimmunediseases are end products of very complex mechanisms, other genes in the mousestrains could explain the outcome of these experiments as well. With the availability of V~6monoclonalantibodies, the issue can be clarified (137). Nonobese

Diabetic

Mouse

The nonobese diabetic (NOD)mouse is a model for spontaneous-onset, insulin-dependent diabetes mellitus (138). At least three recessive genes are involved in the disease process, one of which maps to the MHC,probably the class-lI l-A-region (139-142). NODmice do not express I-E molecules; no mRNA for the/-E~-gene is detectable (139). The disease is mediated by T cells, and both CD4+ and CD8+ cells are required for induction of disease by adoptive transfcr into irradiated healthy animals (143, 144).

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Recent experiments have shown that crossing NODmice with transgenic mice expressing functional I-E~ genes and backcrossing these mice to NOD results in protection from invasion of immunecells into the pancreatic islet cells (insulitis) (145). It was argued that I-E is involved in mediating suppression of the immuneresponse. This result was surprising because backcross studies of NODmice with C57.BL6mice, which do not express I-E~, also show a recessive gene mappingto the MHCwhich is needed for disease to occur. Therefore the expression of I-E must operate at a different level. At least two explanations are possible: (a) The transgene was inserted close to one of the other recessive susceptibility genes of the single I-Etransgenic C57.BL6mouseused in the crosses, and (b) expression of I-E results in clonal elimination of TCRmolecules involved in the disease process. Amongthe six V~-genes analyzed V~I 1 and V~17are depleted in I-E expressing mousestrains (18, 19; Ed Palmer, personal communication). Therefore, it is possible that one or several of the TCRelements whichare eliminated in I-E-expressing mice are involved in pathogenesis.

T-Cell Vaccination It was shownby Cohenand colleagues that it is possible to use autoimmune T-cell clones or lines as immunogensto prevent or revert autoimmune diseases (see Table 5). Either doses of T-cell clones which are below the threshold for inducing disease, or irradiated, fixed or pressure-treated T cells exerted this effect (146-149). These animals were protected for prolonged times, and disease could not be induced with these T-cell lines or T-cell clones, or with antigen. This protection is specific in that animals protected from adjuvant arthritis are still susceptible to EAE.This protection can be transferred to unprotected animals with lymph node cells. T-cell clones specific for autoimmuneT cell clones have been isolated from rats that recovered from EAE. These T-cell clones are either CD4+or CD8÷ T cells. The CD8÷ T cells lyse their target cells specifically, and this cytotoxicity is not blockable with anti-CD4, anti-CD8,anti-class I, or anti-class II antibodies. Whentransferred together with the disease-causing clones, they protect from disease (150, 151). It remains to be determined whether the protective T-cell clones recognize TCRor another specific Tcell surface marker expressed on EAE-causing T-cell clones but not on adjuvant arthritis-causing T-cell clones.

OTHER FORMS OF SPECIFIC INTERVENTION

IMMUNE

The interaction between T cells, antigen-presenting cells and antigen initiates normal immuneresponses as well as autoimmune responses.

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Therefore several approaches involving immuneintervention in autoimmunediseases have focused on molecules that play a role in this interaction. The results are summarizedin Table 5.

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Class-H

Molecules

Treatment protocols with anti-class II-specific antibodies have been shown to partially deplete class II--expressing ce~ls in vivo (152). Successful prevention of autoimmune diseases was achieved in many murine autoimmunediseases (see Table 5) (153-161, Boitard et al, submitted). form of treatment results in a general immunesuppression. In the human population most individuals are heterozygousfor class-II alleles. In studies using F1 mice expressing different class-II antigens, it was shownthat immuneresponses linked to the first allele, despite the depletion of the majority of the cells expressing the second class-II allele, remainedintact (162). Utilization of noncytotoxic antibodies should reveal whether this approach can be used to prevent or revert autoimmunediseases without depletion of cells. These approaches not only interfere with antigen presentation but also result in a suppression of the autoimmuneresponse (163166).

Table5 Attempts in prevention andtreatment of autoimmune diseases Autoimmune disease EAEmouse(PL/J,

T cell Anti-class II Anti-CD4Anti-TCRvaccination Peptides TNF-c¢ Anti-inf-7 + +

+ +

+ +

ND

+

ND

ND

+ + + + + + +

+ + + + + + + R +

+ ND ND ND ND

ND + ND ND ND

+ + ND ND ND ND

ND ND ND ND ND ND

ND ND ND ND ND ND

ND

ND

ND

÷

+

ND

ND

++

++

ND

ND

ND

+

+ +

+

ND

ND

+

ND

ND

ND

B10.PI~) EAEmouse(SJL) EAE(rat) EAMG BBrat NODmouse Collagen-induced arthritis (mouse) Adjuvantarthritis (rat) (NZB x NZW)FI Lupusnephritis EAT(mouse)

Forreferencessee text. +, preventionof autoimmune disease. + +, preventionand reversal of clinically detectable autoimmune disease. R, preventionand reversal of histologically detectable autoimmune disease. -, no effect on either preventionor reversal of autoimmune disease. ND,not done.

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T-Cell

393

Subsets

Treatment of mice with the rat monoclonal antibody GKI.5 results in rapid depletion of CD4+T cells (167). CD4+T cells represent about 60%of the T-cell population which primarily interact with peptides bound to class-II molecules. Depletion of these T cells results in protection from or reversal of many autoimmunediseases (see Table 5) (167-178). several antigen-recognition systems it was shownthat administration of antigen shortly after depletion of CD4+T cells can result in induction of tolerance toward this antigen for a prolonged time. This includes tolerance to the xenogeneic rat antibody GK1.5(179-181). The protocols used for prevention of autoimmune disease used antibody treatment for limited time periods and nevertheless resulted in prolonged protection from autoimmunedisease in autoimmunediabetes (178). It is not clear yet whether the long lasting effects are simply due to the tolerance induction toward the target antigens or whether other mechanismsare involved. Recently it was shownthat application of anti-CD4 antibodies which do not deplete the CD4+ population had similar effects (182, 183). Interleukins After initiation of immuneresponses different interleukins are produced. If an individual produceslowlevels of a specific interleukin, administration of this factor could prevent autoimmunedisease. This was shownin the case of lupus nephritis in (NZB × NZW)F1mice using TNF-a (184). Applicationof interferon-3, resulted in precipitation of the disease in several autoimmune diseases: (185-188). Depletion of interferon-~ with monoclonal antibodies has been shown to have a protective effect in lupus nephritis in (NZB× NZW)F1mice (189). Peptides Application of low doses of peptides or application of the antigen intradermally (which at higher doses can induce an autoimmune disease) resulted in lifelong protection from future induction of disease in adjuvantinduced arthritis and EAEin rats (62, 113, 190-192). Analysis of similar approaches in other systems in which the target antigens are well characterized is necessary. Because the reagents used for prevention have the potential to trigger an autoimmuneresponse, care must be taken in applying this approach in humans. Careful analysis of the mechanismof this effect is needed. In EAEand arthritis, induction of tolerance towards the autoantigen prevented future induction of the autoimmunedisease (193195). Anotherapproach is based on finding specific altered peptides which bind very strongly to specific MHC proteins and prevent presentation of self antigens by competitive interactions or induction of tolerance.

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CONCLUSIONS The new approaches with anti-TCR-antibodies use more specific reagents for interfering with autoimmunediseases than do those employed previously. EAErepresents the first disease modelin which the heterogeneity of TCRin autoimmunedisease has been analyzed in detail. In mice of the H-2U-haplotype and in Lewis rats, a very limited heterogeneity of TCRs was found to be involved in the autoimmune process. In both models depletion of these cells prevented autoimmunedisease (90, 128-130). SJL mice (H-2~) a more complexpattern was observed. A series of at least three encephalitogenic nested epitopes are located in a region encompassing 20 amino acids of the MBPmolecule (119-121). This situation reminiscent of results obtained with model peptides (27, 45, 53-60a). remains to be determined whether the autoantigen MBPis unusual in this respect or whether such simple patterns are found in other autoimmune diseases as well. Indirect evidence for such limited heterogeneity in other spontaneous or induced autoimmunediseases has been found in collageninduced arthritis, experimental allergic thyroiditis (EAT), diabetes (NOD mouse), and adjuvant arthritis (134-136, 145-151). What triggers an autoimmunereaction is not fully understood. Is autoimmunitythe result of exposure of a very specific autoantigenic peptide to the immunesystem or a more general breakdownof tolerance mechanisms? Furthermore, it remains to be determined whether in the original autoimmunereaction a similar complexity of antigens and TCRsis involved at different stages of the disease, e.g. whenit becomesclinically apparent or chronic. It is possible that a specific T cell starts a chain reaction and that at a later stage the autoimmunecells do not reflect the composition of TCRof the original T-cell population. After destruction of target cells, newautoantigen-reactive T cells could be activated due to the presentation of autoantigens by APCs. The results from treatment of EAEwith antiTCRantibodies are encouraging in that removal of specific T cells during ongoing disease reverses clinically apparent symptoms.According to these results, the immunesystem does not escape when the majority of autoreactive cells is depleted. While leaving 5-20%of the encephalitogenic T cells in PL mice intact after antibody treatment, mice remain protected (90). On the other hand, removal of only 50%of autoreactive T cells the SJL mousehad no effect on the course and intensity of the disease (120). Using different combinations of monoclonal antibodies which can removespecific subsets of T cells will allow examination of whether more than one population of T cells bearing certain TCRV genes must be targeted.

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Treatment of autoimmunediseases with monoclonal antibodies is not without problems. Most of the available antibodies are isolated from mice, rats, rabbits, or hamsters. These antibodies can elicit an immuneresponse in a xenogenic host, and repeated application can lead to anaphylactic shock or rapid elimination of the antibody (196). Recent approaches tailored the CD-regions of the murine antibodies into human immunoglobulin molecules or made chimeric antibodies with constant regions from human and variable regions from mice (197-201). A better understanding of the role of various constant regions will allow utilization of the optimal reagents. The optimal duration of treatment is unknown.If such treatment can be stopped without reoccurrence of the autoimmune response, fewer problems might ensue. Lessons from previous depletion experiments with anti-I-A or anti-CD4 antibodies showed that removal of specific populations of lymphocytes influences the entire immunesystem. Althoughit is clear that mousestrains lacking certain TCRV regions are perfectly healthy, it has to be determined whether depletion of specific V-regions alter the immuneresponse of an individual. This can be easily addressed in mice by depletion of specific V regions in adult mice and analysis of the immuneresponse and tolerance patterns in vivo and in vitro. As shown in the comparison of the I-Au and I-As mouse strains for EAE,different peptides and different TCRmolecules are involved in the autoimmune response. How could anti-TCR treatment be valuable for autoimmune diseases in humans, given the heterogeneity in MHCantigens? Autoimmunediseases often are tightly MHC-linked. In insulindependent diabetes mellitus, for example, about 50%of patients express only one type of susceptibility mediating class-II molecules (for review, see 202). In pemphigusvulgaris, close to 100%of patients have at least one of two class-II antigens responsible for susceptibility (203). In these diseases and many others, the target antigens are not yet known, and further research is needed to evaluate whether such a treatment procedure with a single anti-TCR antibody or pools of anti-TCR V-region antibodies could be useful. On the other hand, if limited TCR-heterogeneityis found in the response to certain autoantigens, treatment could be tried without knowledgeof the autoantigen. Thus "reverse genetics" might be employed for treatment of autoimmunedisease. Due to the clonal deletion process genetic correlations cannot always be expected between certain autoimmune diseases and TCRV regions when the germ-like repertoire is examined. Additional experiments addressing the repertoire of T cells in the periphery are needed. Oneof the possible explanations for dominant protection by certain MHCantigens

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such as DR2 from insulin-dependent diabetes mellitus (IDDM)(for review, see 202) could be due to tolerance to cross-reactive determinants or depletion of self-reactive T-cell clones. In humans, analysis of the knowntarget antigens in myasthenia gravis, rheumatoid arthritis, and thyroiditis will help understand these patterns. In a recent report, TCRrearrangements were analyzed from T cells isolated from joints of rheumatoid arthritis patients. Using constant region probes of the TCR,one specific band was detected in southern blot analysis for each patient (204). Specific bands under these conditions are indicative usage of the same V and J region. The same rearrangements were found in different joints of the same patient but not in the periphery. In a study in multiple sclerosis, restricted heterogeneity of TCRgenes was found in the spinal fluids of patients (205). In another study certain TCRV and RFLPswere found in increased frequencies in multiple sclerosis patients (206). By utilizing antibodies to TCR,highly specific reagents could be used which would impair only a minor part of the T-cell repertoire. What is neededin the near future is the careful analysis of T-cell receptors involved in different aut~immunediseases. The results of these studies will be valuable in deciding which form of therapy is appropriate for a particular autoimmunedisease. ACKNOWLEDGMENTS

Wethank Ed Palmer, Ellen Heber-Katz, and Hans Hengartner for communication of results prior to publication. The critical reading of the manuscript by Theresa Lopez, Alexandra M. Livingstone, and David C. Wraith is highly appreciated. The work was supported by National Institute of Health grants RO1NS18235 and AI,07757, the National Multiple Sclerosis Society, the SwimFoundation, and the Rosenthal Foundation. H. Acha-Orbea was supported by the Juvenile Diabetes Foundation and the Swiss National Science Foundation. Literature Cited 1. Moeller, G. Ed. 1983. HLAand disease susceptibility, lmmunol.Rev. 70 la. Nepom, G. T. 1988. Immunogenetics of the HLA-associated diseases. Concepts Immunopathol. 5:80-105 2. Unanue, E. R., Allen, P. M. 1987. The basis for the immunoregulatoryrole of macrophagesand other accessory cells. Science 236:551-57 3. Dialynas, D. P., Wilde, D. B., Marrack, P., Pierres, A., Wall, K. A., .Havran,

W., Otten, G., Loken, M. R., Pierres, M., Kappler, J., Fitch, F. W. 1983. Characterization of the routine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK1.5: Expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHCantigenreactivity. Immunol. Rev. 74:29-56 4. Shimonkevitz, R., Kappler, J., Marrack, P., Grey, H. 1983. Antigen recog-

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Ando, D. G., Sercarz, E. E., Hood, L. 1988. Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy. Cell 54:577-92 129. Epplen, J. T., Chluba, J., Steimle, V., Hinkkanen, A. 1988. T cell receptors in autoimmunedisease. J. Cell. Biol. Suppl. 12E: 18 (Abstract R204) 130. Owhashi, M., Heber-Katz, E. 1988. Protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotope on rat T cell receptors specific for myelinbasic protein. J. Exp. Med. In press 131. Behlke, M. A., Chou, H. S., Huppi, K., Loh, D. Y. 1986. Murine T-cell receptor mutants with deletions of//-chain variable region genes. Proc. Natl. Acad. Sci. USA 83:767-71 132. Fritz, R. B., Skeen, M. J. 1987. Influence of the H-2u haplotype on immune function in F1 hybrid mice. II. F1 antiparent mixed lymphocyte reactivity. Immunogenetics 25:161q56 133. Wooley, P. H., Luthra, H. S., Stuart, J. M., David, C. S. 1981. TypeII collagen-induced arthritis in mice. I. Major histocompatibility complex (I-region) linkage and antibody correlates. J. Exp. Med. 154:688-700 134. Banerjee, S., Haqqi, T. M., Luthra, H. S., Stuart, J. M., David, C. S. 1988. Possible role ofVt~T cell receptor genes in susceptibility to collagen-induced arthritis in mice. J. Exp. Med. 167:832-39 135. Banerjee, S., Behlke, M. A., Dungeon, G., Loh, D. Y., Stuart, J., Luthra, H. S., David, C. S. 1988. Va6 gene of T cell receptor maybe involved in type II collagen induced arthritis in mice. FASEBJ. 2(4) (Abstr. 2120) 136. Anderson, G. D., Banerjee, S., Stuart, J. M., Luthra, H. S., David, C. S. 1988. Interaction of MHCand TCRloci in susceptibility to collagen-induced arthritis in mice. FASEBJ. 2(4) (Abstr. 2121) 137. Payne, J., Huber, B. T., Cannon,N. A., Schneider, R., Schilham, M. W., AchaOrbea, H., MacDonald, R. H., Hengartner, H. 1988. Twomonoclonal rat antibodies with specificity for the V~6 region of the routine T cell receptor. Proc. Natl. Acad. Sci. USA 85:7695 138. Makino, S., Kunimoto, K., Muraoka, Y., Mizushima,Y., Katagiri, K., Tochino, Y. 1980. Breeding of a non-obese, diabetic strain of mice. Exp. Anim. 29: 1-13 139. Hattori, M., Buse, J. B., Jackson,

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TCR IN AUTOIMMUNE DISEASE R. A., Glimcher,L., Dorf, M.E., Minami, M., Makino, S., Moriwaki,K., Kuzuya,H., Imura,H., Strauss, W.M., Seidman,J. G., Eisenbarth,G. S. 1986. The NODmouse: Recessive diabetogenic gene in the majorhistocompatibility complex.Sciene23 I: 733-35 140. Prochazka,M., Leiter, E. H., Serreze, D. V., Coleman,D. L. 1987. Threerecessiveloci requiredfor insulin-dependent diabetes in nonobesediabetic mice. Science 237:286-89 141. Wicker,L. S., Miller, B. J., Coker, L. Z., McNally,S. E., Scott, S., Mullen, Y., Appel,M.C. 1987.Geneticcontrol of diabetesandinsulitis in the nonobese diabetic (NOD)mouse.J. Exp. Med. 165:1639-54 142.Acha-Orbea, H., McDevitt, H. O. 1987. Thefirst external domainof the non-obesediabetic mouseclass II Aa chain is unique Proc.Natl. Acad.Sci. USA84:2435-39 143. Miller, J. B., Appel,M.C., O’Neill,J. J., Wicker,L. S. 1988. Both the Lyt2+ and L3T4+ T cell subsets are requiredfor the transfer of diabetes in nonobesediabetic mice. J. Immunol. 140:52-58 144 Bendelac,A., Carnaud,C., Boitard, C., Bach, J. F. 1987. Syngeneictransfer of autoimmune diabetes from diabetic NODmice to healthy neonates. Requirement for both L3T4÷and Lyt2+ T cells. J. Exp. Med.166:823-32 145. Nishimoto, H., Kikutani, H., Yamamura,K.-I., Kishimoto,T. 1987. Prevention of autoimmuneinsulitis by expression of I-E molecules in NOD mice. Nature 328:432-34 146. Ben-Nun,A., Wekerle,H., Cohen,I. R. 1981. Vaccination against autoimmune encephalomyelitis with T-lymphocyte line reactive against myelinbasic protein. Nature292:60--61 147. Holoshitz, J., Naparstek,Y., Ben-Nun, A., Cohen,I. R. 1983. Lines ofT lymphocytes induce or vaccinate against autoimmune arthritis. Science219: 5658 148. Lider, O., Karin, N., Shinitzky, M., Cohen,I. R. 1987. Therapeutic vaccination against adjuvantarthritis using autoimmuneT cells treated with hydrostatic pressure. Proc.Acad.Sci. Natl. USA84:4577-80 149. Maron,R., Zerubavel, R., Friedman, A., Cohen,I. R. 1983. T lymphocyte line specific for thyroglobulinproduces or vaccinates against autoimmune thyroiditis in mice.J. Immunol.131:2316 150. Lider, O., Reshef,T., Beraud,E., BenNun, A., Cohen, I. R. 1988. Anti-

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idiotype network induced by T cell vaccination against experimental allergic encephalomyelitis. Science239: 181-83 151. Sun, D., Qin, Y., Chluba,J., Epplen, J. T., Wekerle,H. 1988. Suppression of experimentally induced autoimmune encephalomyelitisby cytolytic T-Tcell interaction. Nature332:843-45 152. Waldor, M. K., Hardy, R. R., Hayakawa, K., Steinman,L., Herzenberg,L. A., Herzenberg, L. A. 1984. Disappearance and reappearanceof B cells after in vivo treatment with monoclonal anti-I-A antibodies. Proc.Natl. Acad. Sci. USA81:2855-58 153. Steinman, L., Rosenbaum, J. T., Sriram, S., McDevitt,H. O. 1981. In vivo effects of antibodies to immune response geneproducts: Preventionof experimental allergic encephalomyelitis. Proc. Natl. Acad.Sci. USA78: 7111-14 154. Adelman, N. E., Watling, D. L., McDevitt, H. O. 1983. Treatment of (NZBx NZW)F1 disease with anti-I-A monoclonalantibodies. J. Exp. Med. 158:1350-55 155. Waldor,M. K., Sriram, S., McDevitt, H. O., Steinman,L. 1983.In vivo therapy with monoclonalanti-I-A antibody suppressesimmune responsesto acetylcholine receptor. Proc. Natl. Acad. Sci. USA80:2713-17 156. Sriram, S., Steinman,L. 1983. Anti-1A antibody suppresses active encephalomyelitis: Treatment model for diseases linked to IR genes. J. Exp. Med. 158:136247 157. Wooley,P. H., Luthra, H. S., Lafuse, W.P., Huse,A., Stuart, J. M., David, C. S. 1985. TypeII collagen-induced arthritis in mice. III. Suppressionof arthritis by using monoclonal andpolyclonalanti-Ia antisera. J. Immunol. 134: 2366-74 158. Boitard, C., Michie,S., Serrurier, P., Butcher, G. W., Larkins, A. P., McDevitt, H. O. 1985. In vivo prevention of thyroid and pancreatic autoimmunity in the BBrat by antibody to class II majorhistocompatibilitycomplex gene products. Proc. Natl. Acad. Sci. USA82:6627-31 159. Sriram, S., Topham,D. J., Carroll, L. 1987.Haplotypespecific suppressionof experimental allergic encephalomyelitis withanti-IAantibodies.J. Immunol. 139:1485-89 160. Vladutiu, A. U., Steinman, L. 1987. Inhibition of experimental allergic thyroiditis in micebyanti-I-A antibodies. Cell. Immunol.109:169-80

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161. Jonker, M., Mitchell, D., Steinman,L. Treatment of EAEby anti-MHCclass II specific monoclonalantibodies in Rhesus monkeys. In Immunologyof HLA,ed. B. Dupond.In press 162. Rosenbaum,J. T., Adelman, N. E., McDevitt,H. O. 1981. In vivo effects of antibodies to immune responsegene products. I. Haplotypespecific suppression ofhumoral immuneresponses with a monoclonalanti-l-A. J. Exp. Med. 154:1696702 163. Perry, L. L., Greene,M. I. 1982. Conversion of immunityto suppression by in vivo administration of I-A subregion-specific antibodies. J. Exp. Med. 156:480-91 164. Williams, I. R., Perry, L. L. 1985. Regulation of transplant immunity in vivo by monoelonalantibodies recognizinghost class II restriction elements. II. Effects of anti-Ia immunotherapy on host T cell responses to graft alloantigens. J. Immunol.134: 2942-47 165. Aoki,I., Idhii, N., Minami,M., Nagashima,Y., Misugi,K., Okuda,K. 1987. Induction of suppressor T cells by intraveneous administration of monoclonal anti-I-A antibodies. Transplantation 44:421-25 166. Broder, S., Mann,D. L., Waldman, T. A. 1980.Participation of suppressor T cells in the immunosuppressive activity of a heteroantiserum to human Ia-like antigens(p23, 30). J. Exp.Med. 151:257-62 t67. Waldor,M. K., Sriram, S., Hardy,R., Herzenberg,L. A., Herzenberg,L. A., Lanier, L., Lim,M., Steinman,L. 1985. Reversal of experimentalallergic encephalomyelitis with a monoclonal antibody to a T-cell subset marker (L3T4). Science 227:415-17 168. Deleted in proof 169. Brostoff, S. W., Mason,D. W.1984. Experimentalallergic encephalomyelitis: successfultreatmentin vivo with a monoclonalantibody that recognizes T helper cells. J. Immunol.133:1938 42 170. Wofsy,D., Ledbetter, J. A., Hendler, P. L., Seaman,W.E. 1985. Treatment of murinelupus with monoclonalantiT cell antibody. J. Immunol.134: 85257 171. Ranges, G. E., Sriram, S., Cooper, S. M.1985. Preventionof type II collagen-induced arthritis by in vivotreatmentwith anti-L3T4.~LExp. Med.162: 1105-10 172. Wofsy,D., Seaman,W. E. 1985. Successful treatment of autoimmunityin

NZB/NZW F1 mice with monoclonal antibody to L3T4.J. Exp. Med. 161: 378-91 173. Christadoss,P., Dauphinee,M.J. 1986. Immunotherapy for myastenia gravis: A murine model. J. Immunol. 136: 2437~40 174. Like, A. A., Biron, C. A., Weringer, E. J., Byman,K., Sroczynski, E., Guberski, D. L. 1986. Prevention of diabetes in Biobreeding/Worcester rats with monoclonalantibodies that recognizeT lymphocytes or natural killer cells. J. Exp. Med.164:1145-59 175. Wang, Y., Hao, L., Gill, R. G., Lafferty, K. J. 1987. Autoimmune diabetes in NODmouse is L3T4T-lymphocyte dependent. Diabetes 36: 53538 176. Koike,T., Itoh, Y., Ishii, T., Ito, I., Takabayashi, K., Maruyama, N., Tomioka,H., Yoshida, S. 1987. Preventive effect of monoclonalanti-L3T4 antibody on developmentof diabetes in NODmice. Diabetes 36:539-41 177. Wofsy,D., Seaman,W.E. 1987. Reversal of advanced lupus in MZB/NZW F1 mice by treatment with monoclonal antibody to L3T4. J. Immunol.138: 324~53 178. Shizuru, J. A., Taylor-Edwards,C., Banks, B. A., Gregory, A. K., Fathman, C, G. 1988. Immunotherapyof the nonobesediabetic mouse:Treatmentwith an antibodyto T-helperlymphocytes. Science 240:659~62 179. Gutstein, N. L., Seaman,W.E., Scott, J. H., Wofsy,D., 1986. Induction of immune tolerance by administration of monoclonal antibody to L3T4. J. Immunol. 137:1127 32 180. Benjamin, R. J., Waldman,H. 1986. Induction of tolerance by monoclonal antibody therapy. Nature 320:449-51 181. Goronzy,J., Weyand,C. M., Fathman, C. G. 1986. Long-term humoral unresponsivenessin vivo, induced by treatment with monoclonal antibody against L3T4.J. Exp. Med.164: 91125 182. Waldor, M. K., Mitchell, D., Kipps, T. J., Herzenberg,L. A., Steinman,L. 1987. Importance of immunoglobulin isotype in therapy of experimental allergic encephalomyelitiswith monoclonal anti-CD4 antibody. J. lmmunol. 139:3660-64 183. Charlton, B., Mandel,T. E. 1988. Progressionfrominsulitis to fl-cell destruc+T tion in NODmouserequires L3T4 lymphocytes.Diabetes 37:1108-12 184. Jacob, C. O., McDevitt, H. O. 1988. Tumornecrosis factor-~ in murine

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TCR IN AUTOIMMUNE DISEASE autoimmune "lupus" nephritis. Nature 331:356-58 185. Engelman,E. G., Sonnenfeld,G., Dauphinee,M., Greenspan, J. S., Talal, N., McDevitt,H. O., Merigan,T. C. 1981. Treatment of NZB/NZW F 1 mice with mycobacteriumbovis strain BCGor type II interferon preparations accelerates autoimmune disease. Arthritis Rheum.24:1396-1402 186. Heremans,H., Billan, A., Colombatti, A., Hilders, J., DeSomer,P. 1978. Interferon treatment of NZBmice: Ac~ celerated progression of autoimmune disease. Infect. Immunol.21:925 187. Segiescu,D., Cerutti, I., Efthymiou, E., Kahan, A., Chany, C. 1979. Adverse effects of interferon treatmenton the life span of NZBmice. Biomed.Exp. 31:48 188. Parsitol, H.S., Hirsch,R. L., Haley,A. S., Johnson,K. P. 1986.Exacerbations of multiplesclerosis in patientstreated with gamma interferon. Lanceti: 89394 189. Jacob, C. O., van der Meide, P. H., McDevitt,H. O. 1987. In vivo treatment of (NZB×NZW)F1lupus-like nephritis with monoclonalantibody to y-interferon. J. Exp. Med.166:798-803 190. Alvord,E. C., Shaw,C. M., Huby,S., Kies, M. W. 1965. Encephalitogeninduced inhibition of experimental allergic encephalomyelitis: Prevention, suppression and therapy. Ann. NY Acad. Sci. 71:142230 191. Einstein,E. R., Csejty,J., Davis,W.J., Ravch,H. C. 1968.Protective action of encephalitogen and other basic proteins in experimental allergic encephalomyelitis. Immunochemistry5: 567-75 192. Higgins,P. J., Weiner,H. L. 1988.Suppressionof experimentalallergic encephalomyelitisby oral administrationof myelinbasic protein andfragments.J. lmmunol. 140:440-45 193. Sriram,S., Schwartz,G., Steinman,L. 1983. Administrationof myelin basic protein-coupledspleen cells prevents experimental allergic encephalitis.Cell. Immunol. 75:378-82 194. McKenna, R. M., Carter, B. G., Paterson, J. A., Sehon,A. H. 1983.Thesuppressionof experimentalallergic encephalomyelitisin Lewisrats bytreatment with myelinbasic protein-cell conjugates. Cell. Immunol.81:391-402 195. Schoen, R. T., Greene, M. I., Trentham,D. E. 1982.Antigen-specificsuppression of type II collagen-induced arthritis by collagen-coupledspleen cells. J. Immunol.128:717-19

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196. Chatterjee,S., Bernoco,D., Billing, R. 1982.Treatmentwith anti-Ia and antiblast/monocytemonoclonalantibodies can prolongskin allograft survival in nonhumanprimates. Hybridoma1:69 197. Morrison,S. L., Johnson,M. J., Herzenberg,L. A., Oi, V. T. 1984.Chimeric human antibody molecules. Mouse antigen binding domain with human constantregion. Proc.Natl. Acad.Sci. USA 81:6851-55 198. Boulianne, G. L., Hozumi,N., Shulman,M.J. 1984.Productionof functional chimeric mouse/human antibodies. Nature 312:643-46 199. Roberts, S., Cheetham, J. C., Rees,A. R. 1987. Generation of an antibody with enhancedaffinity and specificity for its bindingby protein engineering. Nature 328:731-34 200. Verhoeyen,M., Milstein, C., Winter, G. 1988. Reshapinghumanantibodies: Graftingan antilysozymeactivity. Science 239:153635 201. Riechman,L., Clark, M., Waldman, H., Winter, G. 1988. Reshapinghumanantibodies for therapy. Nature332:323-27 202. Todd, J. A., Acha-Orbea,H., Bell, J. I., Chao, N., Fronek, Z., Jacob, C. O., McDermott,M., Sinha, A. A., Timmerman,L., Steinman, L., McDevitt, H. O. 1988. A molecularbasis for MHC class II-associated autoimmunity. Science 240:10034 203. Szafer, F., Brautbar, C., Tzfoni, E., Frankek G., Sherman,L., Cohen,I., Hacham-Zadeh, S., Aberer, W., Tappeiner, G., Holubar, K., Steinman, L., Friedman,A. 1987. Detection of disease-specific restriction fragment length polymorphismsin pemphigus vulgaris linked to the Dqw1 and DQw 3 alleles of the HLA-D region. Proc. Natl. Acad. Sci. USA84:6542-45 204. Stamenkovic, I., Stegagno,M., Wright, K. A., Krane, S. M., Amento,E. P., Colvin, R. B., Duquesnoy,R. J., Kurnick, J. T. 1988. Clonal dominance amongT-lymphocyteinfiltrates in arthritis. Proe. Natl. Aead.Sei. USA 85:1179-83 205. Hailer, D. A., Duby,A. D., Lee,S. J., Benjamin,D., Seidman,J. G., Weiner, H. L. 1988. Oligoclonal T lymphocytes in the cerebrospinalfluid of patients with multiple sclerosis. J. Exp. Med. 167:1313-22 206. Oksenberg,J., Bernard, C., King, M. C., Erlich, H., Cavalli-Sforza, L., Steinman,L. 1988.V~andC, alleles of the T cell receptorlinkedto multiplesclerosis and myastheniagravis. Proc.Natl. Acad.Sci. USA.In press

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Ann. Rev. lmmunol. 1989. 7:407-44 Copyright © 1989 by Annual Reviews Inc. All rights reserved

MANIPULATION OF T-CELL RESPONSES WITH MONOCLONAL ANTIBODIES Herman Waldmann ImmunologyDivision, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom

INTRODUCTION T cells are central to all immuneresponses and are therefore a prime target for any therapeutic intervention designed to control immunefunction. For many years polyclonal antilymphocyte and anti T-cell sera have been investigated as immunosuppressive agents. The recent advent of monoclonal antibody technology (1, 2) and the development of a wide range of xenogeneic monoclonal antibodies (Mabs) to T-cell surface molecules have together rekindled interest in using antibodies to regulate the immune response. In animal models therapy with Mabs to T cells has provided new knowledgeon the roles of T cells and their subsets in immunityand immunopathology.In clinical practice we have witnessed the evolution of a newgeneration of therapeutic agents of substantial promise. In theory Mabsmaybe used to enhance, suppress, or alter the quality of an immuneresponse. The outcome maybe restricted to the response to a specific antigen, or to sets of antigens, or it could be directed to the immuneresponse in general. The field of monoclonal antibody therapy is still in its early stages, and mostavailable informationdeals with the global effects of therapy. In time, more knowledgeof the structure and function of the immunesystem maymake it routinely possible to achieve antigen° specific or function-specific regulation. This review, although not comprehensive, summarizes what has been learned about Mabtherapy in animal models, reflects on the impact of Mabs in human transplantation, and speculates on how Mabs may be 407 0732-0582/89/0410-0407502.00

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used to establish antigen-specific immunologicaltolerancefor therapeutic purposes.

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IMMUNOSUPPRESSION Mabs may immpnosuppress by eliminating vital cells or by perturbing the function of critical receptors, adhesion molecules or their respective ligands. The degree to which Mabscan eliminate cells will depend on their capacity to recruit the natural effector systems either humoral (complement) or cellular (e.g. K-cells or other cell types with a range of receptors). The rules by which Mabscan harness these systems are best studied in vitro, and the present state of knowledgeas relevant to therapy is summarized below. The Complement

System

The complementcascade is initiated by the binding of C1 to the antigenantibody complex. This binding occurs through the Clq subcomponent. The bulk component of the cascade is C3 which when bound to cells can result in cell elimination by at least two mechanisms. One involves the assembly of a series of "terminal" components in the membrane,and the other involves clearance by cells of the macrophage/granulocyte series which possess receptors (CD1la/CD18) for bound C3. To exploit or avoid complementactivation, it is essential to knowwhat factors govern the ability of Mabsto activate the process. Information, albeit incomplete, is available for three categories of Mabs: mouse, rat, and human. The major variables seem to be antigen density, antibody isotype, and someas yet ill-defined property of the target antigen itself. ~SOT,~’EThe impact of antibody isotype is ideally demonstrated by the use of class-switch variants or chimeric recombinant antibodies although there is abundant supporting data from myelomaproteins and Mabs. For mouseIgG the subclass hierarchy for lysis seems to run in parallel with Clq binding as mIgG2a= or > IgG2b > IgG3 > IgG1 (3, 4, 5). For human Igs the hierarchy is hu’IgG1 > IgG3 > IgG2 > IgG4 for lysis (6) and IgG3 > IgG1 > IgG2 > IgG4 for Clq binding (6,7). IgG3 less effective than IgGl in the activation of C4 for reasons whichare as yet unclear (8). In the case of rat immunoglobulins,Fust et al (9) demonstrated consumption of hemolytic complement by aggregated myeloma proteins in the order rIgG2b > IgG2a > IgG2c > IgGl. Hughes-Jones et al (10) showedthat rat Mabsof the ’Ig2b subclass were more efficient than other IgG subclasses for Clq binding and lysis. Recently Bruggemannet al (11)

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using a set of matchedchimeric molecules have demonstrated a hierarchy for lysis of rIgG2b > IgG1 > IgG2c or IgG2a compared to IgG2b > IgG2c > IgG1 > IgG2a for Clq binding. The reasons for variation in C1 q binding betweenisotypes with identical specificity is unknown as all share a commonClq-binding motif (12). It maybe that interactions between antibody Fc regions mayhelp create a favorable juxtaposition of antibody pairs to create Clq binding sites and also that the different isotypes mayvary for this property. THETARGET ANTIGEN The importance of antigen-specificity in complement activation is documentedby Bindon et al (13). The special feature of "good" antigens was that they permitted Mabs to bind and activate more C1 than did "poor" antigens. Again the reasons are unclear but are consistent with notions of Fc-Fc interactions. The proportion of human lymphocyte-surface molecules that are "good" (i.e. support lysis with humancomplementis small). A particularly effective set of antigens is family of glycoproteins defined by the rat IgM MabCAMPATH-I (14). The ability of CAMPATH-1 antibodies to lyse lymphocytes with human complement has been particularly valuable for purging of T cells from donor marrowin clinical bone marrowtransplantation (BMT)(see later). SYNERGISTIC ANDMONOVALENT MABSIt has been possible to improve complement lysis for some of the "poor" antigens. One approach has involved the use of synergistic pairs of Mabsdirected to different epitopes on the same molecule (10, 15, 16). Synergy works best with pairs of rat IgG2bs or mouseIgG2as or a combination of both (10, 15). One possible therapeutic application where trials are underwayis the use of synergistic pairs of CD45Mabs for eliminating passenger leukocytes from organ grafts. The leukocyte commonantigen defined by CD45Mabs is present on all leukocytes. Passenger leukocytes have been implicated as the major immunogenicelements of tissue grafts (17), and their removal should reduce graft immunogenicity. SomeMabsrapidly redistribute their target antigen on the cell surface (18). This mayproduce resistance to complementlysis. Sometimesthe rate of modulation can be reduced and lysis achieved by use of monovalent Mabs with one active and one inactive F(ab) arm (19). This has shown (Figure 1) for a rlgG2b CD3 Mab (CAMPATH 3) and may widely applicable. The monovalent CD3Mablike its wild-type or OKT3 is active in vivo and can, like its parent, reverse acute rejection episodes in renal transplantation (20). It is not clear howimportant complementactivation is, nor which parts of the pathwayare relevant to the elimination of cells in vivo. A detailed assessment of the in vitro complementactivating properties of particular

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Biv(~lent

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"Inactive"

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100

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1 0"1 -1 Antibodyconcentration I pg.m[

Fi#ure1 Complement lysis by a monovalentCD3antibody. A hybridomaproducinga rlgG2bCD3Mabwasfusedto a cell line producingan irrelevant light chain. Thethree possiblechaincombinations wereseparatedbyHPLC, andfractionstestedfor lysis of human T-cellblasts withhuman complement by a chromium releaseassay (fromRef. 20).

isotypes and their mutants to defined target antigens will allow assignment of in vivo function with defined residues and will permit selection or creation of the appropriate antibodies for the desired therapeutic effect.

Elimination of Cells by Mechanisms Involving Fc Receptors Knowledgeof the diversity and function of Fc receptors in mouse and humanis accumulating rapidly. However, it is still not clear which Fc receptors either alone or in concert may participate in destruction of cells in vivo. Again the availability of class-switch variants, recombinant antibodies and the mutants thereof, as well as of cloned Fc receptor genes, should make it possible to define which Fc receptors and which accessory cell types are responsible for depletion of lymphocytes and other immunerelated hemopoieticcells in vivo. MOUSE FC RECEPTORS Three distinct

Fc receptors for IgG-Fc have been

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T-CELL

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described (21-23): a high affinity trypsin-sensitive receptor for IgG2a (moFcyRI);a low-affinity trypsin-resistant receptor for mIgG1,mIgG2b, and mIgG2a(mo FCyRII), and a receptor for IgG3 (moFCyRIII). moFC~RI and III are found on cells of the monocyte/macrophagelineage, while Fc~RIIis widely distributed on manycells types. Recently, two genes have been identified as encoding the Fc),RII receptor (21). These encode transmembrane proteins that have near-identical extracellular domains but that differ in the transmembrane and cytoplasmic domains. The FcR~ transcript is expressed only in macrophages,while the FCR/~transcript is found in both macrophages and lymphocytes. Both forms of receptor participate in phagocytosis and are inducible with ~IFN. Although it is uncertain which Fc receptors are responsible for eliminating Mab-coatedcells in vivo, it is widely accepted that antibody-dependent cellular cyotoxicity (ADCC)is probably a good in vitro correlate for those in vivo FcR-dependent effector systems. Where mouse Mabs have been studied in conjunction with mouse effector systems, both moFcRyI and moFcRylIhave been implicated in ADCC (24, 25). There is too little information on interactions of rat lg isotypes with mouseFc receptors to explain whythe rat isotypes vary in their ability to deplete cells in vivo. HUMAN FC RECEPTORS There are at least three receptors on human leukocytes for humanIgGs. The huFc),RI is a high affinity receptor found on cells of the monocyte/macrophageseries and is also inducible on macrophages. HumanIgs are thought to bind in the rank order IgGl = or > than IgG3 >IgG4 >> IgG2 (26, 23). Residue 235 (Leu) on huIgG3 been implicated as crucial to the interaction with huFcTRI(27). A role for huFc3,RI in ADCC has been suggested both from the use of conventional assays and from use of heteroconjugates (i.e. antibodies with dual specificity, whereone of the specificities is directed to the Fc receptor itself). huFcyRIis also 7IFN inducible. The situation for huFcTRII (CDw32)is similar to the mouse. The huFcTRIIis a widely expressed low affinity receptor with a well-defined polymorphism. Three huFc),RII cDNAshave been isolated (28, 29), again with extensive homologyin the extracellular domainsbut with differences in the intraeytoplasmic regions. The binding of humanIgGs ranks huIgG 1 = IgG3 >> IgG2 and IgG4, although there is some uncertainty about the ranking of IgG3 (29, 30). This pattern of reactivity of the different human isotypes with the different Fc receptors suggests that the exact site on humanIg which interacts with huFcTRII may differ from that bound by huFcyRI (30). Like moFcyRII, the huFcTRII binds mIgG2band mIgG1, and it has been suggested that this receptor maybe involved in the T-cell triggering generated by mIgG1 Mabs to human CD3, polymorphism in

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this receptor accounting for triggering failures in 30%of normal individuals. There is insufficient data concerning interactions with rat Mabs; but as all the rat Ig genes have been cloned (31, 32) and expressed, this information should soon be available. The huFcTRIII(CDI 6) is a low affinity receptor found on neutrophils, eosinophils, natural killer cells, and a subset of T cells. Recently Simmons & Seed (33) have described a eDNAclone encoding this receptor, and observed a close relationship with.the form of the moFC?,RII.The binding hierarchy of hulgGs with, transfected receptor was huIgG3 -- IgG1 >> IgG4 and IgG2, and mIgG3=IgG2a > IgGl >> IgG2b. Several groups have studied ADCCusing human K-cells and murine Mabswith therapeutic relevance in mind. The ranking observed was essentially mIgG3> IgG2a > IgG2b with IgG1 apparently inactive (34, 35). Recent studies with heteroconjugates with one specificity for huFc7RIII showed that neutrophils could also be triggered to ADCCactivity. Hale et al (36) using a wide selection of rat Mabshave shownthat rlgG2b are far more potent than rIgG2a and rIgG2c for ADCC.Recent work with recombinant (chimeric) Mabsconfirms an isotype hierarchy for rIgGs rlgG2b;. IgG1. >> IgG2a > IgG2c (11). Lymphocyte

Depletion

In Vivo

RODENa" MODELS The injection of Mabsin vivo can affect immunefunction by depletion of cells or perturbation of functional molecules. It is desirable to determine what features of Mabsallow cell depletion and ~ whether, depletion is relevant to an observed influence on the immuneresponse. Most data in this area have accumulated from rat Mabsinjected into mice, and this is the major topic of the section. There are four rat I~Gsubclasses; the order of their CHgenes is 72c ~2a ~1 ~,2b (31). Class-switch variants (37) and recombinant antibodies nowbe isolated and should allow detailed analysis of differences of subclass function in vivo without variations due to differences.in fine-specificity. However,the available published data deal with collections of Mabsreactive with the same antigen, but differing in their variable regions, Cobbold(38) and Ledbetter &Seaman(39) first observed that rat IgG2b Mabsdirected to the Thy-1 antigen were able to deplete T cells in vivo. The rIgG2b Mabs were more effective than rIgG2a, IgG2c, or IgM both for.depletion and immunosuppression. Ledbetter & Seaman noticed that T cell depletion with rIgG2a anti-Thy 1 was delayed compared to the IgG2b. Both groups were careful to~ avoid measurement artefacts due to modulation. Le Gros et al (40) described a rIgG2a Mab, a single intraperitoneal injection of which could remove(surprisingly) all detectable T cells from lymphoid organs but not from the thymus. Thierfelder

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et al (41) precoated allogeneic marrowand spleen cells with nine different anti-Thy-1. Mabsprior to transplantation, into irradiated recipients. Only two of these Mabs were able to prevent GVHD.Both were rlgG2b, while the ineffective Mabs were IgM (5.Mabs) and IgG2a (2 Mabs). recently Cobboldet al (42) compareda series of class-switch variants and three different IgG2cMabsdirected to Thy-1. Of the class-switch variants, IgG1 and IgG2b could deplete whereas the IgG2a could only modulate the surface antigen. Of the three IgG2cMabs,one could deplete while the others modulated. Three rat IgG2b Mabs to CD5(Lytl), CD4(L3T4), and CD8(Lyt2) have been compared to IgG2a Mabs for acute depletion (43, ,~4, and Qin et al, unpublished data), rlgG2b were always superior. The issue is by no means trivial because, in a number of publications, rlgG2a Mabshave been used with intent to "deplete" lymphocytes, and in somecases (e.g. for Mab53.6 widely used as a CD8Mab)contradictory claims (depletion or not) have been made. It is conceivable that the antiimmunoglobinresponse that manycell-binding Mabselicit may actually "develop" depletion and explain the delayed cell clearance seen by Ledbetter. Resolution of the contradictions is important because eventually one would like to use a Mabto block function without resorting to monotonous preparation of antibody fragments. What can probably be concluded is that rlgG2b and possibly rlgG1 (for which too little information isavailable) are the most effective of rat Mabsfor in vivo depletion of mouse lymphocytes. Synergistic pairs of Mabs may be more effective than single antibodies in vivo. Qin et al (45) demonstrated that pairs IgG2b Mabsto non-overlapping epitopes of the CD4molecule were more lytic in vitro and in vivo than either one was alone. Comparablelevels of lymphocytedepletion are also possible in the rat (46), but the role of mlgGisotypes has been less clearly defined. A clearer picture of the rules for depletion in these,-.rodent modelswill emerge with use of panels of class-switch variants or recombinant Mabs and more knowledgeof the effector, mechanismsresponsible for destruction of antibody coated cells in vivo. SUBHUMAN PRIMATES ANDHUMAN Surprisingly there is little data available to demonstrate effective depletion of lymphocytes by Mabs in humanor other primates. V.ery often lymphocytes are reported coated but not depleted (47), and other times, modulation is-observed. Antigenic modulation is due to a redistribution of antigen on and from the cell surface brought about by interaction with bi- or multi-va!ent antibody .as,,for example, after therapy wj~h CD3and CD5therapeutic Mabs(48). Perhaps the most lytic~..am.~!ymph0cyte.~an.tibodyso far described is the panlymphocyte rIgG2bMab’ C~AMPA~H:IG (37). It is one of a panel

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rat Mabsof different isotypes but with similar specificity, all knownto lyse lymphocytes with homologous (human) complement in vitro. These exhibit a hierarchy of complement lysis rIgM > IgG2b = IgG2a > IgG~ > IgG2c. The rIgG1 and IgG2b versions were derived as classswitch variants from the rIgG2a form. In a recent study (Figure 2) (49) a patient with an advanced prolymphocytic leukemia was treated sequentially with the IgM; IgG2a, and then the rIgG2b. The IgM Mab was shown to consume hemolytic complement but could produce only a transient clearance of lymphocytes from blood with no long-term depletion, while the rIgG2a was totally ineffective even at very high doses. However the rIgG2b variant produced profound depletion of lymphocytes in blood, spleen and bone marrow with no detectable change in the hemolytic complementlevels. The extent of depletion in subsequent patients was equally profound. Clearly CAMPATH IG (rIgG2b) is unusually lymphotoxic reagent with "debulking" properties that predict a broad application in serotherapy of leukemia, and in immunosuppression. Recently a huIgG1 (CAMPATH-IH)form has been obtained by the

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Day Number Fiyure 2 The effects of Mabs of different isotypes against the CAMPATH1 antigen expressed on lymphocytes. This patient with BCLLwas treated unsuccessfully in 1985 with rlgG2a and the rlgM CAMPATH1. By 1987 the disease had progressed into a prolymphocytic transformation, and further therapy was instituted. Substantial clearance of the blood lymphocytes was obtained. The arrows refer to occasions when the stated number of mgs of Mab were injected (from Ref. 49).

64

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recombinant DNAapproach (50), and this too has exquisite lymphocytotoxic function in vivo (160). The unusual potency of CAMPATH IG and CAMPATH 1H may depend on features of both the antigen and antibodies. The antigen density on lymphocytes is high (about 5 x 105 molecules per cell); and the antigen is not modulated by antibodies. This and the strong interaction between the Fc regions of rlgG2b or hulgG1, and humaneffector systems seem to guarantee potency. Of course the prospect of debulking the lymphocyte pool in a large animal like humanis a different proposition than for the rodent. In the rodent, short courses of Mabscan achieve more than 2 logs of depletion with striking effects on immunity.The samelevel of depletion in the human would probably only have a small-term impact, given the remaining large pool of cells that could easily be recruited into an immuneresponse. For "good" antigens (like the CAMPATH-1 target) that do not modulate with prolonged therapy, it is very likely that extended therapy possible with humanized Mabs (160) will guarantee maximal use of effector systems with the prospect of high level depletion.

MonoclonalAntibodies as Ablative Ayents to Establish the Functions of T-Cell Subsets The immuneresponse to any antigen has particular cell requirements for its induction and others for its effector phase. Research into the cellular basis of these two phases of a response has traditionally depended upon adoptive transfer and/or in-vitro culture systems. The availability of Mabs that could ablate T cells or subsets has made it possible to use "immunological surgery" to study T-cell function in vivo. In the same way that endocrinologists had been able to infer function from ablation of particular endocrine tissues, it becamepossible to achieve this for T cells. StmSEa’-OEI~LETEO ~CE In 1984 Cobbold et al (43) described a way constructing subset-depleted mice (long-term depleted of CD8, CD4, or all T-cells) by injection of high doses of rlgG2b Mabsinto adult thymectomized mice. Adult thymectomy precluded any de novo reconstitution ofT cells from primary lymphoid sources. Both CD4- and CD8depleted animals remained depleted for at least 100 days, with very little restoration of the function associated with the depleted subset (43, and Qin unpublished). A la carte subset-depleted animals have been used to provide a ready source of CD4-and CD8- cells for in vitro or adoptive transfer studies, and for determining the functions of each subset in the induction of responses to simple antigens, grafts, and microorganisms, and in autoimmunity and immunopathology.It has also been possible to produce rats long-term depleted of CD8cells, but it has been harder to

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construct rats long-term depleted of CD4cells because the Mab-treated animals seem gradually to reconstitute from the small residual pool of CD4cells (D. Mason, personal communication). The difference between mouseand rat maysimply be a question of the size of the lymphocytepool that Mabsneed to deplete. Probably too few CD4cells are left in a welldepleted mouseto be able to reconstitute the animal. CD4- mice were shown to be unable to make IgG responses to Tdependent antigens (Figure 3) and to viral antigens even after boosting (51, 52); CD4- mice were unable to generate DTHto herpes simplex virus (52) and were less effective at rejecting skin grafts betweenboth whole "major" and multiple "minor" mismatch combinations; they could not easily be primedto exhibit secondset rejection (53). A striking finding with these mice is the variable dependence of CD8 cells for CD4"help." There are manyinstances where CD8T-cell functions could be elicited without CD4T-cell help. For example, it was easier to generate class I-restricted cytotoxic cells to herpes simplex virus in CD4animals than in controls (51). Similarly, the absence of CD4cells did not

LOG 2 Agg[utingtiontitee p~eo,m~

CD5 ~ ~

i~

"-

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Figure 3 The anti-SRBCantibody responseof thymectomizedmice that have beentreated with various monoclonalantibodies and then challenged with SRBC someweekslater, The antibodies and their isotype are displayed on the ordinate, The total and mercaptoethanolresistant (hatched) antibody titres are shownon the abscissa. Apart from the CD8Maball the r]gG2b Mabswere able to immunosuppress(adapted from Ref. 43). The symbol* indicates significant difference to the positive control; while ** indicates no significant difference from the preimmune serum.

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affect the development of CD8lymphocyte-dependent choriomeningitis with LCMvirus (52). In experimental transplantation, CD4-mice can often reject grafts. This has been shownfor grafts across complete MHC barriers (53), second set rejection of minors(53), resistance to bone marrowgrafts (67, 83), in the rejection of allogeneic tumors (42). The participation of either both of CD4and CD8cells in tumor allograft rejection is consistent with other model systems where both subsets have been separately implicated in rejection of tissue allografts (38, 54). Kauffmannhas highlighted a numberof models ofintracellular bacterial infection where CD4- mice exhibited hitherto unsuspected protective functions for CD8cells in the antibacterial response (55, 56, 57). CD8- mice survive in a remarkably healthy state--a somewhat disconcerting fact for those whohave argued so forcefully for a critical role of lyt2 + cells in immunoregulationand self-tolerance. I-Iumoral responses in such animals were equivalent to controls (58) both in quality and quantity, although very little has been done to examinethe fine details of the response (e.g. the isotype pattern). Sequential antigenic competition, once suggested as mediated by CD8+ cells (59) was as active in CD8mice as in controls (59). Similarly, one cannot persuade nonresponder H-2 (C57BL/10) CD8- mice to become responders to hen egg lysozyme (HEL) after ablation of CD8cells (H. Waldmann,unpublished data). had been suggested that the N-C"suppressogenic" peptide of HELmaintains unresponsiveness to remaining parts of the molecule through suppressive CD8cells (60). Given the large number of complex regulatory circuits involving CD8+ cells that have colored the literature, it wouldbe helpful to establish their importance and relevance in CD8-mice. It is sad that suppressor enthusiasts have not taken the bait. One report from Sedgwick (46) has shown that CD8- animals acquire and can recover from acquired allergic encephalomyelitis(active or passive)just like normal animals. Following spontaneous recovery of CD8-- animals, like their normal counterparts, showed no signs of relapse and remained resistant to further disease induction, ruling out a role for CD8cells in immunoregulation in this disease model. By using mice that were CD4-, CD8-, and both CD4- and CDS-, Cobbold et al (53) were able to demonstrate that both CD4and CD8 cells could contribute collaboratively and also independently to allograft rejection and to GVHD (see also below). ANTIBODY ABLATION IN EUTHYMIC MICEEuthymic mice have the capacity to regenerate new T cells from the thymus. Although parenterally administered Mabsenter the thymus the effector mechanismsnecessary for cell

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ablation are lacking in that organ. Despite this, functional blockade is still possible. However,acute experinaents that examinethe function of peripheral cells are probably not prone to any changes that might be induced in thymic T cell development. In contrast, long-term administration of Mabsmayhave an effect on differentiation in the thymus, which may in turn influence the quality and repertoire of exported T cells. Indeed, this maybe a useful way of analyzing the stages of T-cell development(61, 62). SUBSETS REJECTING SKINALLOGRAFTS In the early 1980s a great deal of discussion occurred as to whichT-cell subsets were responsible for allograft rejection (63, 64, 65). The work of Mason and Loveland & McKenzie had highlighted an important role for CD4(rat) and lytl + CD8- cells (mouse). In contrast counterarguments maintained that the experimental systems used (adoptive transfer models) were never free of CD8+ cells that could have been the real effectors, especially as studies with cloned CD8+ cells indicated that these could bring about "rejection" if injected directly into the graft (66). The Mabablative approach provided an ecumenical solution--that both T-cell subsets could effect rejection either independently or in collaboration, and that the fine details of strain combinations determined which had the dominant role (53, 67). Cobbold al (67) examined skin graft rejection across complete major and minor differences (BALB/cskin to CBA/Ca)and multiple minors (B10. BRskin to CBA/Ca).Animals received multiple injections of depleting cocktails of CD4, CD8, or both Mabs. In the full mismatch CD8Mabs did not delay rejection while CD4Mabsproduced a significant delay of about 9 days. The combination of both Mabs delayed the rejection process by a further 30 days (Figure 4). This was a clear demonstration that both major subsets of T cells could reject skin grafts independently. In this particular strain combination the natural rate of rejection of the CD4subset was faster than the CD8subset. Similar studies for second set rejection showed that CD8cells played a dominantrole following priming, and that priming was dependent on the presence of CD4cells. Similar experiments in "minor" mismatch combinations failed to uncover an independent role for CD8cells in first set rejection but showedthat both subsets contributed to rapid secondset rejection (53). Graft survival in the primedanimals that had received a short course of both CD4and CD8Mabs was remarkably prolonged, extending far beyond the time needed to reconstitute the depleted T cells. If in the complete mismatchcombinations, Mabswere injected at different stages of the rejection process, it was possible to showthat CO8Mabs alone could delay rejection over a whole MHC difference. It was concluded

Annual Reviews 419

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Figure 4 Both CD4and CD8cells can participate independently in graft rejection. Adult CBA/Camice received BALB/cgrafts and regular injections of antibodies. The CD4therapy significantly delayed the rejection process, but CD8Mabtherapy alone did not. The combination of both Mabswas the most effective producing a substantial delay in graft survival (adapted from Ref. 67).

that once CD4cells had begun to respond to the graft they would nowbe able to help CD8cells enter the rejection process faster than the CD8cells might have done independently. These studies highlighted the opportunistic nature of the rejection process and demonstrated that CD8cells could be triggered without help from CD4cells, but that they could also accept help from CD4cells if this was available. Woodcocket al (68) independently reached similar conclusions for the role of CD4cells in rejection and also showed that CD8cells could be triggered to cytotoxic function without CD4help. Subsequently, Wheelan & McKenzie(69) performed similar ablative experiments although they used rlgG2a Mab53.6.72 as a CD8reagent, and a rat IgG2a MabH129.19 as a CD4reagent. The CD4Mabdespite its isotype was claimed to be extraordinarly effective at depletion (the effect lasting 45 days) as was the particular CD8Mabin their hands. They were able to confirm that both CD4and CD8cells could mediate rejection. For minor mismatches or a class-I MHCmismatch, they demonstrated a dominant and independent role of CD8cells. In the combinationBml2to B6 (class-II difference) they showed that both CD4and CD8Mabscould delay rejection, and that the best immunosuppression could only be obtained with a combination of both Mabs. They failed to achieve any effects on graft survival in mismatches involving both class I and class II. This probably reflected the quality of the particular Mabs used. More recently Auchincloss et al

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(70) using a pair of rIgG2b Mabswere able to confirm the ability of combination of CD4 and CD8 Mabs to delay MHCmismatched grafts (BALB/c skin onto thymectomized C57/BL6). They too concluded that help for rejection, could be elicited without CD4cells. comparison~w4th ¯ skin grafts, vascularized heart grafts and allogeneic or xenogeneic isletg have been relatively easy to immunosuppress with CD4and CD8Mabs (71-74). Even ifthe mismatch were whole MHCeither of CD4or CD8 Mabscould produce:prolonged graft survival (71)! Bone-marrowgrafts across MHC barriers tend to engraft with difficulty even in lethally irradiated recipients. However,therapy of recipients with CD4and CD8Mabs prevented rejection, even in animals subjected to lower levels of irradiation (600 rads; see Figure 5) (67). Mabsto both the subsets were required. Neither alone was effective. In a multipleminor mismatched combination, engraftment of marrow could be achieved without any irradiation if recipients were pretreated with both CD4and CD8 Mabs but not when treated with any one Mab (75). In marrow rejection, unlike that of skin, CD8cells reactive with minor antigens seem to be able to reject without CD4"help" (75), In general the .results ffom Mabablation complementthe recent data

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SURSETS INVOINED IN REJECTION OF OTHER TISSUES ]~y

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Figure 5 Marrowgraft rejection in irradiated, T-cell subset~lepleted mice. Adult CBA mice were given CD4, CD8, or both CD4and CD8Mabs, irradiated with 600 rads and then given allogeneic T cell~zlepleted BALB/cmarrowand spleen cells. Chimerismwas established at the time periods sh3q~iaS D3norchimerism was (a) virtually complete in recipients that had been conditioned Witfi b0th~antibodies, (b) hardly detectable in CD8treated,-and,,(e) mixed in CD4treated recipidnts (adapted from Ref. 67).

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arising fromadoptive transfer of purified subsets of T cells in defined strain combinations (76, 77). CD4cells are biased to recognize MHC class IIassociated determinants, and CD8cells of class I-associated determinants. The rejection potential of CD4-CD8-T cells is still uncertain, but in the systems discussed above they appeared to have little if any impact. An effective rejection response requires T-cell collaboration. It wouldseem that CD4cells have a special propensity to. give help both to themselves and to CD8cells. However, CD8cells are not always dependent on CD4 help, and there clearly are opportunities for collaborative ventures between CD8cells themselves, which depend upon the particular tissue and the antigens concerned. The immunological issues in resolving the cellular basis of GVHD were similar to those in skin grafting, and in part based on the confusion from use of anti-lyt 1 antibodies as markers for cells that were later defined by CD4Mabs. In 1980 Korngold & Sprent (78, 79) showed that GVHDover certain minor-mismatched combinations was mediated by CD8cells. Mason (80) demonstrated that CD4cells and also, to a lesser extent, CD8cells could mediate GVHD across barriers in the rat. Vallera et al (81) and Korngold&Sprent (82) suggested that lethal GVHD across MHCbarriers involved predominantly lyt 1 + cells and not CD8cells. As all T cells express somelyt 1 (CD5) was possible tht CD8cells were damaged in the complement-dependent lytic procedures adopted. Using opsonizing rIgG2b Mabs to coat the donor marrow, or simply taking donor marrowfrom subset depleted mice, Cobbold et al (83) showedthat CD8cells could be the dominant effectors of GVHDin the combination CBAinto (CBAx BALB/c)F1. They also showed that both subsets could mediate GVHD in a fully allogeneic combination BALB/cinto CBA/Cawhere the recipients had been p.retreated with Mabsto permit engraftment. These results therefore emphasized that both the major T-cell subsets could mediate acute lethal GVHD. In addition, experiments with subset-depleted marrow donors clearly excluded an obligatory need for CD4cells to help CD8cells mediate lethal GVHD.Using purified CD4and CD8cells Korngold & Sprent (84) were also.able to demonstrate differential participation of either CD4or CD8 cells, depending upon t, he.strain combinations used. Hamilton and Korngo!d~ & Sprent-(8.5; 86) have since documenteda variable participation ¯ C-D4cells, in GVHD to non-H2differences, and have also established that (’CD4,.cells mediating lethal GVHD react with MHCclass-II, and CD8cells :- react with class I-associated determinants.The,fagtors ’that.,di.ct.ate which subset dominates in minor or whole MHCmismatched-~ombinations remain unclear. SUBSETS INVOLVEDIN GRAFT-VS-HOSTDISEASE

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The Interaction of Host-versus-graft Reactions in Marrow Transplantation

and Graft-versus-host

Although the alloreactions (GVHand HVG)that confound BMTtend be studied separately, a successful outcometo therapeutic transplantation requires prevention of GVHD and graft rejection in the same patient. Mousemodels have emphasized the interplay between donor T cells reacting against the host and host T cells that have survived irradiation reacting against the donor marrow. The phenomenonhas sometimes been referred to as "reciprocal interference" because alleviation of GVHD enhances rejection and abolition of rejection enhances susceptibility to GVHD. Cobbold et al (67, 83) showed that failures of engraftment across MHC barriers in irradiated recipients could be overcomeby eliminating residual recipient CD4and CD8T cells. This capacity of donor marrowto damage host and of host T cells to damage donor marrow can have dramatic consequences on the balance of donor vs host chimerism that can be achieved (Figure 5). An even more striking interaction was described Harper et al (87) who demonstrated a requirement for donor CD4cells to interact with recipient CD8cells to produce an SLE-like syndrome resembling chronic GVHD in unirradiated recipients. Ablation of recipient CD8cells abrogated disease and indeed improved donor chimerism. T-CELLSUBSETSIN MICROBIAL INFECTIONS The use of Mab ablation to study the roles of the major T cell subsets in microbial infections was initiated by Nash et al (51) in their studies of herpes virus infections in mice. They observed that CD8ablation abolished class I-restricted cytotoxicity but spared DTHand the capacity to produce HSV-specific antibodies. In contrast CD4-depleted animals were unable to make antiviral antibodies and to mount a primary DTHresponse, but could generate augmentedlevels of class I-restricted cytotoxic cells. The ability to clear virus was greatly compromised by CD4depletion. CD8-depleted mice cleared virus normally in the periphery but could not clear it from the nervous system; this suggests that CD4and CD8T cells mayexert different antiviral effects in different tissues. Moskophidiset al (88) determinedthe nature of effector cells that eliminate LCMvirus. During the effector phase of viral clearance the levels both of cytotoxic activity (CTL)and of cells producing antiviral antibodies were high. Treatment with rlgG2b antiThy-1 and rlgG2b CD4Mabsin this effector phase obliterated cytotoxic function and prevented viral clearance. In contrast CD4ablation abolished antibody production but did not affect viral clearance. Unlike the situation with herpes simplex CD8cells were critical to the effector arm of immunity but CD4cells or antibody were not. Bullet et al (89) observed that CD4 ablation did not prevent mice mounting a CTLresponse and recovering

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from infection with ectromelia. They were however unable to confirm that CD8cells were responsible for protection because they used a CD8Mab incapable of depleting CD8cells (the rlgG2a Mab53-6.7). The issue of CD4dependence of CD8responses in this context is probably no different from that discussed previously for GVHD and graftrejection. The dependence on CD4help will predictably vary from virus to virus. Sometimesthe damageelicited by viral infection requires host participation. Mabablation can help define the harmful as well as the protective properties of T cells. This is exemplifiedin the case of Theiler’s virus encephalomyelitis produced by intracerebral inoculation. Mice depleted of CD4cells fail to clear virus and die of an acute encephalitis. Welshet al (90) found that depletion of T cells just before the demyelinating phase of the disease substantially lowered incidence of paralysis. Rodriguez & Sriram implicated CD8cells in the demyelination process following infection (91). Traditional dogmawould not have predicted any protective role of CD8 cells in bacterial and protozoal infections. Recently there have been data supporting such a role for CD8cells, much of this data based on the ablative approach (reviewed by Kauffmann;55). Perhaps the most clearcut situation is that reported for immunityfollowing vaccination with malarial sporozoites; the immunitycould be co~npletely eliminated by therapy with CD8Mab but not with CD4Mabs (92). A role for CD8cells has also been claimed for resistance to mycobacteria (56, 57), listeria (93) leishmaniasis (94). Their role in these infections is uncertain. CD4Mab therapy in a mouse model of cutaneous leishmaniasis has revealed a schizophrenic role for CD4cells in genetically resistant and susceptible strains. CD4Mabtreatment of resistant CBAmice rendered them partially susceptible to leishmania major infection whereas treatment of susceptible BALB/cmice rendered them more resistant (95). Liew et have noted an inverse correlation between IL-3 production and resistance, and they have interpreted the data in the context of a balance between two types of CD4T-cells, one that can promote disease and one that is protective. MONOCLONAL ANTIBODY THERAPY OF AUTOIMMUNITY

Mab therapy has

been helpful in understanding the cellular basis of a range of autoimmune disease models in the mouseand rat, and in the derivation of strategies for treatment of humandisease. Steinmanet al (96) demonstrated that anti-I-A antibodies could be used to prevent and reverse EAEin SJL mice, and this work highlighted the potential value of allele-specific anti-I-A therapy. Theyand others showed

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similar benefit of anti-I-A Mabsin experimental myasthenia gravis and the lupus-like nephritis of NZBmice (97-103). In some of these models the B-cell masswas clearly depleted which mayin part explain the level of immunosuppression. Seaman et al (104) demonstrated that a rIgG2b anti-Thy 1 Mab was able to ameliorate renal disease and lymphoproliferation in MRL/lprmice although the same antibody had little effect on lupus-like disease in NZB/NZW F1 mice (105). In 1984 Brostoff & Mason (106) were able successfully treat a rat model of EAEwith a CD4Mab known to have little depleting ability. Simultaneously Wofsy& Seaman(~07, 108) were able to treat lupus in the NZB/NZW mouse by weekly injections of high doses of rIgG2b CD4Mab. These two publications were followed ~by several that have confirmed the benefit of CD4therapy in models-of collagen-induced arthritis (109), mouse EAE(110), diabetes (111), thyroiditis (112). Clearly the value of CD4Mabtherapy in these disorders is so impressive that it has becomeimperative to establish mechanisms. Carteron, Schiementi, and Wofsy (personal communication), Brostoff al (113), and Steinmann (l 14) have shown that similar effects may obtained with F(ab)2 fragments of CD4Mabs. This shows that. _depletion maynot always be needed to achieve a therapeutic effect. The concept of immunosuppression without depletion is considered further in a subsequent section. A most encouraging outcome to CD4therapy in autoimmunity is the fact that in .two instances of a ."natural disease" prolonged CD4therapy could eventually be stopped without disease recurrence (108, 111). This suggests either that therapy held animals over somecritical risk period or that the immunesystem had somehowchanged to become specifically unresponsive to the culpable antigens. Specific unresponsiveness with CD4 Mabsis considered later in somedept.h. The above results argue for only a minor role of CD8cells in most autoimmunedisorders or alternatively for a critical role of CD4cells alone in initiating the disease. However, Kantwerk et al (115) and Kong al (112) have documentedthe improved therapeutic effect of short-term therapy with both CD4and CD8Mabs, compared to CD4mab alone, in diabetes that develops following low dose streptozotocin or experimental thyroiditis. SELECTIVE STRATEGIES FOR MAB THERAPY IN AUTOIMMUNITYThus

far I

have discussed therapy directed at all CD4cells. In an attempt to focus therapy specifically to the autoimmuneresponse, several laboratories have adopted alternative approaches..One strategy has involved the use of antiIL-2 receptor antibodies expressed somewhatselectively on activated T cells

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(116, 117). These and other activation markers woulddefine T cells that have showntheir cards by participating in the response. If activated cells could be selectively eliminated, then the hope is that newly developing T cells wouldbe subject to natural tolerance mechanismsthat wouldpreclude their involvement in autoimmunity. Although this has been strongly advocated for transplant prophylaxis (117, 118), preliminary data in some models of organ-specific disease are also encouraging (116). Another way to selectively control the reactive T cells has recently been reported by Zamvil et al (119). They show a dominant role of T cells bearing V/~8 the pathogenesis of EAEin the PL/J mouse. Therapy with a V/38 specific Mabwas able to prevent induction of the disease. Recently Acha-Orbeaet al (120) have also been able to reverse ongoingdisease with the same Mab. These data indicate that a T cell receptor-specific approach to therapy can be used with a minimal impairment to overall immunity. These exercises in therapy of autoimmunity have laid downa framework for future research. It isn’t obvious what the advantages of CD4therapy over anti-iA therapy maybe. Advocatesof one argue that the distribution of class-II molecules in vivo is too diverse to be safe; proponents of CD4 therapy voice concern about the risks of infection that may follow ablation of blockade of CD4cells, and the fact that sometimes one may be interfering with a CD4cell with suppressive properties. (CD4Mab ablation of suppressive cells has indeed been demonstrated in mouse modelsof thyroiditis; 121 and 125). As more information becomes available on T cell receptor usage and lymphokine participation in autoimmunity it may be possible to devise some forms of combination therapy that maximize the benefit of inactivating the dominantT cell clones while controlling the others. The ability to achieve immunological tolerance therapeutically will of course be a major goal in the evolution of monoclonal anitbody therapy. In the following section this subject is exploredin somedepth.

MONOCLONAL ANTIBODIES FOR THE INDUCTION OF TOLERANCE Tolerance Induction with Depleting and Nondepleting Antibodies In the course of analyzing the immunosuppressive effects of CD4Mabs, Benjamin& Waldmann(123, 124), and Gutstein et al (124) observed rIgG2b CD4Mabsgiven at higl~ doses produced immunological tolerance to irrelevant rIgG2b immufioglobulifis:’Benjamin et al (122, 126) showed that CD4therapy would also all6w tolerance to humanand rabbit immu-

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noglobulins if these were given simultaneously (Figure 6). Goronczyet (127) noticed that CD4therapy produced a state of long-term unresponsiveness to sperm whale myoglobin, where even primary challenge 4 weeksafter CD4therapy could not elicit a response. This finding, a point the authors discussed, was not obviously compatible with an interpretation of tolerance, and the nature of the unresponsible state they reported has not been resolved. Qin et al (45) observed that synergistic pairs of CD4 Mabsinjected at doses too low to deplete cells could also induce tolerance to HGG.Gutstein & Wofsy (128) and Benjamin et al (126) demonstrated that F(ab)2 fragments of CD4Mabs were immunosuppressive and also capable of permitting tolerance (126, 129). Coullie et al (130), Charlton al (131), and Qin et al (75) have all shown three independent rIgG2a mabs to be immunosuppressive, and recently S. Qin, H. Waldmann,S. P. Cobbold & Y. N. Kong (unpublished) have shown that a rIgG2a CD4 mabmay also permit tolerance without cell-depletion. Recently Benjamin et al (126) have established the cellular basis of this form of tolerance. Tolerance was induced in T helper cells and not in B cells; CD8cells were unnecessary; nor could "suppressor cells" be detected on adoptive transfer. Most interestingly each challenge with the immunogenic form of test antigen reinforced the state of tolerance so that it could be maintained indefinitely (Figure 7). Finally, they observed that "Mabfacilitated tolerance" was not simply a property of CD4Mabs but could also be induced by a non-depleting LFA-I antibody. The conclusion seems to be that CD4cells may be tolerized when confronted with certain antigens in circumstances where the CD4and

1:102~0

aggregafedHGG

(b)

~ 1:2560 >, 1:6/+0 c 1:160

(Weeks) 6

7

Figure 6 CD4Mab facilitates tolerance induction to HGG.Mice were pretreated with 2 mg CD4Mab (small arrows) or saline and HGG(thick arrow). Mice pretreated with (squares) were unresponsive on rechallenge with aggregated HGG,compared to animals that had had no CD4Mab (diamonds) and those that had received CD4therapy only. panel (b) are shownthe titres (on d56) of anti-FGG from HGGtolerant animals and controls (adapted from Ref. 122).

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E

90

120

150

Days aff~r Tolerance Induction Fiyure 7 Antigen challenge reinforces HGGtolerance. All mice were pretreated with 1 mg of CD4Mab on days 0, 1 and 2 and were given HGGor saline as well on day 2. Groups were challenged with aggregated HGGon one of days 30, 60, 90, or 120. After the first challenge the same mice received monthly challenge doses (arrows). The titres were assessed 14 days following each challenge. Exposure to antigen at day 60 elicited no response, but the duration of the animal’s unresponsive state was extended (from Ref. 126).

LFA-1 surface "adhesion" molecules have been perturbed with appropriate Mabs. Similar strategies have recently been applied in attempts to produce transplantation tolerance. Qin et al (132) have used CD4and CD8Mab therapy to avoid GVHDand marrow rejections and have succeeded in achieving something akin to classical transplantation tolerance (133) the adult. Judicious use of appropriate rIgG2b Mabsenabled the establishment ofchimerism across "multiple minor" and class I ÷ minors without the need for any irradiation or chemotherapy (Figure 8). Chimeric animals were able to maintain donor skin-grafts indefinitely. All components of the experiment could be administered together (i.e. antibodies, skin and marrow). In this same model F(ab)2 fragments of CD8Mabswere sufficient to permit tolerance of CD8cells if the CD4cells were ablated. Recently we have also shown (75) that tolerance can be induced in CD4 T cells without any need for their depletion. This was accomplished with high doses of the rIgG2a Mab.If this form of "tolerance therapy" could be extended over stronger antigenic barriers then there would be obvious clinical application in transplantation and correction of autoimmunity. More recently R. J. Benjamin, S. Qin, S. P. Cobbold, H. Waldmann (manuscript in preparation) have succeeded in establishing hemopoietic chimerism and tolerance across complete H-2 differences by using a combination of CD4, CD8, and LFA-1 Mabs together with low dose (300 rads) irradiation. A major goal of future research will be to understand

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[ Mabonly

1

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BIO. BM

I

I

I

I

I

Figure 8 "Classical transplantation tolerance" in the adult animal. CBA/CA mice (n = 6) were prepared by injection with 3 mg/mouse of rlgG2b CD4and CD8. B10.A marrow was transplanted on the first day of treatment and CD4and CD8Mabsover 5 days. Three weeks later donor skin was transplanted. The group that had received both marrow and Mabs becametolerant to B 10.A skin. Animalsreceiving either one alone did not (taken from Ref.

132).

and obviate the need for irradiation to be able to use BMTto achieve transplantation tolerance in the adult. This work demonstrates that CD4/CD8Mabtolerance therapy is not only confined to special protein antigens but is potentially exploitable in transplantation. A more complete understanding of the cellular mechanisms underlying tolerance and immunity may permit the evolution of rationale "tolerance therapies" using Mabs together with other immunosuppressive drugs. In trying to moveto such rational therapies we have proposed a hypothesis of how tolerance may arise in normal development and how monoclonal antibodies maybe used to simulate this process in the adult (134). There are two underlying assumptions to the hypothesis. The first requires that T cell receptor occupancyand the signal provided by recognition of peptide-MHCbe equivalent for both tolerance and immunity. The second assumption is that the critical decision (ONor OFF) is decided by secondary signals in a Bretscher-Cohn manner (135). Wewould suggest that these costimulatory signals arise through multiple cell interactions with each cell contributing some level of cumulative "help" which ultimately reaches a threshold which pushes the collaborative unit and each of its individual cellular componentsto "ON"(i.e. activation). By inference a cell that has adequately bound antigen but is isolated from collaborating

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partners cannot receive costimulatory signals and so defaults to "OFF". In other words the choice "ON" or "OFF"is dictated by the frequencies of antigen-reactive cells, and the chance that these can find each other and form a collaborative unit. For a potent antigen with manyforeign epitopes the chance of such an event would be high. However, any circumstances where antigen-specific T cells might be isolated in space or time would predispose to tolerance. By this token we can explain the ease of tolerance induction in the neonatal mouse, the irradiated animal, and the animal depleted of lymphocytcs by thoracic duct drainage or by administration of ALG. What of normal ’self-tolerance’? Here we have made the analogy of lemmingsfalling over a cliff of self-antigen. In essence we argue that the thymus, the environment where T-cell tolerance is largely generated, is a relatively helpless one. Nosooner can a T cell express an anti-self receptor than it comesto the antigen-cliff and "jumps" or deletes. In this waythere will be a perpetual loss of self-reactive cells one by one. As a result no helpful cells could accumulate to provide costimulator activity to the incoming self-reactive set, and consequently these newcomersdie. The situation would be different in the peripheral lymphoidtissues following exposure to a foreign antigen. MatureT cells are continuously recirculating from blood to lymph. Antigens with multiple epitopes are likely to successfully recruit sufficient specific T cells to generatean active collaborative unit where all the necessary requirements for triggering and growth are met. As a result tolerance is prevented. To someextent bystander responses to other antigens in the vicinity wouldalso tend to prevent tolerance. It can be seen that this hypothesis views tolerance as a process that is normally "prevented" in the encounter with a foreign antigen. Its relevance to Mab therapy is based on the philosophy that Mabs may be used to ALLOW natural tolerance to just happen. Let us consider how tolerance is facilitated by CD4, CD8, or LFA-1 Mabs. If therapy has depleted the CD4cells, then the numberof residual antigen-specific T cells maybe too small to form a collaborative unit allowing tolerance to occur in remaining cells. If antigen persists or is repeatedly injected, then newly formed T cells would be tolerized as they appear. Bone marrowis a self-replicating form of antigen and therefore donor antigen would persist. Protein antigens like HGGwould eventually be cleared to a subtolerogenic level. AnyT cells emergingfrom the thymus at this point wouldnot be tolerized but wouldbe tolerizable if any reexposure to antigen occurred before sufficient T cells had been replaced. This would adequately explain the reinforcement phenomenon(126) described before, without recourse to implicating "suppressor cells" (whatever they

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are). Let me develop the argument further to explain whyit should be Mabs like CD4, CD8, LFA-1, and not many others, that can facilitate tolerance. It is important to realize that blockadeof T cell function is also a subtle wayof isolating T cells from potential collaborative units. While blockaded by CD4and CD8, Mabs T cells simply ignore antigen, because both these two molecules are required to guarantee proper T cell receptor occupancy by antigen (peptide-MHC). Any T cell that had evaded depletion wouldstill be subject to Mabblockade. It is only whenT cells emerge from Mabblockade (e.g. after therapy is stopped) that tolerance may occur. This exodus from blockade will be a gradual process which might result in a staggered exposure of T cells to persisting antigen. The otherwise competent T cell would find itself isolated and "helpless" and consequentlytolerance susceptible. Tolerance wouldarise as a result of too few T cells being available to establish a collaborative unit. For complex antigens or antigens that are rapidly cleared it maybe hard to achieve this staggered exit from blockade, and so immunity would not be prevented. On this model, depletion would not be essential if the blockade and exit therefrom were carefully orchestrated. It is likely that the sameprinciples might apply to tolerance with LFA-1and anti-IL2 receptor Mabs. In the latter case one source of help would be compromised. This model would argue that interference with other growth factor receptors and other adhesion molecules would also increase the chance of isolating antigenspecific T cells from "help" and thus enable the induction of tolerance. Future Prospects for Tolerance Therapy The basis of the abovehypothesis is that a T cell is tolerizable if it can be isolated from other helpful cells while contacting antigen on appropriate APCs.The ease of such an isolation will depend upon a numberof factors. For example memorycells may be more frequent, may be upregulated for desirable adhesion molecules, and maybe poised to express other critical growth factor receptors in a way that increases chances of effective collaborations. It maybe possible to use combinations of methods to try to protect or isolate memorycells from sources of help to determine if they too are tolerizable. If memory cells are tolerizable then "tolerance therapy" with minimal cell depletion may be a therapeutic goal in autoimmunity. In translating the rodent work to human we should remember that the starting T cell pool is many orders of magnitude greater in humans. Consequently, the task of preventing collaborative events will be that much harder. Controlled debulking of T lymphocytes with antibodies like the humanized form of CAMPATH-1 may establish a platform onto which nondepleting antibodies directed to CD4, CD8, LFA-I and IL-2 receptor maybe effectively exploited for tolerance induction.

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THE ANTIGLOBULIN RESPONSE TO THERAPEUTIC ANTIBODIES It is a little disconcertingthat experimentalanimalsandpatients can make such good antiglobulin responses towards the very same immunosuppressiveMabswith whichthey are treated. These responses can be fast andcan be directed to both constant and idiotypic determinants(136). Clinically they are probablyundesirable,if oneis to avoidsensitization to future Mabtherapy, let alone to guaranteea prolongedperiod of treatment. Benjaminet al (123) showedthat a large numberof rat Mabs mousehemopoietic cells were very immunogenic.However,high doses of rIgG2b CD4Mabs were completely non-immunogenicand induced tolerance to rIgG2bimmunoglobulins (123, 124). This state of tolerance to a therapeutic antibody undoubtedlybenefited the Wofsy(108) and Shizuru(111) studies on long-term therapy of mouselupus and diabetes. Micetolerant to rIgG2bwereable to generate antiidiotypic responsesto other rIgG2bMabsdirected to cell surfaces but not to non-cell binding Mabs(123) (Figure 9). There are somefundamentalquestions to be sweredhere. Whatis the cellular basis for the anti-idiotypic response? Whatdeterminantsare recognizedby helper T cells? Whyis it so hard to tolerize to idiotypic determinantson cell-binding antibodies but not to constant region determinants? More recently Benjamin et al (126) have shown that an immunosuppressive rat LFA-1Mabis also non-immunogenic in mice, so "silence" is not a property unique to CD4Mabs. 1:102k0 ,~, 1:2560 °.2 1:6~0

~ <

1:160

~ I ~,0 Immunising YTH YTH YTH Thyl CO8 £D5 Antibody 2~.’5 12.5 3-2.6 ’-- non cell-binding -~’---~ cell-binding --’ Fi.oure 9 The anti-globulin response of mice tolerant to rlgG2b. The influence of antibodies binding to cells. Mice were tolerized with deaggregated rIgG2b (closed bars). Controls received saline. Mice were then immunized with a set of cell-binding Mabs (CD5; CD8; anti-Thy 1) or irrelevant non-cell binding Mabs. Antiglobulin responses to cell bound Mabs were shownto be largely anti-idiotypic (adapted from Ref. 123).

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If the problemsof the antiglobulin response are to .be minimizedthen there are available a numberof steps that can be taken. Theseinclude: (a) the search for Mabsthat are sufficiently immunosuppressive or the use of combinationtherapy that can immunosuppress or "tolerize" for the Mab (137); (b) the sequential use of a series of Mabsthat will preempt response to the preceding ones (138); (e) the developmentof a better understandingof mechanismsto allow appropriate intervention; (d) the conversion,of the antibody to be compatiblewith the species. [There has beenmuchinterest of late in the use of CDR grafting (139, 50) to generate humanizedV-regions that can be associated with humanconstant region cDNA segmentsin appropriate expression systems. There are still some theoretical problemsrelated to allotypy of the heavyand light chains and noclear biologicalreasonfor thinkingthat an anti-idiotypic responseis not possible. However,our recent extended use of humanizedCAMPATH-1 in the serotherapy of two patients with leukemia(4 to 6 ,weeks) without detectable antiglobulin responses(160) suggests that a humanizationmay be a valuable development];(e) exploiting the finding. There is evidence that certain antigens attached to Mabsto cell surfaces mayevokestrong immuneresponses (140, 141), an observation that could be advantageous for vaccination. As there is a lack of useful adjuvantsfor humanvaccine, antigens associated with humanizedV-regionstargeted to. cell surfaces maybypass such a requirement. ¯

CONCLUSIONS

OF THE EXPERIMENTAL

STUDIES

Potent immunosuppressive effects clearly can be achievedwith.Mabsthat deplete andwith th0~se.,that simplyblock or perturb functional molecules. I have focussed largely on CD4, CD8, and LFA-1Mabs, but probably the same principles underlie therapy with anti-IL-2 receptor Mabsso extensivelystudied by Stromandhis colleagues(116-118),as well as with CD3(142) Mabswhich have recently entered animal studies after the enormouspre-veterinary workup in humans(143). Withthis basic knowledge it should be possible to approachthese sameexperimentalsystems with newreagents and strategi~s~ to manipulateimmuneresponses with a greate~degreeof finesse than,hashitherto -beenpossible. ENHANCING THE IMMUNE TO ANTIGENS ON CELLS

RESPONSE

Thus, far: I.,have consideredwaysof preventingT-cell responses. Monoc, lonal~ ant~b, odies, qanalso be used to enhanceresponsesby focussingT

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cells onto targets (for example tumor cells) which they might otherwise ignore. Twodirections hve been taken. I shall refer to these as effector cell retargeting (ECR)and xenogenization.

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Effector’ Cell Retargetin9 The fact that CD3Mabsare capable of triggering T cells to .divide and to kill targets to which they maybe focussed (143-145) has led to .the, construction of chemically induced heteroconjugates and more recently of bispecific hybrid antibodies with dual specificity for both the target and for the T cell receptor complex(146-148). If such therapy is to be successful, then T cells must be effectively drawnto their targets without risk of lysis by the antibodies themselves. One straightforward approach has been to prepare F(ab’)2 fragments of bispecific antibodies. Another has been modify the constant regions of the bispecific Mabso that complementlysis and ADCCwere virtually inactivated. This was achieved by creating hybrid antibodies between parental immunoglobulinsof different isotypes (rIgG2b/rIgG2c and rIgG2b/IgG2a) (148). The resulting Mabs extremely effective targetting agents in vitro and exhibited a therapeutic ratio (relative target-to-effector cell lysi~) between30- and 300-fold higher than rIgG2b/rIgG2b bispecifics. The process of constructing bispecifics of desirable therapeutic ratios is a laborious one. Recently Gilliland et al (149) have developeda bispecific antibody that can be used to target manytumors. Using a bispecific Mab with specificity for CD3in one arm and rat kappa light chain in the other arm, they demonstrated that ECRcould be achieved as a two-stage process. This bispecific Mabcould direct cytotoxic cells to any target coated with a rat kappa bearing Mab. Concern about indirect systems of this type dwell on potential blockade by free anti-target Mab. However the cell interactions here involved are very muchbased on multivalency and certainly blockade in vitro has not-been a significant problem (149). The special advantage of the idealized anti-globulin bispecific.antibody depicted in Figure 10 is that it does not preciS’de recruitment of other effector mechanisms.

Xenoyenisation Recently Lanzavecchia et al (150, 151) have shown that xenogeneic monoclonals processed by the tumor target can themselves be a target of immuneT cell attack. T cell responses to xenogeneic immunoglobulins might also generate "help" for weak tumor antigens,, More knowledgeon. the processin:g.pathWays followed by Mabsto different surffice:an:t~gen~ maypermit a f6ll6rexploitation of this "strategy of n’at~re."

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Indirect

ECRInd ADCC:

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TWOMechani|ml for T&~ge! Cell

~c ~llo~

ADCC

Figure10Idealizedtumorcell therapywithbispecific antibody.Theuse of a twostage "indirect"effectorcell retargeting allowsoneto protecttheeffectorcell (byusingbispecific antibodiesthat cannotkill the effectorcells) whileallowingother effectormechanisms to simultaneously target the tumor(takenfromRef. 20).

CLINICAL

PERSPECTIVES

Despite the depth of data on Mabimmunosuppression that has accumulated in rodent models and the valuable primate studies of Jonkers and her colleagues (152, 153), it is no simple matter to transplant that information onto current therapy, for both practical and ethical reasons. In clinical practice a transplant is performed because of discase, and this can mean

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that simple rules of immunologyderived from work on inbred strains of animals in good health may not transfer without some modifications appropriate to the clinical setting. This is perhaps best exemplified in the practice of allogeneic BMTin leukemia. Allogeneic BMTis used as a "rescue" procedure to restore hemopoiesis after maximal anti-leukemic therapy. The donor marrow supplies multipotential stem cells that should replenish the host. Marrowcollections are always heavily contaminated with T cells from blood and following marrow transfusion these T cells can produce GVHD.Without immunosuppression GVHD would occur in virtually all HLA-matchedsib transplants. Even with conventional drug immunosuppression, acute and chronic GVHD are a commonand a significant cause of morbidity and mortality. Indeed the severity of GVHD has precluded transplants across other than weak MHCbarriers. One of the first therapeutic applications to which Mabscould be applied with some optimism was in T cell purging. Many Mabs and purging strategies were used (154, 155), but one of the simplest involved the use the rIgM Mab CAMPATH-1M which had the distinctive property of lysing humanlymphocytes with human complement (i.e. complement from the marrowdonor) (155 158). T cell purging has undoubtedly reduced incidence of GVHD but introduced new problems which need solution for the full benefit of purging to be realized. This is exemplified in our own multicenter study with CAMPATH-1M (158) which broadly reflects the general experience. Of 441 patients transplanted in manycenters in Europe, the incidence of severe acute GVHD (grades 3-4) was 8% (335 evaluable patients), and of severe chronic GVHD was 3 % (274 evaluable patients)well below the figure expected with cyclosporine therapy alone. There were 181 patients whohad received no cyclosporin prophylaxis, and these still exhibited this low incidence of GVHD. However, in contrast to unpurged transplants where the incidence of marrowrejection is less than 1%, the incidence of complete rejection in purged transplants was 15%. Clearly, prevention of GVHreactions had tipped the balance of alloreactivity in such a way that residual recipient T cells held the upper hand, and in some patients complete marrowrejection resulted. Perhaps not unrelated in the light of the previous discussion of competition between donor and host hemopoietic systems, the incidence of relapse in at least one form of leukemia (chronic myeloid leukemia-CML)was increased (Figure 11). best correlates of relapse risk in this group of CMLpatients were lack of any GVHD (Figure 1 l a) and time to engraftment (Figure I l b). In of the intimate association between GVHD and HVG,it is not easy to determine whether T cell purging removed an anti-leukemic function, or whether purging tipped the balance of hemopoiesis such that some donor

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~0

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2~

(b)~.ee

~(83)

GVH-

P=0.02S

~.000

Figure 11 Probability of remaining in remission for patients with CGLtransplanted in first chronic phase. (A) Influence of graft versus host disease. Patients with any degree of GVH are compared to patients without GVHD.(B) Influence of the rate of engraftment. Patients who reached a neutrophil count of 0.5 × 109/1 before day 26 (<26 days) are compared with those who engrafted more slowly (> 25 days). These data are taken from a study of 140 patients who received matched sibling allografts (Ref. 158). Fifteen patients were excluded from analysis because they did not engraft, or died before engraftment. The quoted P values are from log-rank analysis of the actuarial curves; similar results were obtained by a Cox multifactorial linear regression analysis (taken t¥om Ref. 158).

stem cells (including leukemic cells) could effectively competefor growth, or indeed whether both explanations were valid. If T-cell purging is to have a useful future, then the clear goals are to find better anti-rejection and anti-leukemic therapy. The lympholytic and immunosuppressive rIgG2b form of CAMPATH-1 may have a role in preventing marrowrejection, and it is currently being tested for its impact on graft rejection in HLA-matchedbut unrelated donor-recipient combinations. If marrowrejection could be avoided, then it maybe easier to

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THE APPLICATION OF MABS TO ORGAN GRAFTING Current practice in renal transplantation is moderatelysuccessful. It is not easy, therefore, to measurethe impact of a new treatment strategy unless randomized trials are performed. Undoubtedly the mIgG2a Mab OKT3 (143) is an effective agent for reversing rejection episodes, albeit with certain side effects and a measurableinfection risk (159). The key questions are whether this Mabis better than any other available, and if potentially better Mabsappear, will there be opportunities for proper comparison? The ability of a pharmaceutical companyto supply Mabsfor trials can to a large extent determine current usage. It is hoped that the overiding wish to achieve immunologicaltolerance with limited drug therapy will dictate which Mabs and which biological strategies are ultimately adopted for routine transplantation. ACKNOWLEDGMENTS

I thank Geoff Hale, Mike Clark, Steve Cobbold, Richard Benjamin, Shixin Qin, and Carol Bindon for the discussions and exchanges of ideas that color myviews. Our work is supported by the Medical Research Council, United Kingdom, the Cancer Research Campaign and Wellcome Biotech. Ltd. CAMPATH is a trademark of Wellcome Biotech. Ltd. Literature Cited 1. Kohler, H., Milstein, C. 1975. Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256:495 2. Galfre, G. C., Milstein, C., Wright, B. 1979. rat x rat hybrid myelomasand an anti-Fd portion of mouse IgG. Nature 277:131 3. Klaus, C. G. B., Pepys, M. B., Kitajima, K., Askonas, B. A. 1979. Activation of mouse complement by different classes of mouse antibody. Immunology 38:687 4. Neuberger, M. S., Rajewsky, K. 1981. Activation of mouse complement by mouse monoclonal antibodies. Eur. J. Immunol. 11:1012 5. Oi, V. T., Vuong, T. M., Hardy, R., Reidler, J., Dangl, J., Herzenberg, L. A., Stryer, L. 1984. Correlation between segmental flexibility and

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T-CELL FUNCTIONS IN VIVO autoimmunethyroiditis: L3T4cells as mediators of both thyroglobulin-activated and TSH-induced suppression. Clin. Immunol. Immunopath.In press 126. Benjamin, R. J., Qin, S.-X., Wise, M. P., Cobbold, S. P., Waldmann, H. 1988. Monoclonal antibodies for tolerance induction: a possible role for CD4 (L3T4) and CDI la (LFA-1) ecules in self-non-self discrimination. Eur. J. lmmunol. 18:1079 J., Weyand, C. M., 127. Goronczy, Fathman, C. G. 1986. Long tcrm humoral unresponsiveness in vivo induced by treatment with monoclonal antibody to L3T4. J. Exp. Med. 164: 911 128. Gutstein, N. L., Wofsy, D. 1986. Administration of F(ab),_ fragments monoclonal antibody to L3T4 inhibits humoral immunity in mice without depleting L3T4+ cells. J. lmmunol. 137:3414 129. Carteron, N. L., Wofsy, D., Seaman, W. E. 1988. Induction of immunetolerance during administration of monoclonal antibody to L3T4 does not depend upon depletion of L3T4cells. J. Immunol. 140:713 130. Coullie, P. G., Coutelier, J.-P., Uttenhove, C., Lambotte, P., van Snick, J. 1985. In vivo suppression of T-dependent antibody responses by treatment with a monoclonal anti-L3T4 antibody. Eur. J. Immunol. 15:638 131. Charlton, B., Burkhardt, K., Mandel, T. E. 1988. Howimportant is the L3T4 antigen to L3T4+ function in-vivo. Immunol. Today 9:165 132. Qin, S,-X., Cobbold, S. P., Benjamin, R., Waldmann, H. 1988. Classical transplantation tolerance in the adult. Submitted 133. Billingham, R. E., Brent, L., Medawar, P. 1953. Actively acquired tolerance of foreign cells. Nature 172:603 134. Waldmann,H., Cobbold, S. P., Benjamin, R. J., Qin, S. 1988. A theoretical framework for tolerance and its relevance to the therapy of autoimmune disease. J. Autoimmunity.In press 135. Bretscher, P., Cohn, M. 1970. A theory of self-non-self discrimination. Science 169:1042 136. Jaffers, G. J., Fuller, T. C., Cosimi,A. B., Russell, F. S., Winn,H. J., Colvin, R. B. 1986. Monoclonal antibody therapy. Antiidiotypic and non-anti-idiotypic antibodies to OKT3arise despite intensive immunosuppression. Transplantation 41:572 137. Jonker, M., den Brok, J. H. A. M. 1987. Idiotype switching of CD4specific

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monoclonal antibodies can prolong the therapeutic effectiveness in spite of host anti-mouse IgG antibodies. Eur. J. Irnmunol. 17:1547 138. Lowder, J. N., Miller, R. A., Hopps, R., Levy, R. 1987. Suppression of antimouse immunoglobulin antibodies in subhuman primates receiving monoclonal antibodies against T-antigens. J. Immunol. 138:401 139. Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S., Winter, G. 1986. Replacing the complcmentarity-dctermining regions in a human antibody with those from a mouse. Nature 321: 522 140. Carayanniotis, G., Barber, B. H. 1987. Adjuvant free IgG responses induced with antigen coupled to antibodies against Class II MHC.Nature 327:59 141. Kawamura,H., Berzowsky, J. A. 1986. Enhancement of antigenic potency in vitro and immunogenicity in vivo by coupling the antigen to anti-immunoglobulin. J. Immunol. 136:58 142. Hirsch, R., Eckhaus, M., Auchincloss, H. Jr., Sachs, D. H., Bluestone, J. A. 1988. Effects of in-vivo administration of anti-T3 monoclonal antibody on Tcell function in mice. 1. Immunosuppression of the transplantation response. J. Immunol. 140:3766 143. Ortho Multicenter Transplant Study Group. 1985. A randomised trial of OKT3monoclonal antibody for acute rejection of cadaveric renal transplants. N. Engl. J. Med. 313:337 144. Kranz, D. M., Tonegawa,S., Eisen, H. 1984. Attachment of an anti-receptor antibody to non-target cells renders themsusceptible to lysis by a clone of cytotoxic T-lymphocytes. Proc. Natl. Acad. Sci. USA 81:7922 145. Perez, P., Hoffman, R. W., Shaw, S., Bluestone, G., Segal, D. M. 1985. Specific targeting of cytotoxic T-cells by anti-T3 linked to anti-target cell antibody. Nature 316:354 146. Staerz, U. D., Bevan, M. J. 1986. Hybrid antibodies producing a bispecific monoclonalantibody that can focus effector T-cell activity. Proc. Natl. Acad. Sci. USA 83:1458 147. Lanzavecchia, A., Scheidegger, D. 1987. The use of hybrid hybridomas to target human cytotoxic T-lymphocytes. Eur. J. Immunol. 17:105 148. Clark, M. R., Waldmann, H. 1987. Tcell killing induced by hybrid antibodies. Comparison of two bispecific monoclonal antibodies. J N C I 79: 1393 149. Gilliland, L. K., Clark, M. R., Wald-

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mann, H. 1988. Universal bispecific antibody for targeting tumour cells for destruction by cytotoxic T-cells. Proc. Natl. Acad. Sci. USA 85:7719 150. Lanzavecchia, A. 1988. Exploiting the immune system’s own strategies for immunotherapy. ImmunoL Today 9: 167 151. Lanzavecchia, A., Abrignani, S., Scheiddegger, D. 1988. J. Exp. Med. 167:345 152. Jonkers, M. 1987. Immunosuppressive therapy by monoclonal anti-T-lymphocyte subset antibodies. In Leucocyte Typing llI, ed. A. J. McMichael, P. C. L. Beverley, S. P. Cobbold, M. J. Crumpton, W. Gilks, F. Gotch, N. Hogg, M. Horton, N. Ling, I. C. M. MacLennan,D. Y. Mason, C. Milstein, D. Speigelhalter, H. Waldmann,p. 923. Oxford: Oxford Univ. Press 153. Van Lambalgen, R., Jonker, M. 1987. Experimental allergic encephalitis in rhesus monkeys. II. Treatment of EAE with anti-T-lymphocyte subset monoclonal antibodies. Clin. Exp. Immunol. 68:305 154. Reading, C. L., Takaue, Y. 1986. Monoclonal applications in bone marrow transplantation. Biochim. Biophys. Acta 865:141 155. Waldmann, H., Or, R., Hale, G., Weiss, L., Cividalli, G., Weshler, Z., Samuti, S., Manor, D., Brautbar, C., Rachmilewitz, E. A., Polliak, A. M.,

Slavin, S. 1984. Elimination of graft versus host disease by in-vitro depletion of alloreactive lymphocytes using a monoclonal rat anti-human lymphocyte antibody (CAMPATH-1).Lancet ii, 483 156. Waldmann,H., Hale, G., Cobbold, S. 1987. The immunobiology of bone marrowtransplantation. See Ref. 152, p. 932 157. Hale, G., Cobbold, S. P., Waldmann, H. 1988. T-cell depletion in allogeneic marrow transplantation. Transplantation 45:753 158. Hale, G., Waldmann, H. (for CAMPATHusers). 1988. CAMPATH 1 for prevention of GVHD and graft rejection. Summaryof results from a multicentre study. Bone Marrow Transplant. (3 suppl.) 1 : 159. Oh, C. S., Stratta, R. J., Fox, B. C., Sollinger, H. W., Belzer, F. O., Maki, D. G. 1988. Increased infection risk associated with the use of OKT3for treatment of steroid resistant rejection in renal transplantation. Transplantation 45:68 160. Hale, G., Dyer, M. J. S., Clark, M. R., Phillips, J. M., Marcus, R., Reichmann, L., Winter, G., Waldmann, H. 1988. Remission induction in NonHodgkin’s lymphoma with the reshaped human monoclonal antibody CAMPATH-1H. Lancet (In press)

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Ann. Rev. Immunol.1989. 7.’445-80

CLONAL EXPANSION VERSUS FUNCTIONAL CLONAL INACTIVATION: A Costimulatory Signalling Pathway Determines the Outcomeof 1T Cell Antigen Receptor Occupancy Daniel L. Mueller, Marc K. Jenkins and Ronald H. Schwartz Laboratory of Cellular and Molecular Immunology,National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 INTRODUCTION The rapid developmentof a T-cell proliferative response to foreign antigens is one essential determinant of the capacity of the immunesystem to eliminate a pathogen that has invaded an organism. The expansion of + inducer phenotype is critical in the antigen-specific T cells of the CD4 generation of delayed-type hypersensitivity responses (DTH)as a result the ability of these cells to secrete lymphokines(including interferon-v) that attract and activate macrophagesat the site of antigen deposition. + cells are also critical for the developmentof cytotoxic responses to CD4 virus-infected cells, malignant cells, and allogeneic tissue, either directly through lymphotoxin release (TNF-/~), or by the lymphokine-mediated expansion of cytotoxic T lymphocytes. Finally, these T cells also provide help for antibody responses through the secretion of interleukins during interactions with antigen-specific B cells. ~ The USGovernmenthas the right to retain a nonexclusive license in and to any copyright covering this paper.

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Becauseof the pivotal role of the T cell in these situations, T lymphocyte proliferation mustbe tightly regulated to protect against the development of inappropriate immune responsesto self-antigens, or exaggerated,pathologic responsesto foreign antigens. Deletionof autoreactiveT-cell clones during maturation in the thymus,and induction of antigen-specific regulatory or suppressive T-cell populationsboth appearto represent control mechanisms capable of modifyingthe potential for T-cell responsiveness to antigen. In addition, the antigenresponsivenessof anyparticular mature T cell mayhe dependentuponits previous experience with antigen and accessorycells, such that subsequentresponsescould vary in intensity and nature. In this reviewweexaminethe control of T-cell proliferation at the level of the antigen-presentingcell (APC).In particular, wefocus on the nature of the critical biological andbiochemicalsignals required to activate the T cell, as well as the consequencesof incompletesignalling. Finally, we proposea three-signal modelto explain our observations, and wesuggest howthis modelcould be applicable to clonal deletion and clonal anergy mechanisms of tolerance induction in vivo. A BIOLOGICAL TWO-SIGNAL T-CELL ACTIVATION

MODEL OF

A solution for the problemof self-nonself discrimination was one of the driving forces behindthe development of clonal selection theories in immunology (1-3). Theexistence of preformedcells--each with a unique receptor--allowed the system to purge itself of autoreactive clones by eliminatingcells bearinganti-self receptors. Theearliest explicit modelto speculate on howthis deletional process might occur was put forth by Lederberg(4). He postulated a temporal modelof lymphocytesignalling in whichantigen receptor occupancywasidentical in both immatureand mature cells, but coupling of receptor engagementto more distal biochemicalpathwaysvaried with the state of maturity of the cell. Receptor engagementby self-antigens during early developmentgave a negative signal to the cell, causing it to die. Subsequentmaturationof surviving lymphocyteschangedthe biochemicalcoupling mechanism inside the cells in such a way that later receptor engagementby foreign antigen was perceivedas a positive signal for the cell to respond(i.e. with secretionof antibody or lymphokine). Bretscher & Cohn(5) subsequently recognizedthat this modelwas not sufficient to accountfor B-cell self-tolerance, giventhe propensityof the immunoglobulingenes in activated B lymphocytesto undergo somatic

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hypermutation. Through this process, receptors on mature B cells could acquire the capacity for recognition of self-antigens, which upon engagement would lead to autoreactivity in the Lederberg model. There could be no escape by reversion of the cells to an immature state, because in the adult this wouldallow foreign antigen the opportunity to induce tolerance. As a solution to this problem, Bretscher &Cohn proposed the first twosignal model of lymphocyte activation. Signal one was postulated to be occupancyof the antigen-specific receptor (membraneIg on B cells). Alone, this signal led to inactivation of the lymphocyte. If, however, the cell received a second signal, simultaneous with antigen-receptor occupancy, then the combination of signals would activate the cell and induce a positive response to the antigen. The second signal in their model was formulated in terms of the emergingconcept of T-cell help (hapten-carrier effects) (6); today it might take the form of T cell-derived lymphokines (7). This model was capable of dealing with the problem of continually arising autoreactive clones in the mature B-cell population, as such cells would be turned off when they encountered self-antigens alone, in the absence of autoantigen-stimulated T-cell help. Foreign antigens, however, would remain immunogenic,as simultaneous T-cell stimulation by carrier determinants would provide the responding B-cell population with a second signal. Thus, the modelpredicted a form of competition at the level of antigenic stimulation betweenactivation and inactivation of the mature, responding lymphocyte.Despite the fact that tolerance at the B-cell level no longer appears absolutely necessary--i.e. T-cell receptor genes do not undergosomatic hypermutation(8), and, therefore, T-cell tolerance should be sufficient to control B-cell responses, the general outlines of the model have been substantiated for multivalent antigens (reviewed by Nossal in Annu. Rev. Immunol.; 9). The extension of the concept of a two-signal modelto T-cell activation was primarily the workof Lafferty and coworkers (10, 11). They postulated from their studies on the generation of cytotoxic T lymphocyte (CTL) responses that the alloantigens found on most cells in a transplanted tissue were incapable of eliciting an immuneresponse; only hematopoietic stimulator cells carried within the transplanted tissue provided both allogeneic major histocompatibility complex (MHC)antigens and what they termed an inductive stimulus (second signal) required for the initiation a T-cell response. Consistent with this modelwas the work of Bach et al (12), which demonstratedthat UV-irradiation of allogeneic stimulator cells resulted in a failure to elicit a CTLresponse to class-I determinants in a primary mixed lymphocyte culture (MLC). Although Bach suggested that the UV-sensitivity of the stimulator cell was at the level of presentation of class-II determinants (as the addition of normal class-II disparate stimu-

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lators could restore the response), Lafferty &Woolnoughinterpreted the result as evidence that UV-irradiation reduced the metabolic activity of the stimulator cell population and prevented the delivery of a costimulatory signal (signal two). Theywent on to suggest a soluble nature for the second signal, given the ability of concanavalin A (Con A)-stimulated spleen cell supernatants to restore the response to UV-treated stimulator cells. Our current understanding of CTLgeneration would suggest that CD4+ T cell-derived lymphokines such as interleukin 2 (IL-2) make the soluble factors within a Con-Asupernatant capable of supporting CTL growth (13, 14). Nonetheless, we might predict that the failure to produce + helper T cells in a mixedlymphocyteculture directed against IL-2 by CD4 UV-irradiated stimulator cells stems from an inability of these treated stimulatory cells to deliver a secondsignal. The role of the accessory cell in T-cell activation has also been defined through the use of mitogens such as Con A and phytohemagglutinin (PHA). These molecules are capable of stimulating T-cell proliferation the absence of MHC molecule recognition by the antigen-specific receptor, presumably by interacting directly with the CD3complex (15). When accessory cells are carefully depleted from T-cell populations, a significant reduction in mitogen responsiveness is observed, consistent with the need for accessory cell~lerived second signals to stimulate a proliferative response (16). The ability of IL-1 in manycases to replace the requirement for accessory cells in both the generation of CTLin mixed lymphocyte cultures and in mitogen-induced proliferation has led to the hypothesis that this monokinemay represent the soluble second signal (17, 18). information accumulates, however, to suggest (a) that IL-1 receptors are + T-cell populations (19), (b) that the effects not present on all murine CD4 of IL-1 on the responsive population of murine T cells maybe limited to enhancement of lymphokine response rather than lymphokine production (20), and (c) that the effects of IL-1 mayin somecases be limited to actions on the accessory cells in the population (21), it seems unlikely that IL-1 alone plays a direct role in alloreactive or mitogen-inducedresponses as the actual second signal for manyT cells. Nevertheless, IL-1 mayaugment the ability of accessory cells within the population (or contaminants within an accessory cell-depleted population) to deliver the second signal to cells. All of the studies described above support the notion of a general biological two-signal modelfor T-cell activation. The modelof Bretscher & Cohn, however, makes a unique prediction regarding the potential effects of isolated T-cell antigen receptor (TCR)occupancyin the absence of an adequate accessory cell-dependent signal two, i.e. signal one alone should inactivate the cell. Experimental evidence consistent with this was

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first reported by Clamanand coworkers (22, 23). In their studies, intravenous injection of haptenated spleen cells induced a state of tolerance, as measured by the subsequent failure of the animal to develop contact sensitivity upon reexposure to the hapten. The ability of simultaneous Con-Aadministration to prevent this induction of antigen-specific unresponsiveness and, in fact, to lead to the sensitization of the animal to the hapten suggested that two signals were operative in the control of contact sensitivity. In their model, the haptenated spleen cells provided one signal (antigen/MHC) to the responding T cells, which alone was tolerogenic, and the Con A provided a second signal that converted the first signal to a positive stimulus. Can a biological two-signal model explain both the requirement for accessory cells in the induction of T-cell responses and the control of Tcell responsiveness to subsequent stimulation? Workin our laboratory over the past three years has directly demonstrated that some T cells can enter a state of reduced responsiveness upon encountering processed antigen presented by Ia molecules in the absence of a functional accessory ÷ T-cell clones with peptide cell (24). Incubation of murine Type I CD4 antigens presented in association with purified Ia molecules in a planar membrane(25) or presented by chemically fixed APC(26) failed to stimulate proliferation and instead induced a state of proliferative nonresponsiveness. The effect was both antigen and MHC specific, suggesting that TCRoccupancy was required. The state of clonal anergy induced by the incubation was long-lived, lasting greater than seven days, and was not associated with accelerated mortality, as the cells remainedcapable of proliferating if stimulated with exogenousIL-2. Flow cytometric analysis of the ceils stained with anti-TCR monoclonal antibody demonstrated comparable levels of receptor expression on normal and nonresponsive T cells (25). Thus, reduced expression of the TCRat the cell surface was not the reason for the nonresponsiveness. Analysis of T-cell lymphokine production revealed that the inability of these lymphocytesto proliferate in response to further antigen stimulation appeared to stem from a lymphokine production defect--in particular, the failure to secrete sufficient IL-2 to drive proliferation (25, 27). Production of IFN-y and IL-3 were also reduced by the incubation; however, the defect was not as profound as that observed for IL-2 synthesis (M. K. Jenkins, unpublished observations). Thus, occupancyof the TCRwith antigen in the presence of fixed APCresulted in a functional inactivation of the clone at the level of IL-2 production. Lamb& Feldmann and their colleagues (28, 29) have observed what appears to be a similar state of antigen unresponsiveness in normal human T-cell clones incubated with high doses of free peptide antigen in the

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absence of accessory cells. In contrast to murine T cells, humanT-cell clones express class-II MHC molecules capable of presenting peptide antigens. Such antigen/Ia complexes could occupy the TCRon these cloned cells (signal one) and, in the absence of an exogenousaccessory cell population capable of providing a second signal, induce an unresponsive state. Experiments by their group, however, have suggested that the unresponsive state results from downregulation of antigen receptor expression (30, 31). This is clearly not the case for the murine clones. In addition, others have found some Ia-bearing human T-cell populations competent to present antigen and induce the proliferation of T cells specific for the antigen (32). Possibly this inconsistency is related to the state of activation of the T cell presenting the antigen. An activated T cell may possess costimulatory activity, whereas the rested, cloned T cell maynot (see below). Further studies will be required to determine howapplicable this humanin-vitro modelof T-cell tolerance is. Manydetails regarding the nature of the unresponsive state in the murine system have yet to be explored. Nonetheless, its induction appears to depend on the presence or absence of an accessory cell~lerived costimulatory activity. Several laboratories have found that during stimulation of T cells with peptide antigen and chemically fixed syngeneic APC,the addition of a normal allogeneic accessory cell population (itself incapable of presenting the antigen because of the lack of an appropriate Ia molecule) allows a proliferative response to the antigen (33-35) and antagonizes the induction of unresponsiveness (36). Figure 1 demonstrates this ability normalaccessory cells to induce a proliferative response to peptide antigen presented on paraformaldehyde-fixed APC. Neither fixed APCalone nor fixed APCin the presence of lymphokine(in this case rlL-6) are sufficient to induce proliferation to the antigen in the absence of a normal accessory cell population. In our hands, low-density splenic accessory cells have been the most potent source of this costimulatory activity. The high-density fraction of T-depleted spleen (resting B cells) works to a lesser extent, and its activity is radiosensitive (37). Resting splenic T cells have costimulatory activity, although activated T cells may acquire it (see below). Unlike the soluble activity suggested by Lafferty, culture supernatants (and recombinant IL-1 and IL-6) do not replace the costimulatory activity of intact accessorycells, and separation of the viable accessorycell population from the responding T cells by a permeable millipore membrane prevents the addback effect (36). IL-2 is capable of driving the cell clone to proliferate in the presence of peptide antigen plus fixed APC or planar membranes(as the clone constitutively expresses high affinity receptors for IL-2); however, the presence of exogenous IL-2 can not prevent the induction of the antigen-unresponsive state (25). Thus, these

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3O 25

O NormalAPC [] PF-fixed APC ¯ PF-fixed APC+normal allogeneicaccessorycells

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¯ PF-fixed APC+ rlL-6 50 0

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[Peptide Figure I Normal allogeneic

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+ T cell clone A.E7(2 × 104 per to stimulate a proliferative response.ThemurineTypeI CD4 well) wasincubatedwith the pigeoncytochromee peptide 81-104in the presenceof either normalsyngeneicB10.Aspleen cells (5 × l0~) (open circles), or in the presenceof 0.5% paraformaldehyde-fixed (PF) syngeneicspleen cells alone (5 × l05) (open squares) combination with normal allogeneic B 10 T-depleted low-density spleen cells (2 × 105) (closed squares) or recombinant IL-6 (100 u/ml) (closed circles). Thymidine incorporation measured at 60 hr following a 16 hr pulse and is expressed as the mean cpm of duplicate samples.

experiments appear to define a cell-bound or short-range acting costimulatory activity required for the delivery of signal two. What is the nature of this costimulatory activity? At the present time we only know what it isn’t: IL-1, IL-2, IL-3, IL-4, IL-6, IFN-y, GM-CSF, TGF-fl, and TNF-~ all fail to reconstitute a proliferative response, and none is capable of blocking the induction of unresponsiveness. Kurt-Jones et al (38) have described a membrane-bound form of IL-1 capable providing a costimulatory signal in the proliferative response to antigen of Type II CD4+ T cells. However, we have found that receptor occupancy of Type II CD4+ T cells in the absence of IL-1 or costimulatory signals does not induce a state of unresponsiveness (M. K. Jenkins, unpublished observations). Only Type I CD4+ T cells show this effect. Furthermore, membrane IL-1 is incapable of substituting for intact accessory cells in our system. Recently, Weaver et al (39) have described experiments demonstrating that although antigen and fixed resting B cells fail to stimulate proliferation in a Type I CD4+ T-cell population (identical to our system), proliferation is induced with antigen presented by chemically fixed B ceils pre-activated with IFN- 7 and anti-IgM antibody. Presumably the

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pretreatmentof the APCpopulationresulted in the elaborationof a surface moleculewith costimulatoryactivity, resistant to fixation, that wascapable of providingthe secondsignal to T cells respondingto the peptide antigen/MHC complexes.Thus, the molecule(s) is not always constitutively expressed on the APCsurface, and probablynormallyit requires a T-cell interaction to induceit. Fromall these experiments, it seemsreasonable to suggest that the Bretscher& Cohnmodelof B-cell activation and inactivation is applicable ÷ T cell clones (Figure 2). Signal one is definedby occupancy to TypeI CD4 of the T-cell antigen receptor by a complexformedbetween antigenic peptide and an Ia moleculeon the APCsurface, and this signal alone is capable of inducing a nonresponsivestate. Signal two is a costimulatory factor or cell interaction molecule, rapidly inducedon the APCsurface, whichthen binds to a distinct receptor on the T-cell surface and acts in synergy with signal one to stimulate the T cell to makeIL-2 and to proliferate. Howthen are these two signals transducedinto the cell, and what are the biochemicalconsequences? A BIOCHEMICAL TWO-SIGNAL MODEL OF T-CELL IL-2 GENE ACTIVATION Fisher & Mueller (40) showedin 1968 that phyto hemagglutinin stimulation of normalhumanperipheral blood lymphocytesinduced the rapid incorporation of radioactive orthophosphateinto phosphatidyl inositol (PI). Basedon substantive workat that time in other hormone/receptor systems(41; also reviewedby Hokinin 42), the authors suggestedthat PHA stimulates the interconversion of phosphatidic acid and PI in lymphocyte membranes.Later work in other systems emphasizedthe importance of

COSTIMULATORY PEPT1DE ANTIGENMOLECULE COMPLEX COSTIMULATORY ACTIVITYRECEPTOR

PEPTIDE AN’TIGF~Nla MOLECULE COMPLEX T CELLANTIGEN RECEPTOR

Figure 2

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PI breakdownas the initial event in cell activation, rather than the observed reaccumulation of PI in the membrane(43). Soon thereafter, lymphocyte proliferative responses to PHAwere found to be inhibited by nontoxic concentrations of citrate, EDTAor EGTA, consistent with a dependency of the response on calcium ions (44, 45). PHA-induceduptake of radioactive calcium into lymphocytes was demonstrated about the same time (46, 47), and in recent years numerous studies have demonstrated the correlation between increases in the intracellular calcium free ion concentration ([Ca+2]i) and the induction of IL-2 secretion and/or T-cell proliferation by various stimuli including antigen and antigen-presenting cells (27, 35, 48-50). The developmentof the calcium ionophore, A23187, subsequently allowed one to directly test the mitogenicproperties of increasing [Ca+2]i. It was initially reported that the ionophore alone was capable of inducing somedegree of proliferation by human lymphocytes (51); however, the general consensus was that increased [Ca+2]i alone was insufficient for a full proliferative response. More recent studies have demonstrated that calcium ionophores in combination with protein kinase C (PKC)-activating phorbol esters have potent mitogenic activity (52). More importantly, phorbol esters have appeared to substitute for accessory cells in the response of purified T cells to mitogens or treatment with anti-CD3 antibody (49, 53-55). The requirement for PMAin the response to mitogen was found to be at the level of IL-2 gene transcription and protein synthesis, as well as at the level of IL-2 receptor expression (56). Recently, Berridge & Irvine (57) and Nishizuka (58, 59) have proposed a hypothesis that explains the associations between PI metabolism, increased [Ca+2]i, and PKCactivation. Agonist-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) results in the production of the two second-messengers,inositol 1,4,5-trisphosphate (IP3) and 1,2-sn-diacylglycerol (DAG),both of which wouldplay a critical role in the induction of cell proliferation. Utilizing permeabilized cells or microinjection techniques, IP3 and its metabolic products were demonstrated to be capable of increasing the [Ca+2]i by initiating an influx of Ca+2 into the cytoplasm, both from intracellular stores within the endoplasmic reticulum and from the extracellular milieu. DAG is thought to be active in the stimulation of the Ca+2-dependent protein kinase C (PKC), based on in vitro studies with DAGanalogs. The importance of PIP2 hydrolysis and the initiation of this bifurcating pathway of signal transduction in the induction of cellular processes is underscored by multiple observations of synergy between calcium ionophores and PKC-activating phorbol esters in different biological systems (recently reviewed by Berridge in 60). Experiments performed in T-cell systems have in general supported such

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a hypothesis. Imboden & Stobo (50) stimulated the humanT-cell tumor line Jurkat with anti-CD3 antibody and demonstrated the production of IP3 as well as increases in [Ca+2]i, consistent with the modelthat T cell receptor occupancyresults in the hydrolysis of PIP2. Similar increases in inositol phosphates have been demonstrated in normal T cells in response to antigen plus syngeneic APC(27, 61). Kuno, Gardner, and their colleagues (62, 63), following their finding of a PHA-activated, non-voltage gated, calcium channel in normal T-cell plasma membrane,used a patchclamp technique to show that IP 3 (or a metabolite) could open these channels. Finally, several laboratories have demonstrated PKCtranslocation to the plasma membrane (a marker of PKCactivation) (64, or PKC-dependent phosphorylation of the CD3gammachain following stimulation of T cells with antigen and APC,mitogen, or anti-CD3 antibody (66-68), suggesting the endogenous production of DAG. The concordanceof the T-cell findings with other biological systems led Weiss et al (69) to propose a biochemical two-signal model for activation of the IL-2 gene in T lymphocytes. They suggested that accessory cells present antigen and occupy the T cell antigen receptor (TCR)in a manner that can be mimicked by mitogen or anti-CD3 antibody plus a phorbol ester. The result of the perturbation of the receptor is the hydrolysis of PIP2 and generation of IP3 and DAG.The accumulation of these second messengersthen leads to a rise in [Ca÷2]i and activation of PKC,respectively. Takenin light of the observation that the combination of calcium ionophore plus phorbol ester is sufficient to induce T-cell proliferation, this modeldefined increases in [Ca+2]i and PKCactivation as the requisite second messengers for IL-2 gene transcription. For purposes of general discussion, this biochemical two-signal model will be considered the current dogma(Figure 3). Doesthis biochemicalmodelsuffice to explain signal transduction within the framework of the biological two-signal model of T-cell activation described earlier? We have demonstrated that TCRoccupancy in the absence of the accessory cell-derived costimulatory signal is not sufficient for the induction of IL-2 synthesis and that in fact it renders the cell unresponsive to further stimulation. Others have demonstrated a requirement for a phorbol ester in T cell proliferative responses to anti-CD3 antibody or mitogens in the absence of accessory cells. The biochemical two-signal model, on the other hand, suggests that TCRoccupancy is competent to induce the hydrolysis of PIP2 and the production of both IP3 and DAG.This would predict that TCRoccupancy by antigen/Ia complexesalone (in the absence of the biological second signal) should competent to induce T-cell IL-2 secretion. Weiss and his colleagues are aware of this paradox and have offered two possible explanations: (a)

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3"~-g--’~ PImP 2

TRANSCRIPTION

455

~o~Y IONOMYC~

COSTIMULATORY ACTIVITY RECEPTOR

T CELLANTIGEN RECEPTOR

Figure 3

TCRoccupancy alone may never generate the degree of PKCactivation induced by phorbol ester; if so, then the accessory cell costimulatory signal would be required to obtain the high level of PKCactivity needed prior to the initiation ofIL-2 genetranscription. In support of this, recent studies have suggested that IL-1 can act by increasing the level of DAGin T lymphocytesthrough the induction of phosphatidylcholine hydrolysis (70). (b) PMAstimulation of T cells mayresult in effects independent of PKC activation; it is one of these other effects that mimicksthe secondmessenger cascadeinitiated by the accessory cell costimulatoryactivity. In the remainder of this review we address this question and propose that increases in [Ca+2]~and PKCactivation are in fact insufficient second messengers for + T cell the induction of IL-2 secretion and proliferation by Type I CD4 clones. A third biochemicalsignal appears to be required; its identity is at present unknown.

A BIOCHEMICAL THREE-SIGNAL MODEL OF T-CELL IL-2 GENE ACTIVATION Increases in [Ca+2]i CanOccur Independently of the Costimulatory Signal + T cells with As discussed earlier, stimulation of murine Type I CD4 antigen and chemically modified syngeneic APCfails to elicit a normal

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proliferative response and instead induces the T-cell population into a state of antigen unresponsiveness. This effect is both antigen and MHC specific, implying that physiologic occupancy of the TCRand some form of receptor signalling is required. Theability of the samereceptor to signal the developmentof a vigorous proliferative response or to induce a state of unresponsiveness in the absence of proliferation, suggests either that the quality of the signalling can vary, and in turn determine the cell’s response, or that someother intracellular signal is involved. In either case, the accessory cell-derived costimulatory activity would appear to be responsible for determining the outcome. A number of experiments were performed to examine signalling events in T cells following stimulation with antigen in the presence of either normal or chemically modified APC.Measurements of intraccllular calcium ion concentration following stimulation with antigen and fixed APC demonstratedincreased [Ca+Z]i, consistent with intact T cell receptor signalling under these conditions (27). However,the antigen dose response curve demonstrated a requirement for 100-fold more antigen to achieve the same average [Ca+2]~ that one sees upon stimulation with normal APC. In addition, the kinetics of T cell receptor signalling (as measured by calcium influx) were delayed relative to stimulation with normal APC. These findings were compatible with the idea that fixed APCare defective in their ability to present antigen and occupythe TCR;however,the defect does not prevent the induction of an initial increase in [Ca+2]i. It should be noted that recent experiments suggest that a persistence of the increase in [Ca+2]i is a critical factor in the induction of IL-2 secretion (71); have not yet fully explored the possibility that fixed APCare defective in maintaining the increased [Ca+2]i beyond the 20 min period examined in our experiments. Experiments were also performed to determine the effect of antigen and fixed APCon T-cell PI metabolism (27). In this case, IP generation was barely detected, in contrast to the large accumulationsof total IP seen in T-cell populations stimulated with normal APCplus antigen. Similarly, IP generation could not be detected in T cells responding to antigen in the presence of planar lipid membranescontaining Ia-molecule (M. K. Jenkins, unpublished observations). These calcium and IP results initially suggested the interpretation that (a) the developmentof an increase in [Ca+2]i is dependenton the generation of only small amounts of IP3; (b) chemically modified APCand isolated Ia molecules in the presence of antigen are incapable of occupying the TCRin a manner that results in a rapid hydrolysis of PIP2; and (c) failure to activate PKC(secondary to poor DAGproduction) in the face of a significant rise in [Ca+2]iresults in a pattern of T-cell activation that

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is insufficient to induce IL-2 production and proliferation but is sufficient to induce the unresponsivestate. This led to the hypothesis that the balance achieved between PKCactivation and [Ca+2]i increase would determine the response of the T cell to the stimulus. A numberof features madethis hypothesis attractive. First, incubation of the T cells with antigen and fixed APCin the presence of EGTA failed to result in the induction of the unresponsive State, consistent with a requirement for increases in [Ca+Z]i in the response (27). Second, the addition of cyclosporin A during stimulation with antigen and fixed APC prevented the induction of unresponsiveness (36). This result also suggested a role for calcium ions in the response, as cyclosporin A is a potent inhibitor of multiple calcium-dependentearly activation events, possibly by binding and inactivating calmodulin (72-74). Third, the calcium ionophore, ionomycin, was found to be a potent inducer of the unresponsive state, suggesting that a nonmitogenicintracellular calcium rise without PKCactivation was sufficient to induce the state (27). Finally, the hypothesis was entirely consistent with the biochemical two-signal model described above. Such a model would predict that accessory cell-derived costimulatory activity is necessary for maximumTCR-induced PIP2 hydrolysis, DAGproduction, and PKCactivation. In other words, PKC could never be adequately activated in the absence of normal accessory cells. zFurther experiments, however, turned out not to support this model, Careful examination of the kinetics of IP generation suggested that the formation of conjugates between T cells and APCplayed a key role in the observed rate of accumulationof IP. In addition, the delay in calcium rise noted in T-cell populations stimulated with antigen and fixed APCwas also the result of a reduced ability of fixed APCto form conjugates with T cells. Therefore, in subsequent experiments the generation of IP was measuredfollowing pelleting of the cells by centrifugation to assure rapid conjugate formation. These experiments then demonstrated a significant rate of IP generation following stimulation with fixed APCand antigen, although there did remain a clear shift in the antigen dose response curve to higher antigen concentrations when compared to antigen responses in the presence of normal APC.Finally, the addition of a normal allogeneic accessory cell population (incapable of occupying the T cell receptor in the presence of antigen because of the lack of a relevant Ia molecule), during the incubation with antigen and syngeneic fixed APC,resulted in z Mueller, D. L., Jenkins, M. K., Schwartz, R. H. 1989. Anaccessory cell-derived costimulatorysignal acts independently of protein kinase C activation to allow T-cell proliferation and to prevent the induction of unresponsiveness. Submitted.

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no enhancementof IP generation, despite significant augmentation of the proliferative response to the antigen and prevention of the development of the unresponsive state. Attempts to correct the proliferative defect in the response to antigen and fixed APCwith a phorbol ester, on the other hand, met with only partial success (38). A similar need for accessory cells prior to the induction of proliferation in our T-cell clones occurs during activation in response to Con A2 (75). The rate of PIP2 hydrolysis following stimulation with this mitogen in the absence of accessory cells, however, was quite high. The addition of accessory cells to these cultures again had no effect on this rate. Recent experiments by Nisbet-Brownet al (35) also demonstrated an inability accessory cells to influence the effect of anti-CD3monoclonalantibody or antigen and fixed APCon the [Ca+2]i in humanT-cell clones. Thus, occupancy of the TCRalone is sufficient for PIP2 hydrolysis, and the costimulatory signal induced by accessory cells appears to be independent of TCRsignalling. Several important concepts regarding the costimulatory effect of accessory cells have come out of these recent experiments? It appears that a relatively low rate of PIP2 hydrolysis is sufficient to induce a vigorous proliferative response, if an excess of costimulatory activity is present. Experimentally, this is demonstratedby a relative shift of the proliferative dose response curve to lower antigen concentrations, compared to the antigen-induced generation of IP. Under conditions of limited costimulatory activity (either by lowering the density of accessory cells in the culture, or by reducing the costimulatory activity within the accessory cell population with gammaor UVirradiation), proliferation appears more heavily dependenton very high rates of PIP2 hydrolysis. Finally, chemically modified APCappear to possess two functional defects at the level of antigen-presentation. The first is a defect in TCRoccupancyby antigen/Ia molecule complexes. Perhaps fixation-induced damageto the Ia molecule itself or to accessory molecules, responsible for the formation of TceI1/APC conjugates and proper cross-linking of the occupied TCR,resuits in the observed reduction in antigen-induced signalling. The second defect is the loss of the costimulatory activity normally provided by the APCand required by the T cell for proliferation. Only the second defect can be corrcctcd by allogeneic accessory cells. PKC Activation Occurs Independently the Costimulatory Signal

of

Our observation that the rate of PIP2 hydrolysis is not predictive of the ability of a T cell to proliferate in responseto an~, given stimulusis difficult

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to reconcile in terms of the biochemical two-signal modelof activation of the IL-2 gene we have described. One wouldexpect this rate to be directly related to the level of DAGand the degree of PKCactivation, as well as levels of IP3 and [Ca÷2]i The activities of these second-messengersshould then have determined the proliferative response. To address this question directly, we examinedthe level of DAGin a T-cell clone treated with Con A in the absence of accessory cells. 2 Stimulation of these cells resulted in significant increases in DAGby 1 hr of incubation. As predicted by stoichiometry, the Con A-stimulated levels of DAGcorrelated well with the observed increased rates of IP production. Weconcluded from these data that if a costimulatory signal were to play an important role in the proliferative response, and yet act within this two-signal model, its effects would have to be beyond PIP2 hydrolysis, perhaps at the level of direct PKCactivation. To test this, we used the system of endogenous T-cell receptor phosphorylation to assess the activity of PKC. 2 It has been shownthat the CD3-7chain is phosphorylatcd, in response to T-cell stimulation with antigen, at a serine residue indistinguishable from that phosphorylated in response to treatment with a DAGanalog such as phorbol dibutyrate or PMA(76-78). In addition, long-term incubations with phorbol ester under conditions that tend to deplete the cell of PKCactivity result in a failure to phosphorylate the CD3-~chain upon stimulation with antigen (77). By examining the level of CD3-7polypeptide phosphorylation following stimulation of the T cell, we have beenable to determinethe relative activity of PKCunder conditions that either favor the induction of proliferation or the loss of antigen responsiveness. The T-cell clone A.E7 responded to stimulation with antigen in the presence of normal syngeneic APCwith the phosphorylation of CD3-~,demonstrating a rise in PKCactivity during early activation (Figure 4). Fixation of the APCpopulation with 1-ethyl3-(3-dimethylaminopropyl) carbodiimide (ECDI)prior to stimulation nificantly reduced the antigen-induced phosphorylation; this was consistent with the reduction seen at the level of PIP2 hydrolysis. This reduced level of CD3-~phosphorylation was not affected by the addition of normal allogeneic accessory cells. This independence of PKCactivity from the costimulatory signal was confirmed by the observation that Con A stimulation of T cells in the absence of accessory cells resulted in high levels of CD3-~, phosphorylation, without muchproliferation. Thus the low level of PKCactivity induced following stimulation with antigen and fixed AP~2’was sufficient to allow the induction of proliferation, if a source of costimulatory activity was present. These results demonstrated that the costimulatory signal is not being delivered through any mechanismthat increases TCRoccupancy or its observed consequences, and thus it must

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MUELLER, JENKINS & SCHW’ARTZ Group Agalone Ag + Normal APC Ag + ECDI-fixed APC Ag + ECDI-fixed APC + normal AIIo AccessoI~ceils

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GammaPhosphorylation (percent of control) Figure 4 PKC-dependentCD3-7-chain phosphorylation is independent of the costimulatory signalling pathway. [32p]orthophosphate-labeled A.E7 T cells (2.5 × 107 per group) were incubated 45 rain with the pigeon cytochrome c synthetic analog DASP(100 #M) either alone or in the presence of 5 × 107 normal syngeneic B10.A APC, 5 × l07 1-ethyl-3-(3dimethylaminopropyl) carbodiimidc (ECDI)-fixcd B10.A APC, or 5 × 107 ECDI-fixcd B10.A APCplus 2.5 × l07 normal allogeneic BI0 low density T-depleted spleen cells. Cells were lysed in detergent and the CD3-complexwas immunoprecipitated with 145-2C 11 monoclonal antibody (127) and protein A-sepharose. Two-dimensional SDS-polyacrylamide gel electrophoresis analysis of the CD3-7chain was performed and phosphorylations were detected by autoradiograpy of the dried gels. Densitometry of autoradiograms was performed, and results are expressed as the percent increase in gammachain phosphorylation over that seen in the absence of APC(antigen alone).

be acting via an independent intracellular the cell.

second-messenger pathway in

Increases in [Ca+2]i and PKC Activation are Insufficient Second Messengers in the Induction of T-Cell Proliferation Our biochemical data clearly support a role for a costimulatory signalling pathwayin the T cell that is independent of increases in [Ca+Z]~and the activity of PKC.This pathway must be active during antigen stimulation for IL-2 secretion to occur and antigen responsiveness to be maintained. In other words, TCR-mediatedincreases in [Ca÷Z]i and PKCactivation alone are insufficient to induce T-cell proliferation. In an additional series of experiments, we wishedto test this theory, as well as try to reconcile it with the observation that purified T cells will proliferate in response to treatment with the combination of ionomycin and PMA.In particular, we attempted to establish whether there was a true independence of proliferation under these conditions from accessory cell-derived costimulatory signals. Our results argue against such an independence. Incubation of T-cell clones with the combination of ionomycin and PMAat saturating doses consistently resulted in only suboptimal proliferative responses. In

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contrast, incubation with these agents in the presence of an accessory cell population caused maximal proliferation of the T cells (comparable to that achieved with antigen and APC), suggesting a synergistic relationship between costimulatory signals and both increasing [Ca+2]i and PKCactivation 3. Wehave also observed relatively low yet significant levels of proliferation in response to stimulation with either ionomycin or PMA in the presence of syngeneic accessory cells. This finding suggested the possibility that intense stimulation of two of the three biochemical pathways is sufficient to induce a limited degree of IL-2 gene transcription; however, we could not assess the importance of basal PKCactivity or [Ca+2]i, respectively, in these responses. It is important to note, though, that the proliferative response to accessory cells and phorbol ester has been found to be completely resistant to inhibition by cyclosporin A. This is consistent with the hypothesis that the costimulatory signalling pathway operates independently of the effects of [Ca+2]i. More importantly, this effect suggests an identifying characteristic for use in experimentsdesigned to delineate the costimulatory signalling pathway. Workin the human system with the anti-CD28 monoclonal antibody 9.3 has suggested that the CD28molecule is coupled to an alternate activation pathway that, in concert with PKCactivation, is capable of inducing IL-2 synthesis in the absence of a detectable calcium rise and in a cyclosporin A-resistant fashion (79). Thus it is intriguing to suggest that a molecule such as CD28 could be the receptor for the costimulatory activity. Having demonstrated a synergistic relationship between the costimulatory signalling pathway, increased [Ca+2]~and PKCactivation, it was necessary to isolate the T cells completely from any potential costimulatory activity to determine if costimulatory signals were absolutely required for proliferation. Single T cells were found to be incapable of dividing in response to stimulation with only the combination ofionomycin and PMA. This failure to proliferate apparently resulted from a failure to produce IL-2, because cellular division could be induced with the addition of an exogenoussource of IL-2. To reconcile this difference with ionomycinplus PMA-induced proliferation at high T-cell density, T-cell titrations were performed in the presence of ionomycin plus PMA(Figure 5). Log-log plots of thymidine incorporation versus T-cell number established that proliferative responses to ionomycin plus PMAare dependent on cell interactions in the culture. The slope of the plot was approximately two, suggesting that at least two cells must interact to generate the proliferative 3Mueller, D. L., Jenkins, M. K., Chiodetti, L, Schwartz, R. H. 1989. Increased [Ca+2]~ and PKCactivation are insufficient second messengers for T-cell proliferation to ionomycin and PMA.In preparation.

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Olonomycin/PMA alone 13+IL-2 ¯ + Accessory ceils

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T CELL NUMBER Figure 5 Proliferation of T cells in response to ionomycin and PMAis dependent on cellcell interactions. A.E7T cells were titrated into a microtitre plate in 2-fold dilutions from 2~5 cells/well. Ionomycin 0.5 #Mand PMA10 ng/ml were added to all cultures. Incubations

werecarriedout either withoutaddition(opencircles), in the presenceof exogenous IL-2 (2%MLA supernatant)(opensquares),or togetherwith5 × 102cells of the B cell hybridoma LS(following10,000R gamma irradiation and mitomycin C treatment)(closed circles). Cultureswerepulsedat 48hr andthymidineincorporationwasmeasured at 60hr. Results are expressedas the meancpmof six replicatesandplottedin log-logfashion.Theoretical curves with slope of one or two are indicated by the dashed lines.

response. This requirement for cell interactions could be satisfied by the addition of an accessory cell population (at constant density) that contained a good source of costimulatory activity. It also could be overcome by the addition of exogeneousIL-2. Finally, in a separate series of experiments, we found we could inhibit ionomycin plus PMA-induced proliferation with the addition of a fixed splenic B-cell population. Thesecells appeared to be capable of competing with the delivery of costimulatory signals by interacting with T cells in a nonstimulatory fashion--in essence behaving like cold-target inhibitors in a CTLassay. These results suggest that the cell interactions required for proliferation to ionomycinplus PMA involve the delivery of the costimulatory signal to the T-cell population. Similar results have been obtained with T cells stimulated with an antiCD3monoclonal antibody coated on a platc. The identity of the costimulatory activity donor in this response appears to be the activated T cell itself, althoughwe can not rule out the possibility of an extremely potent contaminating accessory cell. Resting splenic T cells are not a source of costimulatory activity; however, we have found ++ that the Type II CD4 T-cell clone D10.G4will allow our Type I CD4 T cell clone A.E7 to proliferate in response to concanavalin A or antiCD3antibody, in the absence of any other accessory cell population. Therefore, there is reason to think that an intensely activated T cell might

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be capable of providing a nearby cell with the costimulatory signal it requires to proliferate. This interpretation could also reconcile the observation that someIa ÷ humanT cells induce T-cell unresponsiveness in the presence of antigen, while others induce proliferation. Given our observations that T-cell proliferative responses to both antiCD3 antibody alone and to the combination of ionomycin and PMA require the generation of a costimulatory signal, it becomesnecessary to reassess the biochemical two-signal modelfor T-cell activation of the IL2 gene. In particular, one must determine if these data support either of the two hypotheses proposed by Weiss and his colleagues to account for the role of the accessory cell (mimickedby phorbol ester) in the induction of IL-2 gene transcription. The first hypothesis stated that the ability of phorbol ester to substitute for an accessory cell population in the response to mitogen or anti-CD3 antibody results from the fact that the accessory cells are required for sufficient PKCactivation. Our data demonstrate that this is not the case. PKCactivation is detectable upon stimulation with fixed APCand antigen or Con A, in the absence of normal accessory cells, and the addition of accessory cell-derived costimulatory activity has no effect on the degree of PKCactivation. There is no evidence that IL-1 acts in our system to increase the level of DAGand to enhance the activity of PKC,as we should have noted such effects at the level of CD3-~chain phosphorylation. The second hypothesis suggested that the effects ofphorbol esters were not limited to PKCactivation, and that one of these other activities mimics the costimulatory signal. Wehave found, however, that under conditions of limited costimulatory activity (purified T cells at low density), PMAcannot mimic the costimulatory signal. Thus, it appears that PMAacts indirectly (presumably through intense PKCactivation in the presence of [Ca+2]i) to induce the costimulatory activity within the purified T cell population and allow the generation of the costimulatory signal as a consequenceof T-T interactions.

Activation of the IL-2 Gene Requires Three Biochemical Signals Wethink that only a three-signal biochemical model is compatible with all of the biological observations (Figure 6). Occupancyof the TCRwith an antigen/Ia complexresults in the hydrolysis of PIP2 leading to a subsequent rise in [Ca+2]~ and activation of PKC. In the absence of the biological secondsignal (costimulatory activity), these intracellular second messengers are capable of inducing a state of antigen unresponsiveness and are insufficient to induce significant IL-2 gene transcription or proliferation. Our data wouldsuggest that increased [Ca+2]i is both necessary and sufficient for the induction of antigen nonresponsiveness. Relatively

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[ ~ \ UNRESPONSIVENESS

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~ COSTIMULATORY t f,~-~DAG lIP SIGNAL ¯ /3 (-~PXP

ANTIGEN UNRESPONSIVENESS

IL-2 GENE TRANSCRIPTION

NOIL-2 GENE TRANSCRIPTION COSTIMULATORY ACTIVITYRECEPTOR

T CELLANTIGE RECEPTOR

÷ T cell on the left is stimulated with antigen/Ia molecule complexes in FixTure 6 The CD4 the absence of the costimulatory activity, The T cell on the right is stimulated in the presence of the costimulatory activity.

low PKCactivity may be permissive in the development of this state, whereas intense PKCactivity acts (with increased [Ca+2]i) to increase the costimulatory activity within the T-cell population and antagonize the development of unresponsiveness. In the presence of the biologic second signal, a separate costimulatory pathwayis activated in the T cell which acts to induce the transcription of the IL-2 gene in concert with increases in [Ca+2]~ and PKCactivation. All three signalling pathways must be active for maximumlymphokine secretion and proliferation. In addition, the costimulatory signal is capable of abrogating the induction of unresponsiveness. The biochemical events that occur in the induction of the nonresponsive state have not yet been elucidated. Cycloheximide addition blocks the induction of unresponsiveness, suggesting that new protein synthesis may be required (25). The simplest model would be to postulate that the calmodulin-dependent pathway activated by increases in [Ca+2]i induces the transcription and synthesis of a series of proteins capable of either directly or indirectly inhibiting the transcription of the IL-2 gene (although the cycloheximide experiment does not differentiate this from the possible effects on mRNAstability or the translation of DNAregulatory proteins). The costimulatory signalling pathway would then block the

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synthesis or activities of these inhibitory proteins as well as facilitate transcription of the IL-2 gene in concert with the effects of PKCactivation and increased [Ca+2]i. ÷ The synergistic interactions observed in the stimulation of Type I CD4 T-cell clones by PMA,ionomycin, and accessory cells support the concept of a three signal model. The fact that these three stimuli can mimicfull Tcell activation, however,does not prove that the second messengercascades they induce must occur following antigen-induced activation via receptor occupancy. More complex models, involving other receptor-mediated signals, can be envisioned. For example, recent studies of T-cell hybridomas expressing receptor mutations have demonstrated that anti-Thy 1 antibody-mediated induction of IL-2 gene transcription can occur in the absence of PIP2 hydrolysis (80, 81). In this case, the natural biochemical signals for inducing IL-2 gene transcription may not be PKCactivation and/or increases in [Ca+2]i. Our incomplete understanding of second messenger generation following TCRoccupancy, however, does not reduce the importance of the costimulatory signalling pathway in the regulation of the IL-2 gene. TCRoccupancy may normally lead to both the rise in [Ca+2]i necessary for the induction of the unresponsive state, and the stimulation of another unidentified signalling pathway capable of acting on the IL-2 gene. The costimulatory signal would still be required to block the effects of calcium ions, in order to prevent the induction of unresponsiveness, as well as to synergize in the induction of IL-2 gene transcription. Most of the data supporting our three signal model come from experi+ T cell clones. These cells do not represent ments involving Type I CD4 small resting T lymphocytes. They constitutively express low levels of IL2 receptors and often contain chromosomal rearrangements. Howapplicable, then, is the model to small resting T lymphocytes? Preliminary ÷ T cells suggest that some of the major studies on freshly isolated CD4 features of the modelhold true (M. K. Jenkins, D. L. Mueller, unpublished observations). For example, the proliferative response of antigen-primed draining lymph node T cells can be completely eliminated by an 18 hr exposure to ionomycin. Furthermore, ionomycin, PMA,and accessory cells synergize in the stimulation of a proliferative response from mesenteric lymphnode T cells. In particular, the expression of the IL-2 receptor also appears to be optimal only in the presence of all three signals. Whether this is a direct effect on the IL-2 receptor gene or an indirect effect via activation of the IL-2 gene, to give IL-2-induced up-regulation of its receptor, remains to be determined. These experiments suggest that the three signal modelmaybe applicable to the activation of IL-2-producing, small resting T cells.

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ROLE OF THE COSTIMULATORY SIGNAL IN THE REGULATION OF T-CELL RESPONSES IN VIVO Experimentally Induced T-Cell Tolerance to Foreign Antigens A number of experimental observations in vivo support the idea that responsiveness to a foreign antigen by mature T cells can be blocked. As early as 1946, Chase (82) demonstrated that contact sensitivity dinitrochlorobenzene in guinea pigs could be inhibited by the prior oral administration of the chemical. Battisto & Bloom(83) later showedthat the development of a DTHresponse to bovine gammaglobulin (BGG) guinea pigs was significantly reduced by pretreatment of the animals with an intravenous infusion of BGG-coupledspleen cells. This experimentally induced tolerance was both antigen-specific and long-lasting. Althoughit nowappears certain that this type of unresponsiveness to foreign antigens + T-cell population, the actual mechanism develops at the level of the CD4 of tolerance can not be ascertained from these data (23). Somehave suggested a role for antigen-specific suppressor cells in the maintenance of this form of experimentally induced T-cell tolerance. A discussion of suppression is beyondthe scope of this review. It should be noted, however, that Miller et al (84) were able to demonstrate that suppression could not completely account for the tolerance induced with hapten-modified spleen cells. They showedthat elicitation of 2,4-dinitro-1-fluorobenzene contact sensitivity was blocked by prior intravenous infusion of dinitrophenylated spleen cells. The induction of tolerance was rapid, antigen-specific, and MHC-restricted,and initially not associated with transferable suppression, suggesting a state of clonal anergy. Furthermore, treatment of the animals with cyclophosphamide prior to the infusion of hapten-modified spleen cells completely abrogated the induction of suppressor cells; yet, tolerance to the hapten was still induced. Based on these observations, and as an extension of previous studies examiningthe specificity of T-cell proliferative responses, our laboratory examined the influence of intravenous administration of pigeon cytochromec, covalently cross-linked with ECDIto the surface of syngeneic antigen-presenting cells (APC), on the priming of a T-cell proliferative response to the antigen (26). Four days following intravenous challenge with the cross-linked antigen/APC, subcutaneous immunization with antigen in complete Freund’s adjuvant was found to be ineffective at priming the T-cell population, i.e. the in vitro secondary T-cell proliferative response to peptide fragments of the antigen was greatly reduced. Immunity to another antigen (purified protein derivative of tuberculin) was not affected. A detailed analysis of the requirements for inducing the non-

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responsive state revealed that the sameantigen and Ia moleculespecificities were needed as those required for T-cell priming. These results suggest that TCRsignalling occurs during both T-cell priming and the induction of specific T-cell unresponsiveness to antigen in vivo. In the case of antigen covalently cross-linked to chemically fixed APC,we assume that the intravenous infusion of antigen/Ia complexes(at the surface of the APC)results in the occupancy of the TCRon antigenspecific lymphocytesin the absence of a source of costimulatory activity. Consistent with the biological two-signal model, antigen responsive clones would not be expanded by this type of stimulation; rather, these T cells would be functionally inactivated, resulting in a failure to detect them upon priming in the standard fashion. This simple application of our in vitro model of T-cell inactivation to the in vivo findings, however, is not sufficient to explain a numberof puzzling observations. First, fixed APCrequire preprocessed antigen in vitro in order to form antigen/Ia molecule complexes, yet coupling the whole molecule to spleen cells was adequate for the in vivo effects. How does the antigen get processed? Are some T-cell determinants generated by denaturation during coupling or by the action of serum proteases at the surface of the fixed cell? Anotherparadox is that in vitro, fixed APC can be prevented from inducing T-cell nonresponsiveness by the addition of normal allogeneic accessory cells. Whydon’t the normal accessory cells in vivo do the same thing? Is this a quantitative effect and does it depend heavily on where the fixed cells home? These and other questions are currently being investigated. The experimental tolerance studies described above do appear to suggest a modelin which the route of entry of the antigen and the physical state of the APCinfluence the outcome of T-cell activation in vivo (clonal expansion versus functional clonal inactivation). Possibly the two factors-location of the antigen and attributes of the available APC--areassociated, i.e. the site at which antigen is encountered under ordinary conditions could determine the type and number of accessory cells available for antigen presentation. The subcutaneous appearance of antigen, with subsequent transport via veiled cells through tissue lymphatics to the lymph node, might assure a rich supply of potent bone marrow-derived accessory cells (e.g. dendritic cells, macrophages)capable of providing the costimulatory activity required for the induction of a proliferative response within the antigen-specific T-cell population (85, 86). In contrast, the oral or intravenous introduction of antigen might result in the presentation of antigenic peptides by APCthat possess little costimulatory activity (87). The examination of Ia molecule expression in various tissues has deter-

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mined that manynonhematopoietic cells can express Ia molecules; furthermore, in vivo treatment with IFN-7 induces the expression of Ia molecules on such cells (88). ÷ fi broblasts, ke ratinocytes, an d th yroid fo llicular epithelial cell lines all fail to induce normal T-cell proliferative responses to peptide antigens in the absence ofa costimulant such as PMA, normal bone marrow-derived accessory cells, or the anti-CD28 antibody 9.3 in vitro (34, 89-91). In the case ofla ÷ keratinocytes, Gaspari et al (92) ÷ T-cell clones in the presence of find that incubation with Type I CD4 peptide antigen results in the induction of proliferative unresponsiveness in the T cell. Recently, Markmannet al (93) have shownthat pancreatic islets taken from transgenic mice that express the I-E MHCmolecule transgene exclusively on their beta cells are also capable of inducing proliferativc unresponsivenessin T-cell lines in the presence of antigen. These results are consistent with the idea that some MHC-bearingcells may occupy the TCRin the absence of a costimulatory signal under physiologic conditions and so may induce clonal anergy. In vivo experimentalevidence in support of the critical role for accessory cells in the regulation of T-cell responsiveness can be found in studies of tolerance induction in adults by the parenteral administration of monomeric preparations of mammalianserum proteins. Dresser (94) showed that immuneparalysis to BGGcould occur after intraperitoneal injection ofa deaggregated preparation of the antigen. Thorbeckeand her colleagues (95) later confirmed the result by demonstrating that bovine serum albumin (BSA) that had been "filtered" by passage through a rabbit (and subsequently recovered in the serum) would induce tolerance to BSAin naive animal following intravenous administration. They suggested that "filtered" BSAis depleted of the "phagocytizable" components necessary for the recruitment of macrophagesinto the response, an event required for sensitization to the antigen. Chiller et al (96, 97) demonstratedthat similar state of tolerance to humangammaglobulin (HGG),following the intravenous infusion of the deaggregated-form of the antigen, resulted from the development of unresponsiveness in both the B-cell and T-cell compartments. Weigle et al (98) have since found that the induction T-cell unresponsiveness by treatment with deaggregated HGGcan be prevented by the simultaneous administration of IL-I. These results are consistent with the notion that the parenteral introduction of antigen in a form that neither induces its uptake and presentation by potent accessory cells of the reticuloendothelial system, nor induces the costimulatory activity within the APCpopulation it encounters, results in presentation of antigen to T cells in the absence of a costimulatory signal. The ability of IL-1 to modulate the T-cell response to antigen in this form appears to confirm the relative lack of costimulatory activity available to the T cell.

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IL-1 treatment in vivo could potentially augmentthe delivery of the costimulatory signal by increasing the expression of surface adherence molecules and upregulating T-cell/accessory cell interactions (99) and/or enhancing accessory cell potency (21). Taken together, these data suggest that the APCpopulation encountered at the site of antigen introduction mayplay an important role in determining the response of the antigenreactive T cell by up- or down-regulating antigen responsiveness.

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T-Cell Unresponsiveness Self-Tolerance

in the Maintenance

of

The models of experimentally induced tolerance to specific antigens in adult animals discussed above suggest that functional T-cell inactivation might play an important role in the generation of this in vivo state. For tolerance to self-antigens, however, recent experiments using the Vfll7aspecific anti-TCR antibody K J23 have established that deletion of autoreactive clones (anti-I-E k, in this particular case) occurs during maturation of T cells in the thymus(100). Similar evidence for clonal deletion has been found in the maintenance of self-tolerance to Mls determinants (101,102) and the male (H-Y) antigen (103). Thus, the costimulatory signal would be importantin natural tolerance only if it played a role in clonal deletion or in the functional inactivation of autoreactive clones that escaped deletion. Within the frameworkof a biological two-signal model of T-cell activation, one could propose that clonal deletion is the direct result of thymocyte antigen receptor signalling in the absence of activation of the costimulatory signalling pathway. This situation could result either from an inability of the immatureT cells to respond to a costimulatory signal at an early stage in their development, or because of an inability of the thymic APCto deliver a costimulatory signal. Recently, Matzinger & Guerder (personal communication) have examined the induction of T-cell tolerance in explanted thymic lobes and found that the addition of normal allogeneic splenic dendritic cells, a potent APCin the generation of in vitro T-cell proliferative responses, resulted in tolerization of the allo-specific CTLprecursors. T cells that matured in organ cultures responded to thirdparty allogeneic stimulator cells but failed to develop a CTLresponse to stimulator cells syngeneic with the donor dendritic cell population. If the biological two-signal modelis applicable, this result suggests that immature thymocytes are incapable of responding to the costimulatory activity, or that the thymic microenvironmentprevents the delivery of such a signal. + (double positive) thymocytes (presumably the ~° Since CD4÷CD8 subset) are capable of responding to antibody-induced TCRcross-linking with a small rise in [Ca+2]i (105, 105a), antigen receptor occupancyin the absence of a costimulatory signal might be sufficient to kill the cell, con-

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sistent with the role of calcium ions in the functional inactivation seen with our mature T cells, in vitro. Recent work in our laboratory has demonstratedthat clonal deletion in the thymusis sensitive to cyclosporin A treatment in vivo (106). Strains of mice that bear V~17a T cell re ceptor gene and express an I-E encoded Ia molecule normally fail to generate significant numbersof matureVfl 17a ÷ T cells becausethese T cells are often specific for determinants associated with I-E molecules and are deleted. Treatment with cyclosporin A during syngeneic bone marrow recon÷) mice resulted in a stitution of lethally irradiated C57Br (V~17a÷,E~ failure to delete entirely the newly arising, Vfll 7a-expressing maturethymocyte population. Similar observations have been made by Gao et al (107) for Vfll 1-expressing cells. Since manyof these thymocytes should bear autoreactive T-cell receptors, these results mayexplain the previous observation that cessation of cyclosporin A therapy following irradiation and bone marrowreconstitution of rats (108, 109), mice (110), and humans (111) results in the developmentof graft-v-host disease. Autoreactive thymocytes developing in the presence of cyclosporin A would fail to be deleted and would then move to the periphery and subsequently mediate the disease, once they were released from the cyclosporin A signalling block. In terms of our discussion, such data suggest that signalling via increases in [Ca+2]i within the thymocyte population maybe necessary for deletion of autoreactive T-cell clones. In support of this notion, Wyllie et al (Ilia) have shown that the calcium ionophore, A23187, can induce DNAdegradation and cell death in rat thymocytes, in a manner that is blocked by cycloheximide. Given the concordance of these data with the in vitro modelofT-cell unresponsiveness, it is possible that this mechanism of thymic tolerance induction may rely on T-cell receptor occupancy in the absence of a second signal. Experiments performed with Ft ~ parent bone marrow chimeras have demonstrated the elimination of thymocytes reactive with any of the MHC antigens expressed on the surface of donor-derived cells, suggesting an important role for the bone marrow~terived thymic dendritic APCin the normal induction of self-tolerance within the T-cell population (112114). In contrast, auto-MHCantigens expressed only on non-bone marrow-derived tissues appear to elicit a different and, at times, incomplete form of tolerance. For example, adult-thymectomized SJL mice (Vf117a+,E~-) that have been lethally irradiated and reconstituted with syngeneic bone marrowand then engrafted with a deoxyguanosine-treated (BALB/c× SLJ)F1 thymus, fail to delete the Vfll7a + population of newly developedT cells (114). In this case, expression of an I-Ed molecule exclusively on the thymic stromal elements was not sufficient to delete physically the potentially autoreactive Vfll 7a+ T cell population; nevertheless, these

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animals did tolerate their thymic graft. Similar evidence for peripheral tolerance to tissues that bear MHCmolecules not expressed on bone marrow-derived cells has been observed in other systems: in (a) chicken chimeras resulting from the transplantation of embryonic quail thymic stromal elements into a chick embryo (115), in (b) Xenopus chimeras created by joining the anterior one-third of a 24 hr old embryo to the posterior two-thirds of an allogeneic embryo(116), in (c) parent ~ F~ marrowcimeras (117) and (d) in transgenic mice that express a particular Ia molecule exclusively on thymic epithelium (114, 118) or peripheral tissues (pancreatic beta cells and kidney tubular epithelium) (119). The basis for this peripheral tolerance in the absence of clonal deletion could be clonal inactivation. It is possible that the T cells, specific for these MHCmolecules expressed only on non-bone marrow~tcrived elements, have had their TCRoccupied by the autoantigen, and a failure of these MHC-bearingcells to provide the costimulatory activity resulted in the functional inactivation of the clones, instead of activation and expansion. This modelwould predict a reduced capacity of T cells from these animals to display autoreactivity to the MHC antigens in mixed lymphocytereaction (MLR)or cell-mediated lympholysis (CML)assays. In the case of the transgenic mice described above, an MLRfailed to demonstrate any significant reactivity to the Ia molecule encoded by the transgene, consistent with a complete functional inactivation of autoreactive clones (in the demonstrated absence of clonal deletion of at least the V/~17a+ population) (114, 118, 119; personal communication, L. Burkly). The parent~ Flbone marrow chimeras demonstrated significantly reduced MLR reactivity relative to third party stimulators and an absent CMLresponse (117). In several of the other models, however, a full response in MLRor CMLwas reported (116, 120-122). Thus, although functional inactivation maybe playing somerole in the peripheral tolerance, it is clear that other regulatory mechanisms are also likely to be involved. Finally, the "veto cell" phenomenon(124, 125), observed in vitro and in vivo, mayalso be mediated by a functional inactivation mechanism.In experiments performed by Rammensee& Bevan (126), infusion of class incompatible spleen cells into mice resulted in the specific loss by the recipient of class I-alloreactivity to the donor strain. The mice were found to be chimeric, as donor-derived CTLcould be demonstrated in an MLC response to third-party allostimulators (but not to stimulators syngeneic with the host). Theseresults suggest that T cells from each strain, specific for the other haplotype, were functionally inactivated. It is possible that this occurred as a result of allospecific T cells recognizing the class-I MHC antigen on the T cell of the other strain, as originally proposed by Miller

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(124). Since we have found T cells to have no detectable costimulatory activity in the resting state, recognition of antigen on the surface of such a cell would result in TCRoccupancy of the responding T cell in the absence of a second signal, the prerequisite for functional inactivation. Thus, the experiments described above suggest that in situations where the physical deletion of T cells specific for self-antigens has not occurred, tolerance to these autoantigens can still be maintained. While it is likely that clonal deletion of autoreactive T cells during developmentrepresents the most important means for achieving self-tolerance, functional inactivation could provide one type of ’second level’ protection against the development of a pathological autoimmuneresponse by clones that escape deletion. Potential

Clinical

Relevance

T-cell unresponsiveness may also be important in the maintenance of peripheral tolerance in clinical situations. Experimentsperformed with the in vivo administration of anti-CD3 monoclonal antibody by Bluestone and his coworkers demonstrated that mice given intravenous anti-CD3 antibody develop rapid suppression of allogeneic skin graft rejection (127, 128). The long-lasting immunosuppressionobserved in this model was not simply due to a loss of alloreactive T cells or CD3modulation, but instead appeared to reflect some functional defect in the T-cell population. It is possible that this element of immunosuppression following anti-CD3 therapy represents the developmentof an antigen-unresponsive state in the ¯ T-cell population, following TCRsignalling by the monoclonalantibody in the absence of a costimulatory signal. Support for this notion comes from findings in vitro on the induction ofantigen-nonresponsiveness in T-cell clones treated with anti-TCR monoclonal antibody in the absence of accessory cells (129). In recent experi+ T-cell clones stimulated ments performed in our laboratory 4, Type I CD4 with anti-CD3 monoclonal antibody (in solid-phase, coated to a plate) proliferated suboptimally and entered a state of reduced antigen-responsiveness following the stimulation. The series of inductive events at both the biochemical and lymphokineproduction levels appears similar to what has been observed with antigen and ECDI-treated APCor Ia molecules in planar membranes.Thus, coating of T cells in vivo with anti-CD3 could lead to the cells being sequestered at sites where Fc receptor-mediated cross-linking wouldtrigger their functional inactivation. 4 Jenkins, M. K., Chen, C., Jung, G., Mueller, D. L., Schwartz, R. H. 1989. Inhibition of antigen-specific proliferation of Type I murine T cell clones following stimulation with immobilized anti-CD3 monoclonal antibody. In preparation.

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The effects of donor blood transfusions on allograft survival might also work through a functional inactivation mechanism(130, 131). Infusions of hematopoietic cells, depleted of costimulatory activity either by treatment of the population with an inhibitor of metabolic activity (e.g. UV light, chemical fixatives), or by removalof potent accessory cells from the population, have been shown to suppress MLRresponses and to prolong allogeneic graft survival (132-135). Donor resting T cells and red blood cells mayinactivate class I-specific alloreactive T cells in a "veto’-like manner;resting B cells, recently shownto be poor stimulators in a primary response in vivo (136), might inactivate class II-specific alloreactive cells. Finally, studies by Lafferty and others (137-140) have demonstrated that depletion of donor APCfrom allogeneic tissue prolongs the survival of the graft. This can be interpreted in terms of the biological two-signal model as a failure of this transplanted tissue to activate an immune response. It is conceivable, however,that the success of this manipulation also depends on the process of inactivation. CTLprecursors entering the nonspecific inflammatory sites (induced by the surgical procedure) may encounter allogeneic class-1 molecules on parenchymal cells incapable of delivering costimulatory signals, and may thus become functionally inactivated. Similarly, if IFN-yis released at these sites from sources such as natural, killer cells, it is possible that endothelial and epithelial cells would begin to express class-II molecules and thus provide a means of + T cells. If these mechanismsdo play a role, then eliminactivating CD4 ination of"passenger leukocytes" with anti-class II antibodies (139) might prove to be a less effective meansof reducing graft rejection than administering antibodies directed against moleculesspecifically expressed on the surface of potent APC, or depleting these APCin some other specific fashion (138).

CONCLUSION Proliferation of T lymphocytes relies on the simultaneous occupancy of the T-cell antigen receptor and delivery of an accessory cell~terived costimulatory signal. T-cell receptor occupancy alone is read as a signal to down-regulate further antigen responsiveness and possibly to maintain self-tolerance. The presence of bone marrow-derivedaccessory cells capable of delivering a costimulatory activity during occupancyof the antigen receptor results in the proliferation of the T cell and development of effector cell function. At the biochemical level, T-cell receptor occupancy by peptide antigen/Ia complexesis sufficient to induce the hydrolysis of PIP2. Increases in [Ca+2]i, as well as activation of PKC(as measured by phosphorylation

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of the CD3-7polypeptide) are directly correlated with the induction of PIPE hydrolysis and occur independently of the presence of accessory cellinduced costimulatory signals. Thus, there is no evidence for a direct involvement of the costimulatory signalling pathway in the generation of increased [Ca+2]~ and PKCactivation. Increased [Ca+2]i and PKCactivation are not sufficient biochemical signals to induce T-cell proliferation. Proliferation requires the generation of a costimulatory signal acting independently on the IL-2 gene. Proliferation of T cells in the absence of accessory cells in response to stimulation with anti-CD3 monoclonal antibody or the combination of ionomycin and PMAresults from the induction of costimulatory activity within the T-cell population and delivery of the activity during T-T interactions. Thus, T-cell IL-2 gene activation and subsequent proliferation are dependent on at least three biochemical signals. Increased [Ca+E]~(following T cell receptor occupancy)is sufficient + T cell clones. These functionally inactivate Type I (but not Type II) CD4 clones develop a state of proliferative unresponsivenessthat is long-lived and stems from an inability to produce IL-2 upon antigen restimulation. The cells maintain their viability in vitro and remain capable of proliferating in response to exogenousIL-2. In contrast, the presence of an accessory cell-induced costimulatory signal in parallel with antigen receptor occupancy prevents the calcium-dependent induction of unresponsiveness and synergizes with the rise in [Ca+2]~ and increase in PKCactivity to induce the transcription of the IL-2 gene. These observations suggest that the original biological two-signal model proposed by Bretscher &Cohnfor B-cell activation is applicable to T-cell activation. Signal one is occupancyof the antigen-specific receptor and alone is capable of inducing the functional inactivation of mature T cells and possibly clonal deletion of immature thymocytes. Signal two is the costimulatory signal; it functions as a gate on the first signal to determine its outcome.The presence of signal two leads to a full proliferative response by the T-cell following occupancyof its antigen receptor; the absence of signal two leads to a state of proliferative nonresponsiveness.

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using the concept of clonal selection. Aust. J. Sci. 20:67-69 4. Lederberg, J. 1959. Genes and antibodies. Science 129:1649-53 5. Bretscher, P., Cohn, M. 1970. A theory of self-nonself discrimination. Science 169:1042~49 6. Mitchison, N. A. 1971. The cartier

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COSTIMULATORY SIGNAL & T-CELL ACTIVATION effect in the secondary response to hapten-protein conjugates. Eur. J. lmmunol. l: 18-27 7. Coffman, R. L., Seymour, B. W. P., Lehman, D. A., Hiraki, D. D., Christiansen, J. A., Shrader, B., Cherwinski, H. M., Savelkoul, H. F.J., Finkelman, F. D., Bond, M. W., Mosmann, T. R. 1988. The role of helper T cell products in mouseB cell differentiation and isotype regulation. Immunol. Rev. 102:5-28 8. Davis, M. M., Bjorkman, P. J. 1988. Tcell receptor genes and T-cell recognition. Nature 334:395402 9. Nossal, G. J. V. 1983. Cellular mechanisms of immunologic tolerance. Annu. Rev. Immunol. 1:33~52 10. Lafferty, K. J., Woolnough,J. 1977. The origin and mechanismof the allograft reaction, lmmunol. Rev. 35: 23162 11. Lafferty, K. J., Prowse, S. J., Simeonovic, C. J. 1983. Immunobiology of tissue transplantation: A return to the passenger leukocyte concept. Annu. Rev. Immunol. 1:143-73 12. Bach, F. H., Grillot-Courvalin, C., Kuperman, O. J., Sollinger, H. W., Hayes, C., Sondel, P. M., Alter, B. J., Bach, M. L. 1977. Antigenic requirements for triggering ofcytotoxic T lymphocytes. Immunol. Rev. 35:76-96 13. Raulet, D. H., Bevan, M. J. 1982. A differentiation factor required for the expression of cytotoxic T-cell function. Nature 296:754-57 14. Wagner, H., Hardt, C., Rouse, B. T., Rollinghoff, M., Scheurich, P., Pfizenmaier, K. 1982. Dissection of the proliferative and differentiative signals controlling murine cytotoxic T lymphocyte responses. J. Exp. Med. 155: 1876-81 15. Schmitt-Verhulst, A.-M., Guimezanes, A., Boyer, C., Poenie, M., Tsien, R., Bufcrnc, M., Hua, C., Leserman, L. 1987. Pleiotropic loss of activation pathways in a T-cell receptor or-chain deletion variant of a cytolytic T-cefl clone. Nature 325:628 31 16. Rosenstreich, D. L., Mizel, S. B. 1978. The participation of macrophages and macrophagecell lines in the activation of T lymphocytes by mitogens, lmmunol. Rev. 40:102-35 17. Mizel, S. B. 1982. Interleukin 1 and T cell activation. Imrnunol. Rev. 63: 5172 18. Durum, S. K., Schmidt, J. A., Oppenheim,J. J. 1985. Interleukin 1: An immunological perspective. Annu. Rev. Immunol. 3:263-87

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19. Greenbaum, L. A., Horowitz, J. B., Woods,A., Pasqualini, T., Reich, E., Bottomly, K. 1988. Autocrine growth ÷ T cells. Differential effects of of CD4 IL-1 on helper and inflammatory T cells. J. Immunol. 140:1555-60 E. A., Hamberg, S., 20. Kurt-Jones, Ohara, J., Paul, W. E., Abbas, A. K. 1987. Heterogeneity of helper/inducer T lymphocytes. I. Lymphokine production and lymphokine responsiveness. J. Exp. Med. 166:1774-87 21. Koide, S. L., Inaba, K., Steinman, R. M. 1987. Interleukin 1 enhances Tdependent immuneresponses by amplifying the function of dendritic cells. J. Exp. Med. 165:515-30 22. Cleveland, R. P., Claman, H. N. 1980. T cell signals: Tolerance to DNFBis converted to sensitization by a separate nonspecific second signal. J. Immunol. 124:474-80 23. Claman, H. N., Miller, S. D., Conlon, P. J., Moorhead,J. W. 1980. Control of experimental contact sensitivity. Adv. Immunol. 30:121 57 24. Jenkins, M. K., Pardoll, D. M., Mizuguchi, J., Quill, H., Schwartz, R. H. 1987. T-cell unresponsiveness in vivo and in vitro: Fine specificity of induction and molecular characterization of the unresponsive state. Immunol. Rev. 95:113-35 25. Quill, H., Schwartz, R. H. 1987. Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes: Specific induction of a long-lived state of proliferative nonresponsiveness. J. Immunol. 138:3704-12 26. Jenkins, M. K., Schwartz, R. H. 1987. Antigen presentation by chemically modified splenocytes induces antigenspecific T cell unresponsivenessin vitro and in vivo. J. Exp. Med. 165:302-19 27. Jenkins, M. K., Pardoll, D. M., Mizuguchi, J., Chused, T. M., Schwartz, R. H. 1987. Molecular events in the induction of a nonresponsivestate in interleukin 2-producing helper T-lymphocyte clones. Proe. Natl. Acad. Sei. USA 84:5409 14 28. Lamb, J. R., Skidmore, B. J., Green, N., Chiller, J. M., Feldmann,M. 1983. Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J. Exp. Med. 157: 143447 29. Feldmann, M., Zanders, E. D., Lamb, J. R. 1985. Tolerance in T-cell clones. Immunol. Today 6:58-61 30. Zanders, E. D., Lamb, J. R., Feld-

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COSTIMULATORY SIGNAL & T-CELL ACTIVATION inates T cells specific for Mls-modified products of the major histocompatibility complex. Nature 332:35~.5 102. MacDonald, H. R., Schneider, R., Lees, R. K., Howe,R. C., Acha-Orbea, H., Festenstein, H., Zinkernagel, R. M., Hengartner, H. 1988. T-cell receptor Va use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature 332:40~5 103. Kisielow, P., Bliithmann, H., Staerz, U. D., Steinmetz, M., von Boehmer, H. 1988. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333:742-46 104. Deleted in proof. 105. Havran, W. L., Poenie, M., Kimura, J., Tsien, R., Weiss, A., Allison, J. P. 1987. Expression and function of the CD3÷ antigen receptor on murine CD4÷8 thymocytes. Nature 330: 170-73. 105a. Finkel, T. H., McDuffie, M., Kappler, J. W., Marrack, P., Cambier, J. C. 1987. Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking. Nature 330: 179 81. 106. Jenkins, M. K., Schwartz, R. H., Pardoll, D. M. 1988. Effects of cyclosporine A on T cell development and clonal deletion. Science 241:1655-58 107. Gao, E.-K., Lo, D., Cheney, R., Kanagawa, O., Sprent, J. 1988. Abnormal differentiation of thymocytes in mice treated with cyclosporine A. Nature 336:176-79 108. Glazier, A., Tutschka, P. J., Farmer, E. R., Santos, G. W. 1983. Graft-versushost disease in cyclosporin A-treated rats after syngeneic and autologous bone marrow reconstitution. J. Exp. Med. 158:1-8 109. Sorokin, R., Kimura, H., Schroder, K., Wilson, D. B. 1986. Cyclosporineinduced autoimmunity. Conditions for expressing disease, requirement for intact thymus, and potency estimates of autoimmune lymphocytes in drugtreated rats. J. Exp. Med. 164: 161525 110. Cheney, R. T., Sprent, J. 1985. Capacity of cyclosporine to induce auto-graft-versus-host disease and impair intrathymic T cell differentiation. Transplant. Proc. 17:528-30 111. Hood, A. F., Vogelsang, G. B., Black, L. P., Farmer, E. R., Santos, G. W. 1987. Acute graft-vs-host disease. Development following autologous and syngeneic bone marrow transplantation. Arch. Dermatol. 123: 74550

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11 la. Wyllie, A. H., Morris, R. G., Smith, A. L., Dunlop, D. 1984. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142:67-77 112. Blackman, M. A., Kappler, J. W., Marrack, P. 1988. T-cell specificity and repertoire. Immunol. Rev. 101:5-19 113. Sprent, J., Lo, D., Gao, E.-K., Ron, Y. 1988. T cell selection in the thymus. Immunol. Rev. 101:173-90 114. Marrack, P., Lo, D., Brinster, R., Palmiter, R., Burkly, L., Flavell, R. H., Kappler, J. 1988. The effect of thymus environment on T cell development and tolerance. Cell 53:627 34 115. Ohki, H., Martin, C., Corbel, C., Coltey, M., Le Douarin, N. M. 1987. Tolerance induced by thymic epithelial grafts in birds. Science 237:1032-35 116. Flajnik, M. F., DuPasquier, L., Cohen, N. 1985. Immune responses of thymus/lymphocyte embryonic chimeras: Studies on tolerance and major histocompatibility complex restriction in Xenopus. Eur. J. Immunol. 15:540-47 117. Sprent, J., von Boehmer,H., Nabholz, M. 1975. Association of immunity and tolerance to host H-2 determinants in irradiated F~ hybrid mice reconstituted with bone marrow cells from one parental strain. J. Exp. Med. 142:321-31 118. Widera, G., Burkly, L. C., Pinkert, C. A. Bittger, E. C., Cowing,C., Palmiter, R. D., Brinster, R. L., Flavell, R. A. 1987. Transgenic mice selectively lacking MHCclass II (I-E) antigen expression on B cells: An in vivo approach to investigate Ia gene function. Cell 51:175-87 119. Lo, D., Burkly, L. C., Widera, G., Cowing,C., Flavell, R. A., Palmiter, R. D., Brinster, R. L. 1988. Diabetes and tolerance in transgenic mice expressing class II molecules in pancreatic beta cells. Cell 53:159-68 120. Jenkinson, E. J., Jhittay, P., Kingston, R., Owen,J. J. T. 1985. Studies of the role of the thymic environment in the induction of tolerance to MHCantigens. Transplantation 39:331 33 121. von Boehmer, H., Schubiger, K. 1984. Thymocytes appear to ignore class I major histocompatibility complexantigens expressed on thymus epithelial cells. Eur. J. Immunol. 14:1048-52 122. von Boehmer, H. Hafen: K. 1986. Minor but not major histocompatibility antigens of thymus epithelium tolerize precursors of cytolytic T cells. Nature 320:626-28 123. Deleted in proof.

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124. Miller, R. G. 1986. The veto phenomenon and T-cell regulation. Immunol. Today 7: 112-14 125. Fink, P. J., Shimonkevitz,R. P., Bevan, M. J. 1988. Veto cells. Annu. Rev. Immunol. 6:115-37 126. Rammensee,H.-G., Bevan, M. J. 1987. Mutual tolerization of histoincompatible lymphocytes. Eur. J. Immunol. 17:893-95 127. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., Bluestone, J. A. 1987.Identification ofa monoclonalantibody specific for a murine"1"3 polypeptide. Proc. Natl. Aead. Sei. USA84.’ 1374-78 128. Hirsch, R., Eckhaus, M., Auchincloss, H., Sachs, D. H., Bluestone, J. A. 1988. Effects of in vivo administration of anti-T3 monoclonal antibody on T cell function in mice. I. Immunosuppression of transplantation responses. J. Immunol. 140:3766-72 129. Tomonari, K. 1985. T-cell receptor expressed on an autoreactive T-cell clone, Clone 4. I. Induction of various T-receptor function by anti-T idiotypic antibodies. Cell. Immunol. 96: 14762 130. van Es, A. A., Bainer, H. 1979. Effect ofpretransplant transfusions on kidney allograft survival. Transplant. Proc. 11: 127-37 131. Opelz, G., Terasaki, P. I. 1980. Dominant effect of transfusions on kidney graft survival. Transplantation 29: 1538 132. Ryan, J. J., Gress, R. E., Hathcock, D. S., Hodes, R. J. 1984. Recognition and response to alloantigens in vivo. II. Priming with accessory cell-depleted donor allogeneic splenoeytcs: Induction of specific unresponsiveness to

foreign major histocompatibility complex determinants. J. Immunol. 133: 2343-50 133. Lau, H., Reemtsma, K., Hardy, M. A. 1983. Pancreatic islet allograft prolongation by donor-specific blood transfusions treated with ultraviolet irradiation. Science 221:754-56 134. Pepino, P., Chabot, J. A., Tannenbaum, G., Oluwole, S., Fawwaz, R., Reemtsma, K., Hardy, M. A. 1988. Effect of carbodiimide donor-specific .blood transfusion on cardiac allografts ~n rats. Transplantation 45:669-70 135. Faustman, D., Lacy, P., Davie, J., Hauptfeld, V. 1982. Prevention of allograft rejection by immunization with donor blood depleted of Ia-bearing cells. Science 217:157-58 136. Lassila, O., Vainio, O., Matzinger, P. 1988. CanB cells turn on virgin T cells? Nature 334:253-55 137. Lafferty, K. J., Cooley, M. A., Woolnough, J., Walker, K. Z. 1975. Thyroid allograft immunogenicity is reduced after a period in organ culture. Science 188:259~51 138. Schreiner, G. F., Flye, W., Brunt, E., Korber, K., Lefkowith, J. B. 1988. Essential fatty acid depletion of renal allografts and prevention of rejection. Science 240:1032-33 139. Bach, F. H., Sachs, D. H. 1987. Current concepts: immunology. Transplantation immunology. N. Engl. J. Med. 317:489-92 140. H~iyry, P., von Willibrand, W., Parthenais, E., Nemlander, A., Soots, A., Lautenschlager, I., Alfoldy, P., Renkonen, R. 1984. the inflammatory mechanisms of allograft rejection. Immunol. Rev. 77:85-142

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Ann. Rev. Immunol. 1989. 7.’481-511 Copyright © 1989 by Annual Reviews Inc. All rights reserved

IMMUNOGENETICS OF HUMAN CELL SURFACE DIFFERENTIATION Wolfgan9 J. Retti 9 and Lloyd J. Old MemorialSloan-Kettering Cancer Center, 1275 York Avenue, NewYork, NewYork 10021 INTRODUCTION The basic structure of mammalian plasma membranes is a lipid bilayer with intercalated proteins and carbohydrateside chains extending from the external membranesurface. Chemicalstudies with SDS-polyacrylamidegel electrophoresis, lectin chromatography, and glycolipid analysis have shownthat plasma membranes isolated from various tissues differ widely in their protein and carbohydrate composition. However,by far the most detailed picture of plasmamembrane diversity, especially with regard to the diversity displayed on the external membrane surface, has comefrom immunologicalstudies. With the advent of monoclonalantibodies (mAbs),hundreds of newhumancell surface antigens have been defined, anda large numberof these havebeenstudied in sufficient detail to justify a series of separate reviews.Ratherthan taking suchan antigenby-antigen approach, wehave chosen to survey current knowledgeabout the antigenic structure of the cell surface froma moreglobal perspective, with the intention of illustrating techniques,approaches,andconceptsthat are advancingour understandingof howthe surface of cells is constructed. IMMUNOGENETIC CLASSIFICATION CELL SURFACE DIVERSITY

OF

Fourgenetically distinct categories of mammalian cell surface diversity have been identified with serologic and cell-mediated immune reactions. First, the discovery of the humanABO blood groups and mouseH-2histo481 0732~)582/89/0410-0481 $02.00

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compatibility antigens established precedents for allogenetic diversity (1, 2). Allogenetic diversity is specified by genetic polymorphism,i.e. the presence and Mendelianinheritance of different alleles of a commongene in membersof the samespecies. There is evidence that the allelic variation mayreside in (a) the structural genefor a cell surface protein, (b) the activity of an enzymeinvolved in carbohydrate synthesis, or (c) a regulatory gene or DNAsequence controlling the expression of a structural gene (3-8). Second, xenogenetic diversity reflects genotypic differences between membersof different species and includes instances in which homologous cell surface molecules in disparate species showstructural diversity and instances in which an antigen of one species has no apparent homologue in another species. A special case of xenogenetic diversity on mammalian cells is antigens encodedby viral genes. Despite the clear genetic distinction, the observed patterns for viral antigen expression maybe deceptively complex, as illustrated by the murine l~ukemia virus (MuLV)-encoded gp70 cell surface antigens of mouse leukemias (9). MuLV genetic information is ubiquitous in the mouse, but whether these endogenous viral genes are expressed or not is determined by a variety of viral and host gene functions. For example, the expression of specific gp70 determinants differs between mousestrains with low and high leukemia incidence and between differentiated normal tissues in the same animal, thus mimicking the.distribution of alloantigens or restricted differentiation antigens encoded by host genes. Third, clonogenetic diversity is determined by somatic changes in gene structure, such as mutations and recombinational events. Genetic recombination is a normal step in immunoglobulin(Ig) and T-cell receptor (Tcr) gene expression, but mutations due to hits by external mutagensor errors in replication mayaffect any surface antigen gene in somatic cells. Such changeswill generally escape notice unless an affected cell undergoesclonal expansion. For example, malignant transformation and antigen-driven expansion of individual B-cell or T-cell clones have allowed serologic detection of cl0notypic determinants on surface Igs and Tcrs (10, 11). The individually distinct tumor rejection antigens of chemically induced sarcomas of mice (12), the unique antigens of humanmelanomadetected by autologous typing (13, 14), and the turn- antigens of mutagenized mouse tumors (15) may represent clonogenetic diversity of this sort nonlymphoidcells. The fourth category of cell surface diversity can be referred to as epigenetic ("differentiation-related") diversity and reflects differences in gene expression, rather than disparity of gene structure. (In this context the term differentiation can be used for all processes that lead to diversity in the patterns of proteins synthesized by cells harboring the same genome,

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including such diverse biological phenomenaas embryonicand fetal development, normal self-renewal in adult tissues, regeneration, woundhealing, inflammation, and other reactive and neoplastic processes.) The first examples of cell surface antigens that distinguish different normalcell types in the same animal were discovered some 20 years ago in the search for tumor-specific antigens of mouse leukemias (16). At that time, H-2 was the only knowncell surface antigen on these tumor cells, and the results of transplantation tests, in particular the induction of tolerance to skin allografts following tolerization with hemopoieticcells of the samedonor, had been taken as evidence that little surface antigenic diversity exists between different cell types in a single animal. However,specific immunization and testing schemesdevised to obviate anti-H-2 responses, led to the detection of several new antigenic systems, including TL, Thy-1, Lyt-1, and Lyt-2,3. Like H-2, these antigens were found both on leukemic cells, which in the mouseare commonlyof T-cell origin and arise in the thymus, and on normal thymocytes. Unlike H-2, the newly defined antigens showed a restricted distribution amongother lymphoid cells and in nonlymphoid tissues and came to be knownas "differentiation antigens." Theterms used here to classify cell surface diversity apply to the epitopes defined by specific antibodies or T cells rather than to entire molecules. Anyindividual cell surface molecule, be it a differentiation antigen or not, maycombine structural features that are xenogenetic, allogenetic, and clonogenetic in nature.

EMERGING VIEW OF CELL SURFACE DIFFERENTIATION The importance of differentiation antigens for normal cell function was first inferred from the analysis of mouselymphoid cells for TL, Thy-1, Lyt-1, and Lyt-2,3 expression. Thesestudies indicated that within the selfrenewing lymphoid system, distinct cell lineages are marked by unique surface antigens (lineage markers) and discrete phases in these lineages are markedby differential expression of additional antigens (phase markers). Enormousinterest was generated by the finding that specific cell surface phenotypes not only permitted a distinction between T and B cells, but also betweenT-cell subsets with cytotoxic (Lyt-2,3+) and helper cell (Lyt2,3-) activities (17). These and subsequent studies in mice and other species led to the notion that cell lineage and phase patterns provide the general organizing principles for differentiation-related cell surface diversity, both in the context of terminal differentiation in the adult lymphoidsystem, and in the context of developmental pathways in the embryo and fetus. The expec-

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tation was that distinctive lineage and phase markers would also be found for other self-renewing adult tissues and embryological lineages and that they wouldserve cell functions unique to the respective lineages and phases. One early exception to this concept was the mouseThy-1 antigen, which was found not only on thymocytes and peripheral T cells but also on fibroblasts, in brain tissue, and on a few additional cell types (5). Thus, Thy-1showsthe restricted expression of a differentiation antigen, yet its tissue distribution is complex, overlapping several distinct embryological lineages and showingdifferences in phasing and modesof regulation within these lineages. Since the introduction of hybridomatechnology (18), several hundred differentiation antigens have been defined on the surface of mammaliancells. Although many of these antigens have been studied through a narrowwindowof interest (single cell type or lineage) with little or no attention to expression in other cell lineages, the vast majority of the knowndifferentiation antigens shows a remarkable reluctance to fit into established cell lineage patterns. Thus, tissue-specific expression patterns cannot be extrapolated from tests with a few key tissues but have to be established through comprehensive mapping studies. With recent developments in serologic, biochemical, and genetic techniques and test systems, it has becomepossible to generate these comprehensivemaps and to study the biochemical and genetic mechanisms underlying complex surface phenotypes. The questions that can now be addressed in this way include the following: (a) Whatportion of the genomeis represented on the cell surface? (b) Whichsurface molecules are constitutively expressed on all cell types and which are differentially expressed? (c) Of the differentiation antigens, howmanyfollow a single lineage or phase pattern and how many show complex patterns? (d) Howare surface antigen genes organized in the genomewith regard to each other (clustering) and with regard to genes encoding other differentiated traits? (e) Are the repertoires of differentiation antigens conserved amongdisparate species, and are the respective homologuesexpressed with the sametissue specificity? (f) Whatis the hierarchy of intrinsic and extrinsic factors that control cell surface differentiation and the coordinate expression of multiple antigenic systems? (9) Whatis the significance of quantitative variations in the expression of individual surface antigens on different cell types? (h) Dothe various antigens that makeup the cell surface have preferential locations as part of a prescribed organization of surface elements, and

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what information is encoded by these supramolecular associations? (i)Whichalterations in surface phenotype are associated with malignant transformation and other perturbations of normal cell growth and function?

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DEVELOPMENT OF TEST SYSTEMS FOR HUMANCELL SURFACE ANTIGENS The initial studies on differentiation antigens were carried out with mouse lymphoidcells (9, 16). As one reason for this choice of test system, mouse thymocytes and T-cell leukemias could be readily prepared in free-cell suspension and are highly susceptible to complement-dependentlysis by antibody. Thus, they were perfect targets in the cytotoxic test that was used to defilae the entire first generation of mousedifferentiation antigens. In addition, the availability of inbred and congenic mousestrains allowed production of monospecificalloantisera that were invaluable for specificity analysis and genetic linkage and chromosome mapping studies (17). Recent technological advances have made similar studies possible in humans. First, hybridoma technology permits sampling of commonand rare immuneresponses and permits rapid analysis of large numbers of antibodies without manyof the elaborate specificity controls required for complex hetero- or xenoantisera. Second, indirect immunofluorescence staining combined with cytofluorometry and fluorescence-activated cell sorting (FACS)(19) and other techniques allow detailed analysis of hemopoietic cells and other tissues that can be prepared as free-cell suspensions. However, cytofluorometry and FACSdo not lend themselves to the analysis of manysubstrate-adherent cultured cells and are not useful for the analysis of intact tissues. To test adherent cells, procedures such as mixed hemadsorption (MHA), radiobinding, immunocytochemical and enzymelinked immunosorbent assays (ELISA) are useful. Of these, MHAand radiobinding assays are generally most sensitive; MHA and immunocytochemical assays allow single-cell analysis and assessment of antigenic heterogeneity in test cultures, and radiobinding assays permit determination of numbers of antibody binding sites and binding affinities. For solid tissues, highly sensitive immunohistochemicalprocedures have been developed (20), including the indirect immunofluorescence, peroxidaseantiperoxidase, avidin-biotin complex, and alkaline phosphatase (ALP)antiALP procedures. These largely replace absorption assays and other methodsthat do not permit antigen localization to specific cell types or subcellular structures within tissues. Third, a large number of human tumorcell lines have been established, representing a wide range of distinct cellular lineages. They are used as standard typing panels for antigen

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specificity and as a. source of homogeneous cell populations for biochemical and genetic studies. Muchprogress has also been madein culturing normal humancells. As a consequence, lymphocytes, melanocytes, keratinocytes, kidney and bladder epithelial cells have becomeavailable for serologic analysis. Fourth, the development of sensitive immunochemicaltechniques in conjunction with mAbshas led to the definition of a host of plasma membraneproteins, glycolipids, and carbohydrates, and it has permitted their chemical characterization. For a numberof proteins, this analysis had yielded information on their primary structure and the nucleotide sequence of their coding genes. For cell surface carbohydrates, it has identified some 50 distinct sugar moieties that reside on plasma membrane glycolipids and glycoproteins (reviewed in Ref. 21). Finally, serologic analysis of rodent-human somatic cell hybrids for expression of human cell surface antigens and the use of cloned surface antigen genes for somatic cell genetic and cytogenetic studies have been so successful that more is known about the humangenomic map for cell surface antigens than for similar maps in any other mammalianspecies (22). MAPS FOR ANTIGENIC

INDIVIDUAL SYSTEMS

SURFACE

In the infant days of cell surface serology, the tissue-specificity of differentiation antigens was frequently defined with just a handful of cell types, and the findings were extrapolated on the basis of cell lineage concepts or correlated with other characteristics of the antigen-expressing cells, e.g. fetal vs adult, normal vs cancer. According to the expanded view of cell surface differentiation, such extrapolations have little predictive value, and comprehensive maps of antigen distribution are required before conclusions can be reached with regard to cell type, tissue type, or cancer specificity. The following mapsprovide the essential information that now goes into a comprehensivespecificity analysis. Cultured

Cell

Map

Cultured cell maps are constructed with tumor cell lines and short-term cultures of normal cells, and Figure 1 illustrates mapsfor five prototype surface antigens. The MC25(23, 24) and K4 (25) antigens show highly restricted distribution and mark individual tumor types. In contrast, Thy1 (26, 27) and MC139(26) are expressed on cells of various unrelated lineages but distinguish related cell types derived from a common lineage. Finally, the J143 antigen (24, 28-31), also referred to as VLA-3(32), expressed independent of cell lineage derivation and simply distinguishes

Annual Reviews CELL SURFACE DIFFERENTIATION Cell surfaceantigen

Targetcells Cell type Designation N~aroe~odenw~l lamors

487

MC26

K4

Thy-1 MC139 VLA-3a

Neurobl~omaSMS-SAN, ~[][]~ Maim Mill ~KAN,:MSN,.KCN lill PNET, ES TC-215,-135,-71, IARC-EWl SK-MG-2,-5,-IO, U251MG ~ ~;;; loll IIII ~ma Melanoma MeWo, SK-MEL-28,-29,-113 Mb,Rb TE671, Y79,Wed ~ ;~ I1~ liB

[]~D~ IIII m~;

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~m~ RMS TC-206,-280,-212, SMS-CTR Sags2,U20S O=eo~oma TE85,TC-256, Flbmsa~oma8387,Hs913,HT-I~O Epithelial Germ gall Tera-l,-2,833KE, PAl ~ Kldn~ SK*RC-2,-6,-28,-29 ~ Bladder T24,RT4,VM-CUBL 639V ~ Colon SW480,~1083,-1417, SK-CO-1 Breast AIAb,MCF7, SK-BR-7, BT-20 Lun~ SK:LC-I,-2,-LL,-9 SK-OV-3,-4,-6 O~W Le~keml~,I~phom~ T-cell MOLT-4, T45,P12,CCRF-CEM B-cell SK-LY-16,-18, DAUDI, ~JI Myelold HL-60,K562

Bill ~ ~

nil ~ ~

lab ~ ~B

Ill iggg liMB

No~alcello Melanoc~esshod-term culture Meningeal cellsshod-term culture Fibmbla~ shod-term culture Kidn~ shod-term culture Bladder shod-term culture Lymphoc~esuncultured FiDure 1 Cultured cell map for human cell surface antigens MC25, K4, Thy-l, MC139, and J143 (VLA-3~) determined by mixed hemadsorption assays. Filled squares, antigen-positive; open squares, antigen-negative; cross-hatched squares, weak antigen expression; half-filled squares, antigen expression restricted to a subset of cells in the test sample.

cells that grow adherent to tissue culture plastic (J143+) from cells that grow loosely adherent or in suspension culture (J143-). Differentiation antigens like Thy-1 and MC139may be mistaken for single lineage or phase markers if tested only on a narrow range of cell types. Furthermore, antigens that cotype amongcells of one lineage (e.g. MC139cotypes with other T-cell markers on hemopoietic cells) maydiffer clearly whentested on additional cell types. Thus, cotyping analysis on discriminating cell panels provides the most widely applicable means of distinguishing antigenic systems. This principle has been exploited in our studies with mAbs raised against neural, melanocytic, mesenchymal,and epithelial cell types (23-26, 31, 33-36). The CD-classification of about 50 surface antigens also uses cotyping as the major distinguishing characteristic, but the cell panel only gives information about the hemopoietic lineage (37). There is evidence from systematic mappingstudies and anecdotal reports that a large

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proportion of differentiation antigens initially thought to be restricted to hemopoietic lineages are also expressed on other cell types, and extended cell mapswill undoubtedlyreveal moreexamplesof differentiation antigens that cross single cell lineage and phase patterns.

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Somatic,

Developmental,

and Neoplastic

Tissue

Maps

Since cultured cell mapsare largely defined with tumorlines, it is important to distinguish the contributions madeto each cell line phenotypeby (i) the differentiation program of the corresponding normal cell, (ii) malignant transformation, and (iii) cell selection and adaptive changes in tissue culture. MAbsto cell surface antigens readily permit immunohistochemical analysis of normal and tumor tissues, thus avoiding several limitations of the cultured cell map. Three complementary maps of antigen distribution in vivo are generally considered: The somatic tissue mapfor antigen expression in normal adult tissues, including such reactive states as inflammation, woundrepair, and regeneration; the developmental mapfor antigen distribution at various stages of fetal development; and the neoplastic tissue map,whichis most closely related to the cultured cell map, for antigen expression in a wide range of tumor tissues. Howdo tissue maps compare to cultured cell maps? Although the two maps appear to be congruent for some antigens, incongruencies are so commonthat the maps have to be established independently. For one group of antigens, one of the two mapsreveals antigen expression in cell types that are not available in the other map. Our finding that the K4 antigen [sialolyl-LNT (25, 38)] of cultured teratocarcinomas (Figure 1) also expressed by normal brain astrocytes (39) exemplifies in vivo antigen detection in cells which, in humans,cannot generally be tested in culture. Conversely,somecells are so rare in the organismor restricted to such early developmental stages that their antigenic phenotype cannot be ascertained from conventional tissue maps. Tumorcell lines mayfaithfully reflect such normal phenotypes and provide convenient models for serologic analysis. The definition of stem cell and progenitor cell phenotypes in the hemopoietic (40), melanocytic (41), and neuronal pathways (23, based on cell line studies illustrates this added reach of the cultured cell map. For a second group of antigens, the cultured cell and tissue mapsare incongruent, due to selection of specific cell types or phenotypicadaptation of cells in vitro. Twoformsof selection occur. First, a single cell type in a normal or neoplastic tissue mayovergrow other cell types and dominate the final culture phenotype. For example, epithelial cells of normal kidney differ in surface antigen phenotype, with Lex and F23 being restricted to a single portion of the nephron, the proximal tubule (42, 43); yet normal

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CELL SURFACE

DIFFERENTIATION

489

+ (25, 43), suggesting kidney epithelial cultures are uniformly LeX+/F23 selective outgrowth of proximal tubular cells. Second, amongtissue specimensobtained from different individuals (e.g. specimens of a particular tumortype), only a small subset maygive rise to cell lines, and this subset maydiffer in surface phenotype from the subset that does not survive in culture. One example is the expression of MC25in only a subset of uncultured neuroblastomas but on virtually all established neuroblastoma lines (23). Adaptive changes in surface phenotype have been described for mesenchymal, neuroectodermal, and epithelial cells. These include the acquisition of F19 on cultured fibroblasts and melanocytes (33, 34), and the loss of CALLA and Thy-1, two proximal tubule markers in vivo, in cultures of normalkidney epithelial cells (27, 44). Specific alterations in cell surface structure are likely to accompany malignant transformation and to participate in the uncoupling of tumor cells from normal extrinsic growth control and their invasive and metastatic potential. However,it is not knownwhetherthese alterations involve the expression of tumor-specific antigens or altered patterns of expression or membraneassembly for antigens that are also expressed in normal cells. The notion of "tumor-specific antigens" has inspired the longest uninterrupted line of inquiry in tumor immunology,but it is important to note that the fascination with this type of antigens stems from their potential use as targets for cancer therapy rather than from any specific theory about the etiology or pathogenesis of cancers that would demandtheir existence. Muchof the current interest in tumor-specific antigens focuses on thc individually distinct tumor rejection antigens of mouse sarcomas (12), the turn variants ofmutagenized mousetumors (15), and the unique antigens of humanmelanomas(14), all of which probably reflect clonogenetic diversity of commoncell surface components recognized by the host immunesystem. The characterization of human tumors with murine mAbshas not so far identified any tumor-specific antigens. Instead, virtually all mAbdefined cell surface antigens knownon humantumors are differentiation antigens that are also found on certain normal cell types, a fact that is often obscured by incomplete or inadequate analysis. While it has been suggested that tumors express differentiation antigens in an "inappropriate" or "ectopic" fashion, our knowledgeof normal cellular phenotypes is not comprehensiveenough to justify such terms. Similarly, it has been suggested that tumors reexpress molecular and biological traits of fetal cells that are absent from normaladult cells. This theory has not held up for any antigenic systemwhenthe analysis has been sufficiently detailed. Antigens that fit an "oncofetal" pattern in one system are invariably found in other systems with a different modeof expression and control.

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Furthermore, manyantigens expressed on normal fetal, but not adult, cells are absent from corresponding tumors, and antigens that are expressed on tumors may not be found on corresponding fetal cells. To mention only two well-studied examples--antigen K21 [lacto-N-tetraose (25, 38)] expressed in normal fetal colon and bronchus and is absent from the correspondingnormaladult tissues, yet it is not generally expressedin colonic and bronchial carcinomas (45); second, the mel-CSPGantigen has been characterized as an "oncofetal" antigen of melanocytic cells (46) but expressed on fibroblasts, visceral and vascular smoothmuscle, and skeletal muscle with no apparent difference between fetal and adult stages (47). Similar departures from the presumed modes of expression have been found for other categories of "transformation-related," "proliferationrelated," and "activation" antigens of cancer. In instances in which tumor cells differ in surface phenotype from matched normal cells (e.g. neuroblastoma vs sympathetic neuron; melanomavs melanocyte; fibrosarcoma vs fibroblast), it is not knownwhether the matched normal cells truly reflect the lineage and phase coordinates and the physiological state of the actual target cell for transformation (histogenetic phenotype) or the nor: real differentiation programwhich the tumor imitates after transformation (histotypic phenotype). The popularity of the term "tumor-associated antigen," which admits all antigens found on tumors and excludes none, epitomizes this dearth of knowledge. Chromosome

Map

Mouse mAbsagainst human cell surface antigens generally recognize xenogcnetic determinants and, consequently, can be applied to gene mapping studies in rodent-humansomatic cell hybrids. This allows questions to be asked about the numberand distribution ofloci in the humangenome that code for cell surface antigens. GENEMAPPING STUDIESIn our gene mapping studies, carried out in conjunction with cell line and tissue mapping(25, 26, 29, 31, 35, 48-53), we found that 42 of the 43 antigens examinedshowed informative patterns of expression amongrodent-human hybrid clones. Comparison of antigen expression and the distribution of individual humanchromosomesin the panel, determined by karyotype and isozyme analysis, permitted chromosomeassignments for all but one of the antigens (see MSKseries in Table 1). Gene mapping studies in other laboratories have provided assignments for additional cell surface antigens (summarizedin Ref. 22), and use of hybrid clones retaining deleted or rearranged copies of specific human chromosomes in the absence of normal homologues has provided subchromosomal assignments for most of the mapped surface antigen genes (Table 1).

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Withthe cloning of an increasing numberof genes coding for cell surface proteins, alternative gene mappingprocedures have becomefeasible that do not dependon antigen expression in somatic cell hybrids. These include Southern blot analysis of genomic DNAextracted from chromosomally defined rodent-humansomatic cell hybrids, in situ hybridization of radiolabeled DNAprobes to metaphase chromosomes, and family studies based on restriction fragment length polymorphisms (54). Gene assignments obtained with DNAprobes reflect the map positions of structural genes for cell surface proteins, whereas those based on serologic analysis of hybrid cell phenotypes have several alternative explanations. For glycoproteins defined with mAbsagainst polypeptide epitopes, it is likely that the mappedloci are also the structural genes for the respective membrane proteins. In the case of carbohydrate epitopes on glycoproteins or glycolipids, including W6/34,HNK1,and Lex, the assigned loci maycode for glycosyltransferases that are unique to the humanfusion partner and that control a critical step in antigen synthesis. The apparent isolation of a human W6/34 gene by DNA-mediated gene transfer (55) may permit identification of its gene product and its role in the synthesis of the W6/34 glycolipid moiety. Analysis of the Lex and HNKIcarbohydrate antigens is more complexsince rodent cells as well as humancells can express these antigens (56, 57) and the available mAbsdo not discriminate between humanand mouseantigens on hybrid cells. Cell-type specific variations in the patterns of enzymatic and genetic complementation may explain why Lex segregates with human chromosome 11 in mouse-human myeloid hybrids (58) but not in mouse-humanneuroblastoma hybrids (53). OF CELL SURFACE ANTIGEN GENES Most human chromosomes have been shownto code for multiple cell surface components, but generally the coding genes do not cluster but mapto distinct subchromosomal regions (Table 1). Onlya small numberof gene clusters for surface antigens has been defined. The most extensive family of structurally related surface antigens knownin humansis the Ig superfamily (59). Within this family, the MHC class-I and class-II molecules, the Ig heavy and light chains and the Tcr alpha and beta chains are encoded by gene clusters, and this arrangementis critical for the generation of allogenetic diversity of MHC genes, clonogenetic diversity of Ig and Tcr genes, and Ig class-switching during B-cell maturation. The other membersof the Ig superfamily are widely distributed in the gcnome(Table 1), and the few examplesof genes that map closely together on specific chromosomes(THY1, CD3E,CD3D, and NCAMon chromosome 1 lq22-q23; IGK and CD8 on chromosome 2p12) may reflect linkage groups maintained during evolution from commonancestral gene (59). FAMILIES

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Table I Genomicmap for humancell surface antigen genes Cell surface antigen

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MAb

Gene symbol

Regional assignment

gp 140 gp60 LFA-3 (gp60) CD45(gp200) CD1(gp43-49) CD2(gp50) CD35(p220) CD21(gpl40) ALPL

MSK1 MSK2

p22 q32-qtcr

CD45 CD1 CD2 CR1 CR2 ALPL

p 13 q32 q32 p36.1 -p34

gp145/100 p35 CD8(1332/33) Ig L kappa ALPI ALPP

MSK8 M1C18 CDSA IGK ALP1 ALPP

p23-qtcr

gp85 heat-labile heat-labile gp 140 CD10 (gpl00) TFRC gp97 (rat MRC-OX2)

MSK9 MSKIO MSK33 MSK32 MSK42 TFRC MF12 MOX1

pter-p 14 pter-p 14 pter-p 14 pter-q21

p45

CD38

gp200 (VLA-1~) CD14 (gp55) PDGF receptor

MSKll CD14 PDGFR

q23-q31 q31-q32

HLAclass I

HLA-A

p21.3

(L243)

HLAclass II

p21.3

A42 MG2

gp57 gpl00 IFN-~, receptor

HLA-B HLA-C HLA-DR HLA-DP HLA-DQ MSK29

Chromosome 1 AJ9 T87 TS2/9 4C a(NA1/34) (anti-T 11) (E 11) (OKB7) Chromosome 2 L230 AUAI (Leu2A)

Chromosome 3 K 15 AJ425 SR3 K66 A J8, NL-1 OKT9 133.2 Chromosome 4 OKTI0 Chromosome 5 SR84 (MOS39) Chromosome 6 (W6/32)

Description

MSK28 1FNGR

p12 p12 q34-q37 q34-q37

q26.2-qter q28-q29

q12-q15 q21 -qter q

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Table 1 (continued) Cell surface antigen MAb

Gene symbol

Regional assignment

EGF receptor Tcr beta

EGFR TCRB

pl 3op12 q32 or q35

Chromosome 9 CNT/6

gp90

MAK34

Chromosome 10 A- 1A5, A J2 (anti-Tac)

gp 130 (VLA-fl) IL-2 receptor

MDF2 IL2R

pter-q22 p15-p14

glycolipid gp80-105

ME R 1 MIC4 MICll MER2

gp25 gp90 gp70 heat-labile gp85/40 heat-stable gp35 gpl30 heat-labile CD5 (956/62) Thy-1 (gp25) p 100 carbohydrate CD26 (9250) Lex (X hapten) N-CAM CD3delta (gp20) CD3epsilon (p20)

MSK21 MSK14 MSK35 MSK24 MD U1 MSK22 MSK25 MSK13 MSK39 CD5 THYI MIC9 LEU7 CD26 CDl5 NCAM CD3D CD3E

p 13 p13 p13 pter-p 15 p p p p cen-q 13 cen-q 13 q 13-qter ql3-qter q 13-qter q 12-q 13 q22.3 q22-qter

CD4 (p55) CD9 (924) gp45/30 heat-stable gpl40 gp 100 p37

CD4 CD9 MSK4 MSK7 MSK27 MSK36 MIC17

Chromosome 7 EGFR 1

Description

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Chromosome 8

Chromosome 11 W6/34 F10.44.2 16.3A5 1 D12 G344 JF23 SR27 NP 13 4F2, T43 A 124 MC139 QI4 5.1H11 T 101 K117 4D 12 HNK 1 B 1.19.2 RIB- 19 (SP-64) (SP-6) Chromosome 12 Leu3A BA-2, M68 K152, A127 VI MG6 CNT/11 BB1 MEgp30-60

q23 q23 q23

pter-p 12 p cen-ql3 ql 3oqter q 13-qter q 13-qter ql2-q 14

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Table 1 (continued) Cell surface antigen MAb

Description

Gene symbol

Regional assignment

Chromosome 13

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Chromosome 14

Chromosome 15 SV13 AO122, B5 F23 28.3.7 30.2A8 BBM.1 (My7) (aiR3) Chromosome 16 TS1/22 (OKM1) (SHCL-3) Chromosome 17 J143, A99 ME20.4 3A1

Tcr alpha Ig H

TCRA IGH

q 11.2 q32.3

gpl05 mel-CSPG gp 140 p95

CD13 (gp 150) IGF-I-R

MSK15 MSK16 MSK17 MIC7 MIC12 B2M CD13 IGF1R

q13-q26 q13-q25 q 13-q26 ql 1-q22 ql 1-q22 q21-q22.2 q25-q26 q25-q26

CD1la (gp180) CD1lb (gpl70) CD1lc (gp150)

CDllA CDI1B CDllC

pl 1-p13 pl 1-p13 pl 1-pl 3

gp140/30 (VLA-3~) NGFreceptor CD7 (p41) p185

MSK18

q22-q23

NGFR CD7 NGL

q22

gp50 gp95 gpS0 gp67 CEA Ins receptor

MSK20 MSK19 MSK37 CD33 CEA INSR

pter-p 13.2 q12-q13.2 q12-q13.2

heat-labile

MSK38

CD18 (gp90) 1FN-~ receptor IFN-/~ receptor

CD18 IFNAR IFNBR

gp45 heat-labile Ig L lambda

MSK40 MSK41 IGL

/~z-m (p 12)

ql 1-q12

Chromosome 18 Chromosome 19 F 10 F8 $7 My9

Chromosome 20 05 Chromosome 21 TS1/18

Chromosome 22 V35/9 E3

q13.1-q13.3 p 13.3-p 13.2

q22.3 q21-qter q21-qter

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Table1 (continued) Cell surfaceantigen MAb

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ChromosomeX 12E7,O13 R1 ChromosomeY 12E7,O13

Description

Genesymbol

Regionalassignment

gp30/25

M1C2 MIC5

pter-p22.32 q27-q28

gp30/25

MIC2

p

~Antigens listed without corresponding mAbsor with mAbsgiven in parentheses have been mapped with DNAprobes and the available mAbshave not been tested for surface reactivity with somatic cell hybrids. Abbreviations: p, polypeptide; gp, glycoproteln: ALPL,ALPI, ALPP.liver, intestinal and placental forms of alkaline phosphatase; lg L, IgH, Ig light and heavy chains; TFRC,transferrin receptor; VLA, very late activation antigens (32); CEA,carcinoembryonicantigen; IFN, interferon; IGF-1-R,insulin-like growth factor-1 receptor; Ins, insulin. Because of space limitations, references for mAbsand chromosome assignments are not listed individually but can be retrieved from Refs. 22, 35, 37, 101-103. Chromosome assignments for CD10 and 5.1 HI 1 are based on our unpublished data for antigen expression on somatic cell hybrids. Becauseof incomplete analysis, a few antigen pairs listed in the table, including F23/CD13, K152/ME491,and T87/LFA-3, cannot be distinguished at present and may represent single antigenic systems.

A second family of cell surface antigens, integrins, is defined by a combinationof structural and functional criteria and includes several cellcell and cell-substrate interaction molecules (60). For humancells, two sets of cell surface heterodimers that are included in the integrin family are the CDI8/CD11glycoproteins (61) and the AJ2/VLAglycoproteins (32). Like the Ig H/L chain, CD3/Ti, HLAclass I/fi2-m, and CDl/fl2m cell surface complexes, the CD18/CDll and AJ2/VLAantigens are multimeric cell surface complexeswhose subunits are encoded by different chromosomes. The CD18/CD 11 heterodimers (LFA- 1, Macl, gp 150/90) consist of a commonfl-chain (CD 18), encoded on chromosome21, which is associated on the surface of leukocytes with three types of structurally related a-chains, encoded on chromosome16pl 1-p13 (62). The three types of CD18/CDI1 heterodimers have a similar tissue distribution but are differentially expressed amongleukocyte subsets (37). In contrast, the AJ2/VLA heterodimers are muchmore diverse in tissue distribution, structure, and chromosomal location. Each of the AJ2/VLAheterodimers comprises a commonfl-chain [recognized by mAbsA J2 (44) and A-1A5 (32)] and one of at least five distinct a-chains (29, 32). In collaboration with M. Hemler(Dana-Farber Cancer Institute, Boston) we have used mAbA J2 and mAbsto four different a-chains to establish the cultured cell and tissue maps of the respective antigens. Our results showunique patterns for each a-chain, with SR84(VLA-la), 12F1 (VLA-2a), and

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(VLA-3~; cf. Figure l) widely expressed on cultured neuroectodermal, mesenchymal,and epithelial cells; BSG10(VLA-4~)expressed on cultured hemopoietic, mesenchymal, and neuroectodermal cells (unpublished results); and A J2 expressed on every cell type tested to date. Somaticcell genetic analysis has shown that the fl-chain gene (MSK12or MDF2)is located on chromosome10pter-q22 (35), the J143 gene (MSKIS)is located on chromosome 17q22-q23 (35) and the SR84 gene (MSKll) is located on chromosome5 (29). Continued structural analysis will undoubtedly reveal other examplesof cell surface antigen families in the humangenome. At present there is no evidence that gene clustering in specific chromosomal sites is of general importancein the coordinate expression of differentiated traits in specific cell types.

Functional Mapsof Cell Surface Differentiation Antigens Cell surface antigens with restricted distribution are commonlythought to serve functions unique to the antigen-positive cells while broadly distributed antigens are thought to serve general housekeeping functions. However,a single antigen may serve diverse functions in different cell types, and similar functions maybe mediated, in parallel or alternatively, by separate molecular mechanisms.Thus, the construction of cultured cell and tissue maps by itself does not give a clue as to the function of an antigen. However,the opposite is true also, since the presumedfunction of a cell surface componentin one cell type does not predict its pattern of expression in other cell types or its possible role in additional antigenexpressing cells. This fact is illustrated by the tissue distribution of the humanNGFreceptor, which is found not only on certain neural cells, but also on subsets of lymphoid, epithelial, and mesenchymalcell types (63, 64), and is consistent with recent suggestions about the cell type-specific pleiotropic functions of various cytokines and their cell surface receptors (65). It is tempting to speculate that manycomplexbiological functions the cell surface are not assigned to individual antigens. They are the responsibility of integrated circuits comprising multiple components,both in the membraneand inside and outside of the cell, in which individual cell surface antigens serve as structural and functional modules. The same module may be used over and over in different cell types and different circuits to serve different functions.

EXTRINSIC AND INTRINSIC CONTROL OF CELL SURFACE DIFFERENTIATION Differentiation-related diversity in cell surface phenotype does not generally involve loss or irreversible repression of surface antigen genes.

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Instead, surface phenotypes are reversibly controlled by extrinsic signals, provided by the external environment of the cell, and intrinsic signals, provided by the genetic differentiation program of the cell. Molecular analysis of these extrinsic and intrinsic signals in humansdependslargely on the availability of in vitro test systems. Withregard to extrinsic signals, progress has been made in identifying the role of soluble growth and differentiation factors in the induction of surface antigen changes on a wide range of cell types (66-69). In addition, an alternative modeof surface antigen control has been identified, whichis triggered by specific cellular interactions with extracellular matrix (ECM)proteins (31, 49). Thus the only antigens shown to be regulated in this way by ECMare the mel-CSPGchondroitin-sulfate proteoglycan (49), and the A42 and J143 glycoproteins (30); J143 (VLA-3~)is the humanequivalent of the avian ECM-receptor CSAT(32) and its induction by ECMmay represent positive feed-back loop for cell attachment. Since manyof the extrinsic signals act through cell surface receptors, their effects are conditional on the intrinsic differentiation programsthat determine receptor expression and receptor-linked signalling pathwaysin individual cell types.

Intrinsic Control The forces maintaining distinctive patterns of gene expression in somatic cells are not known. However,regulatory complementation analyses with somatic cell hybrids (synkaryons), heterokaryons, transfectants, transgenic animals and rare variant cell lines are beginning to unravel the regulatory networksthat underlie cell type-specific surface differentiation. SOMATIC CELLHYBRIDSWith the chromosomal assignment of loci for humancell surface differentiation antigens, questions can be asked about the regulation of antigen expression in hybrids which contain the coding humanchromosomesand are formed between antigen-positive or antigennegative humancells and rodent cell types representing matched or mismatched cell lineages. Findings with more than a dozen such antigenic systems have been reported (26, 27, 29, 31, 49, 50, 70, 71), and four types of outcomecan be distinguished for individual antigens: Antigen-positive humancells fused with different rodent cell types can give rise to hybrids that express the particular humanantigen (permissive mode) or hybrids that lack antigen expression (nonpermissive mode). Conversely, antigennegative humancells can give rise to antigen-positive (inducing mode) or antigen-negative hybrids (non-inducing mode), depending on the rodent fusion partner. Figure 2 illustrates these different modesof control for four differentiation antigens in hybrids constructed with neural, mesenchymal,epi-

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RETTIG & OLD Humancell surface antigen expression

Fusion partners human rodent 1""

Neuroblastoma Neumblastoma

~

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Neuroblastoma 1500-, i

=

Rodent program influence

Hybrids

U Thy-l: permissive [] MC139:permissive i"] 5.1Hl1: permissive [] VLA-3~x:non-inducing

L cell fibroblast

~

¯ Thy-l: non-permissive [] MC139:nnn-pnrmimve [] 5.1n11: permissive ¢~1VLA-3a:inducing

Fibroblast RAG renalcancer

~ ¯ ’~

Kidney epith.

¯ Thy-l: permissive [] MC139:non-inducing I-’l 5.1Hl1:inducing [] VLA-3e:permissive

Lcellfibroblast [] [] []

~

1500 1

÷ Bcell EBV

MC139: non-inducing 5.1Hl1:inducing VLA-3o,:permissive

Chinese hamster fibroblast I~l MC139;non-inducing f’-I 5,1H11:inducing [] VLA-3a:inducing

,-,~.<

T cell leukemia

BW5147thymoma i ~ [] []

Thy.l: non-inducing MC139:non-permissive 5,1Hl1: non-inducing VLA-3~,:non-inducing

Figure 2 Expression of human cell surface antigens Thy-1, MC139, 5.1Hll, and Jl43 (VLA-3ct)on humanand rodent cells and on derived somatic cell hybrids retaining the coding human chromosomes (chromosome 11 for Thy-l, MC139and 5.1H11; chromosome 17 for VLA-3c 0.

thelial, and lymphoidcell types. It is apparent that fusion of humanand mouseneuroblastoma cells results in hybrids that faithfully reflect the humanparental phenotype, showing permissive and non-inducing modes of expression for individual antigenic systems. In contrast, fusions between similarly matched mesenchymal,epithelial, and lymphoid cell types and fusions between mismatchedcell types result in novel patterns of antigen display. Thus, while the differentiation programof the rodent fusion partner clearly affects humanantigen expression in the hybrids, this influence is difficult to predict on the basis of cell morphologyand other general attributes of the differentiated state, and it differs for individual antigenic systems and hybrid combinations. Thy-1 expression in hybrids demonstrates that some humanantigens do not simply follow the distribution of their rodent homologues(27). For

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example, human Thy-1 shows permissive and inducing modes of expression in hybrids formedwith Thy-1- rodent cells, and a non-inducingpattern in hybrids formed between Thy-1- humanT-cell leukemias and Thy-1 + BW5147mouse thymoma cells. The suppression of human Thy-1 in hybrids formed between Thy-1 + humanfibroblasts or neuroblastomas and Thy- 1 - mouseL-cell fibroblasts (27) is the best-studied exampleof a nonpermissive interaction for a surface antigen in interspecific hybrids, and our findings are consistent with findings for mouseThy-1expression in intraspecific hybrids (72). The nonpermissive modeappears to be as antigenspecific as the permissive and inducing modesfor Thy-1expression in other hybrid combinations, and the same L-cell program that suppresses Thy-1 expression is permissive or inducing for other antigens such as 5.1Hll, J143 (VLA-3~), NGFRand mel-CSPG(Figure 2 and Refs. 31, 49, 50). A highly predictable pattern of expression in hybrids has been observed with J143 (VLA-3~).Similar to its distribution in the humancultured cell map, this antigen is found on all substrate-adherent hybrids containing human chromosome 17q22-q23, including hybrids derived from J143human lymphoid cells and neuroblastomas (31). J143 shows the same ECM-dependent expression in ECM-responsive hybrids that was noted for humancells (31). Thus, the J143 glycoprotein is not only faithfully expressed in hybrids and capable of associating with the rodent homologue of the AJ2/VLA fl-chain (30), it is also regulated and mayfunction normally in a rodent cell background. J143 is presently the best example of a humancell surface antigen that is reprogrammedfollowing somatic cell hybridization. Several other humandifferentiation antigens are also induced after cell fusion with appropriate rodent cell types, indicating that their coding genes are not irreversibly inactivated in non-expressor human cells. Weknowof two antigens, Q14and SV13, that behave differently in this regard and whose genes may be inactivated in a more stable fashion (Ref. 29, unpublished results). Both antigens are expressed in hybrids formed between mouseL-cells and antigen-positive humancell types (permissive mode) but not in hybrids formed between L-cells and antigennegative humancell types (non-inducing mode). This constellation permissive/non-inducing modes of expression in hybrids formed with a single rodent cell type reflects inactivation of antigen expression in nonexpressor humancells that is much more resistant to reprogramming in somatic cell hybrids than is the inactivation of antigens like J143. It will be of interest to determine whether this lack of plasticity in antigen expression results from developmental changes in the chromatin structure of the coding genesin specific cell types. HETEROKARYONS Heterokaryons are transient

cell fusion products that

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contain intact parental nuclei with complete sets of chromosomes.Thus, regulatory complementationanalyses can be carried out in heterokaryons without prior chromosomeassignment for the traits under study. Unlike rodent-human synkaryons, in which human chromosomes are preferentially lost and the hybrid phenotypeprogressively shifts towards the phenotype of the rodent parent, heterokaryons formed between different cell types have an indeterminate phenotype. Blau and colleagues have constructed heterokaryons between a mouse muscle cell line and various human nonmuscle cells and found that expression of two human cell surface antigens, 5.1H11 and 24.1DS,is induced, with different kinetics of induction dependent on the choice of humanfusion partner and the nuclear compositionof the heterokaryons (73). While these results were interpreted as an induction of humanmuscle-specific traits in fusions with a mouse muscle cell line (73), a more complexpicture has emergedfrom our studies with 5.1H 11 in synkaryons. First, 5.1H 11 is not muscle-specific in humans but is expressed on a range of neuroectodermal, mesenchymaland epithelial cell types (Figure 2 and unpublished results). Second, 5.1H11expression is permissive in mouse-humanneuroblastoma hybrids and is induced in synkaryons formed between nonexpressor humancells (e.g. fibroblasts, kidney epithelial cells, lymphocytes) with such nonmusclerodent cells as mouse L-cell fibroblasts, Chinese hamster fibroblasts, and even mouse RAGrenal cancer cells (Figure 2). Thus, detailed knowledgeof the cultured cell and tissue mapsof humancell surface antigens and analysis of hybrids formedwith rodent cells representing different lineages can yield a broader interpretation of regulatory complementationpatterns. TRANSFECTANTS Cotransfection of high-molecular weight human DNA and selectable markers into rodent cells has been widely used to isolate genes coding for humancell surface antigens (74, 75), but less for the analysis of intrinsic regulatory factors. This is due in part to the uncertain role of exogenous promoter and enhancer sequences in cell type-specific gene expression and the potential for selecting rare phenotypes that result from high gene copy numbers or fortuitous integration of the transfected genes into transcriptionally active sites (74-77). Nevertheless, studies with class-II gene constructs illustrate the use of cotransfectional analysis in defining distinct cis-regulatory elements for humancell surface antigen genes (78). TRANSGENIC MICETransgenic mice have been constructed for only a few humancell surface antigens (79-83). The studies with Thy-1 transgenic mice have been particularly instructive, since the tissue distributions of the mouse and human homologues are well known and differ widely, thus allowing an unusual look at the relative contributions of trans- and cis-

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acting signals to the respective tissue maps. Gordon et al (79) have described two types of transgenic mouse lines for Thy-1. One type was constructed by introducing a large genomic DNAfragment encompassing the mouse Thy-1.1 gene into the germ line of Thy-1.2-homozygousmice, and the other type by similarly introducing the humanThy-I gene. The resulting transgenic mice were examine~lin detail for the tissue distribution of the various Thy-1 proteins, using mAbsspecific for the Thy-l.1 and Thy-l.2 mouse alloantigens and human Thy-1 in immunohistochemical tests. The endogenous Thy-1 genes and the transgenes were expressed in a tissue-specific manner, and Table 2 summarizes the respective tissue maps. It is apparent that the mouseThy-1.1 transgene is expressed with a pattern closely following the endogenous Thy-l.2, whereas humanThy-I expression in the mousetissues is almost identical to the Thy-1 pattern seen in normal humantissues. Two exceptions were noted. First, the mousetransgene was not expressed in a small numberof tissues where the endogenous Thy-1 gene was normally expressed. These included splenic T cells, fibroblasts, and a subset of neural fibers in the molecularlayer of the cerebellum and in cortical white matter. Second, the humanThy-1 gene was expressed in the kidneys of transgenic mice in additional portions of the nephron that are Thy-1 - in the adult humanbut Thy-1 + in fetal human kidney (33). The most plausible explanation for these findings in transgenic mice and for the species-specific differences in Thy-1 expression is that the Thy-1 gene uses multiple cis-acting sequences in a tissue-selective manner to determine its complex pattern of expression (79, 80). Most likely, the numberor combinationof these eis-acting elements has been altered experimentally in the DNAfragments and gene constructs used to produce Thy1-transgenic animals, and has changed, in a very different fashion, during the evolution of an ancestral Thy-1 gene into the present mouseand human genes. These findings with transgenic mice are in general agreement with the somatic cell genetic observations showingdissociation between mouse and human Thy-1 expression. However, no in vivo counterpart has yet been defined for the action of the trans-acting negative regulators described for L-cell hybrids. GENETICVARIANTS ANDINBORNERRORSOF SURFACE PHENOTYPE Genetic abnormalities leading to changes in surface antigenic phenotypein vivo or in vitro provide important tools for structural and regulatory studies. As one example, leukocytes derived from patients with leukocyte adhesion deficiency lack cell surface expression of all three types of CD18/CDll heterodimers (LFA-1, Macl, p150/90), due to structural defects in the gene for the commonCD18subunit (84). In this respect, the defect

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expression

of human and mouse Thy-1 in

Thy-1 Expression

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Tissue Cerebral cortex Gray matter White matter Cerebellum Molecular layer Granular layer White matter Brain stem Spinal cord Retina Inner layer Outer layer Peripheral nerves Adrenal gland Cortex Medulla Thymus Cortex Medulla Lung Trachea Bronchus Liver Pancreas Endocrine Exocrine Stomach Mucosa Smooth muscle Ganglion cells Kidney Glomerulus Tubules Henle’s loop Urothelium Blood vessels Endothelium Smooth muscle

Human

Mouse

Normal Transgenic

Normal Transgenic

+ + + + +

+ + + + +

+++

+ + + + + + + + + + + + +

+ + + + + + + + + + ND

+++ +++ ++ ++ +++

-+++

ND ND + + +

+ + ÷ -+ + +

+++ --

ND ND

+/-

+/-

-/+

-/+

+++ ++

+++

++

++/-

+++

-/+

-

-

-

--

-

+/+ + +

+/ND

++

++

+ +/-

+ + + + -

++

++

+ + -- / +

+ + - / +

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Table 2 (continued) Thy-1 Expression

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Tissue Skeletal Cardiac Connective tissue Splenic T cells Skin fibroblasts

Human

Mouse

Normal Transgenic

Normal Transgenic

muscle muscle + +

.... .... + +

+ + + +

-

Thy-| expressionwasdeterminedby indirect imnaunofluorescence staining of cryostat sections, isolated splenic T-lymphocytes,and cultured fibroblasts (Gorden et al (79); Rettig et al (33); and P. Garin-Chesea, W.J. Rettig, J. Silver, J. W.Gordon,unpublishedresults). + + +, + +, +, -, strong, moderate, weak,and negative staining; + +/-, heterogeneityin antigen expression;ND,not determined.

similar to the lack of HLAclass-I expression on the DAUDI cell line, which results from a deletion in the gene for the HLAclass I-linked beta2-m subunit (85). Consequently, expression of the intact subunits (CD11 and HLAclass I, respectively) is restored through structural complernentation in fusions with rodent cells expressing hornologues of the missing subunits (86, 87). A second surface antigenic system that has been studied with mutant cell lines is the group of HLAclass-II molecules. For example, several in vitro variants have been derived from class II-positive humanB-cell lines which contain intact class-II genes but lack regulatory factors necessary for class-II gene expression (88, 89). Complementation analyses with heterokaryons formed between these variant lines and similarly class II-deficient humanlymphoblastoid cells from patients with "bare lymphocyte syndrome" [an inherited immunodeficiency syndrome associated with lack of class-II expression (90)], has identified at least two distinct trans-regulatory elements for class-II expression (91). In addition, fusion of one of the class II-negative humanvariants, RJ2.2.5, with mouse B cells has been shownto restore expression of humanclass-II antigens. This regulatory activity has been mappedto a locus on mousechromosome 16, designated air-1 (92). The relationship between mouse air-1 and the humantrans-acting regulators defined through complementation analysis in heterokaryons has not yet been determined. Finally, class-II expression in the RJ2.2.5 variant is partially restored by transfection with activated ras genes (93). The apparent multiplicity of cis- and trans-acting regulatory factors mayhelp explain whyclass-II antigens are constitutively expressed

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in somecell types, such as B cells, macrophages, melanomas, and cultured melanocytes transfected with activated ras genes(but not in nontransfected melanocytes) (94, 95), but are expressedonly in responseto specific extrinsic signals in other cell types, suchas mitogen-and antigen-stimulatedT cells andIFN-stimulatedfibroblasts and endothelial cells (96, 97). Adifferent type of variant formation is seen in humanneuroblastoma cell lines. Thesecells showa reversible but heritably stable transition betweentwo distinct differentiation programs,referred to as N/Stransdifferentiation (98), whichleads to a coordinatechangein the expression of morethan a dozencell surface differentiation antigens (Figure 3) (24), Neuroblastoma variants interneuroblastic mediate epithelial/fibroblastic

~z~ Antigen

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~,

,

.o- ~lnll ilnnnn,

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~1500 ~1500

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, nnnnnuuuu ,~1500 o ~’~oo° nNNnNnNNN InI.--II1~ q2 ~

lml.ll~~

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,,. °NNIluununInnnuIIIaNIn~;~ .o,. °NuNnNNNnNNNNNNNN_nnnN Figure 3 Coordinate change in cell surface antigen expression during N/S transdifferentiation of humanneuroblastoma cells. Neuroblastic, intermediate, and epithelial/ fibroblast-like variants derived from a single tumor share a commonclonal origin, and interconversion is reversible in several of these cell lines (24). Filled boxes indicate homogeneous antigen expression within a culture; cross-hatched boxes indicate heterogeneity in antigen expression with < 30%of antigen-positive cells.

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in addition to other biochemical and structural changes (98, 99). These changes apparently occur as all-or-none phenomenawith no evidence of intermediate forms; thus, they most likely represent transitions between various neuroectodermal lineages or alternative phenotypic programs characteristic for distinct neuronal, glial, melanocytic and ectomesenchymal cell types (24, 98-100). Since the antigens that are coordinately modulated during N/S transdifferentiation are encoded by genes located on at least five different chromosomes (24), it is possible that their expression on neuroblastoma cells is controlled by commontrans-acting regulatory factors. SUMMARY

AND

PERSPECTIVE

Ideas about the surface antigenic structure of mammaliancells have evolved through several phases during the last three decades. Prior to the impact of serological analysis, there was a general notion coming from transplantation studies with tolerant and chimeric animals that cells from different lineages shared a commonsurface antigenic phenotype. The discovery of differentiation antigens, such as TLand Lyt, gave rise to the nowwidelyacceptedidea that the surface of cells reflects their differentiated state and that cells undergoing distinct pathwaysof differentiations have unique surface markers. With the greatly expanded analysis of the cell surface now possible with mAbsand new techniques for antigenic analysis of tissues, there has been ample confirmation of the idea that the cell surface is a highly differentiated structure. However,lineage-restricted antigens turn out to be the exception, and antigens like Thy-1, whose distribution does not conformto embryonicderivation or lineage are the ones commonly found. Despite detailed mappingof over 100 antigenic systems, no general rules or organizing principles have emerged that would allow predictions about newly defined antigens. Although coordinate expression of two or more differentiation antigens can be seen within a particular lineage, these coordinate expression patterns are generally not observed whenother lineages are examined. In addition, some humanchromosomescarry a large number of coding genes for cell surface antigens, but with the notable exception of MHC,Ig, and Tcr genes, genomicclustering of genes for differentiation antigens is not the rule. Even in those cases where a function has been assigned to a surface molecule, e.g. NGFreceptor or EGFreceptor, unexpected cross-lineage patterns of tissue expression have been observed. Understanding this observed complexity in cell surface phenotype is unlikely to comefrom defining further examplesof differentiation antigens, but will require decoding the signals that initiate, regulate, and maintain

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the differentiated state. In fact, the cell surface maybe the modelpar excellence for unravelling the complexity of differentiation. Somatic cell hybridization and gene transfer provide powerful approaches to defining the regulatory signals controlling surface antigenic phenotype. Four modes of such genetic control have been defined through the study of hybrids of humanand rodent cells having congruent or incongruent differentiation programs: permissive, nonpermissive, inducing, and non-inducing. In this way, intrinsic signals can be defined and subsequently’isolated. Extrinsic signals such as ECMand cytokines also modulate the surface phenotype of cells, but the analysis of these influences and the numberof antigenic systems investigated are too limited to draw any general conclusions about in vivo importance. What lessons then can be drawn at this stage of the analysis? (i) From the large pool of genes coding for cell surface antigens, distinct but widely overlapping sets are activated during differentiation. (ii) The distinctive surface phenotype of differentiated cells is achieved not by the display of unique lineage-restricted antigens but rather through unique combinations of antigens that are drawn from a commonpool. (iii) Althoughthe surface phenotypeof differentiated cells is quite stable, there is considerable flexibility in reprogramming individual antigens. Surface antigens are not irreversibly silenced or expressed during differentiation but can be induced or suppressedby specific extrinsic/intrinsic signals. (iv) Individual surface antigens can be viewed as modular elements that together generate complex and unique surface patterns. This concept of modularity in the construction of cell surfaces could also explain unique functions achievable with commonelements, since the same module in different cell types would comeunder different regulatory control and could impart different information.

Literature Cited 1. Landsteiner, K. 1901. Qber Agglutinationserscheinungen normalen menschlichen Blutes. Wien. klin. Wochschr. 14:1132-34 2. Gorer, P. A. 1937. The genetic and antigenetic basis of tumor44:691 transplantation. J. Pathol. Bacteriol. 3. Nathanson, S. G., Uehara, H., Ewenstein, B. M., Kindt, T. J., Coligan, J. E. 1981. Primary structural analysis of transplantation antigens of the murine H-2 major histocompatibility complex. Ann. Rev. Biochem. 50: 102552 4. Lopezde Castro, J. A., Barbosa, J. A., Krangel, M. J., Biro, P. A., Strominger,

5.

6. 7. 8.

J. L. 1985. Structural analysis of the functional sites of class I HLA antigens. Immunol. Rev. 85:149-68 Williams, A. F., Gagnon, J. 1982. Neuronal cell Thy-1 glycoprotein: homology with immunoglobulin. Science 216:696-703 Watkins, W. M. 1966. Blood group substances. Science 152:172-81 Ginsberg, V. 1972. Enzymatic basis for blood group types in man. Adv. Enzymol. 36:13149 Chen, Y.-T., Obata, Y., Stockert, E., Takahashi, T., Old, L. J. 1987. Tlaregion genes and their products. Immunol. Res. 6:30-45

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nomenclature committee. Cytogenet. 9. Old, L. J., Stockert, E. 1977. Immunogeneticsof cell surface antigens of Cell Genet. 46:29-101 mouseleukemia.~Inn. Rev. Genet.11: 23. Rettig, W.J., Garin Chcsa, P., Jen127-60 nings, M. T., Spengler, B. A., Mela10. Allison.J. P. 1987.Structure,function, reed, M. R., Oettgen, H. F., Biedler, and serology of the T-cell antigen J. L., Old,L. J. 1985.Cellsurfaceantigen of humanneuroblastomasis relareceptor complex.Ann. Rev. Immunol. ted to nuclear antigen of normalcells. 5:503-40 I1. Levy,R,, Levy,S., Cteary, M.L., CarProc. Natl. Acad. Sci. USA82: 689498 roll, W., Kon,S., Bird, J., Sklar, J. 1987. Somatic mutations in humanB24. Rettig, W.J., Spengler, B. A., Garin cell tumors. Immunol.Rev. 96:43-58 Chesa,P., Old,L. J., Biedler,J. L. 1987. 12. Srivastava,P. K., Old,L. J. 1988.IndiCoordinatechangesin neuronal phenovidually distinct transplantationantitype and surface antigen expression gens of chemically induced mouse in humanneuroblastomacell variants. tumors. Immunol.Today 9:78-84 CancerRes. 47:1383-89 13. Old, L. J. 1981. Cancer immunology: 25. Rettig, W.J., Cordon-Cardo,C., Ng, Thesearchfor specificity. CancerRes. J. S. C., Oettgen, H. F., Old, L. J., Lloyd, K. O. 1985. High-molecular41:361-75 14. Real, F. X., Furukawa,K. S., Mattes, weight glycoproteinsof humanteratocarcinoma defined by monoclonal M.J., Gusik,S. A., Cordon-Cardo, C., Oettgen,H. F., Old,L. J., Lloyd,K. O. antibodies to carbohydrate deter1988. Class 1 (unique) tumorantigens minants. CancerRes. 45:815-21 of humanmelanoma:identification of 26. Rettig, W.J., Dracopoli,N. C., Garin unique and common epitopes on a 90Chesa,P., Spengler,B. A., Beresford, kDaglycoprotein. Proc. Natl. Acad. H. R., Davies,P., Biedler, J. L., Old, Sci. USA85:3965-69 L. J. 1985. Role of humanchromosome 15. DePlaen, E., Lurquin,C., VanPel, A., 11 in determining surface antigenic phenotype of normal and malignant Mariame, B., Szikora,J.-P., W61fel,T., Sibille, C., Chomez, P., Boon,T. 1987. humancells. Somaticcell geneticanalyImmunogenic turn- variants of mouse sis of eight antigens,includingputative tumor P815: cloning of the gene of humanThy-1. J. Exp. Med.162: 1603tum- antigen P91Aand identification 19 of the tum mutation. Proc. Natl. 27. Rettig, W. J., Nishimura, H., YenAcad. Sci. USA85:2274-78 amandra,A., Seki, T., Obata,F., Bere16. Boyse, E. A., Old, L. J. 1969. Some sford,H. R., Old,L. J., Silver, J. 1987. aspects of normal and abnormalcell Differential expression of the human Thy-1 gene in rodent-humansomatic surface genetics. Ann. Rev. Genet. 3: 269-90 cell hybrids. J. Immunol.138:4484-89 17. Boyse, E. A., Old, L. J. 1978. The 28. Fradet, Y., Cordon-Cardo,C., Thomimmunogenetics of differentiation in son, T., Daly,M. E., Whitmore,W.F., the mouse.HarveyLect. 71:23-53 Lloyd, K. O., Melamed,M. R., Old, 18. K6hler, G., Milstein, C. 1975. ConL. J. 1984.Cell surfaceantigensof hutinuous culture of fused cells secremanbladder cancer defined by monoting antibodyof predefinedspecificity. clonal antibodies. Proc. Natl. Acad. Nature 236:495-97 Sci. 81:224-28 19. Hulett, H., Bonner,W., Barrett, J., 29. Rettig, W.J., Dracopoli,N. C., GoetzHerzenberg, L. 1969.Cell sorting: autoger, T. A., Spengler, B. A., Biedler, mated separation of mammalian cells J. L., Oettgen,H. F., Old, L. J. 1984. as a function of intracellular fluorSomaticcell genetic analysis of human escence. Science 166:747-49 cell surface antigens: chromosomal 20. Polak, J. M., VanNoorden,S. 1983. assignmentsand regulation of antigen Immunocytochemistry. Practical appliexpression in rodent-human hybrid cations in pathology and biology. cells. Proc. Natl. Acad. Sci. USA81: Wright,pp. 1 6437-41 21. Magnani, J. L. 1987. Mouseand rat 30. Kantor, R. R. S., Bander, N. H., monoclonalantibodies directed against Finstad, C. L., Graf, L. H., Lloyd, carbohydrates. Meth. Enzymol. 138: K. O., Old, L. J., Albino,A. P. 1987. 484-92 DNA-mediatedgene transfer of a 22. McAlpine,P. J., VanCong,N., Bouhumancell surface 170-kilodaltonglycheix, C., Pakstis, A. J., Doute,R. C., coprotein. Evidencefor association Shows,T. B. 1987. The1987catalog of with an endogenous murineprotein. 3". mapped genes and report of the Biol. Chem.262:15166-71

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31. Rettig, W. J., Murty, V. V. V. S., Mattes, M. J., Chaganti, R. S. K., Old, L. J. 1986. Extracellular matrix-modulated expression of humancell surface glycoproteins A42and J143. Intrinsic and extrinsic signals determine antigenic phenotype. J. Exp. Med. 164: 1581-99 32. Hemler, M. E. 1988. Adhesive protein receptors on hematopoietic cells. Immunology Today 9:109-13 33. Rettig, W. J., Garin-Chesa, P., Reresford, H. R., Oettgen, H. F., Melamed, M. R., Old, L. J. 1988. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells. Proc. Natl. Acad. Sci. USA85: 311214 34. Rettig, W. J., Garin Chesa, P., Beresford, H. R., Feickert, H.-J., Jennings, M., Cohen, J., Oettgen, H., Old, L. J. 1986. Differential expression of cell surface antigens and glial fibrillary acidic protein in humanastrocytoma subsets. Cancer Res. 46:6406-12 35. Rettig, W. J., Grzeschik, K. H., Yenamandra, A. K., Garcia, E., Chaganti, R. S. K., Old, L. J. 1988. Definition of selectable cell surface markers for human chromosomes and chromosome segments in rodent-human hybrids. Somat. Cell Mol. Genet. 14:223-31 36. Rettig, W. J., Cordon-Cardo, C., Koulos,J. P., Lewis, 3. L. Jr., Oettgen, H. F., Old, L. J. 1985. Cell surface antigens of human trophoblast and choriocarcinoma defined by monoclonal antibodies. Int. J. Cancer 35: 469-75 37. McMichael,A. J., Beverley, P. C. L., Gilks, W., Horton, M., Mason, D. Y., Cobbold, S., Gotch, F. M., Ling, N., Milstein, C., Waldmann, H., Crumpton, M. J., Hogg, N., MacLennan, I. C. M., Spiegelhalter, D. 1987. Leucocyte Typing III. White Cell Differentiation Antioens. Oxford: Oxford Univ. Press 38. Fukuda, M. N., Bothner, B., Lloyd, K. O., Rettig, W.J., Tiller, P. R., Dell, A. 1986. Structures of glycosphingolipids isolated from human embryonal carcinoma cells. J. Biol. Chem. 261: 5145-53 39. Rettig, W. J., Garin-Chesa, P., Beresford, H. R., Melamed, M. R., Old, L. J. 1987. Definition of an extracellular matrix protein in rostral portions of the human central nervous system. Brain Res. 438:312-22 40. Greaves, M. F. 1982. "Target" cells, cellular phenotypes,and lineage fidelity

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CELL SURFACE DIFFERENTIATION mosome17 and regulation of receptor expression in somatic cell hybrids. Somat. Cell Mol. Genet. 12:441-47 51. Brook, J. D., Beresford, H; R., Shaw, D. J., Old, L. J., Rettig, W. J. 1987. Cytogenet. Cell Genet. 45:156-62 52. Dracopoli, N. C., Rettig, W. J., Albino, A. P., Esposito, D., Archidiacono, N., Rocchi, M., Siniscalco, M., Old, L. J. 1985. Genes controlling gp25/30 cellsurface molecules map to chromosomes X and Y and escape X-inactivation. Am. J. Hum. Genet. 37: 199207 53. Rettig, W. J., Yenamandra,A., Grzeschik, K.-H., Martiniuk, F., Hirschhorn, R., Jennings, M., Chaganti, R. S. K., Old, L. J. 1987. Genetic characterization of humancell surface antigens: general application of erythrocyte rosetting assays to hybrid cell analysis. Cytogenet. Cell Genet. 46:681 54. Ruddle, F. H. 1981. A new era in mammalian gene mapping: somatic cell genetics and recombinant DNAmethodologies. Nature 294:115-20 55. Bill, J., Palmer, E., Jones, C, 1987. Molecular cloning of MER-2a human chromosome1 l-encoded red cell antigen using linkage of cotransfected markers. Somat. Cell Mol. Genet. 13: 55361 56. Solter, D., Knowles,B. B. 1978. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA1). Proc. Natl. Acad. Sci. USA 75: 5565-69 57. Schuller-Petrovic, S., Gebhart, W., Lassmann, H., Rumpold, H., Kraft, D. 1983. A shared antigenic determinant between natural killer cells and nervous tissue. Nature 306:179 81 58. Geurts van Kessel, A., Tettero, P., van Agthoven, T., Paulussen, R., van Dongen, J., Hagemeijer, A., yon den Borne, A. 1984. Localization of human myeloid-associated surface antigen detected by a panel of 213 monoclonal antibodies to the ql2-qter region of chromosome I1. J. Immunol. 133: 1265-69 59. Williams, A. F., Barclay, A. N. 1988. The immunoglobulin superfamily. Domainsfor cell surface recognition. Ann. Rev. lmmunol. 6:381-405 60. Hynes, R. O. 1987. Integrins: a family of cell surface receptors. Cell 48: 54951 61. Springer, T. A., Dustin, M. L., Kishimoto, T. K., Marlin, S. D. 1987. The lymphocyte function-associated LFA1, CD2, and LFA-3 molecules: cell adhesion receptors of the immune

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Webster, S. G., Miller, S. C., Webster, C. 1985. Plasticity of the differentiated state. Science 230:758-66 74. Kavathas, P., Sukhatme, V. P., Herzenberg, L. A., Parnes, J. R. 1984. Isolation of the gene encoding the human T-lymphocyte differentiation antigen Leu-2 (TS) by gene transfer and cDNA subtraction. Proc. Natl. Acad. Sci. USA 81 : 7688-92 75. Littman, D. R., Thomas, Y., Maddon, P. J., Chen, L., Axel, R. 1985. The isolation and sequence of the gene encoding TS: a molecule defining functional classes of T lymphocyes. Cell 40:237 46 76. Kavathas, P., Herzenberg, L. A. 1983. Stable transformation of mouseL cells for human membrane T-cell differentiation antigens, HLAand/~2-microglobulin: selection by fluorescence-activated cell sorting. Proc. Natl. Acad. Sci. USA 80:524 77. Rabourdin-Combe, C., Mach, B. 1983. Expression of HLA-DR antigens at the surface of mouseL cells cotransfected with cloned humangenes. Nature 303: 670-74 78. Boss, J. M., Strominger, J. L. 1986. Regulation of a transfected human class-II major histocompatibility complex gene in humanfibroblasts. Proc. Natl. Acad. Sci. USA 83:9139-43 79. Gordon, J. W., Garin Chesa, P., Nishimura, H., Rettig, W. J., Maceari, J. E., Endo,T., Seravalli, E., Seki, T., Silver, J. 1987. Regulation of Thy-1 gene expression in transgenic mice. Cell 50: 445-52 80. Kollias, G., Evans, D. J., Ritter, M., Beech, J., Morris, R., Grosveld, F. 1987. Ectopic expression of Thy-1 in the kidney of transgenic mice induces functional and proliferative abnormalities. Cell 51:21-31 81. Kollias, G., Spandoulou, E., Grosveld, F., Ritter, M., Beech, J., Morris, R. 1987. Differential regulation of Thy-I gene in transgenic mice. Proe. Natl. Acad. Sci. USA 84:1492-96 82. Kievitz, F., Ivanyi, P., Krimpenfort,P., Berns, A., Ploegh, H. L. 1987. HLArestricted recognition of viral antigens in HLAtransgenic mice. Nature 329: 447-49 83. Dill, O., Kievits, F., Koch, S., Ivanyi, P., Hammerling, G. J. 1988. Immunological function of HLA-Cantigens in HLA-Cw3transgenic mice. Proc. Natl. Acad. Sci. USA 85:5664~8 84. Kishimoto, T. K., Hollander, N., Roberts, T. M., Anderson, D. C., Springer, T. A. 1987. Heterogeneous

mutations in the/3 subunit commonto the LFA-1, Mac-l, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell 50:193-202 85. Ploegh, H. L., Cannon, L. E., Strominger, J. L. 1979. Cell-free translation of the mRNA for the heavy and light chains of HLA-Aand HLA-Bantigens. Proc. Natl. Acad. Sci. USA 76: 227377 86. Marlin, S. D., Morton, C. C., Anderson, D. C., Springer, T. A. 1986. LFA1 immunodeficiencydisease: definition of the genetic defect and chromosomal mapping of alpha and beta subunits of the lymphocyte function-associated antigen 1 (LFA-1)by complementation in hybrid cells. J. Exp. Med. 164: 85567 87. Arce-Gomez, B., Jones, E. A., Barnstable, C. J., Solomon, E., Bodmer, W. F. 1978. The genetic control of HLAoA and B antigens in somatic cell hybrids: requirement for/32-microglobulin. Tissue Antigens 11:96-112 88. Gladstone, P., Pious, D. 1980. Identification of a trans-aeting function regulating HLA-DRexpression in a DRnegative B cell variant. Somatic Cell Genet. 6:285-98 89. Accolla, R. S., Scarpellino, L., Carra, G., Guardiola, J. 1985. Trans-acting element(s) operating across species barriers positively regulate expression of major histocompatibility class II genes. J. Exp. Med. 162:1117-33 90. Griscelli, C., Durandy,A., Virelizier, J. L., Hors, J., Lepage, V., Colombani, J. 1980. Impaired cell to cell interactions in partial immunodeficiencywith variable expression of HLAantigens. In Primary Immunodeficiencies, ed. M. Seligrnan, H. Hitzig, pp. 499-503. Amsterdam: Elsevier 91. Yang, Z., Accolla, R. S., Pious, D., Zegers, B. J. M., Strominger,J. L. 1988. Two distinct genetic loci regulating class II gene expression are defective in humanmutant and patient cell lines. EMBOJ. 7:1965-73 92. Aecolla, R. S., Jotterand-Bellano, M., Scarpellino, L., Maffei, A., Carra, G., Guardiola, J. 1986. air-l, a newly found locus on mouse chromosome 16 encoding a trans-acting activator factor for MHCclass II gene expression. J. Exp. Med. 164:369 93. Hume,C. R., Accolla, R. S., Lee, J. S. 1987. Defective HLAclass II expression in a regulatory mutant is partially complemented by activated ras oncogenes. Proc. Natl. Acad. Sci. USA84: 8603-7

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CELL SURFACE DIFFERENTIATION 94. Winchester, R. J., Wang,C. C,, Gibofsky, A., Kunkel, H. G., Lloyd, K. O., Old, L. J. 1978. Expression of Ia-like antigens on cultured humanmalignant melanomacell lines. Proc. Natl. Acad. Sci. USA 75:6235-39 95. Albino, A. P., Houghton, A. N., Eisinger, M., Lee, J. S., Kantor, R. R. S., Oliff, A. I., Old, L. J. 1986. Class II histocompatibility antigen expression in humanmelanoeytes transfected with Harvey murine sarcoma virus (HaMSV)and Kirsten MSVretroviruses. J. Exp. Med. 164:1710-22 96. Ko, S.-S., Fu, S. M., Winchester, R. J., Yu, D. T. Y., Kunkel, H. G. 1979. Ia determinants on stimulated human T lymphocytes. Occurrence on mitogenand antigen-activated T cells. J. Exp. Med. 150:246-55 97. Pober, J. S., Collins, T., Gimbrone, M. A. Jr., Cotran, R. S., Gitlin, J. D., Fiers, W., Clayberger, C., Krensky, A. M., Burakoff,J. J., Reiss, C. J. 1983. Lymphocytes recognize human vascular endothelial and dermal fibroblasI la antigens induced by recombinant immuneinterferon. Nature 305:726-29 98. Ross, R. A., Spengler, B. A., Biedler, J. L. 1983. Coordinate morphological and biochemical interconversion of humanneuroblastoma cells. J. Natl. Cancer Inst. 71:741~19 99. Ross, R. A., Biedler, J, L. 1985. Pres-

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ence and regulation of tyrosinase activity in humanneuroblastoma cell variants in vitro. CancerRes. 45: 162832 100. Tsokos, M., Ross, R. A., Triche, T. J. 1985. Neuronal, Schwannian, and melanocytic differentiation of human neuroblastoma cells in vitro. In Advances in Neuroblastoma Research, eds. A. E. Evans, G. J. D’Angio, R. C. Seeger, pp. 55q58. NewYork: Liss 101. Goyert, S. M., Ferrero, E., Rettig, W. J., Yenamandra, A., Obata, F., LeBeau, M. M. 1988. The CD14monocyte differentiation antigen maps to a region encoding growth factors and growth factor receptors. Science 239: 497-500 102. Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V., Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., Koprowski, H. 1988. Molecular cloning and characterization of an antigen associated with early stages of melanomatumor progression. Cancer Res. 48:2955~52 103. Look, A. T., Pciper, S. C., Rcbentisch, M. B., Ashmun,R. A., Roussel, M. F., Lemons, R. S., LeBeau, M. M., Rubin, C. M., Sherr, C. J. 1986. Molecular cloning, expression and chromosomal localization of the gene encoding a human myeloid membraneantigen. J. Clin. Invest. 78:914-21

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Ann. Re~.lmmunol.1989. 7:513-35

PROBING THE HUMAN B-CELL REPERTOIRE WITH EBV: Polyreactive Antibodies and CD5÷ 1’ B 2Lymphocytes Paolo Casali and Abner Louis Notkins Laboratory of Oral Medicine, Building 30, Room121, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

INTRODUCTION According to the clonal selection theory of antibody production, the tremendousantibody diversity found in biological fluids is the expression of the large heterogeneity and variety of antibody-producing cell clone precursors (14). Knowledgeof the diversity of the available B-cell repertoire at the clonal level is, therefore, necessary to attempt a definition of the repertoire that could be expressed at any given time--the actual repertoire. Because of the resting state of most lymphocytesin the normal B-cell repertoire, the use of a polyclonal B-cell activator (PBA)is essential to express the antibody-producing potential of each cell clone. An ideal PBAmust act directly on B cells, independent of any T-cell help, and induce "unbiased" B-cell clonal activation (5). For mouselymphocytes, number of different macromolecules have been used as PBAs, including dextran sulfate, purified protein derivative of tubercule bacteria. Nocardia opaca water soluble mitogen, and bacterial lipopolysaccharide (LPS) (5, ~ The USGovernmenthas the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper. 2 Abbreviations: Ag, antigen; EBV,Epstein-Barr virus; HD,Hashimoto’sdisease; Ig, immunoglobulin; lgH chain, lg heavy chain; Ins, insulin; LPS, lipolysaccharide; mAb,monoclonal antibody; PBA, polyclonal B-cell activator; RA, rheumatoid arthritis; RF, rheumatoid factor; SLE, systemic lupus erythematosus; ssDNA, single-stranded DNA;TT, tetanus toxoid; Tg, thyroglobulin; TMAg,thyroid microsomal Ag.

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6). For humanlymphocytes, however, the only efficient T cell-independent PBAavailable is Epstein-Barr virus (EBV). The main biological activity of EBVis the infection of lymphocytes, particularly B cells (7, 8), which results in blast formation, immunoglobulin(Ig) secretion, and, ultimately, indefinite cell proliferation--a process termed "immortalization" (8). In this chapter, we summarize experiments from this and other laboratories_ concerning: (a) the use of EBVas a tool to probe the human cell repertoire; (b) the enumerationof cell precursors producing antibody to self- and exogenousantigens (Ags) in healthy subjects; (c) the detection and nature of polyreactive antibodies; (d) the identification of the lymphocyte subset (CD5+) committed to the production of polyreactive antibodies; and (e) the study of the B-cell repertoire in autoimmune conditions.

EBV AS POWERFUL TOOL TO PROBE THE HUMAN B-CELL REPERTOIRE The unique advantage of EBVas a tool to study the human B-cell repertoire is that it serves as both a potent B-cell activating stimulus and an efficient transforming agent. Whenused at high multiplicity of infection, EBVinfects at least every second blood B cell in culture (9-11). EBVinduced activation, Ig secretion, and transformation of human B lymphocytes are one-hit and dose-dependent phenomena (8, 9, 12). Two sequential, discrete events take place after incubation of a B cell with EBV. First, the virus attaches to the surface receptor CR2,which also mediates the binding of the complement C3d split product. This receptor for C3d/EBV is present on all mature B cells and late pre-B cells (13). In most cases, subsequent virus internalization leads to infection of the cell, as revealed by the expression of EBV-relatedAgs, and then to cell activation. Second, after viral infection, actual cell transformation mayensue, yielding cell blasts capable of continuous proliferation and steady Ig secretion. Although EBV-transformed B cells are a useful source of monoclonal antibodies (mAbs), they can be cloned only with difficulty at low numbers (less than 5 cells/well), and their growth and antibody production are erratic when cultured beyond a short period (3-4 weeks). Weand others recently overcamethese limitations by using somatic hybridization techniques involving a non Ig-secretor, oubain-resistant and azaserine-sensitive, human-mousefusion partner (11, 14, 15). Fusion of EBV-transformed B cells with a human-mousehybrid provides a very efficient approach to the construction of mAb-secretingcell lines. Indeed, the resulting humanhuman-mousehybrid clones display muchhigher plating efficiency, stab-

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ility, and antibody secretion rate than do their parental EBV-transformed B cells in long-term culture (Figure 1). EBVis equally efficient in transforming B cells bearing surface/~, 7, and a IgH chains to produce IgM, IgG, and IgA, respectively. Indeed, we recently showed that the percentage of peripheral blood B lymphocytes bearing surface #, ~,, and a IgH chain are similar to the percentages of cells producing IgM, IgG, and IgA, respectively, after EBV-infection (Table 1: Cells sorted for surface CD20)(11). Moreover, EBVbinds with equal efficiency to B cells bearing IgH chains of all different isotypes as shown in dual color fluorescence flow cytometry studies (16) (Figure 2). Finally, in cell fractions homogeneous for different surface IgH chain, the class of the Ig produced is highly consistent with the IgH chain isotype for which the same cells were selected (Table 1) (11). Therefore, the preponderant igM production found after EBV-infection of a whole B-cell population (17-20) is not due to a skewedtropism of the virus for cells bearing surface /~H chain; it simply reflects the higher frequency of these lymphocytesin the normal B-cell repertoire. Furthermore, the results of our experiments (11) and those by others (12) are inconsistent with any significant rate IgH chain isotype switch as a result of EBV-transformation. Although the above findings show that no restriction exists in EBV

0’~

60-

= _~ ~o

° 20

10

5

2

~

0.5

NUMBEROF CELLS SEEDEDPER WELL

Fi#ure 1 Cloning efficiency of monoclonal EBV-transformedhybrid cells (closed circles) and their parental EBV-transformedcells (open circles). Different EBV-transformedB-cell clones were generated, expanded, and then separately fused with F3B6heterohybrid cells. The resulting human-human-mousecell hybrids were cloned. Parental EBV-transformed B ceils and EBV-transformed cell hybrids were seeded at 20, 10, 5, 2, 1 and 0.5/well containing irradiated feeders. The number of growing cultures was determined 2 and 6 weekslater for EBV-transformedhybrid cells and parental EBV-transformedB cells, respectively. Circles are meanvalues + standard deviation (vertical bars) of the percent of outgrowing microcultures from 6 EBV-transformedhybrid cells and their parental EBV-transformedB cells. (M. Nakamura,P. Casali. 1986, unpublished data).

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aTable 1 IgH chain isotype commitmentof B lymphocytes infected

with EBV

B cells ( × 102) producingb B cells sorted for surface

IgM

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B1 (CD20) #-Ig H Chains 7-Ig H Chains ,-Ig H Chains

IgG

Frequency of Immortalized °Cells (× 102)

IgA

86.3+4.1 5.4+ 1,5 99.6+0.8 0.5+0.2 0.3___0,3 99.6+0.7 0.9___0,4 0.5+0.3

5.4+ 1.0 0.3+0.2 0.1__+0.2 98.6+0.9

3.20+0,35 3,30+0.40 2.90+0,30 4.00+0.50

~Surface?, ~, or ,u Ig Hchain+ B lymphocyteswere purified by FACS to yield homogenous cell fractions, infected with EBV and then cultured in limiting dilution conditionin presenceof irradiated ÷ feeders. Purified B(CD20) cells weresimilarly processed.Aftera 4 w~ekculture, fluids wereassayedfor Ig content. b Lymphocytes producingIg of various classes as a percentageof total Ig-producingB cells (approximately1:2.5 plated cells), as calculated by analysis accordingto Poissondistribution. Meanvalue standarddeviationof three experiments using B cells fromdifferent donors. c Immortalized cells as percentof plated B cells.

10~ -

!

A

~O 10 2 ~r biot-EBV

,0,1............ C’ mAb to 19M

~ 10’ 10~ 10

0’

E’

mAb to IgD

mAb to 19G

mAb to IgA

101 102 10’

101 102 10’

z 101 10= 10

RELATIVE FLUORESCENCE INTENSITY (Green) Figure 2 Binding of biotinylated (biot)-EBV to B lymphocytes bearing surface Ig H chains of different isotypes. Enriched B lymphocytepreparation (containing 70%B cells) from one donor were simultaneously incubated with biot-EBV and mAbsto different IgH chain isotypes. All cell samples were then incubated with phycoerytrin-conjugated (PE-)avidin and fluorcsceinated (FITC-) goat F(ab’)~ fragment anti-mouse Ig and submitted to fluorescence flow cytometric analysis. Red fluorescence (bound biot-EBV) and green fluorescence (bound mAb)intensity of each cell were measured and contourgrams derived. Control samples were incubated with: MOPC21 mAb(mouse IgG1 with irrelevant specificity) (Panel A’) or MOPC21 and biot-EBV (Panel A). Experimental cell samples incubated with biot-EBV and one of the specific mouse antibodies including mAbto/t lg H chain (Panel B), mAbto ~ Ig H chain (Panel C), mAbto ~ lg H chain (Panel D), or a mixture ofmAbsto ~ and c~2 Ig H chains (Panel E). Panels B’ through E’ represent the contourgrams derived from analysis of similar cells incubated with the same mAbs,but in the absence of biot-EBV. Modified from (16).

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PROBING THE HUMANB-CELL REPERTOIRE

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attachmentto andtransformationof different B-cell subsets, different phasesof the cell cycle do affect the ability of the virus to transform B cells. Ourrecentstudies usingpurifiedlabeledvirus andB cells cultured with F(ab’)2fragmentto # IgHchainandT-cell conditionedmedium (16) showedthat whereasEBV does bind to cells in G1phase(activated, low density, premitoticcells) almostas efficiently as it doesto cells in Go (resting, smallsize, highdensitycells) it doesnotbindto andinfect cells in S phase(low density, dividingcells). Disappearance of the cell surface CR2(Figure3) (16) parallels the lack of virus attachmentto andtransformation of proliferatingB cells (Figures3 and4). Thesefindingsprovide an explanationfor the failure of EBVto transformandimmortalizeproliferating B cells, as first suggestedby Aman et al (21) andRoome A

B

300 ~i 200t)//~ 100

m ~ ;

z

300 , 200 i ~

C ,~Ab ’ t

~o,-EBV

anti-CR2

BlmAb

~I ~~

mAb

[ I

biot-EBV

>

~ 2~

<

10 RELATIVEFLUORE~ENCE INTENSITY Figure 3 Binding of biot-EBV to activated and proliferating humanB lymphocytes: relationship to presence of surface CD2] (CR2) and CD20(B1) molcculcs. Ceils werc collected at the beginningandat 24, 48, or 72 hr of incubation from cultures inducedby goat F(ab’)~ fragment to humanIg # H chain and T-~II conditioned medium(TCM), and then analyzedusing flow cytometry for proportion of B lymphocytesbinding specific mAbs or biot-EBV. MAbsused were B1, which recognizes CD20,a pan-Bsurface markerunrelated to the C3d/EBVreceptor (Panel A), and a mAbto the C3d receptor (anti-CR2 mAb)(Panel B, full lines). ~ottedlines depictthe fluorescenceprofiles o£ ~]ls reacted with goat FITCF(ab’)= fragment to mouseIg only as negative controls. Panel C showsthe fluores~nce profile of cells reactedwith biot-EBVandFITC-avidin(full line) or with FITC-avidinonly (dottedline). Modifiedfrom (16).

Annual Reviews 518

CASALI & NOTKINS 1.0 0.8

~

~

I

A

0.6

o

0.2

0

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48h ’ ’

,0~24h

~x

[

~ 0.8

o 0.4 r~

u_ 0.2

0.1 10 25 50

100

200

NUMBER OF CELLS PER MICROCULTURE Figure 4 Immortalization rates of resting and proliferating human B lymphocytes by EBV. Induced B lymphocytes (see legend to Figure 3) plus lymphocytes from the same donor, cultured similarly, but in absence of goat F(ab’)~ fragment to human Ig and TCM(noninduced cells) were used. Cells harvested at 0, 24, 48 and 72 hr after initiation of the cultures were infected with EBVand then distributed at different number in microcultures. After 6 weeks, the fraction ofmicrocultures negative for c~ll transformation of the total microcultures originally seeded with non-induced (Panel A) or induced (Panel B) B lymphocytes plotted against the number of cells plated per microculture (cell dose), and the frequency immortalization of these B lymphocytes was calculated using Poisson distribution analysis (dotted lines show the cell dose at which 37% of microcultures are negative for transformation). For instance, the frequency of immortalization of B lymphocytes at time 0 was 1 : 32. Frequency of immortalization of proliferating B cells at 72 hr was less than 1 : 100,000 cells (too low to be plotted). Modified from (16).

Reading (22). Similarly, antibody-producing cells in terminal differentiation stage lose surface CR2 (23). Lack of EBVbinding to cells S phase or plasma cells could explain the lower rate of EBV-induced immortalization observed in cells spontaneously producing antibody in vivo, as is occasionally the case for cells obtained from subjects with an active autoimmune disease.

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ENUMERATION OF B LYMPHOCYTES PRODUCING ANTIBODY TO SELFAND EXOGENOUS ANTIGENS Havingestablished that EBVis equally efficient in inducing B lymphocytes committedto the production of Ig of different classes, we investigated the humanB-cell repertoire using EBVin limiting dilution assay. Whena single hit activation system is used in which only one cell (e.g. B lymphocyte) sufficient for a positive response (i.e. antibody production) (24, 25), limiting dilution analysis is an efficient and reliable methodto probe the humanB-cell repertoire for the frequencies of cells capable of producing different antibodies. Wemeasured the frequency of cell precursors producing autoantibodies to self-Ags, such as the Fc fragment of humanIgG, ssDNA, purified human thyroglobulin (Tg), recombinant humaninsulin (Ins), and purified humanthyroid microsomal antigen (TMAg),and antibodies to an exogenous Ag, such as tetanus toxoid (TT). All subjects studied had been previously vaccinated but not recently boosted with TT. The procedure involved infection with EBVof fresh lymphocytes and their immediatedistribution in microculture in the presence of irradiated peripheral blood mononuclear cells as feeders (11, 26). After a 4-week culture, the supernatant fluids were investigated for their content in total and Ag-specific IgM, IgG, or IgA. Frequencies of total or specific Igproducing cells were calculated by statistical analysis according to Poisson distribution (11, 12, 24). By using this approach, circulating cells capable of producing different classes of antibodies to self- and exogenousAgs can be readily detected in healthy subjects (11, 26): about 3-13% of IgMproducing cells made an antibody to IgG Fc fragment (rheumatoid factor, RF), ssDNA, Tg, TMAgand Ins, or TT (Table 2). AmongIgG-producing cell precursors, the low number (0.01-0.04%) of lymphocytes committed to the production of antibodies binding to self-Ags contrasts with the relatively higher frequency of cells (0.3%) committed to production antibody to TT. AmongIgA-producing cell precursors, the frequencies of those committed to producing antibodies to self-Ags are slightly higher than those of their IgG-counterparts, but still at least two orders of magnitude lower than those of IgM-cells with the same self-Ag-binding activities (Table 2). These findings argue against hypotheses of clonal abortion of self-reactive B-cell clones (4) and provide an explanation for the relative ease with which cell hybrids producing "autoantibodies" are generated when using lymphocytes from normal mice or humans to construct mAbproducingcell lines (27-32).

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Table 2 Frequencyof circulating cell precursors producingIgM, IgG, or IgA to self- and exogenous antigens in healthy subjects, HDand SLEpatients" B Cells (× 102) producing Ig

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Antigen Healthy Subjects ssDNA Tg TMAg Ins TT HDPatients ssDNA Tg TMAg Ins TT SLEPatients ssDNA Tg TMAg Ins TT

IgM

IgG

5.800+2.500 4.200+2.200 3.200+2.900 4.400___2.300 5.7004441.300

0.037___0.017 0.012+0.008 0.009+0.012 0.011+0.003 0.320+0.100

0.075+0.040 0.066+0.026 0.042+0.019 0.087+0.029 0.140+0.035

4.2274442.228 3.880-I- 1.910 3.210+__1.710 4.455+2.482 b 5.120 + 1.800

0.036 4- 0.008 0.251 +__0.282 0.193+__0.200 0.012+0.007 ¢ ND

0.068 _+ 0.032 0.062-I-0.031 0.050+0.034 0.072+0.034 ND

IgA

5.280 + 2.445 0.553___0.932 0.066 4- 0.035 3.748___2.880 0.007 ± 0.002 0.038 _ 0.025 3.920+3.148 0.011+0.006 0.041±0.021 4.900+3.288 0.0114-0.012 0.066_+0.032 5.200± 2.200 d 0.260__+0.100 d a0.120__+0.062

~B lymphocytesfrom17 healthy subjects, 13 HDand 12 SLEpatients wereinfected with EBVand culturcdin limitingdilution. Dataare meanvalues_+ SDof antibody-producing ccll precursor frequencies expressedas proportion of total lgM-,lgG-, or IGa-produ,cing cells. b Datafrom3 patients. ~ Notdetermined; dDatafrom4 patients.

THE NATURE OF POLYREACTIVE

ANTIBODIES

Theobservedhigh frequencyof B cells committedto the productionof antibodies,particularlyIgM,bindingto self-Agswaslikely dueto presence of a populationof antibodies,whichcan bindto morethan one single Ag. Indeed, in the last few years a numberof mAbsbinding multiple Ags, particularlyself-Ags, includingsolublehormones, nucleicacids, structural cellular constituents,tissue components, etc, havebeengeneratedin this andotherlaboratories(27-32). Although at first these polyreactivemAbs had been found to be produced by B lymphocytes from autoimmune patientsor chronicallyinfected mice(33-35), it became soonapparent that similar "autoantibodies" could in fact be derivedfromhealthysubjects (27, 28, 31). It wasalso clear that whilein a fewcasesthe detectedmultiple Ag-bindingactivity wasdueto the bindingof one single antibodyto an identical epitope presentin twodifferent molecules,in mostcases, the

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antibody recognition involvedtwo different epitopes (31, 36). Moreover, it wasrecognizedthat these polyreactive"autoantibodies"react efficiently also with exogenousAgs(31, 36). Thus, even though B cells committed to the production of polyreactive mAbsrecognizing multiple self- and exogenous Ags are knownto be commoncomponentsof the normal Bcell repertoire, their physiologicalor pathologicalroles are still unknown. IDENTIFICATION OF THE B-CELL TYPE (CD5 PRODUCING POLYREACTIVE ANTIBODIES

+)

Recently we identified the cell type that makesthese polyreactive antibodies. In mice, it has beenreported that Ly-1 + B lymphocytesmake autoantibodies(37). Thehumanequivalentof the mouseLy-1+Bcell is the CD5+B cell. In our studies, CD5+B cells werefoundto constitute a discrete subset in the normalB-cell repertoire, accountingfor up to 25%of total + and CD5-B circulating and splenic B lymphocytes(36, 38, 39). CD5 cells were purified fromhealthy subjects by fluorescence-activatedcell sorter (FACS)(Figure 5), infected with EBV,and cultured in limiting dilution condition. Our studies showedthat the vast majority of lymphocytescommittedto the production of IgM, IgG, and IgA to self-Ags, such as IgG Fc fragment, ssDNA,and Tg, and, also, IgMto TT came + B-cell population(Figure 6) (38, 39). In contrast, the fromthe CD5

uJ :’- 103

t ’~z

101 102

103 104

101 I0 ~ 103 104

10~

102 103

RELATIVEGREENFLUORESCENCE INTENSITY

+ and CD5B cells in a healthy Fiyure 5 FACSanalysis and selection of circulating CD5 subject. EnrichedBcells werereacted with PE-mAb to CD20 (red fluorescent), biotinylated mAbto CD5,and then FITC-avidin(green fluorescent). Double(red and green) fluorescent +, CD5 +) cells constituted CD5 + B lymphocytes.Thesecells, included in the right (CD20 +) dotted rectangle, andtheir (CD20 CD5-counterparts,includedin the left dotted rectangle, were sorted as CD5+ and CD5-B lymphocytes,respectively (Panel A). Contourgrams derived + are depicted in Panels fromthe reanalysis of someof the B cells sorted as CD5-and CD5 B andC, respectively. Insets representthe greenfluorescentprofiles of Bcells analyzedin + and CD5-B contourgramA, B and C. Contourgramsand profiles showthat sorted CD5 cells overlaponly marginally.Modifiedfrom(38).

Annual Reviews 522

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& NOTKINS IgGFc, IgM

2’2 I2.0f

2.2

ssDNA,IgM

¯

2.0

1.1

ssDNA, Ig

1.0 ¯

0,9 0.8

Annu. Rev. Immunol. 1989.7:513-535. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

1.4

1.4

0.8

0.8

0.6

0.6

0.6

|

0.3

t

0.4 0.2 0.0

0"2 f0.~

i + CD~

CD6- Unfractionated

1.1

Tg, IgM

0.0

1.1

1.0

+ CD5

CD5-Unfracfonated

~

¯

0.9

0.9

0.8

0,8

0.7

0.3

0.7

0.2 0.1

~

~

+ CD5

CD~-Unfraction ated

TT, IgM 1.11 1.0 0.9

0.7

I ¯

0.0

0.6

0.6:

0.5

0.5

0.3 i

0.7’

~ 0.0 + CD5-UnfractionCD5 CDS- Unfraction ated ated Figure 6 Antibodies produced by segregated CD5+, CDS-, and unfractionated B lymphocytes infected with EBV. Microculture plates were seeded with EBV-infected cells at various doses in the presence of irradiated feeder layers. After 4 weeks of culture, fluids were tested for Ag-binding activity. Each dot represents the concentration (expressed as absorbance at 492 nm) of antibodies with given Ag-binding activities in the culture fluid from a single microculture well. Approximately 100 microculture wells were assayed in each column. Microculture wells that had been seeded with 1000 cells per well were used, the exception being the assays for IgG to TT and ssDNA, for which plates that had been seeded

o.0

+ CD~

CDS-Unfractionated

with 2000 cells

0.0

+ CD5

per well were used. Modified from (39).

majority of cells committedto the production of IgG to TT segregated within the CD5-Blymphocytepopulation (Figure 6). To see whether these antibodies to self-Ags were polyreactive, continuous mAb-producingcell lines were constructed by EBV-transformation and somatic hybridization techniques using purified CD5÷B

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PROBING THE HUMANB-CELL

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REPERTOIRE

lymphocytesfromhealthysubjects (15). Morethan a dozencell clones, selected for productionof RF(IgMmAbbinding to IgGFc fragment) weregenerated(15, 39). All these mAbs werepolyreactive andbound a dose-dependent fashion to mostAgstested. TheAg-bindingcurvesof a representativepolyreactivemAb,Ab23,are depictedin Figure7, PanelA. Mostof the polyreactivemAbs also bound,althoughto different degrees, to variousbacterial LPSandpolysaccharides(15). In contrast, none the IgMmAbsproducedby the cell lines fromCD5-Bcells boundto any of the Agstested (Figure 7, Panel B). Thepolyreactivity of mAbsfrom ÷ B cells wasconfirmed CD5 in competitiveinhibition assays, in whichthe bindingof a given mAb to a solid-phaseAgwastested in the presenceof both homologous andheterologoussoluble Ags. For instance, bindingof Ab23to solid-phase IgGFc fragmentwasinhibited in a dose-dependent fashionandwithdifferent efficiency, not only by the soluble homologous

C

B

A

100-Fc fiagment

~.o

0.6

0.2

0,0 ~ 10 10 ~ 10 ~

10 ~ 0 10 2 10 ~ 10 ANTIBODY, #g / ml

~ 10

10 ~ 100

~ 10

2 10

.~g OF SOLUBLE LIGAND

Figure 7 Panel A: dose-dependent binding ofmonoclonal Ab23 (produced by a monoclonal cell line derived from CD5÷Bcells of a healthy subject) to solid-phase IgG Fc fragment (©), ssDNA(~), Tg (~,), Ins (0) and TT (I). The Ag-binding activity for each molecule expressed as optical absorbance (at 492 nm). The empty squares indicate the binding bovin serum albumin, which was used in the dilution buffer. Panel B: Failure of monoclonal Ab 207 (produced by a monoclonal cell line derived from CD5-Bcells of the same donor used for the generation of Ab23) to bind to solid-phase self- and exogenous Ags (the same used in experiments of Panel A). Panel C: Dose-dependent inhibition of monoclonal Ab23 mAbbinding to solid-phase lgG Fc fragment by soluble homologous(IgG Fc fragment) and heterologous (ssDNA, Tg, Ins and TT) ligands. Samples of the antibody (0.2 ~g) incubated with increasing amounts (from 0.1 to 200/~g) of soluble Fc fragment, ssDNA,Tg, Ins or TT. After 18 hr the mixtures were transferred into ELISAplates precoated with Fc fragment, and the amount of antibody bound to the solid-phase IgG Fc fragment was. measured as absorbance (at 492 nm). The binding of each antibody observed in the presence of soluble ligand is expressed as percentage of the binding measuredafter incubation of the antibody under identical conditions but in absence of any soluble ligand (100%binding activity), Modifiedfrom (15).

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524

CASALI

& NOTKINS

ligand (50%inhibition by 0.05 #g oflgG Fc fragment), but also, and often more efficiently, by soluble heterologous ligands (50%inhibition by 2.2, 4.0, 20, and 28/~g of soluble ssDNA,TT, Tg, and Ins, respectively) (Figure 7, Panel C). The binding of this mAbto each one of the other solid-phase Ags tested, e.g. ssDNA,Tg, Ins or TT, was also inhibited to varying degrees by different soluble heterologous Ags. The dissociation constant (Kd) values revealed as muchas 1000-fold difference in the functional affinity of polyreactive, not only IgM, but also IgG and IgA, mAbsfor different Ags (Table 3). The mAbsconsistently displayed relative low affinity for IgG Fc fragment (Kd, 1 to 4x 10-~ mole-~) and variable affinities for other Ags(Ka, 10-~ to 10-7 mole-L). +, but Our findings prove at the clonal level (a) that most of the CD5 virtually none of the CD5-, B lymphocytes from healthy subjects are committedto the production of antibodies with RF-like activity, and (b) that these antibodies are polyreactive. The polyreactive RF-like mAbs have the functional features attributed to some monoclonal RFs and/or anti-DNAautoantibodies, and to "natural autoantibodies or natural antibodies" of humans and mice (30-32, 41). Although the natural antibody functions have been traditionally associated with molecules of the IgM class, the above findings clearly establish that polyreactive antibodies can also be IgG and IgA (11). Similar to their IgMcounterparts, our studies show that polyreactive IgG and IgA are the products of CD5+ B cells (38, 39). The broad reactivity of natural polyreactive mAbsis somehow surprising, considering the:fine specificity of antibody molecules generated by active immunization. The molecular basis underlying the behavior of polyreactive antibodies relies mainly on the notion that their Ag-binding sites are considerably larger than that needed to be complementaryto a single epitope and are potentially capable of accommodatingdifferent ligands (43, 44). In fact, such a feature mayapply to the polyreactive RF+ B cells. These mAbsare highly polyreactive like antibodies from CD5 and showdifferential binding activity for different ligands. The high Ka values displayed for IgG Fc fragment are compatible with the low binding affinities attributed to humanRFs, in general from patients with monoclonal gammopathies(45). High Ka values have been also reported in the case of a number of murine natural autoantibodies for certain self-Ags (e.g. Tg or ssDNA)(41). Wehave sought a genetic basis for the functional difference between polyreactive and monoreactive antibodies. Our recent work shows that an important peculiarity of polyreactive mAbsis that they utilize selected VH gene segments, including VHIII, VnIVand Vr~V(45a). These gene segments seem to be most often in unmutatedconfiguration. Utilization of selected, possibly 3’, Vn segmentsis a feature characteristic of lymphocytesof the

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x xZZZZZZ

525

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CASALI & NOTKINS

early B-cell repertoire (46-49) and of chroniclymphocyticleukemia(CLL) cells (50). This suggests that CD5÷B lymphocytesmayrepresent an early stage in the ontogenesisof B cells. Restricted utilization of selected and unmutatedVHand, possibly VL,genesegmentscould provide the structural correlate for the high frequencyof idiotypic cross-reactivity observed amonga large proportion of human(polyreactive) RFs and anti-DNA autoantibodies (32, 45, 51). In fact, murineautoantibodies, likely the products of Ly-1 + B cells and thought to utilize a limited numberof unmutatedVHgene segments,maycontribute to the high degree of idiotypic cross-reactivity (i.e. "connectivity") observedparticularly among youngmice(41, 52, 53).

THE B-CELL REPERTOIREPROFILE IN HUMANAUTOIMMUNEDISEASES: Analysis of Polyreactive and Monoreactive Antibody-ProducingCells Giventhe anti-self Agreactivity of the polyreactive (natural) "autoantibodies," it has beentemptingto speculate that these moleculesmayplay somerole in autoimmune diseases. In fact, using EBVin limiting dilution assays, wehave found that in patients with Hashimoto’sdisease (HD)(an organ-specific autoimmune condition directed to thyroid components)and systemic lupus erythematosus(SLE)(a systemic autoimmune disease) frequenciesof circulating cells capable of producingIgMor IgAto selfAgs, as well as exogenousAgs, are similar to those of cell precursors producing antibodies of the same classes and to the same Agsfound in healthy subjects (Table2) (11). In contrast, in HDpatients the meanvalue of the frequencyof B cells committedto the productionof IgGto thyroid Ags(i.e. Tg or TMAg) is approximately0.2%of IgG-producingcells. This is at least 20 times greater than that found in healthy subjects and SLE patients. Alongthe same line, in SLEpatients the meanvalue of the frequency of B cells committedto the production of IgG to ssDNAis approximately 0.5%of IgG-producingcells which is at least 15 times greater than that in healthy subjects and HDpatients. Byconstructing morethan 30 mAb-producing hybrid cell lines selected for productionof antibodies to Ins, Tg, ssDNA,or TT, wefound that the vast majority of IgM"autoantibodies" detected in HDand SLEpatients and healthy subjects are polyreactive. Thenormalfrequencies of the circulating cell precursors producingthese polyreactive antibodies are con+ B-cell subset in these patients sistent with the normalsize of the CD5 (S. E. Burastero,J. E. Balow,A. L. Notkins,P. Casali, in preparation).

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contrast, the IgG autoantibodies found in the HDand SLEpatients are monoreactive and highly specific for thyroid Ags and ssDNA,respectively (11; S. E. Burastero, J. E. Balow, A. L. Notkins, P. Casali, in preparation). Similar to anti-TT IgG in vaccinated subjects, these monoreactive, highly specific autoantibodies are produced by CD5B cells (38; S. E. Burastero, J. E. Balow, A. L. Notkins, P. Casali, in prep.) and their KdSare always muchlower (10-7 to 10-11 mole-1) (higher affinity) than those displayed the same self-Ag by polyreactive "autoantibodies" from healthy subjects or patients (10 -3 to 10-7 mole ~) (Table 3). The frequency and high affinity of the autoantibodies in HDand SLE patients mimick those of anti-TT and anti-Ins IgG mAbproduced by cell lines constructed using specific B cells from vaccinated healthy subjects and Ins-treated patients with Insdependentdiabetes mellitus, respectively (11). In contrast to SLEpatients, in patients with rheumatoid arthritis (RA), another important systemic autoimmune disease, CD5+ B lymphocytes are strikingly increased in number, accounting for up to 50%or greater of the circulating B cells (54, 55). Our recent experimentsclearly established that most CD5+, but very few CD5-, B lymphocytes from RA patients are low density, large size, and spontaneously proliferating cells (54). Moreover the CD5÷ B cells, but not their CD5- counterparts, spontaneously secrete large amounts of IgM, IgG, and IgA RFs (54). When number of mAb-producing cell lines were derived from these purified ÷ B cells, two different populations of RFs with discrete patterns of CD5 reactivity were identified (54). The first, more numerous, consisted polyreactive monoclonal RF-like antibodies, similar in function to the polyreactive RF-like antibodies produced by CD5÷Bcells from healthy subjects. The second, much less numerous, consisted of monoreactive monoclonal RF autoantibodies binding only to IgG Fc fragment, and it was found exclusively in RApatients (Figure 8). The Kd values of poly-5 reactive RF antibodies for IgG Fc fragment were in the range of 10 mole-1, similar to the Kd values of polyreactive RF-like antibodies from healthy subjects. In contrast, the Kd values of monoreactive RFs were at least two orders of magnitude lower (10-7 mole-1) (Table 4). Thus, in the monoreactivehigh affinity autoantibodyresponse is strictly confined to + B-cell compartment, whereas in SLEpatients and TT-vaccinated the CD5 subjects, the monoreactive high affinity anti-ssDNA and anti-TT antibodies, respectively, are confined to the CD5-B lymphocytecompartment. A question that arises from the above considerations is what could be the role of polyreactive natural "autoantibodies" in autoimmunediseases. RF and anti-DNA autoantibodies have been classically related to the pathogenesis of RAand SLE, respectively, and polyclonal B-cell activation has been proposed as one mechanism accounting for the production of

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274.RA.F11 IgM, ~

1.2

A

ssDNA

1.4

B

274.RA.F1 IgA,

1.2 TT 1.0 -9 T 0.8

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0.6 0.4 0.2

0.2 0.0 10-;’

-~ . 10

°10

~ 10

0.0

~ 10

A

|

IgM, ~ tO0

IgA, ¯ 100

Fc

~.

10-’

o 10

~ 10

20-

10-~

10~

o 10

~ 10

102

SOLUBLE LIGAND,pg

Figure8 Top Panels: dose-dependent binding of representative polyreactive (274.RA.F11 mAb) (Panel A) and monoreactive (274.RA.FI mAb) (Panel B) monoclonal RFs produced ÷ B cells from a RApatient, to solid-phase lgG Fc by cell lines constructed using CD5 fragment (O), Ins (O), ssDNA(/k), Tg (~,), and TT (I). The Ag-binding activity each mAbis expressed as optical absorbance (at 492 nm), Bottom Panels: dose-dependent inhibition of 274.RA.FI 1 (Panel A’) and 274.RA.FI (Panel B’) mAbbinding to solid phase IgG Fc fragment by soluble homologous (Fc fragment) and heterologous (ssDNA, Tg, and TT) ligands. Samplesof each antibody (0.2 #g) were incubated in solution with increasing amounts (from 0.1 to 200 #g) of soluble IgG fragment (O), ssDNA(/k), Tg (~,), and TT(I). After 18 hr, the mixtures were transferred into ELISAplates precoated with IgG Fc fragment, and the amount of antibody bound to the solid-phase IgG Fc fragment was measured. The binding of each antibody observed in the presence of soluble ligand was calculated and is expressed as in the experiments of Figure 7 (Panel C). Modifiedfrom (54).

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Table 4 Dissociation constant (Kd, mole/I) for IgG Fc fragment of human RF mAbs ÷ aB cells of patients with RA generated from CD5 RF mAb

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274.RA.FI 1 274.RA.14 274.RA.31 274.RA.F4 274.RA.F5 274.RA.F1

H chain L chain # # k~ /~ # c~

2 k k k k k

Ag-reactivity Polyreactive Polyreactive Polyreactive Monoreactive Monoreactive Monoreactive

Binding to IgG Fc fragment (Kd) -5 4.0 × 10 -5 3.8 × 10 -5 2.3 × 10 6.0 -7 × 10 -7 3.5 × 10 .7 2.0 x 10

Experimental conditionsused for the determinationof K~values werethose describedin Ref. 15, 54.

autoantibodies in this and other systemic autoimmune diseases (56, 57). Accordingto this model,autoantibodiesproducingB-cell clones wouldbe activated becauseof an impairedT-cell suppressivecontrol. Alternatively, they could expresssuch a high degreeof intrinsic activation as to be able to override an otherwise normalsuppressor T-cell mechanism.In either case, the prediction of this hypothesisis that in autoimmune conditionsB cells spontaneouslyproduceautoantibodies, independentlyof the stimulation of any self-Ag. Moreover,the auto-antibodies producedwouldhave the functional features of the anti-self antibodies that can be found in healthy subjects--that is, as weshowedhere, polyreactivity and relative lowaffinity. In fact, polyreactive"autoantibodies"are spontaneouslyproduced in vivo by activated B lymphocytesin patients with RAand SLE, and the CD5+ B cells are dramaticallyincreasedin numberin RApatients. However, the selective Ag-bindingactivity andrelative high affinity (Kd, 10-7 to 10-~° mole-~) of the autoantibodies to lgG Fc fragment, ssDNA, and Tg (produced by someof the B-cell clones derived from RA, SLE, and HDpatientg, respectively) support the hypothesis that an Ag-driven processof clonal selection and somaticmutationlikely shapesthe anti-self responsein these diseases. Alongthese lines, it wasrecently reportedthat in the MLR/Iprautoimmunemousestrain, the gene segments encoding for a numberof different RFs, and for monoclonalanti-DNAantibodies (58, 59), harbor manysomatic mutations, suggesting that a nonrandom; Ag-drivenclonal selection is operative in the generation of these autoantibodies. B cells, originally committedto the productionof polyreactive antibodies utilizing unmutatedVr~and VLgene segments,mayeventually give rise to cells producingantibodies with selective binding activity through an Ag-drivenclonal selection and somatic mutation process (60). Recent findings in mice(61) suggest that anti-arsonate antibodies inducedafter active immunizationare producedby B cells, thought to be the progenyof

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virgin B lymphocytes, originally making Ig with VHsegments in unmutated configuration and endowedwith multiple "anti-self" reactivities. Maturation of the antibody response to arsonate parallels class switch IgMto IgG, and increasing load of point mutations in the Ig VHgene segments and loss of "anti-self" reactivity. Accordingly, it is tempting to speculate that a process of clonal selection and somatic mutation driven by a selfAg could possibly lead to the generation of monoreactive high affinity, potentially pathogenic autoantibodies. SUMMARY

AND

CONCLUDING

COMMENTS

The balanced activation and amplification of discrete B-cell clones and subsets and the generation of continuous mAb-producingcell lines have proved that EBVis a powerful tool to gain new insight into the diversity of the humanB-cell repertoire. The application of this methodologyto the study of the B lymphocytesfrom normal subjects has allowed us to establish that a high frequency of circulating cell precursors produce polyreactive antibodies. These antibodies bind to both self- and exogenous Ags and are found in all normal individuals. Moreover, by using EBV technology in conjunction with fine cell sorting techniques, we were able +) B-cell subset is responsible for the to determine that a discrete (CD5 production of the polyreactive "autoantibodies". + lymphocytes constitute a major component of the normal human CD5 B-cell repertoire. In healthy subjects, most of these cells are committedto the production of antibodies, mainly IgM, but also IgG and IgA, binding not only to a numberof different self-Ags, but also to biologically relevant exogenous Ags. The label "autoantibody-producing B lymphocytes" that one may be tempted to attach to these cells, and maybe to mouse Ly-1 ÷ ÷B B cells, is, perhaps, not appropriate. In fact, the Ig produced by CD5 cells constitute the class of antibodies more appropriately described as polyreactive or natural antibodies. Polyreactive "autoantibodies" are easily detected in the circulation of healthy humansand mice during the physiological response to exogenous Ags, such as TT, or after injection of bacterial LPS, or during the course of viral, bacterial, or parasitic infections (62-69). Indeed, the number circulating B lymphocytes producing RF-like antibodies increases during the immuneresponse to TT in humans (62). The detectable levels circulating natural antibodies (42) found in healthy subjects suggest that ÷ B cells under normal conditions secrete Ig in at least some of the CD5 ÷ B lymphocytes are large, activated cells vivo. In fact, some humanCD5 (39). Large, activated B cells spontaneously secreting natural autoantibodies have been detected in normal mice (41). A major role for the

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+ B lymantibodies, particularly with RF-like activity, produced by CD5 phocytes is likely that of a first line of defense against invading microorganisms (by enhanced phagocytosis, complement-mediated lysis, etc) and that of enhancementof an ongoing specific immuneresponse. Indeed, polyreactive antibodies derived from CD5÷ B cells bind TT and LPS or polysaccharides from different bacterial strains (15, 38, 39). Moreover, it has been shown that RF IgM can neutralize in vitro preformed immune complexes involving IgG and herpes simplex virus (70), and RFlike IgM completely protects rat pups from infection with Trypanosoma levysi (71). The application of the EBVtechnology to the study of the B-cell repertoire of patients with autoimmunedisease revealed that as in healthy subjects a large proportion of circulating B cells were committedto the production of polyreactive IgMantibodies. In contrast to normal subjects, however, a significantly higher frequency of B lymphocyteswas committed to the production of monoreactive autoantibodies, mainly IgG, but also IgM or IgA (RA patients) relevant to the particular disease. In these patients, monoclonalpolyreactive antibodies were in general low affinity (Kd, 10 -3 to 10 -7 mole-~), whereas monoclonal monoreactive autoantibodies were high affinity (Kd, 10-7 to 10-I~ mole-l). The high frequency and affinity of the monoreactive IgG to ssDNAand thyroid Ags in SLE and HDpatients, respectively, as well as the high affinity of the monoreactive IgM and IgA RFs in RApatients are in the same range of frequency and affinity as that of the monoreactive IgG to TT and Ins produced by B-cell clones from vaccinated healthy subjects and Ins-treated patients, respectively (11). These findings support the hypothesis that the B-cell repertoire in these patients is shaped by Ag-drivenresponses rather than merelyreflecting a polyclonal B-cell activation. In conclusion, it is nowpossible to quantitate and characterize human B cells committedto the production of different types of antibodies; to prepare humanmonoclonal antibodies of predetermined specificity and isotype; and to sequence the humangene segments coding for antibodies to both self-Ags and foreign Ags. This should lead to a better definition of the humanB-cell repertoire and should help elucidate the function and relationship of cells committedto the production ofpolyreactive antibodies as compared to monoreactive antibodies. ACKNOWLEDGMENTS

Weare indebted to Drs. Minoru Nakamura, Samuele E. Burastero, Yuji Ueki, and Giorgio Inghirami for their collaboration in most of the experiments reported here.

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Literature Cited 1. Jerne, N. K. 1955. The natural selection theory of antibody production. Proc. Natl. Acad. Sci. USA 41:849 57 2. Talmage, D. W. 1959. Immunological specificity. Science 129:1643-48 3. Lederberg, J. 1959. Genes and antibodies: do antigens bear instructions for antibody specificity or do they select cell lines that arise by mutation?Science 129: 1649-53 4. Burnett, F. M. 1959. The clonal selection theory of acquired immunity. London: Cambridge Univ. Press 5. Waldmann,T. A., Broder, S. 1982. Polyclonal B cell activators in the study of the .regulation of immunoglobulinsynthesis ~n the humansystem. Adv. Immunol. 32: 1-63 6. Vitetta, E. S., Brooks, K., Isakson, P., Layton, J., Pure, E., Juan, D. 1984. B lymphocyte receptors. In Fundamental Immunology, ed. W. E. Paul. 3: 221-43. NewYork: Raven. 650 pp. 7. Rosen, A., Gergely, P., Jondal, M., Klein, G. 1977. Polyclonal Ig production after Epstein-Barr virus infection in human lymphocytes in vitro. Nature 267:52-53 8. Miller, G. 1985. Epstein-Barr virus. In l~irology, ed. B. N. Fields, 27: 563-589. New York: Raven. 1614 pp. 9. Henderson, E., Miller, G., Robinson, J., Heston, L. 1977, Efficiency of transformation of lymphocytes by EpsteinBarr virus. I"irology 76:15243 10. Bird, A. G., Britton, S., Ernberg,I., Nilsson, K. 1981. Characteristics of EpsteinBarr virus activation of humanB lymphocytes. J. Exp. Med. 154:832-39 11. Nakamura, M., Burastero, S. E., Ueki, Y., Larrick, J. W., Notkins, A. L., Casali, P. 1988. Probing the human B cell repertoire with EBV.Frequency of B cells producing monoreactive high affinity autoantibodies in patients with Hashimoto’s disease and SLE. J. Immunol. 141: In press 12. Yarchoan, R., Tosato, G., Blaese, R. M., Simon, R. M., Nelson, D. L. 1983. Limiting dilution analysis of EpsteinBarr virus-induced immunoglobulin production by human B cells. J. Exp. Med. 157:1-14 13. Cooper, N. R., More, H. D., Nemerov, G. R. 1988. Immunology of the CR2, the B lymphocyte receptor for EpsteinBarr virus and the C3d complement fragment. Annu. Rev. Immunol. 6: 85113 14. Pollack, M., Raubitschek, A. A., Larrick, J. W. 1987. Humanmonoclonal

antibodies that recognized conserved epitopes in the core-lipid A region of lipopolysaccharides. J. Clin. Invest. 79: 1421-30 15. Nakamura, M., Burastero, S. E., Notkins, A. L., Casali, P. 1988. Human monoclonalrheumatoid factor-like antibodies from CD5(Leu-1 ÷ B cells are polyreactive. J. Immunol. 140: 418086 16. Inghirami, G., Nakamura,M., Balow, J. E., Notkins, A. L., Casali, P. 1988. A model for virus attachment: identification and quantitation of EBV-binding cells using biotinylated virus in flow cytometry. J. Virol. 62:2453-63 17. Steel, C. M., Philipson, J., Arthur, E., Gardner, S. E., Newton, M. S., Mclntosh, R. V. 1977. Possibility of EB virus preferentially transforming a subpopulation of human B lymphocytes. Nature 270:729-31 18. Bird, A. G., Britton, S., Ernbert, I., Nilsson, K. 1981. Characteristics of EpsteinBarr virus activation of humanB lymphocytes. J. Exp. Med. 154:832-39 19. Brown, N. A., Miller, G. 1982. Immunoglobulin expression by human B lymphocytes clonally transformed by Epstein-Barr virus. J. Immunol. 128: 24-29 20. Stein, L. D., Ledgley, C. J., Sigal, N. H. 1983. Patterns of isotype commitmentin humanB cells: limiting dilution analysis of Epstein-Barr virus-infected cells. J. Immunol. 130:1640-45 21. Aman, P., Ehlin-Henriksson, B., Klein, G. 1984. Epstein-Barr virus susceptibility of normal human B lymphocyte populations. J. Exp. Med. 159:208-20 22. Roome,A. J., Reading, C. L. 1987. Frequency of B lymphocyte transformation by Epstein-Barr virus decreases with entry into the cell cycle. Immunology60: 195-201 23. Boyd, A., Anderson, K., Freedman, A., Fisher, D., Slaughenhoupt, B., Schlossman, S. F., Nadler, L. M. 1985. Studies on in vitro activation and differentiation of humanB lymphocytes. I. Phenotypical and functional characterization of the B cell population responding to anti-Ig antibodies. J. lmmunol. 134: 1516-23 24. Lefkovits, I., Waldmann, H. 1984. Limiting dilution analysis of the cells of immunesystem I. The clonal basis of the immune response. Immunol. Today 5: 265-68 25. Waldmann, H., Lefkovits, I. 1984. Limiting dilution analysis of cells of the

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PROBING THE HUMAN B-CELL REPERTOIRE immunesystem II: Whatcan be learned? Immunol. Today 5:295-98 26. Casali, P., Inghirami, G., Nakamura, M., Davies, T. F., Notkins, A. L. 1986. Humanmonoclonals from antigen-specific selection of B lymphocytesand transformation by EBV. Science 234: 47679 27. Dighiero, G., Lymberi, P., Mazie, J. C., Rouyre, S., Butler-Browne, G. S., Whalen, R. C., Avrameas, S. 1983. Murine hybridomas secreting natural monoclonal antibodies reacting with self-antigens. J. Immunol. 131: 226772 28. Prabhakar, B. S., Saegusa, J., Onodera, T., Notkins, A. L. 1984. Lymphocytes capable of making monoclonal autoantibody that react with multiple organs are commonfeatures of the normal B cell repertoire. J. Immunol. 133:2815 17 29. Dighiero, G., Lymberi, P., Holmberg, D., Lundquist, I., Coutinho, A., Avrameas, S. 1985. High frequency of natural autoantibodies in normal newborn mice. J, Immunol. 134:765-71 30. Schwartz, R. S., Stollar, B. D. 1985. Origins or anti-DNAautoantibodies. J. Clin. Invest. 75:321-27 31. Ternynck, T., Avrameas, S. 1986. Murine natural monoclonal autoantibodies: a study of their polyspecificities and their affinities. Immunol. Rev. 94: 99-112 32. Bona, C. A. 1988. V genes encoding autoantibodies molecular and phenotypic characteristic. Annu. Rev. Immunol. 6:327-58 33. Dighiero, G., Guilbert, B., Fermand, J. P., Lymberi, P., Danon, F., Avrameas, S. 1983. Thirty-six humanmonoclonal immunoglobulins with antibody activity against cytoskeleton proteins, thyroglobulin, and native DNA:Immunological studies and clinical correlation. Blood 62:264-70 34. Satoh, J., Prabhakar, B. S., Haspel, M. V., Ginsberg-Fellner, F., Notkins, A. L. 1983. Human monoclonal autoantibodies that react with multiple endocrine organs. New Engl. J. Med. 309: 217-20 35. Haspel, M. V., Onodera, T., Prabhakar, B. S., McClintock, K. E., Ray, U. R., Yagihashi, S., Notkins, A. L. 1983. Multiple organ-reactive monoclonalautoantibodies. Nature 304:74-76 36. Casali, P., Prabhakar, B. S., Notkins, A. L. 1988. Characterization of multireacrive autoantibodies and identification of Leu- 1 + B lymphocytesas cells making antibodies binding multiple self and exo-

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genous molecules. Int. Rev. lmmunol. 3:17-45 37. Hayakawa, K., Hardy, R. R., Honda, M., Herzenberg, L. A., Steinberg, A. D., Herzenberg, L. A. 1984. Ly-1 B cells: functionally distinct lymphocytes that secrete IgMautoantibodies. Proc. Natl. Aead. Sci. USA 81:2494-98 38. Casali, P., Burastero, S. E., Nakamura, M., Inghirami, G., Notkins, A. L. 1987. Human lymphocytes making rheumatoid factors and antibodies to single stranded DNAbelong to the Leu-1+ B cell subset. Science 236:77-81 39. Burastero, S. E., Casali, P. 1988. Characterization of human CD5 (Leu-1, OKT1)+ B lymphocytes and the antibodies they produce. In B Cells and B Cell Products, ed. P. del Guercio. Basel: Karger. In press 40. Friguet, B., Chaffotte, A. F., DjavadiOhaniance, L., Goldberg, M. E. 1985. Measurementof true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. 77:305-19 41. Portnoi, D., Freitas, A., Holmberg,D., Bandeira, A., Coutinho, A. 1986. Immunocompetent autoreactive B lymphocytes are activated cycling cells in normal mice. J. Exp. Med. 164: 2535 42. Guilbert, G., Dighiero, G., Avrameas, S. 1982. Naturally occurring antibodies against nine commonantigens in human sera. I. Detection, isolation and characterization. J. Immunol. 128: 277987 43. Richards, F. F., Konigsbert, W. H., Rosenstein, R. W., Varga, J. M. 1975. Onthe specificity of antibodies. Science 187:130-37 44. Amit, A. G., Mariuzza, R. A., Phillips, S. E., Poljak, R. J. 1986. Three dimensional structure of an antigen-antibody complexat 2.8 A resolution. Science 233: 747 53 45. Carson, D. A., Chen, P. P., Fox, R. I., Kipps, T. J., Jirik, F., Goldfien, R. D., Silverman, G., Redoux, V., Fong, S. 1987. Rheumatoid factors and immune networks. Annu. Rev. Immunol. 5: 10926 45a. Sanz, I., Casali, P., Thomas, J. W., Notkins, A. L., Capra, D. J. 1989. Genetic basis of natural autoantibodies: organization, complexity, and mechanisms of diversity of the humanB cell repertoire. J. lmmunol.In press 46. Yancopoulos, G. D., Desiderio, S. V., Paskind, M., Kearney, J. F., Baltimore, D., Alt, F. W. 1984. Preferential utilization of the most JH-proximal VHgene

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segmentsin pre-Bcell lines. Nature311: 727-33 47. Perlmutter, R. H., Kearney, J. F., Chang,S. P., Hood,L. E. 1985. Developmentcontrolled expression of Ig VH genes. Science 227:1597-1601 48. Schroeder,H.W.Jr., Hillson,J. L., Perlmutter, R. M.1987. Early restriction of the humanantibody repertoire. Science 238:791-93 49. Schroeder, H. W., Walter, M. A., Hofker,M. H., Ebens, A., VanDijk, K. W., Liao, L. C., Cox,D. W.,Milner,E., Perlmutter, R. M.1988. Physicallinkage of a new immunoglobulin VHgene family (VH6)to DHand Jr~ gene segments. Proc. Natl. Acad. Sci. USA.In press 50. Humpries,G. C., Shen, A., Kuziel, W. A., Capra,J. D., Blattner, F. R., Tucker, P. W. 1988. A new immunoglobulin VHfamily preferentially rearranged in immature B-cell tumors. Nature 331: 446-49 51. Kipps, T. J., Tomhave,E.,Chen, P. P., Carson, D. A. 1988. Autoantibodyassociatedx light chain variable region gene expressed in chronic lymphocytic leukemia with little or no somatic mutation. Implication for etiology and immunotherapy.J. Exp. Med.167: 84052 52. Painter, C., Monastier, M., Bonin, B., Bona, C. A. 1986. Functional and molecularstudies of V genes expressed in autoantibodies.Immunol.Rev. 94: 7598 53. Holmberg. 1987. High connectivity, natural antibodies preferentially use 7183 and QPC52V~ families. Eur. J. Immunol. 17:399-403 54. Burastero, S. E., Casali, P., Wilder,R. L., Notkins, A. L. 1988. Monoreactive high affinity andpolyreactivelowaffinity rheumatoidfactors are producedby ÷ B cells from patients with rheuCD5 matoidarthritis. J. Exp. Med.168: In press 55. Hardy, R. R., Hayakawa,K., Shimizu, M., Yamasaki,K., Kishimoto,T. 1987. Rheumatoid factor secretion from humanLeu-I÷ B cells. Science 236: 8183 56. Klinman,D. M., Steinberg, A. D. 1987. Systemic autoimmunedisease arises frompolyclonalB cell activation.J. Exp. Med. 165:1755q50 57. Hang,L., Slack, J. H., Amudson,C., Izui, S., Theofilopoulos, A., Dixon,F. J. 1983. Induction of murine autoimmune diseaseby chronicpolyclonalB cell activation. J. Exp. Med.157:874-83 58. Shlomchik,M. J., Marshak-Rothstein,

A., Wolfwicz,C. B., Rothstein, T. L., Weigert,M.G. 1987. Therole of clonal selection andsomaticmutationin autoimmunity. Nature 328:805-11 59. Shlomchik, J. J., Aucoin,A.J., Pisetsky, ID. S., Weigert, M. G. 1987. Structure and function of anti-DNAautoantibodies derived from a single autoimmunemouse.Proc. Natl. Acad. Sci. USA84:9150-54 60. Gearhart, P. J. 1983. The effect of somatic mutationon antibody affinity. Ann. N.Y. Acad. Sci. 418:171-76 61. Naparstek, Y., Andre-Schwartz, J., Manser,T., Wysocki,L. J., Breitman, L., Stollar, B. D., Gefter, M., Schwartz, R. S. 1986. A single germline Vn gene segment of normal A/J mice encodes autoantibodiescharacteristic of systemic lupus erythematosus.J. Exp. Med.164: 614-26 62. Welch,M. J., Fong, S., Vaughan,J., Carson,D. A. 1983. Increased frequency of rheumatoidfactor precursor B lymphocytes after immunizationof normal adults with tetanus toxoid. Clin. Exp. Immunol.51: 299-304 63. Carson,D. A., Bayer, A. S., Eisemberg, R. A., Lawrence,S., Theofilopoulos,A. N. 1978. IgG rheumatoid factors in subacute bacterial endocarditis: relationship to IgMrheumatoidfactors and circulating immune complexes.Clin. Exp. Immunol. 31:100-13 64. Casali, P., Perrin, L. H., Lambert,P. H. 1979. Immunecomplexesand tissue injury. In Immunological Aspects of Infectious Diseases,ed. G. Dick,9: 295342. Baltimore: Univ. Park Press. 524 PP. 65. Steele, E. J., Cunningham, A. J. 1978. High proportion of immunoglobulin producing cells makingautoantibodies in normal mice. Nature 274:483-84 66. Dresser, D. W. 1978. Most IgM-producing cells in the mousesecrete autoantibodies (rheumatoidfactor). Nature 274:480-82 67. Dziarski,R. 1982.Preferential induction of autoantibodysecretion in polyclonal activation by peptidoglycanand lypopolysaccharide.II. In vivo studies. J. Immunol. 128:1026-30 68. Coulie, P., VanSnick, J. 1983. Rheumatoid factor (RF) during anamnestic immune responsesin the mouse.II. Activation of RFprecursorcells in induced by their interaction with immune complexesandcarrier-specifichelperT cells. J. Exp. Med. 161:88-97 69. VanSnick, J., Coulie, P. 1983. Rheumatoid factors and secondary immune responses in the mouse. I. Frequent

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occurrence ofhybridomas secreting IgM anti-IgG antibodies after immunization with protein antigens. Eur. J. Immunol. 13:890-95 70. Ashe, W. K., Daniels, C. A., Scott, G. S., Notkins, A. L. 1971. Interaction of RF with infectious Herpes Simplex

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virus-antibody complexes. Science 172: 176-77 71. Clarkson, A. B. Jr., Mellow,G. M. 1981. Rheumatoid factor-like immunoglobulin Mprotects previously uninfected rat pupsScience and dams from Trypanosoma lewisi. 214:186-88

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Ann. Rev. Immunol. 1989. 7.’537-59 Copyright © 1989 by Annual Reviews Inc. All rights reserved

STABLE EXPRESSION AND SOMATIC HYPERMUTATION OF ANTIBODY V REGIONS IN B-CELL DEVELOPMENTAL PATHWAYS C. Kocks and K. Rajewsky Institute for Genetics, University of Cologne, 5000 Cologne41, Federal Republic of Germany INTRODUCTION: A SCHEME B-CELL DIFFERENTIATION

OF PATHWAYS

The generation of B lymphocytes from stem cells occurs through an ordered program of immunoglobulin (Ig) variable (V) region gene arrangements. As a result, each newbornB cell expresses a particular pair of heavy (H) and light (L) chain V regions (1, 2). This represents molecular basis of the clonal selection theory which postulates that each B cell and its progeny are predetermined for a single antibody specificity (3). The occurrence of somatic hypermutation of antibody V regions the generation of immunological memoryrequires some modification of this theory (see below). The life histories of B lymphocytes may differ from each other dramatically. In Figure 1 we give a schematic outline of B-cell differentiation pathways as we see them today. On the right hand side of the figure are the T cell dependent pathways of acquired immunity. Cells able to enter these pathways are continuously generated from stem cells in the bone marrowthroughout the life of the animal. Most of the newborncells die within a few days. A substantial fraction of the B-cell population in the 537 0732~0582/89/0410-0537502.00

Annual Reviews 538

KOCKS & RAJEWSKY death Stemcells v gene rearrangement

B cells/

Plasma cells ° Annu. Rev. Immunol. 1989.7:537-559. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

B cells ~’(~ (LylB; others?)

1 response

~(~-/ /

Memory~

~

ntigen

Plasmacells Q

cells//~ntigen

~

?1/ ’~ /I ~’

¯ Plasma cells ° 2 response

Natural Immunity AcquiredImmunity Ahypotheticalschemeof B cell differentiation pathways.B cells expressing

Figure 1 unmutated(germline) antibodies are indicated by open circles, B cells expressing somatically mutatedantibodies by filled circles. Arrowsrepresent B cell differentiation pathways. In the pathway of memorygeneration the accumulation of somatic mutations is indicated by black dots. Pathwaysright of the vertical line are T-cell dependent. Pathwayson the left are partly or entirely T-cell independent.

peripheral immunesystem consists of such short-lived cells (4, 5). Some cells are selected into immuneresponses. Here they mayclonally expand and terminally differentiate into antibody secreting plasma cells in the primary response. Other cells enter a pathway in which a new repertoire of antibody V regions is generated through somatic hypermutation (see Figure 1 and discussion below). The result of this process which is also under the control of T helper cells is B-cell memory,the hallmark of acquired immunity: Uponthe next encounter with antigen, the cells selected in this pathwayproduce a secondary response in which large quantities of antibodies with an increased affinity for the antigen are synthesized. In another differentiation pathway(Figure 1, left side) a separate set B cells has been identified, representing only a few per cent of all B cells and called LylB because of the expression of the Lyl surface antigen. These cells which perhaps represent a distinct B-cell lineage, appear to be seeded into the immunesystem early in ontogeny and to be propagated in the animal as mature B cells over long periods of time (6-9) in the absence of somatic hypermutation (see further below). A similar B-cell expressing the homologous Leul (CD5) antigen exists in humans(9, 10). Knowledge

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about the physiological role of Lyl B cells is still fragmentary.In the mouse,mostcells of this subset reside in the peritoneal cavity and seemto producea major fraction of serumimmunoglobulins (notably IgM;7, 11). Functional studies havesuggested that Lyl B cells are selected for the expressionof a restricted repertoire of antibodyspecificities. This repertoire containsspecificities for the bacterial antigens ~l,3-dextran(Dex) (11) and phosphorylcholine (A. Stall, L. Herzenberg, personal communication). Theformer is a T cell-independent antigen of type II, and responsesto suchantigens couldbe typical for B cells in this compartment. Repertoireselection maybe initiated before contact with external antigen, by auto-antigens(9, 12-15)includingantibodyidiotypes (16, 17) expressed within the Lyl B compartment.In Figure 1, this is indicated, and Lyl B cells are hypotheticallycategorizedas carriers of natural immunity.This function Lyl B cells wouldpresumablyshare with other sets of B cells (see, for example,Ref. 7). Thesubject of the present article is the expansion,mutation,andselection of B cells in the variousdifferentiation pathways as outlinedin Figure 1. This issue can be studied by identifying cells throughtheir rearranged antibody V region genes. As we have mentioned, each B cell carries V region generearrangementswhichare characteristic for it andits progeny and thus are a convenient clonal marker. Furthermore, since the rearranged V region genes expressed by the cell determine its antigen bindingspecificity, their analysisdirectly relates to cellular selection. Asa specific aspect, determinationof the nucleotidesequenceof Vregion genes expressedby cells in the variousdifferentiation pathways,in particular by clonally related cells, and the comparison of these sequenceswith those of the corresponding genesin the germlineallow one to investigate the extent to whichand the wayin whichsomatic hypermutationcontributes to the generationof the antibodyrepertoire.~ Asystematicanalysis of B-cell differentiation pathwaysalongthese lines could be undertakenwhenit becamepossible to immortalize individual antibodyproducingcells froma given cell populationwith the cell hybridization methodof K6hler & Milstein (18) and to characterize their rearrangedV region genesby methodsof moleculargenetics. In the present article wereviewthe results obtained to date throughthis approachand try to incorporatetheminto a coherentpicture of B-cell selection in the immunesystem of the mouse. 1 Somatic hypermutation does not include somatic variations generated through V region rearrangement by imprecise joining and de novo nucleotide insertion (see Figure 1). Consequently, we refer to the term "expression of germline," disregarding this kind of somatic variation.

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PATHWAYS FOR THE EXPRESSION OF GERMLINEANTIBODY SPECIFICITIES Selection and Expansionof GermlineSpecificities in the Lyl B-Cell Compartment An example of autoreactive antibodies produced by murine Lyl B cells are antibodies binding to bromelain-treated autologous red blood cells. Large numbers of cells producing such antibodies were found to reside in the peritoneal cavity (19 21) and later were mapped to the Lyl compartment (12). The epitope recognized by these antibodies is phosphatidyl choline (22), a commoncell membrane component. The regions of a collection of such antibodies from two strains of mice were sequenced :~y, Reininger et al (23), and complete identity of both VHand VLwas found~ apart from strain-specific positions. While this strongly suggests but does not prove the germline origin of the VHand VL genes expressed in these antibodies, another study has provided unequivocal evidence for the expression of selected germline V genes in the Lyl B population. In these experiments (8, 24) peritoneal cells from adult CB.20 mice were transferred into (IgH allotype-congenic) newborn Balb/c animals. Whenthe recipients~had grown up, all donor B cells detectable in the animals belonged to the I~.yl B-cell subset, Three main results emerged from the sequence analysis of the V regions of a collection of antibodies produced by such cells: (a) Certain VHand VLgenes were found repeatedly expressed in clonally unrelated cells. (b) Clonally related cells were.repeatedly isolated, indicating substantial clonal expansion (in one case progenyof a clone were identified in spleen and peritoneal cavity). (c) Knowngermline V genes were found expressed in manyof the antibodies and, despite clonal expansion, only a single somatic mutation was seen within a total of 7.8 kb. Similarly, Tarlinton et al (25) found a particular V~ gene repeatedl.y and identically expressed in independent Lyl B-cell clones expandi:ng in the peritoneal cavity of an (NZB× NZW)F1 mouse. Certain germline V regions are thus selectively expanded in the Lyl B compartment, and this expansion does not involve, as in the pathway of memorygeneration in "classical" B cells (see below), somatic hypermutation. Whetherthis holds true for Lyl B cells invo’l*ed in intentionally induced antigen-driven antibody responses has not yet been directly addressed. It is noteworthy, however, that there is no evidence so far for a participation of Lyl B cells in T cell-dependent antibody responses in which the generation of somatic B-cell memorytypically occurs (see below). This contrasts with T cell~ndependent responses like the one to De~~ (26), in which Lyl B cells are knownto be involved (11).

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Waysin which repertoire selection in the Lyl B compartmentcould be controlled are discussed below. This control is of importance not only because of the possible involvement of the Lyl B population in natural defense and, perhaps, autoimmunity, but also because of the frequent development of chronic B-cell leukemia (B-CLL) in the Lyl B compartment (9, 24, 27, 28). Indeed, the propagation of clones of Lyl B ce.lls in the animal over long periods of_time makesit difficult to draw the line between normal and malignant growth, .and it is to be anticipated that normal and malignant Lyl B cells are or have been selected at somestage on the same principles, based upon the specificity of the antibody which they express. The available data on V regions expressed by Lyl B-derived CLLin both mouse and humansupport this concept in that they suggest repeated usage of certain V genes in the absence of somatic hypermutation (24, 29-31, 31 a; the sequencevariations seen by Shenet al (29) may..reflect V gene polymorphism).It is particularly striking that manyof the V genes expressed in the B cells studied by Pennel et al (31a) were also identified in the Lyl B-cell population described in (24). Expression of Germline Specificities Antibody Responses

in Primary

There is ample evidence in the litera.ture that primary antibody responses are generally produced by cells expressing germline encoded antibody specificities, and this subject has been.exhaustively reviewed (32-35). The evidence includes responses to T cell-dependent antigens (in which frequent isotype switching accompanies,. ¢tonal expansion before terminal differentiation) as well as polyclonal activators like bacterial lipopolysaccharide On which isotype switching is more restricted). Somatic mutations were.also not seen in a set of antibodies isolated after immunization with a T cell-independent antigen of type II (36). There is some uncertainty in the latter case whether the antibodies analyzed had been induced by immunization, because serum antibodies obtained in response to the same antigen have different serological and idiotypic properties (37). Overall, the interpretation of:the data is that the B cells in the preimmunerepertoire (and this includes classical B cel.ls as well as Lyl B cells) usually express unmutated antibodies and that cells from this repertoire are selected into the primary response in which they clonally expand, switch isotype, and terminally differentiate, in the virtual absence of V region mutation. The molecular analysis of the antibo.dies expressed by clonally related B cells producing a T cell~dependent primary response in a cell transfer systemhas provided direct evidence for this notiort (38).

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THE PATHWAY OF T CELL-DEPENDENT MEMORY GENERATION: SOMATIC HYPERMUTATION B-cell memory,defined here as the ability of the immunesystem to produce an enhanced antibody response of higher affinity upon repeated contact with antigen, is typical of T cell~lependent responses. At the level of antibody V regions, B-cell memoryis characterized by somatic hypermutation. Secondary and hyperimmuneresponse antibodies against various T cell-dependent antigens have been analyzed systematically by molecular methods. The antigens used in these studies include a variety of haptens (coupled to different carriers) as 2-phenyl-5-oxazolone(394 1), hydroxy-3-nitrophenyl)-acetyl (42-45) and p-azophenylarsonate (46, protein antigens such as influenza hemagglutinin (48-50) and immunoglobulin (50, and a T cell-dependent form of the carbohydrate/%(1,6)galactan (52). All secondary and hyperimmunehybridomas analyzed these systems display somatic mutations in their expressed V regions.

Features of I9 Gene Hypermutation TARGET OF HYPERMUTATION Somatic hypermutation acts specifically on rearranged immunoglobulinV, D, and J gene elements and their immediate 5’ and 3’ flanking regions. Somatic mutations have not been detected in constant region genes of somatically mutated antibodies (43, 53 56) and only rarely outside a region of about 1 kb surrounding rearranged V region gene elements (53, 56). In nonrearranged V genes, mutations have been found only occasionally (57) or not at all (58, 59). The signal for recognition of the mutational target seems to be generated by the rearrangement of V and J gene segments. For full activation of the mechanism, signals associated with both V and J may be required, since DJ rearrangements in inactive IgH loci exhibit about ten times less somatic mutations than do their active VDJcounterparts (32, 60, 60a). Productively and nonproductively rearranged Ig loci containing a V gene element are mutated to a similar extent (54, 59, 61). Curiously, somatic mutations were also detected in a c-myc gene, translocated in the IgH locus, such that all V and J gene elements were removed (62). An explanation based upon the arguments made above would imply that the translocated c-mye gene had undergone a second rearrangement leading to the deletion of signal sequences. The hypermutation mechanism does not depend on the chromosomal locations of the Ig loci, because somatic mutations were found at different transgene-insertion sites in B cells of transgenic mice carrying a rearranged x light chain gene (63).

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The mechanismmay not be equally active on all V gene segments. V21 and V~45.1 gene segments exhibit fewer mutations than do the respective heavy chain region counterparts in many somatically mutated antibodies (33, 44, 45). PATTERN OF MUTATIONS ASearly observations indicated (64-66), point mutations are the main class of somatic mutations seen in murine antibody V regions (39, 44, 49, 67, 68). Additionally, somedeletions (44, 53, 56), one insertion (53), and a few potential recombination (conversion) events (44) have been found. Replacement mutations (expressed as amino acid exchanges) and silent mutations (not expressed at the protein level) spread over the entire V region. The codon usage within V genes varies between framework (FR) and complementarity determining (CDR)regions, affecting the ratio of possible replacement to silent mutations (R/S). This effect is not very pronounced, though. Whereas in the FRs the theoretically expected R/S ratios are generally around 3:1, they range from 6,2:1 to 3,5:1 in the CDRsof a collection of 14 murine VHgenes (T. Simon, personal communication; note, however,that the effect maybe stronger for single CDRs). Even taking the different codon usage into account, replacement mutations are not randomly distributed. They tend to accumulate in CDRs,and a repeated occurrence of certain replacement mutations at the same sites has been observed in several antigenic systems (32, 33, 35, 46). These phenomenahave been interpreted as the result of antigenic selection for better binding antibodies. Certain amino acid exchanges, repeatedly found in anti-hapten antibodies at the same position could indeed be experimentally identified as the cause of an increased affinity for antigen (32, 33, 69; see also below). On the other hand repeats of mutations that cannot be explained by cellular selection have been found in several systems (32, 33, 69, 70). These appear to define hot spots of mutation preferred by the hypermutation mechanism. STEPWlSE INTRODUCTION AND RATE OF MUTATION The

molecular analysis of the V regions of clonally related B cells as first isolated by McKean et al (48) from animals under immunization, has led to important insights into the way somatic mutants are selected. Several groups have isolated cells of this type, carrying identical IgH and IgL rearrangements and sharing somesomatic mutations, but differing in other mutations. V region sequences from such cells can be arranged into genealogical trees, showing that the mutants are derived from a commongermline precursor and that somatic mutations are introduced stepwise during clonal proliferation (45, 48, 49, 51, 52, 60, 71-74; an example of a genealogical tree is shown

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in Figure 2). Extrapolating from this result that somatic mutations are introduced at a constant rate and independently from one another in the course of proliferation, mutation rates can be calculated. Early calculations made further assumptions, namely, onset of mutation after immunization -3 and a cell division time of 24 hr. The resulting rates were close to 1 x 10 per base pair per generation (48, 51). A similar rate (5 x -4 per base pair per generation) was calculated by a methodbased on the increase in silent mutations observed between antibodies isolated on day 7 or on day 14 after immunization (33). One can look at the problem in a simpler way, however.Onlyat a rate in the order of 1 x 10-3 per base pair per generation can nonselectable mutations, as they are seen in secondary and hyperimmuneantibodies, accumulate in a proliferating cell population (see modelcalculations in 75 and 32). At this rate roughly every second progeny cell is hit by a mutation, and cells expressing highly mutated antibodies will thus becomea major fraction of the growing clone. A mutation rate of 10-s per base pair per generation found in a pre-B-cell line (76) is too low to cause the pattern of mutation observed among memoryB cells. Rates higher than 10-3 per base pair per generation would drastically reduce the clone size because of the accumulation of mutilating mutations (see below; 32, 75). The mutation rate may be variable depending on the differentiation

~Xiati~ I

~

i

~

A~0/44~..~,,,~

100!

o-

~-~

~-,

,~.,

Figure 2 Intraclonal a~nity maturation. (Le~) Genealogicaltree o[ B cells connectingthree somatic mutantsto a germline progenitor. V region sequencesare schematically shownwith replacementmutations indicated. (Right) A~nities of anti-idiotypic antibodies expressedby the experimeutally reconstructedprogenitors (1, 2, 3) and the somaticmutantsisolated vJvo (A2/69, A20/44, A8/4). Thea~nities can be read on the abscissaat the point of 50% inhibition by antigen (S43.10Fab).

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stage of the B cells. A rate of 10 5 per base pair per generation at the pre-B-cell stage (76) allows for the occurrence of a few mutants in the preimmuneand early immuneresponse repertoire. Experimental evidence concerning the rare occurrence of mutations in myelomasand hybridomas, i.e. at the plasma cell stage (for a discussion see 77), and the stable expression of V-regiongenes in the effector phase of the secondary response (38) suggest that the hypermutation mechanismis up- and down-regulated in a stage-dependent fashion, possibly determined by the local environment. The rate estimates discussed above do not take into consideration the identification of hot spots of mutation, indicating that the mutation rate differs at different positions in V-region genes. Another problem is that mutations are not necessarily introduced independently of each other (see below). If they are not~ mutation rates referring to the introduction single mutations cannot be calculated. special mechanism of Ig gene hypermutationis clearly required to explain the abundanceof nonselectable mutations in immunoglobulin genes (53). This mechanism introduces mutations into the V regions of the proliferating cells in distinct mutagenic episodes (48, 49, 51, 52, 7l). In the absence of a suitable in vitro model, the molecularbasis of somatic hypermutation remains a matter of speculation. Twotypes of models can be distinguished. In the first, mutations are introduced as errors of DNA reduplication, during either DNAcell division or repair. Mutations are fixed upon replication in the progeny. Brenner &Milstein have proposed a mechanisminvolving V region gene specific error-prone repair (78). There is experimental evidence for error-prone DNApolymerase activity (79) in DNArepair in humancells. In this case more than one mutation may occur during a single mutagenic episode. Clustering of somatic mutations in antibody V regions has been claimed (56), but this issue controversial (75). Interestingly, a high error rate (1.5 × 10-3 per nucleotide) has bccn determined for eucaryotic DNApolymerase/~, believed to play a role in DNArepair (80). Eucaryotic DNApolymerases show sitepreferences for certain types of mutations (79, 80). Such preferences may account for the mutational hot spots that have been observed in V genes (see above). In a second type of model, mutations are introduced by mismatchrepair of misaligned templates such as quasipalindromes (81). Mutations would again be fixed by DNAreplication. Repair of quasipalindromes leads to base substitutions in a bacteriophage gene (82). AntibodyV genes contain more palindromic structures than do other genes (83-85), and in some systems mutational hot spots seem to correlate with nucleic acid secondary structure (33, 70, 84). POSSIBLE MECHANISMSOF HYPERMUTATION A

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TIMING AND ANATOMICALSITE OF SOMATIC HYPERMUTATIONThe available

data suggest that somatic hypermutation is induced by antigenic stimulation. The mutation frequency increases gradually in antibodies from late stages of the primary (39), from the secondary (40, 44, 47), and from tertiary (41) immuneresponse. Data of this kind, as systematically obtained by Milstein and co-workers, suggest onset of hypermutation after immunization. They cannot exclude, however, selection and outgrowth over time of rare, pre-existing mutants through preferential stimulation. Direct evidence that hypermutation occurs predominantly in the antigen-dependent phase of B-cell differentiation has been provided by Manser & Gefter (86): After anti-idiotypic suppression of a germline specificity, a particular mutant phenotype, regularly expressed in the response of unsuppressed animals, could not be generated upon antigenic challenge. Together with the stepwise accumulation of somatic mutations during clonal expansion (see above) and the occurrence of somatic mutations rearranged immunoglobulin transgenes (63), this experimental evidence argues strongly against the hypothesis (58) that hypermutation is linked to DNAreplication during V gene rearrangement. Introduction of somatic mutations into immunoglobulin V regions is not dependent on heavy chain isotype switching (39, 48, 51, 52). The timing of somatic hypermutation relative to isotype switching has been reviewed recently (8). Whetherthe isotype switch has any functional relation to the hypermutationprocess at all is not clear. If it has a relation, it would, contrary to earlier suggestions (42, 67), be the molecular event that terminates the hypermutation process (8). MemoryB-cell generation requires, besides the induction of the hypermutation process, affinity-selection by antigen without induction of terminal differentiation into plasma cells. Possible anatomical microenvironments for memoryB-cell generation are germinal centers, substructures of peripheral lymphoid organs, containing accumulations of proliferating B cells, antigen-presenting cells, and T cells. Germinalcenters build up after immunization with T cell-dependent antigens around the time when mutated antibodies begin to appear in the animal (87, 88). Germinal center B cells initially express IgM. The disappearance of germinal centers with time might coincide with the release of cells that have mutated and switched to form stable memorycells. Mutant Selection

and Affinity

Maturation

RATIOS OF REPLACEMENTTO SILENT MUTATIONSAS INDICATORS OF SELECTiON Ratios of replacement to silent mutations (R/S) have been widely

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used as measures of selection (44, 49, 50, 71-73, 89), operating either positively (e.g. selection of high affinity mutants) or negatively, preserving antibody structure (as often seen in frameworkregions). On this basis has been concluded that selection by antigen is a driving force in the generation of follicular lymphomas (71, 89) and in the propagation of cells producing anti-DNA(73) and anti-IgG (72) autoantibodies. In responses against different determinants of influenza hemagglutinin, R/S ratios indicate that in somecases the light-chain (49), in others the heavy-chain region (50) maybe more important for antigen binding. The main difficulty in evaluating the significance of R/S ratios is that, besides the problem of mutational hot spots, the fraction of "nonpermissible" replacement mutations leading to a structurally nonfunctional antibody is not known. Estimates range from 25% (44) to 50%(73, of all mutations. The latter estimate is based on an evaluation of KabatWuvariability plots, postulating that besides nonsense mutations (leading to stops or frameshifts) any exchanges at sites which are invariant, or nonconservative exchanges at sites that vary only by conservative substitutions, will lead to a structurally nonfunctional V region domain. Recent experimental evidence indicates that the situation is more complicated, however.Whereasantigen- and idiotype-binding can be lost easily by single aminoacid exchanges (77, 90-92), the only direct evidence so far available for a deleterious effect of a point mutation on an antibody V region, leading to a structurally nonfunctional antibody molecule, is a glycine to arginine exchangein codon 15 of a 22 chain (93). This exchange interferes with secretion. Point mutations, resulting in the loss of an invariant tryptophan (position 148) from one allele and an invariant cysteine (position 194) from the other allele of the C~genesof a patient with x chain deficiency, probably represent the molecular defect responsible for the disease (94). A similarly critical role for invariant positions in the V regions should be expected. However,substitution of a heavy chain, variable-region cysteine at position 92, part of an invariant disulfide bridge so far considered crucial for the correct folding and function of the Ig V region, had no influence on antigen-binding or -precipitation (95). Furthermore, exchange by mutagenesis of an invariant tryptophan at position 36 of the second heavy-chain variable region did not have any apparent effect on the binding properties of an antiarsonate antibody (96). Althoughthe latter experiment does not directly address the deleterious effects of single point mutations since it involves an exchange of two nucleotides in one codon, these examples underline the fact that estimates on the fraction of "nonpermissible" mutations are still hypothetical.

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AFFINITY MATURATIONAND SOMATIC MUTATION Most antibodies

isolated

from secondary or hyperimmune responses have an increased affinity for the immunizing antigen as compared to typical primary response antibodies. This finding correlates well with the presence of somatic mutations in the V regions of secondary antibodies in various antigenic systems (39, 44, 47, 97). In two cases unmutated (primary) and mutated (secondary) antibodies can be compared which use identical VHVLgene combinations with identical VHDJH and VLJLjoining regions. The antioxazolone antibody NQ22.15.18 (41) contains 13 replacement mutations, leading to morethan 100-fold higher affinity (33). Antibody107.17, specific for the NP hapten (38), carries seven replacement mutations and has an affinity ten times higher than its unmutated counterpart (C. Kocks, unpublished). In these instances affinity maturation is thus exclusively due to somatic hypermutation. Recently, single replacement mutations resulting in affinity maturation have been identified. Mutagenesis experiments, carried out in a study of affinity maturation of anti-NP antibodies, demonstrated that of all mutations present in the V regions of 21 chain bearing high affinity antibodies, a single exchangein CDR1of the heavy chain is mainly responsible for the affinity increase observed in this antibody population in the secondary response (32, 69). Berek & Milstein (33) mention heavy and light chain recombination experiments in which one particular amino acid exchange (always occurring in high affinity antibodies) leads, together with one other conservative exchange, to most of the increase in affinity of two antioxazolone antibodies tested. In contrast to the situation in the anti-NP response, where only a single mutation capable of improving hapten binding seems to be available in the VH/VLgene combination predominantly used, stepwise affinity maturation through an ongoing process of mutation and selection can also occur (33, 48, 49). Wehave demonstrated stepwise intraclonal affinity maturation directly (98). Through recombinant DNAtechniques, a genealogical tree (5 l, 60) was reconstructed (see Figure 2) whichconnects three mutantcells that had been isolated from an in vivo immuneresponse to a progenitor B-cell expressing a germline encoded antibody. Affinity measurementsof the mutants and the reconstructed antibodies demons’trate that intraclonal affinity maturation occurred stepwise (Figure 2). Affinity maturation started from a germline-encodedspecificity; this further suppOrts~th6concept that somatic hypermutation is induced by the immunizingantigen. Affinity maturation, a classical phenomenonin immunology(99), has been classically interpreted AFFINITY MATURATIONAND CLONAL SELECTION

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B-CELLDIFFERENTIATION PATHWAYS 549 in the frame of the clonal selection theory (3) as preferential selection and outgrowth with time of pre-existing, infrequent clones bearing receptors with high affinity for the immunizingantigen. The data discussed in the previous section lead to a different interpretation: Cells expressing high affinity antibodies are #enerated from pre-existing antigen-specific cells as a consequence of immunization, through clonal expansion, somatic hypermutation, and recurrent selection by the immunizingantigen. While this latter mechanismapparently plays a dominantrole in affinity maturation, the recruitment of rare, pre-existing cells carrying high affinity germline antibodies (or even of rare, pre-existing somatic mutants)--the classical interpretation--could be involved as well. In accord with this notion, differences between the "clonotypes" (a clonotype being define~d by a particular H- and L-chain V-region gene rearrangement) of B cells seen in primary and secondary responses have been reported (40, 44, 100, 101). However,a first direct test of two secondary response "clonotypes" from which somatic mutations had bccn eliminated by genetic engineering suggested recruitment into the memorypathway on grounds other than a high affinity for antigen before somatic hypermutation (69). For further speculations about clonotype selection in primary and secondary responses we refer the reader to the literature (33, 44, 102-104). Memory B Cells

and B-Cell

Memory

On the basis of what has been discussed above we can.define a memoryB cell as a surface Ig expressing B lymphocytewhich has-been selected in a pathway of antigen-driven proliferation and somatic hypermutation and is ready to produce a secondary response upon antigenic challenge. Whether the cell produces such a secondary response (in which hypermutation does not seem to occur--see below) or continues to proliferate and hypermutate (104) may be determined by the microenvironment which the cell happensto be. Alternatively, the cell could reach a stage of differentiation at which the hypermutation mechanism has been irreversibly turned off, perhaps through isotype switch (8). There is evidence that high affinity B cell memoryis carried by cells that have already switched to the isotype later expressed in the secondary response (38, 105107). IgM expressing cells which produce an enhanced response upon in vivo transfer or in cell culture have also been identified in antigen-primed animals (108-110). These cells maybe still in the pathwayof proliferation and hypermutation. They seem to produce low affinity antibodies (107, 109), and it is not clear whether they participate in the secondary response in the intact animal. The persistence orB-cell memorycould involve continuous B cell recruit-

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mentand selection, driven by antigen (87, 111) or controlled by the idiotypic network(4, 112). It couldalso reflect the generationof stable, longlived memorycells. These two mechanismsmight work hand in hand, and consequentlyevidencein favor of one does not necessarily argue against the other. Recent experiments(P. Vieira, K. Rajewsky,in preparation) have shownthat chronic treatment of mice with an anti-T helper cell antibody, at doses blocking primary responses and memoryinduction, does not affect established B-cell memory.Underthese conditions memory (measuredas an adoptive secondaryresponse in the presence of an excess ofT-cell help) remainedquantitatively unchanged over a period of at least four weeks.Theresults argue for the persistence of memory B cells in the absenceof T cell~lependentantigenic stimulation. STABLE EXPRESSION OF MEMORY IN SECONDARY RESPONSE

THE

Proliferation anddifferentiation of B cells into antibodysecretingcells in the secondaryresponse can be regarded as a "readout" of B-cell memory. Recent evidence indicates that the cells selected from the memory compartmentinto the secondaryresponse behavelike B cells in the primary response,in that they proliferate andterminallydifferentiate in the absence of somatic hypermutation.No further somatic mutations were introduced into the V regions of clonally related B cells undergoingan adoptive secondaryresponse (38). Themolecularanalysis of these cells showed addition that the memory cells, triggered by the secondarystimulus, had already switchedisotype, and no further switch rearrangementsoccurred duringextensiveproliferation. This experimentis in line with earlier data, documentingthe stability of the memoryresponse over long periods of time in cell transfer experiments(113). Theextent of B-cell proliferation in the adoptive secondaryresponse mayexceed that in an intact animal undergoinga secondaryresponse. This does not affect the mainpoint of the experimental evidence, namelythat in the appropriate environment hypermutated(memory)cells producea secondaryresponse in the absence of further hypermutation. Thesimilarity of kinetics of adoptiveandin situ secondaryresponsesas well as the absenceof hypermutationin terminally differentiated cells supportsthe interpretation that this pattern is characteristic of the secondary response pathway. Weconclude that in both primary and secondary responses the cells behaveas postulated by the clonal selection theory: each precursor cell and its progeny is predeterminedfor the production of an antibody of a given specificity (3).

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SEPARATE PATHWAYS FOR THE EXPRESSION GERMLINE AND SOMATICALLY MUTATED REPERTOIRES: FUNCTIONAL SIGNIFICANCE Natural and Acquired Immunity

551

OF

If there is evolutionary selection for the expression in the pre-immune repertoire of a set of antibody binding sites with a high protective value and at the same time for maximaladaptability of the system to a particular antigenic challenge, then the existence of separate developmentalpathways for these two functions, namely natural and acquired immunity, seems reasonable (see Figure 1). In the cellular compartmentresponsible for natural immunity,the cells wouldbe selected to express a restricted repertoire of germline V regions specific for microbial antigens as it maybe the case for Lyl B cells (8). The mechanismof repertoire selection still poorly understood and might include cell-autonomous processes like preferential rearrangement of certain V genes or V gene combinations (2, 114, 115) as well as positive V-region selection by receptor-mediated (idiotypic) cell interactions either within the population (17) or between cells and regulatory T cells (116). The evidence in the latter cases relates to selection early in ontogeny and might point to a special mechanismby which a certain pattern of specificities is imprinted into the system for lifetime. Later on, other autoantigens mayparticipate in the propagation of the cells, in concert with microbial antigens entering the body. Autoreactivity of antibodies and their potency in antimicrobial defense maybe interrelated, corresponding to microbial mimicry and adaptation in the interaction of microbe and host: Natural immunity must be targeted to epitopes which microbes are unable to change by mutation for functional reasons. Such epitopes likely include the ones acquired by microbes through mimicry. This would explain why autoreactivity is a dominant feature of the repertoire not only in the Lyl B compartmentbut in natural antibodies in general (117-119). While somatic mutation would be as deleterious to the antibody repertoire expressed in natural immunityas to any other evolutionarily selected structure in the organism, it plays a central role in the major Bcell developmental pathway leading to acquired immunity, namely the acquisition of somatic immunological memory.Here, the central need is adaptation to a particular stimulus. The generation of a new, diverse antibody repertoire from pre-existing binding sites through somatic hypermutation suits this purpose, because the newly generated repertoire is likely to contain an abundanceof antibody mutants which either bind the immunizingantigen with increased affinity (as discussed above) or are able

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to recognizestructurally related antigens. Thelatter is of particular value in microbialinfections in whichthe microbesattemptto escapethe immune system through the generation of mutants. In line with this concept is evidence that uponT cell~tependent immunization(a) somatic antibody mutantswith changedantigen binding specificities can indeedbe isolated fromthe system(38, 120) and (b) idiotypically related antibodies binding to different epitopes of the immunizingantigen or even to none of its epitopes occasionallyappearin the serum(121; for reviewsee 122). Threat and Control of Autoimmunity Thede novogeneration of an antibody repertoire through somatic hyper~ mutation requires an efficient control to protect the organismagainst somaticmutantswhichhappento haveacquired autoreactivity (123, 124). Thekeyto the solution of this problemlies presumably in the fact that the pathway of B memorygeneration is T-helper-cell dependent. B-cell mutantsreactive with an autoantigen will in general not be driven by T helper cells becauseautoreactive T helper cells are knownto be selected against in the thymus(125, 126), and somatic hypermutationseemsto restricted to the B-cell compartment (127). However, negative selection the thymusdoes not completelypurge the p~fipheral T cell compartment from potential autoreactivity (128). In addition, antibody mutants may arise whoseVregions, i.e. idiotypes, happento be recognizedas such(with or without "processing"(129, 130) by helper cells in the environment. such situations mutant, autoreactiveB-cell clones could be triggered into uncontrolled expansion and attack the organism--a scenario described by Weigertand his colleagues in their analysis of autoantibodyproduction in the autoimmune strain MRL (72, 73; see also 131). Positive Selection as a General Principle in B-Cell Development A common feature of the developmentalpathwaysof B cells reviewed in this article is positiveselection throughligandsbindingto the Ig receptor. Thesecould be internal ligands (including antibodyVregions themselves) as is perhaps the case in early (Lyl?) B-cell development,or foreign antigens as in the pathwaysof acquired immunity.A common principle seemsto emerge,valid for both T (132)andB cells, namely,that a cell will only survive if addressed by the environmentthrough its receptor. This principle seemsalso to hold for B-cell tumors. In follicular B-cell lymphomasin whichV-regionhypermutationoccurs, Ig negative variants are rare (R. Levy, personal communication) and strong selection for certain V-regiondeterminantswasseen (71, 89). Theantibodyreceptor is therefore

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not only a determinant of normal, but presumably also of malignant, Bcell growth. The pronouncedselection of certain V regions in chronic Bcell leukemias (24, 29-31) supports this view. The principle of positive selection (and death of non-selected cells) particularly attractive for cells in the pathwayof memory generation. Here, of the abundanceof somatic mutants only those reactive with the antigen (or with antigenic variants) would be retained and expanded--a situation reminiscent of T-cell developmentin the thymuswhere only cells restricted to self-MHCare selected (132). Note, however, that as in the case of cells positive selection does not preclude negative selection operating through the elimination of high affinity self-reactive cells (133-135). Decision

Making."

Cell

Autonomy versus

Local

Environment

One major unresolved problem in B-cell development is how a cell is driven into a particular pathwayof differentiation (see Figure 1). As in all developmentalprocesses, the task is here to identify and distinguish cellautonomousprocesses from processes induced by the environment. There is evidence that Ly I B cells represent a lineage different from classical B cells. It is unclear, however,to which developmentalstage these different lineages can be traced back, i.e. at which point and through which mechanism they separate from each other. In the developmental pathways of classical B cells, decision-making is not understood at all. Whethera B cell participates in a primary antibody response or enters the pathwayof memory generation may depend on the microenvironment in which the cell happens to be. Alternatively, the two pathways mayrepresent developmentalprogramsof different B-cell lineages (135). Similarly, a mutated and antigen-selected memorycell maydifferentiate to a stage where it is programmedto produce a secondary response upon contact with antigen (and a T helper cell). It is possible, however, that in the appropriate microenvironment the cell can again enter the pathway of hypermutation and selection. Withadvancedcell culture systems, artificial lineage markers (136-138) and a variety of models of genetically determined B-cell deficiencies (139) available, these unresolved problems can be experimentally approached. ACKNOWLEDGMENTS

Wethank I. F6rster for help and discussions, T. Simon, P. Vieira, A. Stall, and L. Herzenberg for communicating unpublished results, E. Siegmund for preparation of the manuscript, and U. Ringeisen for the figures. The work from our laboratory was supported by the Deutsche Forschungsgemeinschaft through SFB 74 and by the FAZIT Foundation.

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pression of IgD by murine lymphocytes. Loss of surface lgD indicates maturation of memoryB cells. J. Exp. Med. ~47:984-96 110. Yefenof, E., Sanders, V. M,, Uhr, J. W., Vitetta, E. S. 1986. In vitro activation of murine antigen-specific memory B cells by a T-dependent antigen. J. Immunol. 137:85-90 111. Gray, D., MacLennan,I. C. M., Lane, P. J. L. 1986. Virgin B cell recruitment and the lifespan of memoryclones during antibody responses to 2,4-dinitrophenylhemocyanin. Eur. J. lrnmunol. 16:64148 112. Jerne, N. K. 1974. Towards a network theory of the immune system. Ann. lmmunol. (Inst. Pasteur) 125C: 373-89 113. Askonas, B. A., Williamson, A. R. 1972. Factors affecting the propagation of a B cell clone forming antibody to the 2,4-dinitrophenyl group. Eur. J. Immunol. 2:487-93 114. Yancopoulos, G. D., Desiderio, S. V., Paskind, M., Kearney, J. F., Baltimore, D., Alt, F. W. 1984. Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines. Nature 31 I: 727-33 115. Perlmutter, R. M., Kearney, J. F., Chang, S. P., Hood, L. E. 1985. Developmentally controlled expression of immunoglobulin VH genes. Science 227:1597-1601 116. Marcos, M. A. R., de la Hera, A., Pereira, P., Marquez, C., Toribio, M., Coutinho, A., Martinez, A. C. 1988. B cell participation in the recursive selection ofT cell repertoires. Eur. J. lmmunol. 18:1015-20 117. Ternynck, T., Avrameas, S. 1986. Murine natural monoclonal autoantibodies: A study of their polyspecificities and their affinities. Immunol. Rev. 94:99-112 118. Dighiero, G., Lymberi, P., Holmberg, D., Lundquist, I., Coutinho, A., Avrameas, S. 1985. High frequency of natural autoantibodies in normal newborn mice. J. Immunol. 134:765-71 119. Holmberg,D., Freitas, A., Portnoi, D., Jacquemart, F., Avrameas, S. 1986. Antibody repertoires of normal BALB/c mice: B lymphocyte populations defined by a state of activation. Immunol. Rev. 93:147-69 120. Manser, T., Parhami-Seren, B., Margolies, M. N., Gefter, M. L. 1987. Somatically mutated forms of a major anti-p-azophenylarsonate antibody variable region with drastically reduced affinity for p-azophenylarsonate. J. Exp. Med. 166:1456~3

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B-CELL DIFFERENTIATION 121. Oudin, J., Cazenave, P.-A. 1971. Similar idiotypic specificities in immunoglobulin fractions with different antibody functions or even without detectable antibody function. Proc. Natl. Acad. Sci. USA 68:2616-20 122. Rajewsky, K., Takemori, T. 1983. Genetics, expression and function of idiotypes. Annu. Rev. lmmunol. 1:569-607 123. Diamond, B., Scharff, M. D. 1984. Somatic mutation of the T15 heavy chain gives rise to an antibody with autoantibody specificity. Proc. Natl. Acad. Sci. USA 81:584144 124. Giusti, A. M., Chien, N. C., Zack, D. J., Shin, S.-U., Scharff, M. D. 1987. Somatic diversification of SI07 from an antiphosphocholine to an anti-DNA autoantibody is due to a single base change in its heavy chain variable region. Proc. Natl. Acad. Sci. USA 84:2926-30 125. Von Boehmer, H. 1986. The selection of the c~,/~ heterodimericT-cell receptor for antigen. Immunol. Today 7:333-36 126. Kappler, J. W., Roehm, N., Marrack, P. 1987. T cell tolerance by clonal elimination in the thymus. Cell 49:273-80 127. Toyonaga, B., Mak, T. W. 1987. Genes of the T-cell antigen receptor in normal and malignant cells. Ann. Rev. Immunol. 5:585 620 128. Cohen, I. R. 1986. Regulation of autoimmune disease--physiological and therapeutic. Immunol. Rev. 94:5-21 129. Che~nut, R. W., Grey, H. M. 1986. Antigen presentation by B cells and its significance in T-B interactions. Adv. lmmunol. 39:51-94 130. Tite, J. P., Kaye, J., Saizawa, K. M., Ming, J., Katz, M. E., Smith, L. A., Janeway, C. A. Jr. 1986. Direct interactions between B and T lymphocytcs bearing complementary receptors. J. Exp. Med. 163:189-202 13l. Behar, S. M., Scharff, M. D. 1988. Somatic diversification of the S107 (TI5) VHll germ-line gene that en-

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codes the heavy-chain variable region of antibodies to double-stranded DNA in (NZB × NZW)F1mice. Proc. Natl. Aead. Sei. USA 85:3970-74 132. Kisielow, P., Teh, H. S., BliJthmann, H., von Boehmer, H. 1988. Positive selection of antigen-specific T cells in thymus by restricting MHC molecules. Nature 335:730-33 133. Nossal, G. J. V. A. 1983. Cellular mechanisms of immunologic tolerance. Ann. Rev. Immunol. 1:33-62 134. Goodnow, C. C., Crosbie, J., Adelstein, S., Lavoie, T. B., Smith-Gill, $. J., Brink, R. A., Pritchard-Briscoe, H., Wotherspoon,J. S., Loblay, R. H., Raphael, K., Trent, Ronald J., Basten, A. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenie mice. Nature 334:67f~82 135. Linton, P.-J., Gilmore, G. L., Klinman, N. R. 1988. The secondary B cell lineage. In B Cell Development,ed. O. Witte, M. Howard, N. Klinman, UCLASymp. Mol. Cell. Biol. Vol. 85. NewYork: Alan R. Liss. In press 136. Dick, J. E., Magli, M. C., Huszar, D., Phillips, R. A., Bernstein, A. 1985. Introduction of a selectable gene into primitive stem cells capable of longterm reconstitution of the hemopoietic system of W/Wvmice. Cell 42:71-79 137. Keller, G., Paige, C., Gilboa, E., Wagner, E. F. 1985. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature 318: 14954 138. Lemischka, I. R., Raulet, D. H., Mulligan, R. C. 1986. Developmental potential and dynamic behaviour of hcmatopoictic stem cclls. Cell 45:917 27 139. Schultz, L. D., Sidman, C. L. 1987. Genetically determined murine models of immunodeficiency. Ann. Rev. Immunol. 5:367404

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Ann. Rev. Immunol. 1989. 7.’56l 78 Copyright © 1989 by Annual Reviews Inc. All rights reserved

T-CELL RESPONSES AND IMMUNITY TO EXPERIMENTAL INFECTION WITH LEISHMANIA MAJOR Ingrid Miiller, Thierry Pedrazzini, Jay P. Farrell, and Jacques Louis WorldHealth Organization, Immunology Research and Training Centre, Institute of Biochemistry,Universityof Lausanne,1066Epalinges, Switzerland INTRODUCTION Leishmaniasesare infectious diseases ~f protozoanorigin that represent an increasing public health problemworldwide(1). There are indications that these diseases also affect an increasing number of immunocompromisedpatients in someareas of developed countries (2). The organismsresponsible for these diseases are trypanosomatidprotozoans of the genus Leishmania.Theparasites are transmitted to their mammalian hosts by the bite of an infected phlebotomine sandfly. Thelife cycle of all species of Leishmaniaincludes two developmentallyand morphologically distinct forms:(a) the extracellular, flagellated promastigoteswhichcolonizethe digestivetract of the insect vector, and(b) the sessile amastigotes whichare foundexclusivelyas obligate intracellular parasites within cells of the mammalian reticuloendothelial system(3). Infection of humanswith Leishmaniaresults in a broadspectrumof disease profiles whosefeatures dependuponthe characteristics of the different Leishmaniaspecies and the efficiencyof the host’s immunological responseto the parasite (4). That natural self-healing infection leads to the developmentof immunityto reinfection strongly suggeststhat control of leishmaniasisby prophylactic immunizaton is possible. Since the entire spectrumof clinical manifestations seen in humans 561 0732~0582/89/02~ 10~0561$02.00

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infected with Le&hman& can be reproduced after experimental infection with Leishmania major (L. major) in mice depending upon their genetic background, this modelof experimental infection has been used by several research groups to elucidate the immunemechanismsinvolved in resistance or susceptibility. For example, BALB/cmice, which are susceptible to infection with L. major, the causative agent of cutaneous leishmaniasis, develop severe lesions at the site of inoculation and fatal visceralization, whereas CBA-micedevelop only small cutaneous lesions that resolve spontaneously within several weeks(5, 6). In the mammalianhost, Le&hmanh~ can only live and multiply in mononuclear phagocytic cells, and the activation of parasitized macrophages has been recognized as one of the main mechanismsby which these intracellular parasites are destroyed. Therefore these cells, which are involved in the induction of the immuneresponse (e.g. antigen presentation) as well as the effector phase of the immuneresponse, (that is, responsiveness to T cell~terived cytokines), have a central role in Leishmania infection. Observations show that peritoneal macrophages from resistant mice inhibit proliferation of intracellular L. major to a greater extent than do macrophagesfrom susceptible mice; these observations suggest that the innate ability of macrophagesto destroy intracellular parasites mayplay a role in the outcome of infection (5, 7). Comparedto macrophagesfrom resistant animals, macrophagesfrom susceptible mice are less sensitive to activation by lymphokinepreparations in vitro (8). These results, together with the demonstration in the spleen of susceptible mice infected with L. major (9), of an increased number of immature cells of the macrophagegranulocyte lineage which are presumed to be phagocytic but refractory to activating lymphokines and therefore permissive for parasite growth, indicate clearly that macrophagesare important in the expression of disease induced by L. major. There is little evidence in the murine model of experimentally induced cutaneous leishmaniasis that antibodies, detectable from two to three weeksafter infection, directed against any life-cycle stage of the parasite, play a major protective role during an established infection (10, 11). However, the possibility still exists that antibodies specific for those structures on the parasite’s surface that bind to receptors on the membraneof the macrophagesmight have someprotective function by preventing the initial attachment of Leishmania to macrophages and thereby preventing entry of the parasite and its survival as an amastigote. Twomolecules on the surface of L. major promastigotes have been demonstrated to bind to the macrophage membrane, namely the abundant surface glycoprotein gp63 and the lipophosphoglycan (12, 13). Monoclonal antibodies (mAb) directed against the lipid containing glycoconjugates of the surface of L.

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major have indeed been shownto inhibit the binding of promastigotes to macrophages (13). Furthermore, mAbsspecific for membranecomponents of L. mexicana promastigotes conferred complete protection when inoculated with L. mexicana promastigotes into the footpad of susceptible BALB/cmice (14). Available information strongly indicates that T cell-mediated immunity rather than antibody responses plays a crucial role in the outcomeof infection with Leishmaniaparasites. The aim of this reviewis to summarizeresults, obtained by several laboratories, delineating the role of specific T-cell responses in the course of experimentally induced cutaneous leishmaniasis. Recent results from our research group aimed at analyzing further the characteristics of the T cells triggered during infection are also discussed.

T CELLS AND RESISTANCE TO INFECTION WITH L. MAJOR The pioneering work of the group of G. Mitchell has emphasized the important role of T cells in resistance to Leishmaniainfection by showing that athymic mutant nu/nu mice from either resistant or susceptible strains develop a generalized fatal disease after infection with L. major and that this extreme susceptibility could be fully reversed by adoptive transfer of T ceils from normal syngeneic mice (15). It is interesting that resistance was seen only after transfer of relatively small numbers(10 6) Of normal syngeneic lymphoidcells. In adoptive cell transfer experiments, using normal mice as recipients of spleen cells from genetically resistant mice which had spontaneously recovered from infection, the protective T-cell response is mediated by Lyt 1 +2- (L3T4÷) T cells (16). + T lymphocytesin experiTo evaluate directly the role of specific L3T4 mentally induced murine cutaneous leishmaniasis, the course of infection has been studied in resistant and susceptible mice in which the pool of L3T4÷ T cells had been reduced more than 95%by administration of antiL3T4monoclonal antibodies (mAb)during the entire course of infection with L. major. Virtual elimination of L3T4÷ T cells by anti-L3T4 mAb led to the developmentof severe, uncontrolled lesions in both susceptible and resistant mice (17). Takentogether, these results clearly show that + T cells are essential for effective immunological Leishmania-specific L3T4 control of cutaneous leishmaniasis. The frequency of T cells generated in the lymph nodes draining the lesions, that were capable of transferring specific DTHreactions into naive mice, was determined. Compared to susceptible BALB/cmice, CBAmice produced three times as manyparasite-specific Lyt 2+ cells at a time when lesions begin to resolve in mice of this resistant strain (17). In

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attempt to further evaluate a possible role of Lyt 2+ T cells in the healing of lesions induced by L. major, the pattern of infection was studied in mice treated with anti-Lyt 2 mAb. Depletion of Lyt 2+ T cells in vivo by administration of anti-Lyt 2 mAbbefore and during infection resulted in an exacerbation of lesions both in resistant and susceptible mice. However, resistant mice severely depleted of Lyt 2+ T cells were still capable of healing their lesions (17). Further evidence for a role of specific Lyt + cells in the elimination of the parasite by infected hosts has recently been obtained in a murine model of L. donovani infection (18, 19). Taken together, these results indicate that specific Lyt 2+ T cells, although not the main protective cells, do contribute to the immunological control of experimental leishmaniasis. Since the activation of macrophagesrepresents the main effector pathway by which intracellular L. major are eliminated by infected hosts, the + T cells on the resolution of L. majorcrucial effect of specific L3T4 induced lesions was suspected for some time to be the consequence of their releasing, upon activation, lymphokines required for macrophage activation. Indeed, the ability of mice from inbred strains to resolve spontaneously their cutaneous lesions was directly correlated with the capacity of lymphnode cells draining the lesions to release interferon-y (IFN-7), major macrophage-activating lymphokine, in response to stimulation with parasite antigens in vitro (20). These results were confirmed by observations showingthat, comparedto susceptible mice, significantly greater amounts of IFN-7 mRNA were present, 8 weeks after infection, in the lymphnodes and spleens of genetically resistant mice (21). The important role of IFN-7 in the resolution of lesions is further supported by recent results from our laboratory which have shown that the in vivo administration of anti-IFN-7 mAbduring the course of infection, in mice of either a resistant or a susceptible phenotype, significantly enhancedthe development of lesions (Figure 1). Inasmuch as production of IFN-7 Lyt 2+ T cells has also been documented(22, 23), these cells mayalso able to activate parasitized macrophages,resulting in the destruction of intracellular L. major. The ability of specific Lyt 2+ T cells to lyse infected macrophages could represent another mechanismby which these T cells exert their protective function.

T-CELL RESPONSE AND SUSCEPTIBILITY INFECTION WITH L. MAJOR

TO

÷ T cells also play a role in Experimental evidence indicates that L3T4 determining the susceptibility of the host to infection. Thus early obsero

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3.0

565

[] Control

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[] anti - IFN,y treated

,,, 1.0

N

7 14

28 days after

49 infection

70

Fiyure 1 Developmentof lesions in resistant CBAmice treated with anti-IFN-~ mAb.Five CBAmice infected with 2 × 106 L. major received 500 /~g of anti-IFN-~/ mAb(47) twice weekly for the entire course of the experiment. Five mice similarly infected but not treated with mAbwere used as controls. The size of lesions was determined by subtracting the footpad thickness of the uninfected footpads from the infected ones.

vations from Mitchell’s group showedthat the injection of relatively high numbersof T cells from normal BALB/cmice, in contrast to those from nu/ nu mice of a resistant genotype, into syngeneic nu/nu mice failed to confer resistance. Moreover, lymphoid cells obtained from BALB/cmice infected with L. major rendered syngeneic nu/nu mice more susceptible to cutaneous leishmaniasis and abolished the state of resistance normally conferred by adoptive transfer of relatively small numbersof normal T cells (15, 24). Furthermore, the immunization of mice by the subcutaneous injection of crude Leishmania antigens with or without adjuvant was demonstrated to lead to an exacerbation of lesions induced by subsequent challenge with virulent parasites (25, 26), and this effect was the consequence of the + T cells by this treatment (27). triggering of "disease-promoting" L3T4 + To evaluate further the role of L3T4 T cells in susceptibility or resistance to infection with L. major, the numbers of specific L3T4+ T cells triggered in lymphoidtissues as a result of experimental infection were + T cells were comparedin genetically susceptible and resistant mice. L3T4 scored by their capacity to transfer specific DTHreactions, and their frequency was determined by limiting dilution analysis in vivo. Compared

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÷ T cells mediating to resistant animals, the frequencyof specific L3T4 DTHreactions was up to 50 times higher in lymphnodes draining the lesions of susceptiblemice(28). Therefore,these results revealedthat the immuneresponse of susceptible micewas characterized by the generation ÷ T cells. Basedon these results, we of high numbersof specific L3T4 postulated that protective responsescould dependuponthe actual number ÷ T cells triggered during infection. Accordingto this of specific L3T4 hypothesis, protection or disease-promotion wouldonly reflect quantitative differences in the numberof activated Leishmania-specific T cells in an infected host. Alternatively,it could be postulatedthat twodistinct ÷ T cells are generated: one T-cell type types of parasite-specific L3T4 wouldbe deleterious and promotethe disease (preferentially inducedin susceptible mice), whereasthe other T-cell type wouldbe protective and lead to the resolution of the disease (preferentially inducedin resistant mice). The outcomeof infection wouldtherefore dependuponthe balance of the activities of disease-promoting andprotective T cells. In this vein, during infection and after immunizationof mice with L. major, four ÷ T cells havebeen functionally distinct subsets of parasite-specific L3T4 described(29). ÷ Thedemonstrationof two functionally distinct subsets within the L3T4 T-cell population(30) has recently provideda rational basis for the understanding of the cellular parametersof T-cell responses involved in susceptibility and resistance to Leishmaniainfections. It appears that IFN-v and IL-4 are produced by mutually exclusive subsets of L3T4÷ T cell clones respectively designatedTH~and TH2(31). Elegant studies in which the mRNA, purified fromlymphnodecells of either resistant or susceptible miceinfected with L. major, wasprobedfor the presenceof IL-4 and IFN-v messages revealed a 50-100-fold greater amountof IFN-~, messagein resistant miceanda 50-fold greater amountof IL-4 messagein susceptible mice(21). Theseresults indicate the preferential expansionof TH2cells releasing 1L-4 in lymphnodes of infected susceptible BALB/cmice. In contrast, TH1cells producingIFN-vare preferentially inducedin resistant mice(21). Themechanisms leading to the preferential expansionof protective (TH0or deleterious (TH2)T cells and thereby determiningsusceptibility or resistance to infection are not known. MODULATION OF T-CELL RESPONSE AND INDUCTION OF RESISTANCE TO L. MAJOR IN GENETICALLY SUSCEPTIBLE MICE The elimination, during the course of infection, of only about 60%of L3T4+ T cells from lymphoid tissues by administration of anti-L3T4

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mAbto BALB/cmice has been observed to reverse their susceptibility to infection and even to render them resistant to subsequent challenge with L. major (32). Together with the results mentioned above showingthat the + T cells render those mice extremely susceptible, this elimination of L3T4 ÷ T cells are observation confirms the notion that, although specific L3T4 absolutely required for the immunologicalcontrol of L. major infection, these cells are also instrumental in mediatingsusceptibility. Althoughthese observations as well as those showing an inhibition of parasite-induced lesions in BALB/cmice treated with cyclosporin A (33) could be interpreted as indicating that disease promotion and protection merely reflect + T cells activated quantitative differences in the numberof specific L3T4 + T cells during infection, recent results strongly indicate that distinct L3T4 mediate either resistance or susceptibility to L. majorinfection. Werecently observed that BALB/cmice treated with a single dose of 600 #g of anti-L3T4 mAbGK1.5resolved their cutaneous lesions about 60 days after infection and becomeresistant to subsequent challenge with virulent parasites, provided that this treatment was given early in the infection (T. Pedrazzini, G. Milon, G. Marchal, J. Louis, manuscript in preparation). Administration of anti-L3T4 mAbeither 10 days before or 10 days after infection allowed these highly susceptible animals to resolve their lesions. In contrast, the sameantibodies given 20 days after infection did not influence disease progression. This treatment with anti-L3T4 mAb ÷ T cells GK1.5resulted in a significant but transient reduction of L3T4 in lymphoid tissues, which was maximal48 hr after treatment. Thereafter, the lymphoid tissues were gradually repopulated with L3T4+ T cells and ÷ T cells similar to those seen in after 50 days contained numbersof L3T4 normal mice. It is interesting that at that time, in comparisonto control infected mice, the frequency of L3T4÷ T cells capable of mediating DTHresponses (20-hr reaction) was drastically reduced in the lymph nodes the cured mice, although their lymphnode cells provided similar L. majorspecific helper activity (Table 1). Theseresults suggestthat in this antigenic system, the helper function, and the ability of L3T4+ T cells to mediate DTH-reactions could result from the activity of distinct L3T4+ T cells that are modulateddifferently after treatment in vivo with anti-L3T4 mAb. ÷ The results also suggest that functionally distinct parasite-specific L3T4 T cells are triggered during infection. The ability of anti-L3T4 mAbtreated BALB/cmice to contain L. major infection is also correlated with the emergence, in draining lymph nodes and spleen, of T cells able to release IFN-~after specific restimulation in vitro (34). These findings support the notion that healing of L. major-induced lesions results from the activity of TH~cells (34). They also indicate that precursors of these protective cells are present in lymphoid tissues of susceptible mice but

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Table 1 Functional activities of lymph node cells of normal or anti-L3T4 mAb-treated susceptible mice after infection with aL. major Number of DTH-Mediating cT Cells bTreatment in vivo

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a-L3R4 mAb None

~L3T4

÷ Lyt2

10 1000

15 50

Helper-Activity d(PFC/106Spleen Cells) TNP-L. major TNP-PPD No antigen 1540 884

10 10

45 134

aAll groupsof animals wereinfected subcutaneouslyin the footpad with 2 × 10 6 virulent L. major promastigotes. hMicewere treated with ~-L3T4mAbGKI.5 day and 11 post infection. + or atLyt2 + T10cells c Frequencyof parasite-specific L3T4 capable of transferring DTH-reactionswas determined50 days post infection. LNC of all three groupsof animalswereeither treated in vitro with mAbc~-L3T4and complement,or complement alone and serial dilutions of the selected T populations weretransferredtogetherwitha challengedoseof 2 × 106parasitesinto 10 recipientmice.Specificfootpadswelling wasdetermined16 to 18 hr later. The frequencyof DTH-mediating T cells wascalculated by analysingthe Poisson-distributionrelationship betweenthe numberof nonresponding miceper groupand the number of transferredcells (28). ’~Lymph node cells of either control infected animals, or animalscured as a result of a-L3T4mAb treatmentwereremoved60 days post infection and cultured in vitro for 7 days together with TNP-specific B-cells either with 106 TNP-L.major, or with 2/~g/mlTNP-PPD as antigen or in the absenceof antigen. Helperactivity of the T-lymphocyteswas evaluated by determining the numberof anti-TNPplaqueformingcells.

that either their expansion is hamperedor their effector lymphokinesare neutralized during the course of infection. In this vein, it is interesting that early observations showing that susceptible BALB/cmice can be rendered resistant by sublethal whole body ~-irradiation were interpreted as indi+ cells cating that such treatment interfered with the generation of L3T4 normally capable of preventing the expression of protective T-cell responses in chronically infected susceptible mice (35). The injection of some antigens into mice treated with anti-L3T4 mAb renders them specifically unresponsiveto these antigens (36). It is possible therefore, that a single administration of anti-L3T4 mAbto mice infected with L. major rendered them tolerant to parasite antigens encountered ÷ T cells reat that time by either mature or newly differentiated L3T4 populating the lymphoidtissue after the physical depletion of peripheral + T cells. If this is the case, one might speculate that the beneficial L3T4 effect on the development of lesions by administration of anti-L3T4 mAb during the early phase of infection would also result from the induction of unresponsiveness to someparasite antigens present in the infected host at that time. Since treatment with anti-L3T4 mAbis effective only within 10 days of infection, it can be hypothesized that the parasite antigens processed and presented during the early phase of infection induce T-cell

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responses harmful to the host. Because the majority of living L. major promastigotes injected subcutaneously into mice appear to be destroyed within a few hours by nonimmunemechanisms(37), the antigens presented during the early phase of infection are probably derived primarily from such degraded parasites. In a first attempt to investigate whether or not these antigens elicit a detrimental immuneresponse, we studied the course of infection in mice rendered unresponsive to some of these antigens. Normal BALB/cmice were rendered tolerant by injecting syngeneic splenocytes treated with the cross-linker l-ethyl-3 (-3 dimethyl aminopropyl) carbodiimide (ECDI) soluble Leishmania antigens derived from sonicated L. major promastigotes (38). The level of unresponsiveness was assessed in vitro measuring the intensity of the specific proliferative response of lymph node cells from these mice after immunizationwith the tolerizing antigens emulsified in complete Freunds’s adjuvant (CFA). The specific proliferative response measured in lymph node cells of tolerized mice was reduced by about 60%compared to control mice. Groups of tolerized and normal mice were infected subcutaneously with 2 × 10 6 virulent L. major promastigotes, and the resulting lesions were monitored for a period of 80 days. The results shownin Figure 2 demonstrate that the tolerized mice were able to contain their cutaneous lesions. This beneficial effect of tolerization to someLeishmania antigens on the disease progression was confirmed by counting the viable parasites in the lesion. These findings indicate that some parasite antigens present during the early phase of ÷ T-cell response which favors the growth infection may trigger an L3T4 of parasites to the detriment of the host. In contrast, since treatment with mAbGK1.5administered 20 days after infection had either no effect on the development of lesions or exacerbated disease progression, one might speculate that antigens present at this later stage are different from those seen at the beginning of infection. Theselate-stage antigens are likely to derive from multiplying amastigotes. Indeed, it is possible that processing and presentation of antigens derived from disrupted Leishmania promastigotes are different from those derived from amastigotes multiplying inside macrophages. PARASITE-SPECIFIC EFFECT ON THE

T-CELL LINES DISEASE PROCESS

AND CLONES:

Homogeneouspopulations and clones of parasite-specific L3T4+ T cells have been generated in vitro in an attempt to study more precisely the effects of T cells on the course of experimentally induced cutaneous leishmaniasis.

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[~ Control [] Tolerant

14

35 Daysafter infection

Figure 2 Development of lesions in infected BALB/cmice rendered partially tolerant to soluble Leishmania atttigens. Normal BALB/cmice were rendered tolerant to soluble Leishmaniaantigens by injecting these antigens, covalently coupled to syngeneic spleen cells. Five days later, these animals and normal control mice were infected s.c. into the footpad with 2 x 106 virulent L. major parasites. Lesion size was determined in both groups (5 mice/group) by subtracting the footpad thickness of the uninfected footpads from the infected ones,

+ Exacerbation of Cutaneous Leishmaniasis by Specific L3T4 T Cell Lines + T lymphocyteswere derived from lymphnode Leishmania-specific L3T4 cells of mice that had been either immunizedby the s.c. injection of a crude preparation of killed L. majorpromastigotesemulsified in complete Freund’sadjuvants or infected s.c. with virulent L. majorpromastigotes. + T cells Homogeneous populations and clones of antigen-specific L3T4 were derived from cultures of immunelymphnode cells responding to crude preparations of leishmanial antigens and were maintainedin vitro followinga protocol described in detail elsewhere(26, 39). After their + T-cell lines transfer intravenously into syngeneicrecipients, all L3T4 and clones established following selection by this crude preparation of leishmaniaantigens enhancedthe developmentof lesions and favored the multiplication of parasites in lesions of both genetically resistant and susceptiblemice(26, 37). Exacerbation of cutaneous leishmaniasis by these antigen-specific

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L3T4+ T cells required their localization to the site of infection and their in situ activation by antigen (26; R. Titus, J. Louis, manuscript in preparation). This effect did not require the participation of host T cells, was not the consequence of the production of specific antibodies by the host, and appeared to result from the continuous recruitment to the site of infection of blood-derived phagocytes, in which parasites can multiply (40; S. Mendonca,J. Louis, manuscript in preparation). These specifically activated L3T4+ T cells released molecules which increased the pool of circulating mononuclearcells recruited to the lesion thus favoring the multiplication of parasites. Indeed, after activation in vitro, these short+ T-cell lines have been shownto release Interleukin term cultured L3T4 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GMCSF), which can modulate the proliferation and differentiation of macrophage progenitor cells (41). The involvement of these molecules in promoting the development of lesions is supported by recent observations which showed that treatment of BALB/cmice with recombinant IL-3 or GM-CSF leads to lesions that are larger and contain more parasites (41, 42). Titration experiments with L3T4+ T cell lines that either exacerbated the developmentof lesions or had no effect on the course of disease showed that the T cells capable of promoting the growth of parasites in vivo are homogeneous.Furthermore, several T-cell clones derived from these lines exacerbated cutaneous leishmaniasis after transfer to normal mice (R. + T cells Titus, J. Louis, in preparation). These Leishmania-specific L3T4 have been shownrepeatedly to release substantial amounts of MAFafter specific activation in vitro and to transfer specific DTHreactions to naive mice (26, 43). The macrophage-activating property of supernatant from + T cells was recently shownto be greatly these specifically activated L3T4 inhibited by anti-IFN-~ mAbbut not significantly modified by anti-IL4 mAb(T. Pedrazzini, J. Louis, unpublished). These findings, therefore, makeit difficult to explain whythese T cells are not able to contribute to the elimination of parasites in vivo. Furthermore, these results indicate that some parasite-specific THI cells can promote disease progression. Their failure to eliminate the parasites effectively maytherefore be related to their fine specificity for antigens rather than to their functional proper+ T cells were selected in vitro by a crude preparation ties. Since these L3T4 of Leishmaniapromastigote antigens, it is tempting to speculate that the antigens they recognize are different than those expressed on the surface of parasitized macrophagesin vivo. It is therefore possible that these cells do not see antigens on the surface of infected macrophagesbut rather may recognize antigens either released by infected macrophagesor resulting from the destruction of parasites in the lesions and presented by uninfected

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macrophages. According to this hypothesis, because of their specificity, + T cells fail to contact infected macrophagesand the exacerbating L3T4 ultimately fail to deliver sufficient amountsof IFN-~in their close vicinity. However,the activity of somemolecules released after their activation by antigen, derived from degraded parasites, leads to an accumulation of infectable phagocytic cells in the lesions. This hypothesis is supported by + T cells specific for an antigen unrelated observations showingthat L3T4 to Leishmania, are also able to exacerbate cutaneous leishmaniasis, provided that they localized and were specifically activated at the site of the developing lesions (R. Titus, J. Louis, manuscript in preparation). Parasite-Specific

L3T4+ Cells

That Mediate Protection Recently we obtained two cloned L3T4+ T-cell lines capable of significantly protecting highly susceptible BALB/cmice against challenge with virulent L. major promastigotes (I. Miiller, J. Louis, manuscript submitted). The experimental approach used to generate and select these protective T cells was different from that used for the exacerbating T-cell lines. The protective L3T4+ cloned T-cell lines were derived from spleen cells of BALB/cmice infected intravenously with live stationary phase promastigotes. Twoweeks after infection, spleen cells were cultured with syngeneic accessory cells and live L. major promasti~totes in the presence of IL-2-containing supernatants. After two cycles of enrichment for antigen-specific cells in vitro, T cells were cloned under limiting dilution conditions in the presence of live L. major promastigotes as antigens. + T cells on the disease process, To study the effect of these cloned L3T4 the development of lesions was studied in BALB/cmice adoptively transferred with 3-4 × l0 6 T cells 24 hr prior to subcutaneous infection with 2 × 10 6 virulent L. major promastigotes. Control mice similarly infected received no T cells. Twoof the cloned T-cell lines significantly reduced the developmentof lesions. The result of a representative experimentis depicted in Figure 3, whereit can be seen that, comparedto control mice, the lesions in genetically susceptible animals receiving those T cells were muchsmaller at least up to 100 days post infection. Enumeration of viable parasites present in the lesions of T-cell recipients and control mice, using a limiting dilution assay (37), confirmed these results. For example, in a representative experiment72 days after infection, about 8 × 107 parasites were found in the lesions of control infected micebut only 1.5 × 105in the lesions + of T-cell recipients. The mechanism(s) by which these specific L3T4 protective T cells suppress multiplication of parasites and developmentof lesions, is unknown. Preliminary functional characterization of these

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e--e Tcell recip. CP--~c) Controls

--

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5

T

10

30

50 70 DAYSAFTERINFECTION

_

90

Fi~lure3 Protective capacity of cloned L3T4+parasite-specific T cells. Purified T cells were transferred i.v. into syngeneic naive BALB/cmice (3.4 x 106 T cells/recipient). Twenty-four hours later, the animals were infected s.c. with 2 x l0 6 live stationary phase parasites in one of the hind footpads. The results represent the meanof five animals. Lesion-size was determinedby subtracting the footpad thickness of the uninfected footpad from the infected one.

clones reveals that they release IL-2 uponspecific stimulation in vitro, so that like the previously described exacerbating T cells, they also belong to + T cell subset. It is interesting that the the recently described TH~-L3T4 pattern of antigen-reactivity of the protective T-cell clones was different from that of exacerbating T-cell lines and clones. The protective T-cell clones responded only to live L. major promastigotes as antigen in vitro. In contrast to the exacerbating T-cell lines and clones, they failed to respond in vitro to either crude preparation of frozen and thawed promastigote antigens or to fixed L. major. This unresponsiveness to killed L. major promastigotes is strong evidence that the protective and exacerbating T cells differ in their antigenic specificity. It is temptingto speculate that the ability of protective T cells to recognize antigens expressed by macrophagesharboring living parasites determined their protective capacity. Inasmuchas a T-cell clone, belonging to the THI subset and recognizing a defined leishmanial antigen, has recently been shownto adoptively transfer protective immunity(44, 45),

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it would be interesting to search for the presence of that antigen on the surface of infected macrophages.

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CONCLUDING

REMARKS

It appears that the evolution of infection induced by Leishmania depends uponseveral factors, including the virulence of the parasites, the magnitude of the macrophageinnate microbicidal activity, and its response to T-cell activating signals, this article has only summarizedthe current data and views concerning the T-cell responses triggered by L. major infection in genetically resistant and susceptible mice. Based on experimental evidence the conclusions most investigators draw are that exacerbation and resolution of lesions induced by L. major result, + T cells. at least in part, fromthe activity of distinct parasite-specific L3T4 Thus, the results showthat in susceptible mice, parasite-specific TH2cells are triggered in excess. The mechanisms by which TH2 cells promote susceptibility haye not yet been elucidated. Possibly their activation leads to the release of large amountsof lymphokines capable of increasing the numberof macrophageprecursors and of recruiting these cells to the site of lesions. The presence of considerable numbers of macrophagesin the lesions might simply dilute the effect of activating lymphokines. Another hypothesis wouldbe that THzcells secrete mediators that are neutralizing the macrophageactivating capacity of IFN-7 secreted by protective TH~ cells. Delineating the cellular parameters accounting for the predisposition of susceptible mice to expand Leishmania-specific precursor cells clonally from the THzsubset, and determining whether or not TH2and THI cells are preferentially triggered by some leishmanial antigens, would enhance our understanding of this fascinating host-parasite interaction. There is also evidence that some T cells responsible for susceptibility and resistance to L. major infection belong to the same subset but differ in antigenic specificity. Thus, THIcells recognizing antigens presented by macrophagesinfected with live parasites, mayactivate the cells and destroy the parasites, either through the release of IFN-7 close to the cells or through direct cell-cell interactions (46). In contrast, TH~cells recognizing other parasite antigens presented only by nonparasitized macrophages maynot be able to concentrate the IFN-~, that they secrete on the surface of infected macrophages. Susceptible mice might be high-responders to these antigens. Conceivably, in excess, the latter T cells accumulatein the lesion of host cells necessary for parasite survival without being able to activate parasitized macrophages(Figure 4).

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Fiyure 4 Diagramof hypothesis that those T cells (clear cytoplasm) recognizing L. major antigens displayed at the surface of parasitized macrophagescould lead to parasite destruction whereas those T cells (dark cytoplasm) recognizing parasite antigens processed and presented only by normal (non parasitized) macrophages,fail to assist elimination of parasite and instead exacerbate the developmentof lesions.

Finally, although the involvementof Lyt-2 + T cells in protective immunity to L. majorinfection is still controversial, newresults indicate that they contribute to healing of lesions by unknownmechanisms. ACKNOWLEDGMENT

Workfrom the author’s laboratory described here was supported by grants from the Swiss National Science Foundation and the UNDP/World Bank/WHOSpecial Programme for Research and Training in Tropical Diseases.

1.

M/iller

was supported

Forschungsgemeinschaft.

by a fellowship

from the

Deutsche

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ET AL

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Literature Cited 1. Modabber, F. 1987. The Leishmaniases. In Tropical Disease Research. A Global Partnership. 8th ProgrammeReport of the UNDP/WORLD BANK/WHO Special Pro, q. Training in Trop. Dis., ed. J. Maurice, A. M. Pearce, p. 99-112. Geneva: WHO 2. Fuzibet, J. G., Marty, P., Taillan, B., Bertrand, F., Pras, P., Pesce, A., Le Fichoux, Y., Dujarin, P. 1988. Is Leishmania infantum an opportunistic parasite in patients with anti-human immunodeficiency virus antibodies? Arch. Intern. Med. 148:1228 3. Behin, R., Louis, J. A. 1984. Immune response to Leishmania. In Critical Reviewsin Tropical Medicine, Vol. 2, ed. R. K. Chandra, pp. 141-88. NewYork: Plenum Press 4. Turk, J. L., Bryceson, A. D. M. 1971. Immunological phenomena in leprosy and related diseases. Adv. Immunol. 13: 209 66 5. Handman, E., Ceredig, R., Mitchell, G. F. 1979. Murine cutaneous leishmaniasis: Disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages. Aust. J. Exp. Biol. Med. 57:9 30 6. Behin, R., Mauel, J., Sordat, B. 1979. Leishmaniatropica: Pathogenicity and in vitro macrophagefunction in strains of inbred mice. Exp. Parasitol. 48:8141 7. Mauel, J., Behin, R. 1987. Immunity: Clinical and experimental. In The Leishmaniases, Vol. II, ed. W. Peters, R. Killick-Kendrick, pp. 731-91. London: Academic Press 8. Nacy, C. A., Fortier, A. H., Pappas, M. G., Henry, R. 1983. Susceptibility of inbred mice to Leishmania infection: correlation of susceptibility with in vitro defective macrophagemicrobicidat activities. Cell. Immunol.77:298-307 9. Mirkovich, A. M., Galelli, A., Allison, A. C., Modabber, F. Z. 1986. Increased myelopoiesis during L. major infection in mice: Generation of "safe targets," a possible way to evade the effector immune mechanism. Clin. Exp. Immunol. 64:1-7 10. Mitchell, G. F. 1984. Host-protective immunityand its suppression in a parasitic disease: murine cutaneous leishmaniasis. Immunol. Today 5:224~26 11. Liew, F. Y. 1986. Cell-mediated immunity in experimental cutaneous leishmaniasis. Parasitol. Today 2:264-70 12. Russel, D. G., Wilhelm, H. [986. The involvement of the major surface gly-

coprotein (gp63) of Leishmania promastigotes in attachment to macrophages. J. Immunol. 136:2613-20 13. Handman, E., Goding, J. W. 1985. The Leishmania receptor for macrophages is a lipid-containing glycoconjugate. EMBOJ. 4:329-36 14. Anderson, S., David, J. R., McMahonPratt, D. 1983. In vivo protection against Leishmania mexicana mediated by monoclonal antibodies. J. Immunol. 131: 1615-18 15. Mitchell, G. F., Curtis, J. M., Handman, E., McKenzie, I. F. C. 1980. Cutaneous leishmaniasis in mice: Disease pattern in reconstituted nude mice of several genotypes infected with Leishmania tropiea. Aust. J. Exp. Biol. Med. Sci. 58:521 32 16. Liew, F. Y., Hale, C., Howard, J. C. 1982. Immunologicregulation of experimental cutaneous leishmaniasis: V. Characterization of effector and specific suppressor T cells. J. Immunol. 128: 1917-22 17. Titus, R. G., Milon, G., Marchal, G., Vassalli, P., Cerottini, J. C., Louis, J.+A. 1987. Involvement of specific Lyt-2 T cells in the immunological control of experimentally induced murine cutaneous leishmaniasis. Eur. J. Immunol. 17:1429-33 18. Stern, J. J., Oca, M. J., Rubin, B. Y., Anderson, S. L., Murray, H. W. 1988. + and Lyt-2+ cells in experiRole of L3T4 mental visceral leishmaniasis. J. Immunol. 140:3971-77 19. Kaye, P. M., Roberts, M. B., Blackwell, J. M. 1987. Analysing the immune response to L. donovani infection. Ann. Inst. Pasteur/lmmunol. 138:762~8 20. Sadick, M. D., Locksley, R. M., Tubbs, C., Raft, H. V. 1986. Murine cutaneous leishmaniasis: Resistance correlates with the capacity to generate IFN-~, in response to Leishmania antigens in vitro. J. Immunol. 136:655-61 21. Locksley, R. M., Heinzel, F. P., Sadick, M. D., Holaday, B. J., Gardner, K. D. 1987. Murine cutaneous leishmaniasis: susceptibility correlates with differential expansion of helper T cell subsets. Ann. Inst. Pasteur/Immunol. 138:744-49 22. Kelso, A., Glasebrook, A. L. 1984. Secretion of Interleukin 2, macrophagesactivating factor, interferon, and colonystimulating factor by alloreactive T lymphocyte clones. J. lmmunol. 132: 292431 23. De Libero, G., Kaufmann,S. H. E. 1986. Antigen-specific cytolytic T lymphocytes from mice infected with the intracellular

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T CELLS AND IMMUNITY TO LEISHMANIA bacterium Listeria monocytogenes. J. lmmunol. 137:2688-94 24. Mitchell, G. F., Handman,E., Moll, H., McConville, M. J., Spithill, T. W., Kidane, G. Z., Samaras, N., Elhay, M. J. 1987. Resistance and susceptibility of mice to Leishmania major: A view from Melbourne. Ann. Inst. Pasteur/lmmunol. 138:73843 25. Liew, F. Y., Hale, C., Howard, J. G. 1985. Prophylactic immunization against experimental leishmaniasis. IV. Subcutaneous immunization prevents the induction of protective immunity against fatal Leishmaniamajorinfection. J. Immunol. 135:2095-2101 26. Titus, R. G., Lima, G. C., Engers, H. D., Louis, J. A. 1984. Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed type hypersensitivity. J. lmmunol. 133:1594-1600 27. Liew, F. Y., Singleton, A., Cillari, E., Howard,J. G. 1985. Prophylactic immunization against experimental leishmaniasis. V. Mechanismof the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major. J. Immunol. 135: 2101 7 28. Milon, G., Titus, R. G., Cerottini, J. C., Marchal, G., Louis, J. A. 1986. Higher frequency of Leishmania major specific L3T4÷ T cells in susceptible BALB/c mice than in resistant CBA-mice. J. Immunol. 136:1467 71 29. Liew, F. Y. 1987. Analysis of host-protective and disease promoting T cells. Ann. Inst. Pasteur/Immunol. 138:749-55 30. Mosmann,T. R., Coffman, R. L. 1987. Twotypes of mousehelper T cell clone. Implications for immune regulation. Immunol. Today 8:223-27 31. Mosmann,T. I~., Cherwinski, H., Bond, M. W., Giedlin, M. A., Coffman, R. L. 1986. Twotypes of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. lmmunol. 136:2348-57 32. Titus, R. G., Ceredig, R., Cerottini, J. C., Louis, J. A. 1985. Therapeutic effect of anti-L3T4 monoclonal antibody GK1.5 on cutaneous leishmaniasis in genetically susceptible BALB/cmice. J. ImmunoL 135:2108-14 33. Solbach, W., Forberg, K., Kammerer, E., Bogdan, C., R611inghoff, M. 1986. Suppressive effect of cyclosporin A on the development of Leishmania tropica induced lesions in genetically susceptible BALB/cmice. J. Immunol. 137:702-7

577

34. Sadick, M. D., Heinzel, F. P., Shigekane, M., Fisher, W. L., Locksley, R. H. 1987. Cellular and humoral immunity to Leishmania major in genetically susceptible mice after in vivo depletion of L3T4+ T cells. J. Immunol. 139:1303-9 35. Howard, J. C., Hale, G., Liew, F. Y. 1981. Immunological regulation of experimental cutaneous leishmaniasis. IV. Prophylactic effect of sublethal irradiation as a result of abrogation of suppressor T cell generation in mice genetically susceptible to Leishmania tropica. J. Exp. Med. 153:557~8 36. Benjamin, R. J., Waldman, H. 1986. Induction of tolerance by monoclonal antibody therapy. Nature 320:449-51 37. Titus, R. G., Marchand, M., Boon, T., Louis, J. A. 1985. A limiting dilution assay for quantifying Leishmania major in tissue of infected mice. Parasite Immunol. 7:545 55 38. Jenkins, M. K., Schwartz, R. H. 1987. Antigen presentation by chemically modified splenocytes induces antigenspecific T cell unresponsivenessin vitro and in vivo. J. Exp. Med. 165:302-19 39. Louis, J. A., Moedder, E., MacDonald, H. R., Engers, H. D. 1981. Recognition of protozoan parasites by murine T lymphocytes. II. Role of the H-2 gene complex in interactions betweenantigen-presenting macrophages and Leishmaniaimmune T-lymphocytes. J. Immunol. 126:1661-66 40. Louis, J. A., Mendonqa,S., Titus, R. G., Cerottini, J. C., Cerny, A., Zinkernagel, R., Milon, G., Marchal, G. 1986. The role of specific T cell subpopulations in murine cutaneous leishmaniasis. In Pro9ress in Immunology,VI, ed. B. Cinader, R. G. Miller, pp. 762-69. New York: Academic 41. Feng, Z. Y., Louis, J. A., Kindler, V., Pedrazzini, T., Eliason, J., Behin, R., Vassalli, P. 1988. Aggravationof experimental cutaneous leishmaniasis in mice by administration of interleukin 3. Eur. J. Immunol. 18:1245-51 42. Solbach, W., Greil, J., R611inghoff, M. 1987. Anti-infectious responses in Leishmania major-infected BALB/c mice injected with recombinant granulocytemacrophage colony-stimulating factor. Ann. Inst. Pasteur/Immunol. 138: 75962 43. Lima, G. C., Engers, H. D., Louis, J. A. 1984. Adoptive transfer of delayed type hypersensitivity reactions specific for Leishmania major antigens to normal mice using murine T cell populations and clones generated in vitro. Clin. Exp. Immunol. 57:130-38

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soluble promastigoteextract. J. lmmu44, Scott, P., Pearce,E., Natovitz,P., Sher, A. 1988.Adoptivetransfer of protective nol. 139:3118-25 immunity against lethal Leishmania 46. Wyler,’. D. J., Neller, D. I., Sypek, major infection with an L3T4(THe) J. P. 1987. Macrophage activation for antileishmanial defense by an apparcell clone recognizing a low molecular weightparasite antigen. FASEB J. 2 (5): ently novel mechanism.J. lmmunoL 138: 1246-49 A 1256(Abstr.) 45. Scott, P., Pearce,E., Natovitz,P., Sher, 47. Spitalny, G. L., Havell, E. A. 1984. Monoclonalantibody to mur, ine gamma A. 1987. Vaccinationagainst cutaneous leishmaniasis in a murine model. II. interferon inhibits lymphokine-induced Immunologicproperties of protective antiviral and macrophagetumoricidal activities. J. Exp.. Med.159:1560-65 and nonprotective subfractions of a

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Annual Reviews www.annualreviews.org/aronline

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Ann. Rev. lmmunol. 1989. 7.’579-99 Copyright © 1989 by Annual Reviews Inc. All rights reserved

THE BIOLOGIC ROLES OF CD2, CD4, AND CD8 IN T-CELL ACTIVATION Barbara E. Bierer*~f, Barry P. Sleckman*, Sheldon E. Ratnofsky*, and Steven J. Burakoff*~ *Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, the ~ Department of Medicine; Brigham and Women’s Hospital, Boston, Massachusetts and the Departments of ~" Medicine and :~ Pediatrics, Harvard Medical School, Boston, Massachusetts

INTRODUCTION Activation of T lymphocytesoccurs uponinteraction with cells bearing appropriate antigen in the context of major histocompatibility complex (MHC) proteins. Theextraordinary specificity of the T-cell response conferredby the polymorphic antigen-specificT-cell receptor (TcR).Onthe cell surface, the heterodimericTcRis physicallyassociated with a complex of nonpolymorphic proteins, CD3(T3, Leu4), whichis thought to transducean activating signal fromthe surface to the cytoplasmof the cell. In addition to the antigen-specific TcR,a numberof other cell surface proteins, called accessorymolecules,regulate T-cell activation andimpart sensitivity and plasticity to the immune response. Theidentity and significance of these moleculeswereinitially deducedby the development of monoclonalantibodies (MAbs)whichinhibit their function. Theseinclude the antigens CD2,CD4,CD8,and lymphocytefunction associated antigen (LFA)-I. Theseare non-polymorphic moleculesthat not only increase the avidity with whicha T cell interacts with its antigen presentingcell (APC) or target cell but also mayplay a role in signal transduction. Several of these accessory moleculeshave nowbeen cloned and the cDNAs expressed in recipient cells. Genetransfer technologyhas alloweda clearer defini579 0732~3582/89/0410-0579502.00

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tion of function and identification or confirmation of their respective ligands. There are several mechanism(s) by which accessory molecules modulate the immuneresponse. Certain molecules, such as CD2and LFA-I, clearly function to increase adhesion betweenthe T cell and the APCor target cell. The CD2molecule seems involved not only in antigen-dependent activation but also in an antigen-independent pathway of T-cell activation. CD4and CD8are expressed on mutually exclusive populations of peripheral T cells that bear TcRsspecific for antigen in association with MHC class-II and class-I proteins, respectively. They appear to enhance the avidity with which a T cell binds to its antigen bearing cell and mayalso promote the interaction of the TcRwith its appropriate antigen. Whether this occurs by the formation of a ternary structure (e.g. CD4, TcR, antigen/MHC)or indirectly remains to be identified. Finally, upon binding ligand, the accessory molecule maytransmit a signal which directly impacts upon the signal delivered via the TcR-CD3complex. These mechanisms of action are not mutually exclusive. This review focuses on the role of CD2, CD4, and CD8in T-cell activation. The cluster of differentiation (CD) nomenclature is used to refer to both humanand murine molecules, unless otherwise stated.

THE

CD2

RECEPTOR

The Role of CD2 Bindin9 Activation

to LFA-3 in Adhesion

and

CD2(TI1, Leu 5, LFA-2, the sheep red blood cell (SRBC)receptor) nonpolymorphic glycoprotein of 45-50 kd expressed on all humanthymocytes and peripheral T cells, including the large granular lymphocytes, and most natural killer (NK)cells (1 5). The CD2antigen has cloned, and the predicted anaino acid sequence reveals a transmembrane glycoprotein with homology to immunoglobulin (6-8). The extracellular domainhas three potential N-glycosylation sites, and the large cytoplasmic domain(116 amino acids) is rich in prolines and basic residues. HumanCD2mediates rosetting of T cells to SRBC,a procedure classically used to isolate human T cells from the mononuclear population (1-3, 9, 10). In addition, anti-CD2 MAbsinhibit antigen-specific and -nonspecific conjugate formation (11, 12), and therefore a variety of cell functions, including alloantigen- and mitogen-stimulation of T-cell proliferation, stimulation of lymphokineproduction, and antigen-specific cytotoxic T lymphocyte(CTL)-mediatedkilling (3, 5, 11-14). A ligand for CD2has recently been identified (12, 15). On the surface

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of SRBC,a glycoprotein of 42 kd called TI ITS (T11 target structure) has been found to interact with CD2on the T cell (15). The homologous protein in humansis a broadly distributed glycoprotein of 55-70 kd termed LFA-3(12, 16). MAbdirected against LFA-3inhibit the same range ofT lymphocyte functions as anti-CD2 MAb,but inhibit by binding to the ÷ T cells bind target or stimulator cell, not to the T cell (5, 11, 12). CD2 to purified LFA-3incorporated into liposomes (17) or planar membranes ÷ lymphoid cells and inhibits T-cell (18). Purified CD2binds to LFA-3 rosetting (19). CD2binding to LFA-3 has been shown to mediate CTLtarget cell conjugate formation (12) and thymocyte-thymicepithelial cell binding (20). Independent of antigen recognition by the. TcR, the CD2/LFA-3interaction clearly functions to promote adhesion or conjugate formation and thereby contributes to the avidity with which a T cell binds to its target or stimulator cell. A role for CD2in T-cell activation has been inferred from the observation that certain pairs of anti-CD2 MAbsstimulate T-cell proliferation and effector function (21-23). The binding sites on the CD2molecule a number of these antibodies have been mapped and correlated to LFA3 binding (24). CD2cDNAmutants were selected for loss of binding certain anti-CD2 MAbs(negative selection) but retention of binding other anti-CD2 MAbs(positive selection). In this manner, specific epitope loss mutants of CD2were selected and sequenced. Epitope mapping has " thus defined three regions of antibody binding (24). Region I clusters around amino acid 48, spanning a region of 13 amino acids. Region II clusters around amino acid 95, spanning 13 amino acids. Functionally, anti-CD2 MAbsmappingto region I and region II inhibit T-cell rosetting to SRBC(1, 3, 4, 10, 25). Epitope loss mutant CD2molecules of region ÷ human and of region II transfected into COScells failed to bind LFA-3 erythrocytes (24). Thus, regions I and II appear to define two regions critical for LFA-3binding to CD2. A CD2mutation involving two substitutions at amino acids 140 and 141 defined a third region of antibody binding (24). Region III anti-CD2 MAbsdo not block SRBCrosetting or T-cell conjugate formation (21, 26). The CD2epitope defined by region III is expressed weakly on resting peripheral T cells (2 l, 23, 26); however,activation of resting T cells induced increased expression of the activation-related region III epitope (21, 23, 26). This expression occurred rapidly, at 4°C, and in the presence of inhibitors of protein synthesis, suggesting that the increased expression was due to a conformational change in the CD2molecule upon activation, and not to new protein synthesis (23). Activation of resting T cells via the CD2pathway required incubation with a region III MAbwith certain region I or region II MAbs(21, 22, 26). Alternatively, incubation with

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activation-related region III anti-CD2 MAbwith SRBC,which express an LFA-3homologue, induced T-cell proliferation (16). To further assess the role of the CD2/LFA-3 interaction in T-cell activation, we have developed a model system to express wild type and mutant CD2molecules in a cell line that retains antigen-reactivity (27, 28). Using retroviral-mediated gene transfer, we have expressed humanCD2cDNAs in a murine T-cell hybridoma that produces interleukin-2 (IL-2) upon stimulation with the HLA-DR antigens of the humanEpstein-Barr virus transformed cell line JY, which bears LFA-3(29). The functional responses are compared to cell lines expressing comparable levels of the TcR-CD3 complex and infected with the gene encoding only neomycin resistance (Figure 1). + CD2

Neor

~

TcR-CD3

(~

TcR-CD3 CD2

CD2 eDNA

Moloney LTR

¯~

Moloney LTR

Moloney LTR

CMV e’~t~Y promoter

Anti-TcR MAb

Anti-TcR MAb

Anti-CD2 MAb

Anti-CD2 MAb

Moloney LT~

LogFluorescence Intensity ~ + hybridomas (top) were generated by infecting the parent hybriFigure, 1 Neor and CD2 doma155.16 with retroviral constructs (middle) containing the neomycin resistance (Neo’) geue alone (left panel) or together with the CD2eDNA(right panel). Both the resulting hybridomas express the TcR-CD3complex by indirect immunofluorescence with F23.1 MAb + but not the Neo’ hybridomasexpress CD2on their cell surface (bottom). (bottom). The CD2 Log fluorescence intensity of cells stained with detecting antibody followed by fluoresceinconjugated goat-anti-mouse antibody (FITC-GAM)is shown in comparison to cells stained with FITC-GAMalone.

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Hybridomas expressing CD2were able to respond to region I (9, MT110)plus region III (9-1) anti-CD2 MAbsor to SRBCplus a region III (9-1) anti-CD2 MAbto produce IL-2 (Table 1) (27). Furthermore, murine L cells transfected with and expressing the humanLFA-3molecule were able to replace the requirement for either region I anti-CD2 MAbs or SRBCin the stimulation of IL-2 production. This suggested that LFA3 binding to CD2promotes antigen-independent T-cell activation and is consistent with the observation that purified LFA-3in the presence of suboptimal concentrations of stimulatory anti-CD2 MAbsinduce human T-cell proliferation (30). Analysis of the function of murine T-cell hybridomas expressing mutated CD2cDNAsinvolving single amino acid substitutions within +) (H. L. Wolff et al, either region I (QSIL+) (27) or region II (D92H manuscript in preparation) confirmed the structural analysis of the LFA3 binding site (Table 1) (24). These hybridomas were no longer able Table 1 IL-2 production by murine T-cell hybridomas aIL-2 production (U/ml) 155.16 b CD2+

Stimulation 9-1 9.6+9-1MAbs Nu-Ter+9-1 MAbs MTll0+9-1MAbs SRBC SRBC+9-1 MAb LFA-3/L LFA-3/L+9-1

MAb

Q5IL + D92H + CD2AB+ + CD2AS

....... +++ + + ND ++++ +++ ...... + + + + -

cells

MAb

JY cells HLA-DR LFA-3 (HLA-DR+ LFA-3) liposomes

-

+ +

-

+ ++++ liposomes liposomes -+ +

+

-

+++ ++++ ...... +/++ ...... ...... -

ND +/+/++

-

+ ND ++++ + + + + ++++

+ +

a5 × 104 hybridomacells were cultured with 1 /~g ml t MAb,5 × l0 6 SRBC, 2 × 105irradiated LFA3 L cells, 105JYcells, or liposomesas indicated, in a final volumeof 750,ul RPMI-1640 containing10% fetal calf serum,penicillin and streptomycin.After 24 hr, supernatantsfromculturedcells wereassayed for IL-2 contentby their ability to supportthe growthof an IL-2dependentmurinecell line as described (27). + (expressingthe b Hybridoma cell lines used were155.16(parental antigen-specific hybridoma),CD2 + + wild type CD2molecule), Q5IL (expressing a point mutant at aminoacid 51 in region I), D92H + (expressing a CD2deletion mutant (expressinga point mutantat aminoacid 92 in region II), CD2AB + (expressinga CD2deletion lacking the terminal 100aminoacids of the cytoplasmicdomain),and CD2AS mutantlacking the terminal 42 aminoacids of the cytoplasmicdomain). ¢ Monoclonal antibodies recognizingCD2mapto the following domains:9.6 and MT110 (R.egionI), Nu-Ter(RegionII), 9-1 (RegionIII) as described(24).

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ET AL

bind LFA-3and were unable to utilize SRBCor LFA-3+ L cells plus a region IlI MAbfor stimulation of IL-2 production. They were, however, able to produce IL-2 to appropriate pairs of anti-CD2 MAbsthat retained binding, demonstrating that although unable to bind LFA-3these point mutant molecules were still functional. Construction and expression of a number of cytoplasmic domain deletion mutants of CD2have helped to delineate the structural requirements for stimulation via CD2(Table l). Murine hybridomas expressing a deletion mutant in which the carboxyterminal 100 amino acids of CD2 have been replaced with a serine, CD2AB,no longer responded to SRBC + L cells in the presence of a region III anti-CD2 MAb,nor to or LFA-3 pairs of stimulatory anti-CD2 MAbs(28). The mutant CD2molecule was, however, able to bind LFA-3 comparably to the wild type CD2protein. Thus, the cytoplasmic domainappears to be necessary for the delivery of an activating signal but is not required for adhesion. The complexity of the CD2pathway of activation was demonstrated by expression of a second cytoplasmic domain deletion mutant, CD2AS,in which the terminal 42 amino acids have been removed, including the region with greatest conservation between the mouse, rat, and human species (B. E. Bierer et al, manuscript in preparation). These hybridomaswere able to respond to certain pairs of anti-CD2 MAbs,comparablyto the wild type + hybridomas were markedly CD2+ hybridomas (Table 1). These CD2AS deficient, however,in their ability to respond to other pairs of stimulatory anti-CD2 MAbs. These data suggest that the cytoplasmic domain may have several functional regions, as partial deletions of the cytoplasmic domainappear to result in partial defects in signal transduction. The mechanism of transmembrane stimulation via the CD2pathway has been investigated. Similar to the antigen-specific TcR-CD3pathway of activation, stimulation with appropriate pairs of anti-CD2 MAbs induced a rise in intracellular calcium ([Ca+Z]i) and the generation inositol triphosphates (reviewed in 31). Purified LFA-3plus a single region III anti-CD2 MAbalso induced a rise in [Ca+2]~(32). Stimulatory pairs anti-CD2 MAbsincubated with a nonlymphoidinsect cell line transfected with and expressing CD2were unable to induce a rise in intracellular calcium, suggesting that the CD2molecule is not a calcium channel itself (33). CD2appears to require a lymphoid-related structure for signal transduction. The Interrelationship

of CD2 and CD3

While both the antigen-specific TcR-CD3pathway and the antigen-independent CD2pathway may trigger T-cell stimulation, several lines of evidence suggest that they may be interrelated. Modulation of the TcR-

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ANDCt)8

585

CD3complex with MAbfrom the surface of resting T cells or T-cell lines prevented subsequent stimulation with pairs of anti-CD2 MAbs,perhaps by inducing a state of T-cell refractoriness to stimulation (21, 22, 34, 35). Modulation of the CD2antigen by antibody, however, did not prevent a subsequent rise in [Ca+2]i induced by anti-CD3 MAb(34). Some(35) not all (36) CD3mutant cell lines were refractory to stimulation with + anti-CD2 MAbs.Cumulatively, these data suggest that in mature, CD3 T cells, stimulation via the CD2pathway may depend in part upon the expression of the TcR-CD3complex. Expression of CD2, however, may regulate or augment antigen-dependent T-celt responses. Certain region I and region II anti-CD2 MAb have been shown to inhibit antigen-dependent or anti-CD3 MAb-induced proliferation (5, 13). However,incubation of resting humanT cells with region III anti-CD2 MAbin the presence of a nonstimulatory anti-CD3 MAbstimulated proliferation (23). LFA-3binding to CD2mayparticipate in activation of resting T cells. MurineL cells transfected with and expressing human LFA-3+ L cells in the presence of an anti-CD3 MAbor suboptimal concentrations of phytohemagglutinin induced resting T-cell proliferation (37). The role of LFA-3in antigen-dependent stimulation was studied more directly using the antigen-reactive murine T-cell hybridomas expressing human CD2(Table 1). Expression of CD2 enhanced IL-2 production, comparedto the parent cell line in response to the LFA-3+ antigen-bearing cell JY, and this enhanced stimulation was inhibitable by antibodies directed against CD2or LFA-3 (27, 28). Hybridomas expressing either the region I (27) or region II (H. L. Wolffet al, manuscript in preparation) point mutants of CD2, which are unable to bind LFA-3, did not exhibit an enhanced response to JY; this demonstrated that the antigen-dependent response was contingent upon CD2binding to LFA-3. Furthermore, the wild type CD2+ hybridomas were able to produce IL-2 after stimulation with liposomes containing both purified LFA-3and HLA-DR,the physiological ligands for CD2and the TcR, respectively (28). Neither liposomes containing HLA-DR, a poorer immunogenthan intact cells, nor liposomes containing LFA-3were stimulatory (Table 1). Hybridomas expressing the cytoplasmic domain deletion mutant of CD2, in which 100 amino acids had been deleted (CD2AB), allowed distinction between the role of CD2/LFA-3in adhesion-strengthening and + hybridomas the requirement that CD2transduce a signal (28). CD2AB did not exhibit an enhanced response to antigen nor respond to HLA-DR plus LFA-3incorporated into liposomes (Table 1). These data suggest that signal transduction by CD2is required to obtain an optimal response. Given the requirement that antigen regulate the T-cell response in the

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periphery, the CD2/LFA-3interaction antigen-driven response.

may function to augment a weak

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The Role of CD2in Thymic Ontogeny It has been proposed that the CD2pathwayof T-cell activation functions as an early thymocyteproliferative signal (38). Influencing the development of the T-cell repertoire, the thymic microenvironmentappears to regulate + thymocytes bind to thymocyte proliferation and differentiation. CD2 ÷ LFA-3 thymic epithelial cells, binding that is inhibitable by anti-CD2 and anti-LFA-3 MAb(20). Thymocytes may be stimulated to proliferate + murine L cells (37) in the by thymic epithelial cells (39) or by LFA-3 presence of suboptimal concentrations of phytohemagglutinin. Similarly, thymocyte proliferation maybe induced by purified LFA-3in the presence of a region III anti-CD2 MAband exogenous IL-2 (40). LFA-3binding to CD2alone is insufficient for T-cell stimulation, requiring the presence of a second signal. This second signal may be provided by antigen, an anti-CD3 MAb,or an anti-CD2 MAbdirected to the region III epitope (27, 28, 30). In the periphery, TcR/antigen interactions, even of low affinity, mayprovide the second signal. In the thymus, the nature of this second signal is unknown. A physiologic ligand mimicking the region III anti-CD2 MAbhas yet to be identified. HumanCD2is expressed early in thymic ontogeny, prior to the expression of the TcR-CD3 complex. Ifa second ligand exists in the thymus, CD2mayprovide an essential early proliferative signal for T-cell developmentprior to expression of the TcR. In the mature T cell, the CD2/LFA-3 interaction may function in synergy with the TcR.

THE CD4 AND CD8 MOLECULES Structural Studies and Tissue Distribution of CD4and CD8 The humanCD4molecule is a 55-kd glycoprotein with four extracellular domains, the most membranedistal of which has striking homology to immunoglobulin light chain variable genes (for review, see 41). Comparison of the sequences of the cloned human and mouse CD4cDNAs has revealed 55% amino acid homology, with the cytoplasmic domains exhibiting the greatest homology. The cytoplasmic domaincontains three serine residues that mayserve as substrates for phosphorylation by protein kinase C. The highly conserved nature of the cytoplasmic domain of CD4 suggests that this domain maybe important for the function and/or the expression of the molecule. The murine CD8molecule normally exists as a heterodimer consisting of two disulfide-linked subunits, Lyt 2 (~), 38 kd, and Lyt 3 (/~) 30

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CD4,

AND CD8

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Both molecules are membersof the immunoglobulin supergene family. On peripheral T cells, the human CD8molecule is composed of homomultimers of a single 34 kd subunit, the homologue of murine Lyt 2. Humanthymocytes express homodimers of the 34 kd subunit, as well as heteromultimers of the 34-kd subunit complexed with CD1(T6), a 46-kd protein. Using a rat Lyt 3 cDNAprobe, a human Lyt 3 homologue has been identified. Expression of the humanhomologueof Lyt 3 remains to be demonstrated. It is clear that the CD8or Lyt 2 gene product itself is sufficient for biological activity of both the murine (42) and human(43) molecules. Expression of the CD4and CD8molecules by T cells occurs early in thymic ontogeny. Thymocytes can be subdivided into four populations based on the expression of these molecules. The CD4- CDS- (double + CD8 + (double positive) negative) immature cells give rise to both CD4 cells, which comprise up to 80%of the total population, and the CD4+ and CD4 + CDS-(single positives) mature cells. It appears that the CD8 single positive cells arise from the double positive population rather than from the double negative population. In the periphery, the CD4molecule is expressed on a subset of T lymphocytesdistinct from those that express the CD8molecule.

CD4and CD8Interact with Class II and Class I MHC Proteins, Respectively CD4was initially described as a phenotypic marker for helper T lymphocytes (44). Similarly, CD8expression was correlated with cytotoxic lymphocyte and suppressor cell function. However, Swain et al (45) ÷ ÷ observed that anti-CD8 antibodies inhibited CD8 cytotoxic and CD8 helper T cells specific for class-I MHC antigens without regard to function. Subsequentanalysis demonstratedthat certain cytolytic T cells specific for antigen in association with class-II MHCproteins were CD4+ and that anti-CD4 MAbsinhibited the function of these cells (46~9). It became clear that expression of the CD4and CD8molecules was more closely correlated with the MHC specificity of the T cell than with function. These observations led to the hypothesis that CD4and CD8are receptors that interact with determinants on class-II and class-I MHC molecules, respectively, and that these interactions modulateor augmentthe T-cell response. Whetherthe selection for CD4expression in concert with a class-II, MHCrestricted T cell, and for CD8in concert with a class-I, MHC-restrictedT cell, occurs intrathymically or upon antigen stimulation in the periphery is not known. Both functional and direct binding studies have shownthat the ligands for the CD4and CD8receptors are encoded by MHCclass-II and class-I

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molecules, respectively. MostT cells are MHC class-II restricted, therefore, the ligand for the TcRand the ligand for CD4both lie within MHC classII molecules. However, the CD4+ murine T-cell hybridoma 3DT52.5, d molecules, allowed one to differentiate the ligand specific for class-I H-2D requirement of the CD4molecule from that of the TcR. MAbsto CD4 and to MHCclass-lI proteins were able to inhibit the response of the CD4+ hybridoma only when stimulator cells expressing MHCclass-II antigens were used (50). Furthermore, the CD4+ hybridoma, but not CD4-variants, exhibited an enhanced response to L cells transfected with both the MHCclass-I (H-2Dd) and class-II genes as compared to those transfected with the MHCclass-I gene alone (51). Gay et al (52) shown that expression of the human CD4molecule by 3DT52.5 enhances d expressing cells only if human the ability of this cell to respond to H-2D MHC class-II genes are also expressed by the stimulator cells. Direct proof that MHCclass-II is the ligand for CD4has come from binding studies (53). MHC class-II + but not MHCclass-II- B cells were capable of binding to CV1cell lines transfected with and expressing the human CD4molecule. Both anti-CD4 and anti-class-II MHCMAbsinhibited this binding. The simultaneousaddition of antibodies directed against HLA-DR, HLA-DQ, and HLA-DP was required to inhibit binding, suggesting that the CD4receptor is able to bind to the products of all the genes of the HLA-D region. This confirmed earlier functional studies using the 3DT52.5 hybridoma discussed above (50) and implies that CD4 maybind to a nonpolymorphicregion of the MHC class-II molecule. Functional studies from our laboratory (43) have provided strong evidence that the ligand for the CD8molecule is contained within the MHC class-I protein. By expressing a human CD8cDNAin a murine T-cell hybridoma specific for humanMHC class-II molecules, it was possible to separate the ligand requirements of the TcR from those of the CD8 molecule. CD8+ T-cell lines produced 10-fold greater levels of IL-2 in response to MHCclass-I + and class-II + stimulator cells than did the parent cell line. The CD8-dependentresponse required expression of MHC class-I proteins by the stimulator cell. Similarly, the addition of MAbs directed to MHCclass-I proteins on the stimulator cell reduced the + cells to that of the CD8-controls. response of the CD8 Direct binding between CD8 and MHCclass-I molecules has been demonstratedusing cell-sized vesicles called artificial target cells (ATCs) to identify receptor-ligand pairs involved in cell-cell adhesion. The ability to incorporate a high surface density of receptor or ligand into the ATC membraneallowed the detection of low avidity, multivalent, protein-protein interactions. Rosenstein et al (54) have been able to demonstrate the specific interaction of purified CD8with MHC class-I proteins. This

Annual Reviews CD2,

interaction was inhibitable class-I molecules.

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Functional

with MAbsto either

CD4,

AND CD8

589

the CD8or the MHC

Role of CD4 and CD8

The requirement for CD4 or CD8function may be most clearly demonstrated when T-cell stimulation by antigen is suboptimal, that is, when the affinity of the TcRfor antigen is low or whenantigen is limiting in quantity. The interaction of CD4with class-II MHCmolecules on the APCmayin part serve to augmentthe avidity with which a T cell binds to the APC.T cells bearing low affinity TcRsmaybe more dependent on this increased adhesion (e.g. CD4)for activation and would therefore be more susceptible to inhibition by anti-CD4 MAbs.The ability to inhibit the + T-cell clones with anti-CD4 MAbcorrelated effector function of CD4 not simply with CD4expression but rather with the avidity with which a T cell binds to the antigen bearing cell (49, 55-57). MAbsto CD4were + murine capable of inhibiting the function ofa class-I MHC-specificCD4 T-cell hybridoma, 3DT52.5, only when antigen was limited either by decreasing the number of stimulator cells or by decreasing antigen expression on the stimulator cell (51). The CD8molecule also appears to function under conditions of suboptimal stimulation. Shimonkevitzet al (58) investigated the sensitivity inhibition by anti-murine CD8MAbin a series of H-2Kd specific CTL d antigen. clones. The target cell expressed low basal levels of the H-2K Interferon-gamma treatment of the target cells induced increased expression of H-2Kd. T-cell clones which would lyse uninduced targets were insensitive to anti-CD8 MAbinhibition; however, clones that required high target antigen expression were susceptible to anti-CD8 inhibition. Other laboratories have reported that the cross-_reactive lysis of allogeneic targets by virus-specific or allospecific CTLis CD8-dependent despite CD8independence of the lytic response to the eliciting target antigen (59). Cumulatively, these data suggest that the CD8receptor plays a critical role in T-cell activation under conditions of suboptimal Tcell stimulation. The Association

of CD4 and CD8 with the TcR for Antiyen

The close association of CD4expression with MHC class II-restricted T cells and CD8expression with MHC class I-restricted T cells is striking + although not absolute. Flomenberg et al (60) have isolated both CD4 CD8 and CD4- CD8+ CTL specific for class-I MHCproteins. While the CD8+ clones were inhibitable by anti-CD8 MAbs, the CD4+ clones were not susceptible to inhibition by MAbsto CD4.Furthermore, analysis of T-cell clones expressing both CD4and CD8but specific for class-II

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MHC antigens has shown that these clones are more easily inhibited by MAbsto CD4than by MAbsto CD8(61,62). Aparicio et al (63) generated HLA-B27.1 (MHCclass-I)-specific T-cell clones which were also reactive ÷ (MHC against HLA-DR2 class-II) target cells. Lysis of both target cell types could be inhibited by anti-CD3 MAbs, but anti-CD8 MAbonly inhibited lysis of the MHCclass I-specific response. These data suggest that CD4mayfunction more efficiently in the setting of a class-II MHGrestricted TcR and that CD8mayfunction more efficiently in association with a class-I, MHC-restricted TcR. Recent evidence suggests that the CD4and CD8receptors may be in close physical proximity with the TcR during T-cell activation. Using cross-linked MAbsto the TcR/CD3complex under conditions that were ÷ (or CD8 ÷) T cells, addition of antisuboptimal for stimulation of CD4 CD4(or anti-CD8) MAbsto the cross-linking surface resulted in augmentation of the response (64~68). Synergistic effects of MAbsto both CD4and the TcR-CD3complex on the proliferative response were only observed if both MAbswere immobilized on the same surface, but not if either or both were soluble (66) or bound to separate surfaces (67). addition to CD4, MAbcross-linking the CD3 complex to a number of other T-cell surface proteins (most notably CD8, but also CD2, CD5and CD6) induced a comparable augmented proliferative response (66). possible explanation for these results would be that MAbsto CD4or CD8 serve an adhesion function acting to stabilize the interaction betweenthe TcR/CD3complex and MAbsdirected against this complex. The ability of certain soluble anti-CD4 MAbsto augment the IL-2 response and to increase the calcium flux mediated by anti-CD3 MAbs argues that the function subserved by CD4is more than simply adhesion (S. J. Goldman, S. J. Burakoff, manuscript submitted). One anti-CD4 MAb(Leu 3a) enhanced anti-CD3 MAb-mediated T-cell activation assessed by IL-2 production and [Ca+2]~. A second anti-CD4 MAb (OKT4F)had no direct effect itself on anti-CD3 MAb-mediatedT-cell responses but was able to inhibit the enhanced response induced by antiLeu 3a MAb.These data argue for a functional association and possibly a physical proximity between the CD4molecule and the TcR-CD3complex. Furthermore, these findings demonstrate that certain anti-CD4 MAbact as agonists to activate the cell or, alternatively, as antagonists that bind and inhibit signal transduction. Although immunoprecipitation studies have not demonstrated a direct association between CD4and the TcR (69), several studies have shown comodulation of CD4with the TcR. Saizawa et al (70) demonstrated that incubation of CD4+ T cells with Fab fragments of an anti-TcR MAbthat activate T cells resulted in modulation of a small percentage of CD4

Annual Reviews

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receptors. Nonstimulatory anti-TcR MAbsdid not result in modulation of either the TcR or CD4. Anderson et al (71) demonstrated that crosslinked anti-CD3 MAbsmodulated not only CD3but also a small percentage of CD4from the surface of the cell. In addition, cross-linked antiCD4MAbsinduced modulation of a small percentage of CD3from the cell surface. These data must be interpreted with caution for the conditions used for modulation could result in T-cell activation which may then promote modulation of surface molecules. It has recently been shownthat the CD4receptor is modulated from the surface of the T cell in response to phorbol ester or antigenic stimulation (72, 73). Therefore, T-cell activation may lead to modulation of the CD4receptor and the TcR from the surface of the cell, independentof a physical association. Kupfer et al (74) have shown that interaction of CD4+ T cells with APCsbearing the appropriate antigen/MHCcomplex results in clustering of the TcR and CD4at the T-cell/APC interface. Clustering of CD4does not occur in the presence of an MHC class-II + cell in the absence of the appropriate antigen. Thus, the interaction of CD4with its ligand, MHC class-II proteins is not sufficient for clustering. Stimulation of the TcR by its ligand mayresult in the localization of functionally relevant cell surface proteins at the T-cell/APCinterface. Thus, optimal function of CD4(and CD8)mayrequire its close physical proximity to the TcRat the T-cell/APCinterface. The first receptor-ligand interactions to occur betweenthe T cell and APCor target cell are antigennonspecific. Anti-CD4and anti-CD8 MAbsappear to inhibit both antigennonspecific conjugate formation and antigen-specific T-cell triggering (49, 75, 76) and not effector function such as lymphokinerelease (48, 55). It possible then that the CD4:class-II MHCinteraction preceeds antigenspecific recognition and the formation of a ternary complexof CD4, TcR, and antigen/MHC, CD4:class-II MHCpairing could promote trapping and increase the local concentrations of antigen/MHCon the surface of the APCor target cell, providing sufficient antigen density for the pairing of low affinity TcRand their antigen/MHCcomplex. At the instant of Tcell triggering, then, the observed physical proximity of CD4and the TcR might result from their binding to the same MHCclass-II molecule. This binding of CD4to class-II MHC proteins mayhelp to stabilize the classII molecule, allowing it to interact moreefficiently with the TcR.Additionally, interaction of CD4with either MHC class-II proteins or the TcRmay induce conformational changes in the molecules altering their binding affinity. While CD4may subserve an adhesive-strengthening role, the CD4 receptor may also be capable of transducing intracellular signals that may complement or directly act upon those transduced by the TcR/CD3 complex (see below).

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The Mechanism by Which CD4and CD8Function Wehave expressed the human CD4 cDNAin the murine T-cell hybridoma 155.16 (described above) that produces IL-2 in response to HLADRantigens (29). CD4expression markedly enhances the ability of these cells to produce IL-2 in response to the MHCclass-II +, class-I- Daudi cell line. Anti-CD4 MAbinhibited the enhanced response. In order to determine if CD4expression altered the strength of adhesion between the T-cell hybridoma and the stimulator cell, the force required to separate conjugate pairs of cells was measured (77). A 3-4-fold greater force was required to separate Daudi cells from the hybridoma expressing human CD4than from the CD4- parent hybridoma (K.-L. P. Sung et al, manuscript in preparation). Thus, upon binding its ligand the CD4molecule is capable of increasing the adhesion strength between the T-cell and the antigen bearing cell. Several lines of evidence suggest that besides adhesion CD4maytransmit an intracellular signal. Anti-CD4MAbmayinhibit responsiveness of T cells in the absence of a potential ligand for CD4. Anti-CD4 MAbs inhibit anti-CD3 MAbstimulation of T-cell proliferation in the absence of MHCclass-II + cells (78, 79), suggesting that the anti-CD4 MAb functioning not to disrupt a putative receptor-ligand interaction but to down-regulate T-cell responsiveness directly. MAbsto the CD4receptor inhibit increases in [Ca+2]i induced by mitogen or antigen (79, 80). Furthermore, anti-CD4 MAbinhibited Ca+z +2 influx without affecting Ca mobilization originating from intracellular stores (80). SomeMAbsto the CD4receptor appear to be directly capable of increasing [Ca+2]i (81, 82). Carrel et al (82) have recently described an anti-CD4 MAbwhich itself sufficient to activate CD4~ T cells. Certain anti-CD4 MAbsmaybe demonstrated to augment the rise of intracellular [Ca+2]i stimulated by anti-CD3 MAb(S. J. Goldman,S. J. Burakoff, manuscript in preparation). Thus, anti-CD4 MAbsmay have both positive and negative effects on Tcell activation, independentof their ability to disrupt a putative receptorligand interaction. It is possible then, that a positive signal via the CD4or CD8molecule is the delivery of a timely signal (that is, a signal delivered in synchrony with that of the TcR) upon effective stimulation of the TcRby antigen. negative signal maybe delivered asynchronously or inappropriately with respect to that generated through the TcR, perhaps even resulting in a state of T-cell refractoriness to stimulation. Thus, under physiologic conditions, CD4binding to MHCclass-II or CD8binding to MHCclass-I molecules mayinitiate intracellular signals that either synergize or compete with those initiated by the TcR/CD3complex.

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The mechanismby which CD4mediates or modulates T-cell activation is a subject of present investigation. In addition to an effect on calcium mobilization, it has recently been demonstrated that CD4and CD8may be phosphorylated in response to antigen stimulation of CD4+ + or CD8 T cells, respectively (72). Recently CD4has been shownto be associated with a lymphoid-specific tyrosine kinase lck (pp58) that shares homology with membersof the src family of kinases (83, 87). In addition we have evidence that this association is via the cytoplasmic domainof CD4(A. Viellete, B. P. Sleckman,J. B. Solen, S. J. Burakoff, submitted). The CD8 molecule also appears to be associated with the pp58 tyrosine kinase (87). Functional analysis of various mutants of the CD4molecule suggests that the ability of CD4to generate intracellular signals maybe important for its function. Wehave generated a mutant form of the CD4molecule, CD4A,in which the majority of the cytoplasmic domain, including all potential substrates for phosphorylation, has been deleted. Expression of the CD4AcDNAin the murine T cell hybridoma 155.16 demonstrated ÷ hybridoma at that this molecule is as effective as the wild type CD4 enhancing T-cell responsiveness upon stimulation with cell bound antigen. However, the CD4Areceptor is less efficient than the wild type CD4 molecule if the hybridoma is stimulated by a poor immunogen,such as purified HLA-DR incorporated into liposomes (84). The highly conserved cytoplasmic domain appears to be required for optimal CD4function. Wehave generated a second mutant form of the CD4molecule, CD4PI, in which the transmembrane and cytoplasmic domains of CD4have been replaced with the carboxy terminal end of the phosphatidylinositol-linked form of the LFA-3molecule. This molecule is composedof the extracellular domain of CD4, is missing the transmembrane and cytoplasmic domains, and is linked to the membranevia phosphatidylinositol. The CD4PImolecule is unable to enhance IL-2 production by the murine T cell hybridoma (Y. Rosenstein et al, manuscript in preparation). Cumulatively, these data suggest that the transmembrane and cytoplasmic domains of CD4 play important roles in CD4-dependentresponses and signal transduction. A number of parallel experiments suggest that CD8may generate intracellular signals in T cells. Anti-CD8 MAbhave been shown to inhibit + T cells lectin-dependent and anti-CD3 MAb-inducedcytolysis by CD8 of target cells that lack class-I MHC expression (85, 86). Expression CD8in the murine hybridoma 155.16 markedly enhanced IL-2 production ÷ stimulator cells. To differentiate in response to MHC class-I + HLA-DR the role of adhesion-strengthening from signal transduction, we have + stimuexpressed both the CD8molecule in the MHCclass I- HLA-DR lator cell Daudi and humanMHC class-I molecule in the responder cell, thereby allowing adhesion but preventing signal delivery via CD8to the

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responding cell. The expression of CD8in the Daudi stimulator cell and, correspondingly, the expression of the HLA-A2 molecule in the responser, resulted in adhesion similar to that seen when CD8is expressed in the hybridoma. There was minimal increased responsiveness, however, compared to that seen when CD8was expressed in the responder cell. This wouldargue that, besides adhesion, CD8plays an additional role in T-cell responsiveness. Experiments using antibody blocking and gene transfer both suggest that the CD4- and CD8-dependentresponse is mediated not + or CD8+ T cell and its only by strengthening adhesion between the CD4 antigen bearing cell, but by the delivery of an intracellular signal.

CONCLUSIONS A series of cell surface receptors have been identified that function in concert with the antigen-specific T cell receptor to augmentor modulate the immuneresponse. These accessory molecules, CD2, CD4, and CD8 have been demonstrated to increase intercellular adhesion; however, the mechanisms by which they impact upon T-cell responsiveness extend beyond their roles as adhesion molecules. These receptors appear to be able to transduce intracellular signals that maysynergize with those generated by the T cell receptor upon binding to antigen. Whether these signals are qualitatively distinct from those initiated by the TcRand/or amplify signals initiated by the TcRusing similar pathwaysof activation is unknown.In addition to a dependenceat the level of signal transduction, the spatial requirements for optimal function of the CD2, CD4, or CD8 receptors have yet to be determined. A physical association between the TcRand CD4or CD8at the time of antigen recognition may result in the formation of a ternary complex which amplifies the intracellular signal and results in appropriate T-cell stimulation. The conservation of the cytoplasmic domains of CD4and CD8between human, mouse, and rat molecules suggests that these molecules may also interact with other membraneproteins. The interaction of CD2with its ligand LFA-3functions not only in cellcell adhesionbut also T-cell activation. It is clear that pairs of stimulatory anti-CD2 MAbs, or LFA-3 plus a single anti-CD2 MAbcan stimulate T cells without a concurrent signal delivered by the TcR. No second ligand for CD2has yet been identified. It is therefore not knownwhether the CD2pathway alone, independent of the TcR pathway, functions physiologically. In the thymus, the CD2pathway may function as an early proliferative signal important for the developmentof the T-cell repertoire. In the periphery, however, antigen binding to the TeRconfers specificity

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cm, con, ANt~ CO8595 to the immune response. CD2bindingto the broadlydistributed LFA-3 moleculemayamplifythis responseto functionin synergywiththe antigenspecific TcR. ACKNOWLEDGMENTS

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This work was supported by NIHgrant CA34129 and AI 17258 (S. J. B.). B. E. Biereris a recipientof a Clinician-ScientistAward from the American HeartAssociation. Literature Cited 1. Howard,F. D., Ledbetter, J. A., Wong, J., Bieber, C. P., Stinson, E. B., Herzenberg, L. A. 1981. A human T-lymphocyte differentiation marker defined by monoclonal antibodies that block Erosette formation. J. Immunol. 126: 2117-22 2. Kamoun, M., Martin, P. J., Hansen, J. A., Brown, M. A., Siadak, A. W., Nowinski, R. C. 1981. Identification of a humanT-lymphocytesurface protein associated with the E-rosette receptor. J. Exp. Med. 153:207-12 3. Van Wauwe,J., Goossens, J., Decock, W., Kung, P., Goldstein, G. 1981. Suppression of humanT-cell mitogenesis and E-rosette formation by the monoclonal antibody OKTI1A. Immunology 44:865-72 4. Martin, P. J., Longton, G., Ledbetter, J. A., Newman, W, Braun, M. P., Beatty, P. G., Hansen,J. A. 1983. Identification and functional characterization of two distinct epitopes of the humanT cell surface protein Tp50. J. Immunol. 131:180-85 5. Krensky, A. M., Sanchez-Madrid, F., Robbins, E., Nagy, J. A., Springer, T. A., Burakoff, S. J. 1983. The functional significance, distribution, and structure of LFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-targetinteractions. J. Immunol.131: 611-16 6. Sewell, W. A., Brown,M. H., Dunne, J., Owen, M. J., Crumpton, M. J. 1986. Molecular cloning of the humanT-lymphocyte surface CD2(Tll) antigen. Proe. Natl. Aead. Sei. USA 83: 871822 7. Sayre, P. H., Chang, H.-C., Hussey, R. E., Brown,N. R., Richardson, N. E., Spagnoli, G., Clayton, L. K., Reinherz, E. L. 1987. Molecular cloning and expression ofT11 cDNAsreveal a recep-

tot-like structure on humanT lymphocytes. Proc. Natl. Acad. Sci. USA84: 2941-45 8. Seed, B., Aruffo, A. 1987. Molecular cloning of the CD2antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sei. USA 84:3365-69 9. Lay, W. H., Mendes, N. F., Bianco, C., Nussenzweig, V. 1971. Binding of sheep red blood cells to a large population of human lymphocytes. Nature 230: 53132 10. Verbi, W., Greaves, M. F., Schneider, C., Koubek, K., Janossy, G., Stein, H., Kung, P., Goldstein, G. 1982. Monoclonal antibodies OKTI1 and OKT11A have pan-T reactivity and block sheep erythrocyte "receptors". Eur. J. Immunol. 12:81-86 11. Krensky, A. M., Robbins, E., Springer, T. A., Burakoff, S. J. 1984. LFA-1, LFA2, and LFA-3antigens are involved in CTL-target conjugation. J. Immunol. 132:2180-82 12. Shaw, S,, Luce, G. E. G., Quinones, R., Gress, R. E., Springer, T. A., Sanders, M. E. 1986. Two antigen-independent adhesion pathways used by humancytotoxic T cell clones. Nature 323:262-64 13. Palacios, R., Martinez-Maza, O. 1982. Is the E receptor on human T lymphocytes a "negative signal receptor"? J. Immunol. 129:2479-85 14. Tadmori, W., Reed, J. C., Nowell, P. C., Kamoun, M. 1985. Functional properties of the 50 kd protein associated with the E-receptor on human T lymphoeytes: suppression of IL-2 production by anti-p50 monoclonal antibodies. J. Immunol. 134:1709-16 15. Hunig, T. 1985. The cell surface molecule recognized by the erythrocyte receptor ofT lymphocytes. J. Exp. Med. 162:890901

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16. Hunig, T., Tiefenthaler, G., Meyerzum Buschenfelde, K.-H., Meuer, S. C. 1987. Alternative pathwayactivation ofT cells by binding of CD2to its cell-surface ligand, Nature 326:298-301 17. Takai, Y., Reed, M., Burakoff, S. J., Herrmann, S. 1987. Direct evidence for a physical interaction between CD2and LFA-3. Proc. Natl. Acad. Sci. USA84: 6864-68 18. Dustin, M. L., Sanders, M. E., Shaw,S., Springer, T. A. 1987. Purified lymphocyte function-associated antigen 3 binds to CD2 and mediates T lymphocyte adhesion. J. Exp. Med. 165: 677-92 19. Selvaraj, P., Plunkett, M. L., Dustin, M., Sanders, M. E., Shaw, S., Springer, T. A. 1987. The T lymphocyte glycoprotein CD2binds the cell surface ligand LFA-3. Nature 326:400-403 20. Vollger, L. W., Tuck, D. T., Springer, T. A., Haynes, B. F., Singer, K. H. 1987. Thymocyte binding to human thymic epithelial cells is inhibited by monoclonal antibodies to CD-2 and LFA-3antigens. J. lmmunol. 138:358~3 21. Meuer, S. C., Hussey, R. E., Fabbi, M., Fox, D., Acuto, O., Fitzgerald, K. A., Hodgdon,J. C., Protentis, J. P., Schlossman, S. F., Reinherz, E. L. 1984. An alternative pathwayof T-cell activation: A functional role for the 50 kd T11sheep erythrocytc receptor protein. Cell 36: 897 906 22. Brottier, P., Boumsell, L., Gelin, C., Bernard, A. 1985. T cell activation via CD2 (T, gpS0) molecules: Accessory cells are required to trigger T cell activavation via CD2-D66plus CD2-9.6/Tll (1) epitopes. J. Immunol.135:1624-31 23. Yang, S. Y., Chouiab, S., Dupont, B. 1986. A common pathway for T lymphocyte activation involving both the CD3-Ti complex and CD2sheep erythrocyte receptor determinants. J. Immunol. 137:1097-1100 24. Peterson, A., Seed, B. 1987. Monoclonal antibody and ligand binding sites of the T cell erythrocyte receptor (CD2). Nature 329:842-46 25. Bernard, A., Gelin, C., Raynal, B., Pham, D., Gosse, C., Boumsell, A. 1982. Phenomenonof humanT cells rosetting with sheep erythrocytes analyzed with monoctonal antibodies. "Modulation" of a partially hidden epitope determining the conditions of interaction between T cells and erythrocytes. J. Exp. Med. 155:1317-33 26. Bernard, A., Knowles, R. W., Naito, K., Dupont, B., Raynal, B., Tran, H., Boumsell, L. 1986. A unique epitope on

the CD2molecule defined by the monoclonal antibody 9-1: epitope-specific modulation of the E-rosette receptor and effects on T-cell functions. Hum. lmmunol. 17:388-405 27. Bierer, B. E., Peterson, A., Barbosa, J., Seed, B., Burakoff, S. J. 1988. Expression of T-cell surface molecule CD2and an epitope-loss CD2mutant to define the role of lymphocytefunctionassociated antigen 3 (LFA-3) in T cell activation. Proc. Natl. Acad. Sci. USA 85:1194-98 28. Bierer, B., Peterson, A., Gorga, J. C., Herrmann, S. H., Burakoff, S. J. 1988. Synergistic T cell activation via the physiological ligands for CD2and the T cell receptor. J. Exp. Med. 168:1145-56 29. Sleckman, B. P., Peterson, A., Jones, W. K., Foran, J. A., Greenstein, J. L., Seed, B., Burakoff, S. J. 1987. Expression and function of CD4in a murine T-cell hybridoma. Nature 328:351 53 30. Tiefenthaler, G., Hunig, T., Dustin, M. L., Springer, T. A., Meuer, S. C. 1987. Purified lymphocytefunction-associated antigen-3 and Tll target structure are active in CD2-mediated T cell stimulation. Eur. J. lmmunol. 17: 1847-50 3 I. Alcover, A., Ramasli, D., Richardson, N. E., Chang, H.-C., Reinherz, E. L. 1987. Functional and molecular aspects of human T lymphocyte activation via T3-Ti and T11 pathways. Immunol. Rev. 95:~30 32. Bockenstedt, L. K., Goldsmith, M. A., Dustin, M., Olive, D., Springer, T. A., Weiss, A. 1988. The CD2 ligand LFA3 activates T cells but depends on the expression and function of the antigen receptor. J. lmmunol. 141:1904-12 33. Alcover, A., Chang, H.-C., Sayre, P. H., Hussey, R. E., Reinherz, E. L. 1988. The TII (CD2) cDNA encodes a transmembraneprotein which expresses T 11 ~, T112 and T113 epitopes but which does not independently mediate calcium influx: analysis by gene transfer in a baculovirus system. Eur. J. lmmunol.18: 363q57 34. Pantaleo, G., Olive, D., Poggi, A., Pozzari, T., Moretta, L., Moretta, A. 1987. Antibody-induced modulation of the CD3/T cell receptor complex causes T cell refractoriness by inhibiting the early metabolic steps involved in T cell activation. J. Exp. Meal. 166:619-24 35. Breitmeyer, J. B., Daley, J. F., Levine, H. B., Schlossman, S. F. 1987. The T11 (CD2)molecule is functionally linked the T3/Ti T cell receptor in the majority ofT cells. J. lmmunol. 139:2899-2905

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J. W., Strominger, J. L., Burakoff, S. J. 36. Moretta, A., Poggi, A., Olive, D., 1982. Long-term humancytotoxic T cell Bottino, C., Fortis, C., Pantaleo, G., Moretta, L. 1987. Selection and charlines allospecific for the HLA-DR6 anti+. Proc. Natl. Acad. Sci. acterization of T-cell variants lacking gen are OKT4 molecules involved in T-cell activation USA 79:2365~59 (T3 T-cell receptor, T44, and Tll): 48. Meuer, S. C,, Schlossman, S. F., Reinanalysis of the functional relationship herz, E. L. 1982. Clonal analysis of + amongdifferent pathways of activation. human cytotoxic T lymphocytes: T4 Proc. Natl. Acad. Sci. USA 84:1654~58 and T8+ effector T cells recognize 37. Bierer, B. E., Barbosa, J., Herrmann,S., products of different major histocompatibility complex regions. Proc. Burakoff, S. J. 1988. The interaction of Natl. Acad. Sci. USA79:4395-99 CD2with its ligand, LFA-3,in humanT cell proliferation. J. Immunol.140:3358 49. Biddison, W. E., Rao, P. E., Talle, 63 M. A., Goldstein, G., Shaw, S. 1982. 38. Reinherz, E. L. 1985. A molecular basis Possible involvement of the OKT4molfor thymic selection: regulation of TI 1 ecule in T cell recognition of class II induced thymocyte expansion by the T3HLAantigens. J. Exp. Med. 156: 1065Ti antigen/MHC receptor pathway. 76 lmmunol. Today 6:75-80 50. Greenstein, J. L., Kappler, J., Marrack, 39. Denning, S. M., Tuck, D. T., Vollger, P., Burakoff, S. J. 1984. The role of L3T4 din L. W., Springer, T. A., Singer, K. H., recognition of Ia by a cytotoxic, H-2 Haynes, B. F. 1987. Monoclonal antispecific T cell hybridoma. J. Exp. Med. bodies to CD2 and lymphocyte-func159:1213-24 tion-associated antigen 3 inhibit human 51. Greenstein, J. L., Malissen,B., Burakoff, thymic epithelial cell-dependent mature S. J. 1985. Role of L3T4in antigen-driven thymocyte activation. J. ImmunoL139: activation of a class I-specific T cell hy2573-78 bridoma. J. Exp. Med 162:369-74 40. Denning, S. M., Dustin, M. L., Springer, 52. Gay, D., Maddon,P., Sekaly, R., Talle, T. A., Singer, K. H., Haynes,B. F. 1988. M. A., Godfrey, M., Long, E., Goldstein, G., Chess, L., Axel, R., KapPurified lymphocytefunction associated antigen-3 LFA-3 activates human pier, J., Marrack, P. 1987. Functional thymocytes via the CD2 pathway. J. interaction between humanT-cell prolmmunol. 141: 2980-85 tein CD4and the major histocompati41. Littman, D. R. 1987. The structure of bility complex HLA-DR antigen. the CD4 and CD8 genes. Ann. Rev. Nature 328:626-29 lmmunol. 5:561-84 53. Doyle, C., Strominger, J. L. 1987. Inter42. Dembic, Z., Haas, W., Zamoyska, R., action between CD4and class II MHC Parnes, J., Steinmetz, M., VonBoehmer, molecules mediates cell adhesion. NaH. 1987. Transfection of the CD8gene ture 330:256-59 enhances T-cell recognition. Nature 326: 54. Rosenstein, Y., Ratnofsky, S., Burakoff, 510-1l S. J., Herrmann,S. H. 1989. Direct evi43. Ratnofsky, S. E., Peterson, A., Greendence for binding of CD8to HLAclass stein, J. L., Burakoff, S. J. 1987. ExI antigens. J. Exp. Med. In press pression and function of CD8 in a 55. Wilde, D. B., Marrack, P., Kappler, J., murine T cell hybridoma. J. Exp. Med. Dialynas, D. P., Fitch, F. W. 1983. Evi166:1747 57 dence implicating L3T4in class II MHC 44. Reinherz, E. L., Kung, P., Goldstein, antigen reactivity; monoclonalantibody G., Schlossman, S. 1979. Separation of GK1.5 (anti-L3T4a) blocks class II MHC functional subsets of T cells by monoantigen-specific proliferation, release of clonal antibody. Proc. Natl. Acad. Sci. lymp.hokines, and binding by cloned USA 76:4061~5 munne helper T lymphocyte lines. J. 45. Swain, S. L. 1981. Significance of Lyt Immunol. 131:2178 83 phenotypes: Lyt-2 antibodies block 56. Marrack, P., Enders, R., Shimonkevitz, activities of T cells that recognizeclass I R., Zlotnik, A., Dialynas, D., Fitch, F., major histocompatibility complex antiKappler, J. 1983. The major histogens regardless of their function. Proc. compatibility complex-restricted antiNatl. Acad. Sci. USA 78:7101-5 gen receptor on T cells. II. Role of the 46. Krensky, A. M., Clayberger, C., Reiss, L3T4product. J. Exp. Med. 158: 1077C. S., Strominger, J. L., Burakoff, S. J. 91 + 1982. Specificity of OKT4 cytotoxic T 57. Moretta, A., Pantaleo, G., Mingari, lymphocyte clones. J. Immunol. 129: M. C., Moretta, L., Cerottini, J. C. 2001-2003 1984. Clonal heterogeneity in the require47. Krensky, A. M., Reiss, C. S., Mier, ment for T3, T4, and T8 molecules in

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humancytotoxic T lymphocyte function. J. Exp. Med~159:921-34 58. Shimonkevitz,R., Luescher,B., Cerottini, J.-C., MacDonald,H. R. 1985. Clonal analysis of cytolytic T lymphocyte-mediatedcytolysis of target cells with inducibleantigen expression: correlation betweenantigen density and requirement for Lyt-2/3 function. J. Immunol. 135:892 99 59. MacDonald,H. R., Glasebrook, A. L., Cerottini, J.-C. 1982. Clonal heterogeneity in the functional requirement for Lyt-2/3moleculeson cytolytic T lymphocytes: analysis by antibodyblocking and selective trypsinization. J. Exp. Med. 156:1711-22 60. Flomenberg, N., Naito, K., Duffy, E., Knowles,R. W., Evans, R. L., Dupont, B. 1983.Allocytotoxic T cell clones: both Leu2+3- and Leu2-3+ T cells recognize class 1 histocompatibility antigens. Eur.J. ImmunoL 13: 90511 61. Fazekas de St. Groth, B., Gallagher, P. F., Miller, J. F. A.P. 1986.Involvement of Lyt-2 and L3T4in activation of hapten-specific Lyt-2+ L3T4+Tcell clones. Proc. Natl. Acad.Sci. USA 83:2594-98 62. Jones, B., Khavari, P. A., Conrad, P. J., Janeway,C. A. Jr. 1987.Differential effects of antibodies to Lyt-2and L3T4on cytolysis by cloned, Ia-restricted T cells expressingboth proteins. J. Immunol. 139:380-84 63. Aparicio, P., Jaraquemada,D., Lopezde Castro, J. 1987.Alloreactivecytolytic T cell clones with dual recognition of HLA-B27 and HLA-DR2antigens. Selective involvementof CD8in their class I-directed cytotoxicity. J. Exp. Med.165:428-43 64. Eichmann,K., Jonsson,J. I., Falk, I., Emmrich,F. 1987. Effective activation of resting mouse T lymphocytes by cross-linking submitogenicconcentrations of the T cell antigenreceptor with either Lyt-2 or L3T4.Eur. J. Immunol. 17:643-50 65. Owens,T., Fazekas, de St. Groth, B., Miller, J. F. A. P. 1987. Coaggregation of the T-cell receptor with CD4and other T-cell surface moleculesenhances T-cell activation. Proc.Natl. Acad.Sci. USA 84:9209-13 66. Walker,C., Bettens, F., Pichler, W.J. 1987.Activationof T cells by cross-linking an anti-CD3antibody with a second anti-T cell antibody: mechanismand subset-specificactivation. Eur. J. lmmunol. 17:873-980 67. Anderson,P., Blue, M. L., Morimoto,

C., Schlossman,S. F. 1987.Cross-linking ofT3 (CD3)with T4 (CD4)enhances the proliferation of resting T lymphocytes. J Immunol.139:678-82 68. Emmrich,F., Strittmatter, U., Eichmann,K. 1986. Synergismin the activation of humanCD8T cells by crossfinking the T-cell receptor complexwith the CD8differentiation antigen. Proc. Natl. Acad. Sci. USA83:8298-8302 69. Allison,J. P., Lanier,L. L. 1985.Identification of antigen receptor-associated structures on murineT cells. Nature314: 107-9 70. Saizawa,K., Rojo,J., Janeway,C. A. Jr. 1987.Evidencefor a physicalassociation of CD4and the CD3:~:/~T cell receptor. Nature 328:260-63 71. Anderson,P., Blue, M. L., Schlossman, S. F. 1988. Comodulationof CD3and CD4.Evidencefor a specific association between CD4and approximately 5%of the CD3:T cell receptor complexeson helper T lymphocytes.J. lmmunol.140: 173~37 72. Acres, R. B., Conlon,P. J., Mochizyki, D. Y., Gallis, B. 1986. Rapid phosphorylation and modulation of the T4 antigen on clonedhelper T cells induced by myristate acetate or antigen. J. phorbol Biol. Chem. 261:16210-14 73. Weyand,C. M., Goronzy,J., Fathman, C. G. 1987. Modulationof CD4by antigenic activation. J. Imrnunol.138:135154 74. Kupfer,A., Singer,S. J., Janeway,C. A. Jr., Swain.S. L. 1987. Coclusteringof CD4(L3T4)moleculewith T-cell receptor is inducedby specific direct interaction of helper T cells andantigen presenting cells. Proc.Natl. Acad.Sci. USA 84:5888-92 75. VanSeventer,G. A., VanLier, R. A. W., Spits, H., Ivanyi, P., Melief, C. J. M. 1986. Evidencefor a regulatory role of the T8 (CD8)antigen in antigenspecific andanti-T3-(CD3)-induced lytic activity of allospecific cytotoxicT lymphocyte clones. Eur. J. Immunol.16: 1363-71 76. Blanchard, D., VanEls, C., Borst, J., Carrel, S., Boylston,A., DeVries,J. E., Spits, H. 1987. The role of the T cell receptor, CD8,and LFA-1in different stages of the cytolytic reactionmediated by alloreactive T lymphocyteclones. J. Immunol.138:2417-21 77. Sung, K.-L., Sung, L. A., Crimmins, M., Burakoff, S. J., Chien, S. 1986. Determinationof junctional avidity of cytolytic T cell andtarget cell. Science 34:1405-8 78. Bank,I., Chess,L. 1985. Perturbation

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CD2, CD4, ANDCD8 of the T4moleculetransmits a negative signal to T cells. J. Exp. Med.162:12941303 79. Tite, J. P., Sloan,A., Janeway,C. A. Jr. 1986. Therole of L3T4in T cell activation: L3T4maybe both an Ia-binding protein and a receptor that transducesa negative signal. J. Mol.Cell. Immunol. 2:179-90 80. Rosoff, P. M., Burakoff, S. J., Greenstein, J. L. 1987.Therole of the L3T4moleculein mitogenand antigenactivated signal transduction. Cell 49: 845-53 81. Ledbetter,J. A., June,C. H., Grosmaire, L. S., Rabinovitch,P. S. 1987.Crosslinking of surface antigens causes mobilization of intracellular ionizedcalcium in T lymphocytes.Proc.Natl. Acad.Sci. USA 84:1384-88 82. Carrel, S., Moretta, S., Pantaleo, G., Tambussi, G., Isler, P., Perussia,B., Cerottini, J. C. 1988.Stimulationandpro+ peripheral blood T liferation of CD4 lymphocytes induced by an anti-CD4 monoclonalantibody. Eur. J. Immunol. 18:333-39 83. Rudd, C. E., Trevillyan, J. M., Dasgupta,J. V., Wong,L. L., Schloss-

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man, S. F. 1988. The CD4antigen is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.Proc. Natl. Acad. Sci. USA 85:5190-94 84. Hunig, T. 1984. Monoclonalanti-Lyt2.2 antibody blocks lcctin-dependent cellular cytotoxicityof H-2-negative target cells. J. Exp. Med.159:551-58 85. Sleekman, B. P., Peterson,A., Foran,J. A., Gorga,J. C., Kara,C. J., Strominger, J. L., Burakoff,S. J., Greenstein,J. L. 1988. Functional analysis of a cytoplasmic domaindeleted mutant of the CD4molecule. J. Immunol. 141:4%54 86. Schrezenmeier,H., Kurrle, R., Wagner, H., Fleicher, B. 1985. Activation of humanT lymphocytes.IlI. Triggering of bystandercytotoxicityin cytotoxicT cell clonesby antibodiesagainst the T3antigen or by a calcium ionophore.Eur. J. Immunol. 15:101%24 87. Veillette, A., Bookman, M.A., I-Iorak, E, M., Bolen, J. B. 1988. TheCD4and CD8T cell surface antigens are associated with the interial membrane tyrosine-proteinkinasep56~ck. Cell 55: 3018

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Ann. Rev. ImmunoL1989. 7.’601 24 Copyright © 1989 by Annual Reviews Inc. All rights reserved

ANTIGEN RECOGNITION BY CLASS I-RESTRICTED T LYMPHOCYTES Alain

Townsend and Helen

Bodrner

Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU United Kingdom INTRODUCTION The classic experiments ofDoherty, Zinkernagel, and Blanden revealed the requirements for recognition by cytotoxic T lymphocytes (CTL) induced response to infection by lymphocytic ehoriomeningitis virus (1). 51Chromiumlabelled target cells were lysed by CTLonly if they were infected with LCMV and also shared class-I alleles of the major histocompatibility complex (MHC)with the donor of the CTL(2). This observation soon generalized to the responses to manyadditional viruses and other intracellular parasites, the products of minor histocompatibility loci, tumor antigens, and haptenated cell surfaces; it was then enshrined in the term "MHC-restricted" recognition (3, 4). Although the CTLresponding to a variety of virus infections could be shownto differentiate between viruses, what remained unclear for several years was the nature of the antigens recognized by class I-restricted CTL and how they might interact with an MHCmolecule (3, 4). The evidence prior to the use of recombinant DNAtechniques tended to favor the intuitive idea that CTLrecognized foreign glycoproteins inserted alongside MHC molecules in the membraneof the target cell (5, 6-10). This view illustrated by a recent review by Klein (11). The evidence accumulated during the last four years is not compatible with this concept and suggests that class I-restricted T ceils recognize the degraded fragments of proteins that have passed through the cytoplasm of the target cell (12, 13, 14, 15, 16, 17) either as a result of newsynthesis or by entry from outside through the membrane of an endosome or 601 0732~582/89/0410-0601 $02.00

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pinosome (18, 19). This mechanism maybe quite distinct fromthe typically pH-dependentpresentation of soluble protein antigens that remain topographically outside the cell, and whichare usually recognizedby class IIrestricted T cells (20, 21, 22, 23, 24). Theunifyingconceptthat all T cells recognizedegradedforms of protein antigens boundto class I or II MHC moleculesis consistent with evidencethat both types of cell share a commonpool of T cell receptor Vgenes(25, 26). The data supporting these ideas have comepredominantly from the study of virus infections, particularly influenza, whichforms the central themeof this review.Thediscussionconcernsitself first with the problem of whatoccurs betweensynthesis of an antigen and the exposureofepitopes on the plasma membraneassociated with class-I molecules, and second with a review of the information obtained using synthetic pepfides to analyzerecognitionby class I-restricted T cells. FROM PROTEIN

SYNTHESIS

TO RECOGNITION

Recognition by CTL of the Nonglycoproteins of Influenza Virus Thefirst evidence that influenza-specific CTLcould recognize proteins that were not inserted in the membrane of the infected target cell was provided by experiments using reassortant influenza A viruses--for reviewsof the biology and immunology of influenza see (10, 27, 27a, 28). Influenza has a segmentedgenome(eight RNAsegments coding for ten proteins), and reassortants betweenantigenically distinct isolates can be isolated (29). Byusing target cells infected with characterizedreassortant viruses it could be shownthat the genes coding for a viral polymerase (PB2)and nucleoprotein (NP) were necessary for recognition by a subset of CTL,distinct from those specific for hemagglutinin(HA),the major glycoproteinof the virus (30, 31, 32, 33, 34). These experiments were extended to the recognition of target cells expressingviral proteinsindividually,either as a result of transfection(35, 36) or infection with recombinantvaccinia viruses (37, 38, 39, 40). Althoughhemagglutinincould be shownto be a target antigen (35, 36, 37), transfected L cells expressingNPwerealso recognizedefficiently by class I-restricted CTL,despite the lack of detectableNPat the cell surface using NPspecific antibody (36). These results were reminiscent earlier work with the large T antigen of SV40virus, which was also recognizedefficiently by CTLon transfected cells (41-45). Results analogous to those with NPwere obtained with the other unglycosylated proteins of influenza virus expressedin isolation by recombinantvaccinia,

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each of which was recognized by either murine (39) or humanCTL(40). Additional examples of predominantly cytoplasmic or nuclear proteins from other viruses recognized by class I-restricted T cells have since been described (46, 47, 48, 49). Three points emergedfrom this work. First, expression of a single viral protein in a dividing transfected L cell was sufficient for recognition by class I-restricted CTL(35, 36). Second, in comparative experiments with influenza, a muchgreater proportion of the class I-restricted activity in polyclonal CTLcultures from infected mice was directed at NP or other internal proteins than at hemagglutinin, the major glycoprotein of the virus (36, 34). Third, and most interesting, the level of NPexpression transfected cells detected with antibodies was unrelated to the efficiency of CTLrecognition (35, 36, 41). These results therefore resolved themselves into the question of how, and in what form, the epitopes recognized by CTLwere transported to the surface of the infected or transfected cell (36). Transport of Nucleoprotein Epitopes to the Cell Surface Is Not Dependent on A Signal Sequence The intracellular sorting of manynewly synthesized molecules has been found to depend on an exposed signal sequence (50, 51, 52). Expression glycoproteins at the cell surface generally requires a hydrophobicsequence, often found at the N-terminus of the molecule, which is required for translocation into the lumen of the endoplasmic reticulum and subsequent anchoring in the membraneand transport to the cell surface. Nucleoprotein is synthesized on free ribosomes in the cytoplasm and contains neither a recognizable hydrophobic signal sequence nor a characteristic membraneanchor (53, 54). However, accumulation of NPin the nucleus has been shown to depend on a short sequence between amino acids 327-345(55). Daveyet al demonstrated this by constructing a series of overlapping deletion mutants of an NP cDNAwhich were then expressed in Xenopusoocytes. Only fragments containing the sequence between amino acids 327-345 accumulated in the oocyte nucleus. These experiments showed that expression of short fragments could be used to identify localization signals within NP, and so led to an analogous set of experimentswith transfected L cells to identify potential signals for transport to the cell surface, and regions of the molecule recognized by CTL. Earlier work on the recognition of SV40T antigen had employed a combination of transfected target cells and SV40-adenohybrid viruses (41, 42, 56). Epitopes recognized were localized to different regions of the antigen, but because someof the hybrid viruses expressed fusions between T and adenovirus proteins (which mayhave provided signals for transport)

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the requirements for presentation at the cell surface could not be unequivocally assessed. Regions of sequence containing epitopes of influenza NPwere localized by comparingL (12) and later P815 (57) cells expressing large fragments of N.P (greater than 150 amino adds) that overlapped over short segments. The results on specificity showedthat epitopes from different regions of NPwere recognized in association with distinct class-I molecules by CTL from three inbred strains of mouse, and these were localized to sections of sequence less than 60 aminoacids in length (12). Of particular interest was the observation that no signal-like sequence could be defined because Nand C-terminal fragments of NPthat shared only two amino acids were recognized by CTLfrom H-2k or H-2b mice respectively. This implied that epitopes in the two ends of the NPmolecule could be transported to the plasma membraneindependently of each other. In addition, although all of the fragments of NPin transfected cells could be recognized by specific CTL, some were unstable and no longer detected with antibodies that bound the complete folded molecule. Hypothesis: Recognition of the Degradation Products Protein Antigens by Class I-Restricted CTL

of

The data obtained with transfected cells led to the hypothesis that CTL recognized a degraded form of nucleoprotein at the cell surface that was not detected with antibodies that bind the folded molecule (12). As the fragments of NPwere expected to be synthesized on free ribosomes in the target cell cytoplasm, this was proposed as the site where fragments were degraded to short peptides prior to transport to the cell surface by a signal independent mechanism. Furthermore, the cytoplasmic proteolytic mechanism involved may be no different than that involved in normal protein turnover and the rapid degradation of abnormal proteins (58), the mechanismfunctions efficiently in the uninfected (transfected) L (12), P815 (57), or EL4 (19) cell. Ubiquitin-dependent proteolysis (59) suggested as a possible constitutive pathwayof degradation in the cytoplasm. These ideas lead to someexplanations of previously enigmatic data and to certain predictions (12). If the mechanisminvolved is a continuous "housekeeping" process linked to normal protein turnover, then it should exist in all cells that express class-I molecules and should apply to normal internal cellular proteins not usually detected on the cell surface with antibody. Anycellular protein that naturally undergoes degradation may furnish peptides that are exposed on the cell surface in association with class-I molecules (and perhaps class-II, discussed below). This would explain for instance, whypolymorphicminor histocompatibility genes (60)

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and tumorantigens (61) can be defined by cytotoxic T cell recognition, andyet rarely can be definedwith antibodiesto cell surfacestructures. It wouldalso lead to a readjustmentof the Matsingcr-Bevan hypothcsis(62) on alloreactivity, to allow for the degradationproducts of any cellular protein to associate with the binding site of MHC moleculesand serve as a target structure for alloreactive T cells. Finally the unifyingidea that recognitionby dass-I and-II restricted T cells is mechanisticallysimilar is consistent with the related structures of class-I and-II molecules(63) and the evidencethat T cells of both types select receptors from a common V genepool (25, 26). Peptides Recoynized by Class I-Restricted

CTL

If cytotoxic T cells recognizethe products of protein degradation, the epitopes shouldbe definablewith synthetic pcptides in vitro, as has been demonstratedfor class II-restricted T cells for several years (reviewedin 20). Theexperimentswith transfected target cells identified the regions of nucleoprotein sequencein whichepitopes existed (12, 57, 14). Peptides were synthesizedbasedon this informationand tested in the 51Crrelease assayfor their ability to sensitize target cells for lysis by characterized murineor humanCTL.Four NPpeptides were identified that resulted in specific target cell lysis (13, 14, 57), and each wasrecognizedby characterized CD8positive T cells in association with a different class-I molecule (summarized in Table 1). In both mouse(64, 57) and human(65) predominantepitopes recognizedwere determinedby the class-I phenotype of the respondingindividuals. Targetcell lysis occurredeither whenpeptide waspresent in the medium surroundingtarget and T cell, or whentarget cells were preexposedto peptide, washed,and then exposedto T cells (13, 66). In addition the concentrationsof peptide inducingthe half maximalresponsein the lysis assay weresimilar to those reported for class II-restricted T cells in proliferative assays (13). Theseresults have since been confirmedand generalizedto the class I-restricted T-cell responseto a variety of other proteins expressedby transfection or recombinantvaccinia viruses (6778, 15, 17) (summarizedin Table 1) including the transmembraneglycoproteins HLACw3(15) and influenza hemagglutinin(17) (discussed moredetail below). Theseexperimentsestablished that the epitopes of proteins recognized by class 1-restricted CTLcan be definedin the lysis assay with peptides of 9-25 aminoacids, and they were consistent with the crystal structure of the class-I moleculeHLA A2 whichrevealed a groove on its top surface that has all the properties expectedof a bindingsite for peptides of this

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Table1 Class I-Restricted epitopesdefinedin the lysis assay with synthetic peptides

Source

Aminoacid residues

Length

366-379 335-349 147-158 50-63 59-68 508-530 161-179 278-286 308-322 265-279 369-390

14 15 12 14 10 23 19 9 15 15 22

170-182 170-182 61-85 98-113 56-69

13 13 25 16 14

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Influenza nucleoprotein

Influenza matrix Influenza hemagglutinin Murinecytomegalovirusp89 LCM virus G protein HIV(HTLVIIIB) gpl60 HIV (SF2) GAG Plasmodiumfalciparum CS HLA CW3 HLA A24 H-2 Ld HLA A2

Class-I MHC restriction Reference bH-2 D HLA B37 aH-2 K ~ H-2 K HLA A2/Aw69 H-2 aK H-2 aL b H-2 D H-2 dD HLA B27 H-2 x (allele not determined) H-2 aK H-2 aK ~ H-2 D HLA A2 HLA A2/Aw69

13 13, 57, 14, 73, 17 67 69 70 71

65 66 64 74

72 15, 75 75 76 77, 78 78

size (79, 80). Thecombined evidencesatisfied earlier expectationsbased transfection experiments(12) and supportedthe concept that a mechanism exists for the presentation at the cell surface of peptides derived from degraded cytoplasmic proteins

bound to class-I

molecules.

PROTEIN DEGRADATION AND PRESENTATION OF EPITOPES WITH CLASS-I MOLECULES The Requirement for Degradation Evidencethat degradationof protein antigens is a requirementfor recognition by class I-restricted CTLhas been provided by comparisonsof complete proteins with peptides derived from themin the 4-6 hr 51Cr release assay. Targetcells exposedto purified undenaturedinfluenzanucleoprotein (14), chicken OVA (19), or hemagglutinin (81, 17), were sensitized for lysis by class I-restricted CTLspecific for these proteins under conditions whereequimolarconcentrations of appropriate peptide induced maximum lysis. In each case the CTLrecognized target cells that synthesizedthese proteins, implyingthat newlysynthesizedproteins reacheda degradative compartment that was not accessible to material in the external environmentof the cell. This wasparticularly cogentin the

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exampleof HA(81) because the target cell used was shownto be capable of presenting epitopes of soluble HAto a class II-restricted T cell, in conditions where no presentation occurred with class I. In contrast target cells expressing HAas a newly synthesized molecule, after infection with recombinantvaccinia, were recognized by class I- but not class II-restricted CTL. In the latter experiment infection with recombinantvaccinia resulted in the expression of HAas an integral membraneprotein, and therefore a formal interpretation of this result was that class I-restricted CTLmay have recognized the HAmolecule only when inserted in the plasma membrane, whereas class II-restricted cells recognized soluble HAthat had been degraded in a prelysosomal compartmentas classically described for other exogenousantigens (21). However,target cells expressing a mutant HAthat was prevented from leaving the cytoplasm by deletion of the Nterminal hydrophobic signal sequence (82) were recognized efficiently class I-restricted CTLoriginally induced by influenza infection (16). The mutant HAwas not detectable on the plasma membranewith antibodies to the folded molecule, did not enter the endoplasmic reticulum from the cytoplasm (as indicated by the lack of core glycosylation), and was degraded rapidly. The latter results implied that class I-restricted CTL specific for HAalso recognized a degradation product of the molecule, were consistent with earlier work showing that CTLcould be induced in vitro with a fragment of HA(9, 83), and were extended by the definition of an epitope from the transmembraneregion of HAwith a short synthetic peptide in the lysis assay in vitro (17). An interpretation of these results was that class-I and -II MHC molecules may be physically segregated with the degradation products of newly synthesized and exogenoushemagglutinin, respectively (23, 24, 84, 85). This schemeis consistent with the data derived within the short time scale of the 51Cr release assay in vitro (81, 14, 19), with somepossible exceptions (86, 87, 88, 89, 90) but is less easy to accommodatewith experiments involving longer time courses in vivo and in vitro. There are several reports of soluble or particulate antigens that wouldnot be expected to enter the cytoplasm of host cells that either prime class I-restricted T cells in vivo or restimulate them with variable efficiency in vitro (27a, 9197). These include the influenza HAused by Morrison et al to make the comparison between class-I and -II restricted T cells and influenza nucleoprotein (98, 99, 81, 94, 95). Althoughsomeof these results could be explained simply by artefactual extracellular degradation (19), a more interesting possibility is that cells mayvary in their ability to present exogenousantigens with class-I molecules (24, 100) and endogenouswith class-II (89), dependingon their state

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of differentiation, the time available, and the properties of the antigen. An additional theoretical factor to consider is that the proteases that gain access to endogenous and exogenous protein antigens may have different specificities and generate noncross-reacting peptides (90, 101). If this were so, a T cell primed in vivo to a peptide derived from a newly synthesized protein bound to a class-I molecule (most somatic cells do not express class II) would be expected to recognize this combination selectively in vitro. Further evidence that degradation is required for presentation of epitopes-with class-I molecules has come from work with recombinant vaccinia as a means of expressing proteins recognized by CTL. A comparison of early (E) (active before DNAreplication) with late (L) (active after DNAreplication) vaccinia promoters for expression of influenza HA revealed that despite the presence of serologically detectable HAat the cell surface, presentation of HAepitopes to class I-restricted CTLwas inhibited during the late phase of vaccinia infection (102). A similar but selective inhibitory effect was noted for a C-terminal epitope (365-379) of influenza NP(103). The inhibitory effect did depend on the amount of antigen synthesized but was reversed by expression of rapidly degraded fragments of these proteins in the cytoplasm of the vaccinia infected cell. For HA,deletion of the N-terminal signal sequence (as described above) restored presentation, and for NPeither large N-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein (104) partially reversed the inhibitory effect (103). Although the molecular explanation is not known for the inhibitory effect of vaccinia on presentation to class I-restricted CTL,these results emphasize the association between degradation of endogenously synthesized antigens in the cytoplasm of the target cell with recognition by class I-restricted CTLof epitopes at the cell surface. An interesting and testable possibility is that vaccinia, like other pox viruses (105, 106, 107), mayexpress one or more serine protease inhibitors that could accumulate in the cytoplasmof the infected cell and inhibit host proteases involved in the degradation of antigens recognized by CTL. In addition the expected inhibition of synthesis of host class I molecules associated with vaccinia infection maycontribute to the reduced efficiency of antigen presentation 002). The identity of the proteolytic system involved in the presentation of cytoplasmic proteins remains unknown,although the more effective presentation of the rapidly degraded ubiquitin-NP fusion protein (103) and the lack of an inhibitory effect of lysosomotropic agents (13, 81), raises the possibility that ubiquitin-dependent degradation mayplay a role (104, 59).

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The Role of the Cytoplasm The experiments described above were consistent with newly synthesized antigens gaining access to a cytoplasmic degradation system prior to association with class-I molecules and presentation at the cell surface. It was not clear, however, whether this mechanismwas associated with new protein synthesis or could be reproduced by introducing proteins into the cytoplasm, from outside the cell. Earlier work had shownthat for viruses with fusion activity, target cells could be sensitized with inactivated viral particles (5, 6, 7, 8). This approach has nowbeen extended to heat inactivated influenza virus, which retains fusion activity but is no longer competent to replicate or induce newviral protein synthesis (18). Sensitization of target cells with heat inactivated virus for lysis by NPand polymerase (PBI) specific CTLcorrelated with fusion and injection of viral cores into the cytoplasmof the target cell, as demonstratedby the arrival of stainable NPin the nucleus. These results have been extended by using the technique of pinosome lysis to demonstrate formally that introduction of an exogenousantigen into the cytoplasmof a target cell can result in presentation of epitopes to class I-restricted CTL(19). A cytotoxic T-cell response to chicken OVA can be induced by immunizing mice with EL4 cells transfected with the OVAgene (19). As for influenza NP, the predominant epitope recognized can be defined with a short synthetic peptide in vitro, and target cells exposed to complete undenatured OVAare not sensitized for lysis by class Drestricted CTL. However, recognition by CTLwith subsequent target cell lysis occurs if undenatured OVAis introduced into the cytoplasm of the target cell by pinosomelysis, as described by Okada & Rechsteiner (108). Takenwith earlier work, these results imply that at least for signaldeleted HA, NP, and OVA,presentation to class I-restricted CTLwithin the time period of the SlCr release assay (4-6 hr) requires access to the target cell cytoplasm, and these results are consistent with a cytoplasmic degradation system furnishing the peptides that are subsequently recognized at the cell surface.

Transportof Epitopesto the Cell Surface To be recognized by T cells, epitopes originating in the cytoplasm must presumably cross a membranesomewhere between the endoplasmic reticulum and the plasma membrane, in order to associate with a class-I molecule and to reach the cell surface. Althoughthere is no experimental evidence to indicate howthis might occur, there are at least two alternatives.

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As already discussed, transport of epitopes from both HAand NPis independentof a definablesignal sequence(16, 12). Formallyit is possible that signal independenttransport from the cytoplasmacross a membrane could occur either before or after degradation.For example,ribonuclease A micro-injected into the cytoplasm can be shownto be degraded in lysosomes(109, 110), implyingthat certain proteins can movefrom the cytoplasm across the lysosomal membrane,probably by an auto-phagocytic process.It is of interest that this is to someextentselective, beingmore efficient for ribonucleasethan BSA(11 l, 110), andis partially dependent a characterized sequenceat the N-terminusof ribonuclease (112). This phenomenon might help explain whysomecytoplasmic viral proteins are predominantly presentedto class II-restricted T cells (89, 90). If transport occursafter degradationin the cytoplasm,it is necessaryto predict the existence of a transport systemthat passes peptides fromthe cytoplasmacross the membrane of a compartmentcontaining either newly synthesized class-I moleculesor recycling them. A precedentfor energy dependenttransport of short (2-5 aminoacids) peptides across membranes exists in the oligopeptide permease(Opp) of the bacterium Salmonella typhomurium (113). Consistent with someformof peptide transport is the finding that expressionof a 15 aminoacid peptide derived from influenza NP(amino acids 366-379) in the cytoplasm of recombinant vaccinia infected target cells is sufficient to obtain recognitionby definedclass Irestricted CTL(unpublished results). However,at this time it remains unknown howsignal independenttransport ofepitopes from the cytoplasm to the cell surfaceoccurs. THE DETAILS OF PEPTIDE RECOGNITION BY CLASS I-RESTRICTED CTL The Specificity

of CTL for Peptides

Thedefinition of class I-restricted epitopes with synthetic peptides has alloweddetailed investigation of recognition by CTLat the cell surface. Informationregarding the specificity and sensitivity of CTLfor a peptide can be obtainedby titrating peptides in a 51chromium release cytotoxicity assay and comparingthe dose response curves obtained (e.g. Figure la). Comparablecurves have been obtained in experiments whenpeptide is present throughoutan assay and whentarget cells are pretreated at 37°C and then washedbefore adding to CTLin an assay (I 3, 66). Association of peptides with the cell surface is temperaturedependent,as sensitization of target cells in vitro is muchless efficient at 4°Cthan at 37°C(84, 101). The recognition of synthetic peptides by CTLmaybe length dependent.

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Peptide Concentration(M)

(a) 70-

~.=~,~158

R-

®

Targets: Time (rain.)

of IO-TMPeptide Pulse

Figure 1 A. Optimal target lysis requires lower concentrations of the modified peptide NP147-158R-than the wild-type sequence NP147-158. Dose-response curves were compared d restricted CTLclone T9/13 (killer to target ratio, 2). 5]Cr-labelled for NP-specific, H-2K d) P815 (H-2 target cells and CTLwere mixed, and peptides added to each well to give a final concentration as indicated, according to the methoddescribed (66). C), NP147-158; O,NP147-158R-; &, NP141-161; m, NP147-161. B. Modified peptide, NP147-158R-associates with target cells more rapidly than does the wild-type peptide, NP147-158.In a cytotoxicity assay the NP specific H-2 Kd restricted CTLclone T5/5 (K/T ratio = 4) was assayed for recognition of peptide pulsed 5]Cr-labelled target cells. Target cells were pulsed with I0-7Mpeptide for varying periods of time at 37°C, diluted 30-fold with ice-cold BSS,and washedtwice more in the cold before inclusion in the assay, according to the methoddescribed (66). C), NP147-158;O, 147-158 R-. These figures (66) (copyright Cell Press) are reproduced by kind permission.

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For example, the 15 aminoacid peptide 366-379from influenza A virus nucleoprotein wasrecognizedmoreefficiently by Db restricted CTL,than wereshorter or longer peptides from the sameregion (13). Maximal target lysis was obtained at 5 x 10-SM366-379, while a 104 fold higher concentration of 369-379was required for an equivalent level of lysis. However,the effects of length mayvary for different CTLclones specific for the same region of an antigen. Three humanHLA-A2 restricted CTL clonesspecific for influenzaAvirus Matrixprotein, in the region57 to 68, recognized a set of four peptides from nine to twelve aminoacids long withdifferingfine specificities (114). CytotoxicT cells can also distinguish betweenpeptides that are closely related in sequence.There are several CTLepitopes that span areas of an antigen in whichthere is natural sequencevariation. The region of NP recognized by Db-restricted CTLcontains two positions, 372 and 373, whichvary betweenvirus isolates from the years 1934or 1968(27, 14), and for whichCTLclones were either specific or cross-reactive. Using synthetic peptides with sequencemodifications, a conservative changeat position 372 (asp to glu) wasshownto be critical for recognitionby a 1968 specific clone, while substitution at position 373wastolerated. Similarly, Kd-restrictedCTLwereeither specific or cross-reactivefor the regions 171 to 182 of the humanclass-I molecules HLA-Cw3 or A24, whichdiffer at only oneaminoacid position (75). Thesevariations in fine specificity are likely to reflect differencesin the precisemannerof associationof a T cell receptor (TCR)with an MHC-antigencomplex. Recognition of Peptides Sequence Modification

by CTL Can Be Enhanced by

Theability to increase the affinity of antigen-antibody or enzyme-substrate interactions by mutation or chemicalmodification is well known.There are examplesin class II-restricted recognitionin whicha T cell, derived by primingand restimulation with one antigen, is found to havea stronger or heteroclitic response to a related but different antigen (reviewedby Schwartz;115). There are nowtwo similar examplesin class 1-restricted systems. Substitution of a single aminoacid in the Db-restrictedepitope of influenzaA virus nucleoprotein 365-379 resulted in a 50-fold enhancementof recognition of this peptide by one CTLclone (14). Modificationof the drestricted epitope of NP, NP147-158 (66), by substitution of glycine for threonine at position 157 and deletion of the terminal glycine (position 158) resulted in a thousand-fold enhancementof CTLrecognition over that of the wild-type peptide (Figure la). This modifiedpeptide (147-

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158R-)was recognizedat concentrations as low as 3 x 10-~2M,the optimal concentration being between10-IlM and 10-~°M(Figure l a). Studies of the kinetics of target formationshowedthat this pcptidc was able to associate with target ceils far morerapidly than did the natural pcptidc at the sameconcentration (Figure lb). Oneindication that the enhancedactivity of the peptide wasdue to improvedbinding to the classI moleculeon the target cell wasprovidedby the finding that the mutant pcptidc wasapproximately100-foldmoreefficient than the wild-typepeptide in competitionassays (116)(discussedin detail below). Evidence for Bindin9 of Peptides to Class-I Molecules Several workershave demonstrateddirect binding of peptides to soluble or membraneboundclass-II molecules (117, 118, 119, 120, 121). This binding has beencorrelated with recognition of the peptides by helper T cells in in vitro proliferation assays(122, 120, 119),althoughsomepeptides haveemergedwhichshowrelatively high affinity binding to class-II molecules, but do not appear to be immunogenic (120). Studies have shown correlation betweendirect binding of peptides to class-II moleculesand the ability to inhibit in vitro proliferation of helper T cell hybridsto a referencepeptide (122, 120). As yet, there have been no reports demonstrating direct binding of peptides to soluble class-I molecules.However,in viewof the manysimilarities that exist betweenthe structures of class-I andclass-II molecules (63), bindingof peptidesto class I seemslikely. Indirect lines of evidence include peptide competitionstudies (discussedbelow), recognitionof peptide treated target cells whichhavebeenclass I-matchedby transfection (13), and the genetic control of immune responsesto individual peptides ~ wereable by class I genes. For example,all mice whichexpressedH-2-K to respond to the synthetic peptide 147-161of NP(57). Similarly humans,all those expressing HLA-837or the normal variant of HLAA2, and whorespondto influenza Avirus, respondto the epitopes NP335349 and57-68of the matrixprotein respectively(123, 65). Therecognitionof peptides by class I-restricted CTLis consistent with the crystal structure of the class-I molecule,HLA-A2 as describedin detail by Bjorkmanet al (80, 79). Class-I molecules are composedof a heavy chain spanningthe membrane non-covalentlyassociated with a light chain, ¢/z microglobulin. The structure revealed includes a groove situated betweentwo g-helixes on the membranedistal surface of the molecule, derived from the first two domains(g~ and g2) of the heavy chain and supported by a membraneproximal domain (g3, the third external domain). This groove, incorporating manyof the polymorphicresidues of class I, has beenproposedas a putative peptide binding site. The X-ray

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crystallographicpictures of the moleculedisplayedunidentifiable material in this groovewith a density consistent with a boundpeptide. Competition Between Peptides for Presentation to CTL It has provedpossible to inhibit the recognitionof one class I-restricted synthetic peptide by the addition of certain others in excess. Competition betweenpeptides occurred either whenboth were present in solution in a cytotoxicity assay, or if target cells were pretreated with both peptidcs simultaneouslyand then washedbefore exposureto T cells (75, 116, 124). Theseresults impliedthat competitionoccurredat the level of the target cell andsuggestedcompetitionfor bindingto a single region of the classI molecule. Competitionhas been demonstratedbetweenclosely related peptides, such as the sequences derived from the region 356-380of the 1934 and 1968isolates of influenza NP(differing by two aminoacids) (124), also between peptides 170-182 of HLA-Cw3 and HLA-A24, (which differ by one aminoacid) (75). Peptidesof unrelatedsequence,restricted through the sameclass-I molecule(such as NP147-158 and Cw3171-182recognized by Kd restricted CTL)also competed(Table 2) (116). Finally, peptides unrelated in both sequence and MHC restriction maycompetewith each other in vitro. For example,from influenza nucleoprotein, the sequence 50-63 is recognized by Kk restricted CTL(14), 147-158by d restricted CTL(66) and 365-380by b r estricted CTL (13). However, 50-63 inhibited recognitionof both 147-158and365-380(Table2) (116, 124). This implied that 50-63couldbind all three restriction molecules,despite the fact that natural infection did not generateT cells that recognized50-63in association with either K~ or Db in vitro. In summary,these results implythat the binding specificity of class-I molecules maybe broader than indicated by the immuneresponse phenotype of different mousestrains primedby natural infection. It remainsto be seen whetherdirect primingwith these peptides could elicit a response not inducedby virus. Recently, Lakeyet al (125, 126) demonstratedinhibition of class IIrestricted T-cell proliferation by peptides unrelated in both sequenceand MHC restriction, and they suggested that this mayhave occurred through competition for a non-MHC peptide binding protein. Competitionstudies in class I restricted systemswouldnot supportthis, becauseKO-restricted T cells werenot inhibited by a Db restricted peptide whenpresented by a target cell expressing both class-I molecules(124), showingthat competition for a common peptide binding protein did not occur. Althoughcompetitionoccurredat the level of the target cell, it wasnot possible to block recognition by preincubation of the target cell with

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competitor alone (124), nor was it possible to block recognition of preformedtarget cell (either treated with target peptide or infected with virus). Simultaneouspretreatment appearedto be required. Theseresults could be attributed to a slowoff rate of target peptide boundto MHC, as demonstratedin studies of direct binding to class II (117, 118). Lack inhibition following preincubation with a competitor peptide mayhave been due to failure to saturate all available MHC sites. It is not known howmanymoleculesof peptide needto be present on a target cell in order to trigger lysis by a CTL,but fromthe lowconcentrationsof target peptide requiredfor maximal target cell sensitization, andthe expectedlowaffinity, the level of saturation of MHC required maybe of the order of less than onepercent (13, 66, 124). Thecompetitionso far describedwasat the level of the target cell, and no inhibition was shownfollowing preincubation of the CTL(75, 124). Parham,Clayberger, and colleagues have described a system in whichA2 alloreactive CTLwere inhibited by addition of a peptide derived fromA2 itself (77, 78). This peptidewasable to inhibit recognitionof A2-expressing target cells followingpretreatment of the CTL.A similar phenomenon has bccnfoundwith ccrtain A2-rcstrictcd, influenza-specificCTL(P. Robbins, personalcommunication). Boththese results suggestthat, for these T cells, the blockingmayhave beenat the level of the T cell receptor (TCR). Representation of the In Vivo Processed Frayment by Synthetic Peptides Despite the observation that synthetic peptides can be recognizedby CTL and evidencethat degradationof antigen takes place before presentation to a T cell, there is no direct evidencethat the optimalsize of synthetic peptide in vitro is the actual size of the processedfragment. There are several reports in whichT-cell responses that wereinduced against a synthetic peptide did not cross-react with the wholeantigen. For instance, Carboneet al (127) induceda primaryCTLresponse in vitro, incubating unprimedmousespleen cells, at a high density, with peptide. In this way, H-2-Db-restrictedCTLwereinducedagainst the peptide 365380 of influenza NP and 111-122 of ovalbumin (OVA).While a small proportionof the NPpeptidespecific cells recognizedvirus infected cells, none of the OVA peptide induced CTLrecognized OVA transfected target cells. This implied that the majority of CTLinducedby peptide in vitro underthese conditions, were unable to recognize the naturally processed antigen. Similar results were obtained following in vivo priming with peptide to induceclass II-restricted responses(128). Primingin vivo with peptide to induceclass I-restricted responseshas not beenreported. Further evidencethat the minimalpeptides maynot represent the frag-

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ment naturally produced was demonstrated by HLA-A2restricted CTL specific for the epitope 57-68 of influenza A virus matrix protein. Someof these T cells were able to recognize the synthetic peptide in association with both A2 and with a closely related allele HLA-Aw69. In contrast, whentarget cells were infected with influenza virus only the A2expressing target cell was recognized (74). Furthermore Aw69expressing individuals were not able to respond to this epitope of matrix protein (123). It possible that this low response was due to the lack of the naturally processed fragment of matrix protein being able to associate with Aw69. Interactions

of Peptide

and MHC

Muchcontroversy exists over the conformation that a peptide might adopt when bound to MHC.The dimensions of the putative peptide binding site, as defined by Bjorkmanet al (79, 80) are such as would accommodate peptide of around eight aminoacids as an extended chain, or up to twenty residues in the form of an e-helix. Several workers have suggested that peptides could bind in the form of an e-helix, thus segregating residues in contact with MHC and T cell receptor with a defined periodicity. Studies constructing hybrid peptides on this basis, in class II-restricted recognition systems can be interpreted in terms of a helical conformation of bound peptide (115, 129, 130, 122); however, the effects of single residue substitutions are not universally consistent with a helix (130a, b). Information regarding TCRand MHCcontacts for a class I-restricted epitope has been investigated by systematic substitution of amino acids along a peptide. For example, a set of humanHLAA2restricted, influenza A virus matrix peptide 57-68 specific CTLwas used to study analogues of this peptide (114). There was only one position (glycine 61) where each seven substitutions appeared to prevent the peptide from binding to A2. The effects of substitutions at all the other positions were variable, depending both on the amino acid substituted and, in some cases, on the fine specificity of the particular CTLclone. These findings revealed diversity in recognition of peptide by individual CTLclones but were not interpretable in terms of an unequivocal conformation of bound peptide. The detailed structure of a boundpeptide mayultimately require direct analysis by x-ray crystallography. A number of cases have now been reported in which peptides can be presented to CTLin the context of specific MHC molecules with defined sequence variations in the (z 1 or ~2 domains (131, 123, 132, 74), which extend earlier work with virus infected cells (reviewed in 27, 133). commontheme is that residue changes, which from the three dimensional structure of A2 would be expected to be in the peptide binding pocket or accessible to T cell receptor (80, 79), resulted in altered or lost recognition

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of peptide. Manyof these changes had differential effects on individual Tcell clones.

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SUMMARY The work discussed here offers a unified view of T-cell recognition and suggests that class-I and class-II molecules have a closely related function in the presentation of peptides to T lymphocytes. The epitopes recognized by class I-restricted T cells that have been defined with peptides in the 46 hr lysis assay (Table 1) have all been derived from endogenously synthesized proteins expressed by virus infected or transfected cells. Evidence is accumulating that a cytoplasmic degradation system may be involved in the generation of these epitopes. The analysis of the specificity of CTL responses with synthetic peptides has demonstrated the control of immune responses to isolated cpitopes by class-I genes and the great diversity of the receptor repertoire for individual class-l-restricted epitopes.

Literature Cited 1. Zinkernagel, R. M., Doherty, P. C. 1974. Immunological surveillance against altered self componentsby sensitised T lymphocytes in lymphocytic choriomeningitis. Nature 251: 547 2. Blanden, R. V., Doherty, P. C., Dunlop, M. B. C., Gardner, I. D., Zinkernagel, R. M., David, C. S, 1975. Genes required for cytotoxicity against virus-infected target cells in K and D regions of H-2 complex. Nature 254: 269 3. Zinkernagel, R. M., Doherty, P. C. 1979. MHC-restrictedcytotoxic T cells: Studies on the biological role of polymorphic major transplantation antigens determining T cell restrictionspecificity, function and responsiveness. Adv. Immunol. 27:51 4. Zinkernagel, R. M., Rosenthal, K. L. 1981. Experiments and speculation on antiviral specificity of T and B cells. lmmunol. Rev. 58:131 5. Schrader, J. W., Edelman, G. M. 1977. Joint recognition by cytotoxic T ceils of inactivated Sendai virus and products of the major histocompatibility complex. J. Exp, Med. 145:523 6. Gething, M. J., Koszinowski, U., Waterfield, M. 1978. Fusion of Sendai virus with the target cell membrane is required for T cell cytotoxicity. Nature 274:689 7. Koszinowski, U. H., Allen, H.,

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vaccinia virus recombinants.J. Immunol. 137:3973 50. Sabatini, D., Kreibich, G., Morimoto, T., Adesnik, M. 1982. Mechanisms for incorporation of proteins in membranesandorganelles.J. Cell Biol. 92: 1 51. Schatz, G. 1986.Protein translocation. A commonmechanismfor different membranesystems? Nature 321:108 52. Rothman,J. E., Kornberg,R. D. 1986. Anunfolding story of protein translocation. Nature322:209 53. Winter,G., Fields, S. 1981.Thestructure of the gene encodingthe nucleoprotein of human influenza virus A/PR/8/34.Virology 114:423 54. Huddleston, J. A., Brownlee, G. G. 1982. The sequence of nucleoprotein geneof humaninfluenza A virus, strain A/NT/60/68.Nucleic Acids Res. 1029: 55. Davey, J., Dimmock,N. J., Colman, A. 1985.Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopusoocytes. Cell 40: 667 56. Reddy,V. B., Tevethia,S. S., Tevethia, M. J., Weissman,S. M. 1982. Nonselective expressionof simianvirus 40 large tumour antigen fragments in mousecells. Proc.Natl. Acad.Sci. USA 79:2064 57. Taylor, P. M., Davcy,J., Howland,K., Rothbard, J., Askonas, B. A. 1987. Class I MHC molecules, rather than other mousegenes dictate influenza epitope recognition by cytotoxic T cells. Immunogenetics 26:267 58. Hershko, A., Ciechanover, A. 1982. Mechanismsof intraeellular protein breakdown. Annu. Rev. Biochem. 51: 335 59. Hershko, A., Ciechanover, A. 1986. The Ubiquitin pathway for the degradation of intracellular proteins. Prog.Nucleic Acid Res. Mol. Biol. 33: 19. 60. Simpson,E. 1984. H-2 and non H-2 Ir genes. Anal. Immunol135:410 61. DePlaen, E., Lurquin,C., Vanpel, A., Mariame, B., Szikora,J. P., Wolfel,T., Sibille, C., Chomez, P., Boon,T. 1988. Immunogenic (turn-) variants of mouse tumourP815: cloning of the gene of turn- antigen P91Aand identification of the rum-mutation.Proc.Natl. Acad. Sci. USA85:2274 62. Matzinger,P., Bevan,M. J. 1977. Why do so manylymphocytes respond to major histocompatibility antigens? Cell. Immunol.29:1 63. Brown,J. H., Jardetzky, T., Saper, M.

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A., Samraoui, B., Bjorkman,P. J., Wiley, D. C. 1988. A hypothetical modelof the foreign antigen binding site of class II histocompatibilitymolecules. Nature332:845 64. Pala, P., Askonas, B. A. 1986. Low responder MHC alleles for Tc recognition of influenza nucleoprotein. Immunogenetics23:379 65. McMichael, A. J., Gotch, F. M., Rothbard, J. 1986. HLAB37 determines an influenza A virus nucleoprotein epitope recognized by cytotoxic T lymphocytes.J. Exp. Med.164: 1397 66. Bodmer, H. C., Pemberton, R. M., Rothbard,J. B., Askonas,B. A. 1988. Enhancedrecognition of a modified peptide antigen by cytotoxic T cells specific for influenza nucleoprotein. Cell 52:253 67. Del val, M., Volkmer,H., Rothbard,J. B., Jonjie, S., Messerle,M., Schickedanz. J., Reddehase,M, J., Koszinowski, U. H. 1988. Molecular basis for cytolytic T-lymphocyte recognition of the murinecytomegalovirusimmediate early protein pp 89. J. Virol.: In press 68. Whitton,J., Gebhard,J., Lewicki,H., Tishon, A., Oldstone,M. 1988. Molecular definition of a majorcytotoxic T lymphocyteepitope in the glycoprotein of Virol. lymphocitic choriomeningitisvirus. J. 62:687

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83. Wabuke-Bunoti,M. A. N., Taku, A., Fan, D., Kent,S., Webster,R. G. 1984. Cytolytic T lymphocyteand antibody responses to synthetic peptides of influenza virus hemagglutinin. J. Immunol.133:2194 84. Braciale, T. J., Morrison, L. A., Sweetser, M. T., Sambrook, J., Gething, M.J., Braciale, V. L. 1987. Antigenpresentation pathwaysto class I and class II MHC-restrictedT lymphocytes. ImmunoLRev. 98:95 85. Pernis, B. 1985.Internalization of lymphocyte membrane components. Immunol. Today 6:45 86. Schmitt-Verhulst,A. M., Pettinelli, C. B., Henkart, P. A., Lunny, J. K., Shearer, G. M. 1978. H-2 restricted cytotoxiceffectors generatedin vitro by the addition of trinitro phenyl-conjugated soluble proteins. J. Exp. Med. 147:352 87. Yamada,A., Young,J. F., Ennis, F. A. 1985. Influenzavirus subtype-specific cytotoxic T lymphocyteslyse target cells coatedwith a protein producedin E. Coli. J. Exp. Med.162:1720 88. Sethi, K. K., Naher, H., Stroehmann, I. 1988. Phenotypic heterogeneity of cerebrospinal fluid-derived HIV-specific andHLA-restricted cytotoxicT-cell clones. Nature335:178 89. Sekaly, R. P., Jacobson,S., Rickert, J. R., Tonelle, C., McFarland,H. F., Long,E. O. 1988. Antigenpresentation to HLAclass II-restricted measles virus-specificT cell clonescanoccurin the absence of the invariant chain. Proc. Natl. Acad. Sci. USA85:1209 90. Long,E. O. 1988. Processingrequirementsfor presentationof antigens to T lymphocytes.Curr. Opinion Immunol. In press 91. Bevan,M.J. 1976.Cross primingfor a secondarycytotoxic response to minor H antigens with H-2 congenie cells whichdo not cross react in the cytotoxic assay. J. Exp. Med.143:1283 92. Kaufmann,H. E. 1988. CD8+T lymphocytes in intracellular microbial infections. Immunol.Today9:168 93. Tevethia,S. S., Flyer, D.C., Tjian, R. 1980.Biologyof simianvirus 40 (SV40) transplantation antigen (TrAg). VI. Mechanism of induction of SV40transplantation immunityin mice by purified SV40T antigen (D2 protein). Virology 107:13 94. Wraith, D. C., Askonas, B. A. 1985. Indueation of influenza A virus cross reactive cytotoxic T ceils by a nucleoprotein/haemagglutinin J. Gen.Firol. 66:1327 preparation.

95. Wraith, D. C., Vessey, A. E. 1986. Influenzavirus-specificcytotoxicT-cell recognition: stimulation of nucleoprotein-specific clones with intact antigen. Immunology59:173 96. Stearz, U., Karasuyama,H., Garner, A. 1987. Cytotoxic T lymphocytes against a soluble protein. Nature329: 449 97. YideJin, J., Wai-Kuo, S., Berkower,I. 1988. HumanT cell response to the surface antigen of hepatitis B (HBsAg). Endosomal and nonendosomal processing pathwaysare accessible to both endogenousand exogenousantigen. J. Exp. Med. 168:293 98. Zweerink,H. J., Askonas,B. A., Millican, D., Courtneidge,S. A., Skehel,J. J. 1977. Cytotoxic T-cells to type A influenza virus; viral haemagglutinin induces A-strain specificity while infectedcells confercross-reactivecytotoxicity. Eur. J. Immunol.7:630 99. Braciale,T. J. 1979.Specificityofcytotoxic T-cellsdirectedto influenzavirus hemagglutinin. J. Exp. Med.149:856 I00. Morrison,L., Braciale, V., Braciale, T. 1988. Antigenform influences induction of influenza-specific class I and class II MHC-restrictedcytolytic T lymphocytes.J. Immunol.141:363 101. Townsend,A. R. M. 1986. Recognition of influenzavirus proteinsby cytotoxic T lymphocytes. ImmunologicRes. 6: 80 102. Coupar, B. E., Andrew,M. E., Both, G. W., Boyle, D. B. 1986. Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response. Eur. J. ImmunoL 16:1479 103. Townsend,A., Bastin, J., Gould, K., Brownlee,G., Andrew,M., Coupar,B., Boyle, D., Chan,S., Smith, G. 1988. Defective presentation to class Irestricted cytotoxiclymphocytes in vaccinia-infected cells is overcomeby enhanceddegradation of antigen. J. Exp. Med.In press 104. Bachmair,A., Finley, D., Varshavsky, A.1986.In vivohalf-life of a proteinis a function of its amino-terminal residue. Science 234:179 105. Pickup,D. J., Ink, B. S., Hu,W., Ray, C. A., Joklik, W.K. 1986. Hemorrhage in lesions caused by cowpoxvirus is inducedbya viral proteinthat is related to plasmaprotein inhibitors of serine proteases. Proc. NatLAcad. Sci. USA 83:7698 106. Upton,C., Carrell, R. W., McFadden, G. 1986. A novel memberof the serpin superfamily is encodedon a circular

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CLASSI--RESTRICTED RECOGNITION 623 plasmid-like DNAspecies isolated fromrabbit cells. FEBSLett. 207:115 107. Tomley,F., Binns, M., Campbell,J., Boursnell, M. 1988. Sequenceanalysis of an 11.2 kilobase, near-terminal, BamHIfragment of Fowlpoxvirus. J. Gen.Firol. 69:1025 108. Okada,C. Y., Rechsteiner, M. 1982. Introduction of macromoleculesinto cultured mammalian cells by the osmoticlysis of pinocytotic vesicles. Cell 29:33 109. Backer,J. M., Bourret,L., Dice, J. F. 1983.Regulationof catabolismof microinjected ribonucleaseA requires the amino-terminal20 aminoacids. Proc. Natl. Acad. Sci. USA80:2166 110. McElligott, M. A., Miao,P., Dice, J. F. 1984. Lysosomal degradation of ribonuclease A and ribonuclease Sprotein microinjectedinto the eytosol of humanfibroblasts. J. Biol. Chem. 260:11986 111. Bigelow,S,, Hough,R., Rechsteiner, M.1981. The selective degradation of injected proteins occursprincipally in the cytosol rather than in lysosomes. Cell 25:83 112. Backer,J., Dice,J. 1986.Covalentlinkage of ribonuclease S-peptide to microinjected proteins causestheir intracellular degradation to be enhanced during serumwithdrawal.Proc. Natl. Acad. Sci. USA80:996 113. Hiles, I. D., Gallagher,M.P., Jamieson, D. J., Higgins, C. J. 1987. Molecular characterization of the Oligopeptide Permeaseof Salmonellatyphimurium.J. Mol. Biol. 195:125 114. Gotch, F., McMichael,A., Rothbard, J. 1988. Recognition of influenza A matrix protein by HLAA2 restricted cytotoxic T lymphocytes.Useof analogues to orientate the matrix peptide in the HLAA2binding site. J. Exp. Med.In press 115. Schwartz, R. H. 1985. T lymphocyte recognition of antigen in association with gene productsof the majorhistocompatibility complex. Annu. Rev. Immunol. 3:237 116. Pala, P., Bodmer,H., Pemberton,R., Cerrotini, J., Maryanski,J., Askonas, B. 1988. Competition between unrelated peptides recognised by H-2 Kd restricted T cells. J. Immunol.141:2289 117. Babbit, B., Allen, P., Matsueda,G., Haber, E., Unanue,E. 1985. Binding of immunogenic peptides to Ia histocompatibility molecules. Nature 317: 359 118. Buus,S., Sette, A., Colon,S. M., Jenis, D. M., Grey, H. M. 1986.Isolation and

characterization of antigen-Ia complexes involvedin T cell recognition. Cell 47:1071 119. Buus,S., Sette, A., Colon,S. M., Miles, C., Grey, H. M. 1987. The relation betweenmajor histocompatibility complex (MHC)restriction and the capacity of la to bind immunogenic peptides. Science 235:1353 120. Guillet, J., Lai, M., Briner,T. J., Buus, S., Sette, A., Grey, H. M., Smith, J. A., Gefter, M. L. 1987. Immunological self, nonself discrimination. Science 235:865 121. Watts, T, H., McConnell,H. M. 1986. Highaffinity fluorescentpeptide binding to I-Adin lipid membrances. Proc. Natl. Acad. Sci. USA83:9660 122 Guillet, J. G., Lai, M.Z., Binner, T. J., Smith, J. A., Gefter, M.L. 1986. Interaction of peptide antigens and Class II majorhistocompatibilitycomplex antigens. Nature324:260 123. McMichael,A., Gotch, F., SantosAguado,J., Strominger,J. 1988. The effect of mutations and variations of HLA-A2 on recognition of a virus peptide epitope by cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USAIn press 124. Bodmer,H., Bastin, J., Askonas,B., Townsend, A. 1988. Influenza specific cytotoxicT cell recognitionis inhibited by peptides unrelated in both sequence and MHC restriction. ImmunologyIn press 125. Lakey,E. K., Casten, L. A., Anderson, M. S., Smolenski,L. A., Smith,J. A., Margoliash,E., Pierce, S. K. 1987. T cell activation by processedantigen is equally blockedby I-E and I-A restricted immunodominant peptides. Eur. J. Immunol. 17:1605 126. Lakey,E. K., Margoliash,E., Pierce, S. K. 1987.Identification of a peptide binding protein that plays a role in antigenpresentation. Proc.Natl. Acad. Sci. USA84:1659 127. Carbone, F., Moore, M., Sheil, J., Bevan,M.1988. Inductionof cytotoxic T lymphocytes by primary in vitro stimulationwithpeptides. J. Exp. Med. 167:1767 128. Brett, S. J., Cease,K. B, Berzofsky,J. A. 1988. Influences of antigen processing on the expressionof the T cell repertoire. Evidencefor MHC-specific hinderingstructures on the productsof processing. J. Exp. Med.168:357 129. Rothbard, J., Taylor, W. 1987. A sequence pattern commonto T cell epitopes. EMBO J. 7:93 130. Rothbard, J., Lechler, R., Howland,

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K., Bal, V., Eckles, D., Sekaly, R., rilsky, P. 1987.Specificity of peptide Long, E., Taylor, W., Lamb,J. 1988. presentation by a set of hybrid mouse class I MHC molecules. Nature 330: Structural modelof HLA-DRI rcstricted T cell antigenrecognition.Cell 52: 660 515 132. Hogan,K. T., Shimojo, N., Walk,S. 130a. Allen, P., Matsueda,G. R., Evans, F., Engelhard, ¥. H., Maloy,W. L., Coligan, J. E., Biddison, W.E. 1988. R. J., Dunbar,J. B., Marshall,G. R., Unanue,E. R. 1987. Identification of Mutations of the or2 helix of HLA-A2 the T-cell andIa contact residues of a affect presentation but do not inhibit T-cell antigen epitope. Nature327:713 bindingof influenza matrixpeptide. J. Exp. Med. 168:725 130b.Sette, A,, Buus,S,, Colon,S., Smith, J. A., Miles, C., Grey, H. M. 1987. 133. Nathenson, S. G., Geliebtcr, J., Structural characteristics of an antigen Pfaffenbach, G. M., Zeff, R. A. 1986. requiredfor its interaction with Ia and Murinemajor histocompatibility comrecognition by T cells. Nature 328: plex class I mutants:Molecularanalysis and structure-function implications. 395 131. Maryanski,J., Abastrado,J. P., KouAnnu.Re~. lmtnunol. 4:471

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Ann.Rev. Immunol.1989. 7.’625-55 Copyright©1989by AnnualReviewsInc. All rights reserved

THE BIOLOGY OF CACHECTIN/TNF-A PRIMARY MEDIATOR OF THE HOST RESPONSE Bruce Beutler*

and Anthony Cerami~f

*The HowardHughes Medical Institute, The Department of Internal Medicine the University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050, and ~’The Rockefeller University, NewYork, New York 10021 INTRODUCTION Inflammation, the most ancient aspect of the host immuneresponse, is surely of great value to the host, insofar as agents that suppress inflammation in a nonspecific fashion predispose to infection. Yet, the inflammatory response to invasive organisms mayalso, if sufficiently intense or inappropriately prolonged, cause injury or death. A delicate balance has thus been achieved, one which clinicians strive to maintain through judicious application of anti-inflammatory medications (e.g. glucocorticoids, nonsteroidal anti-inflammatory agents, and cytotoxic drugs). Only recently have we come to understand that manyaspects of this primitive response to host invasion are governed by polypeptide hormones, in turn produced by immuneeffector cells. Moreover, we have come to appreciate the pleiot.ropic properties of these so-called "cytokines." It would appear that a limited number of cytokines are capable of orchestrating disease states that scarcely resemble one another; amongthem, endotoxic shock, graft-vs-host disease, cerebral malaria, and cancer cachexia. Cells of monocyte/macrophagelineage play a central role in cytokine ("monokine") production and so act to modulate many aspects of the inflammatory response. While devoid of specificity and immunologicmem625 0732-0582/89/0410-0625502.00

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ory, macrophagesprofoundly influence the host in the course of an infection. Perhaps for this reason, disparate etiologic agents mayhave very similar clinical consequences. Interleukin-1 (IL-1) -~ and -/~, and a second molecule widely known "cachectin" or "tumor necrosis factor-~" (TNF) are amongthe best studied of all cytokines. Cachectin/TNFis one of the most abundant products of activated macrophages, and the remainder of this review is devoted to a description of the moleculeas it is presently understood, to its discovery, and its manyeffects on host physiology.

FACTOR(S) RESPONSIBLEFOR SHOCK ARE HEMATOPOIETICALLYDERIVED Anaphylactic reactions, cell-mediated hypersensitivity, and complementmediated injury are frequently cited as evidence of the imperfect nature of the immuneresponse. In each case, an inciting stimulus triggers injury caused by an immuneeffector mechanism. Eight years ago, the primary role of lymphoreticular cells in the mediation of host responses to bacterial endotoxin (lipopolysaccharide--LPS) was clearly established (1). resistant mice were shown to be sensitized to the lethal effect of LPS following transplantation of marrow derived from LPSosensitive individuals; conversely, LPS-sensitive mice were rendered LPS resistant by irradiation and reconstitution with marrowderived from resistant donors. Thus, a cell of hematopoietic origin, or factors produced by this cell, appeared to confer sensitivity to LPS. Since "priming" stimuli (e.g. Bacillus-Calmette Guerin, Corynebacterium parvum, or Mycobacterium lepraemurium) capable of causing reticuloendothelial hyperplasia are knownto render animals highly sensitive to the effects of LPS(2-5), the macrophageand its progenitor were regarded as likely participants in the response to LPS. Moreover, endotoxinactivated macrophages were shown to produce, in vitro, a factor capable of killing endotoxin-resistant mice (6). Like the host response to LPS, the wasting diathesis so commonly observed in chronic infectious and neoplastic diseases had long been attributed to an endogenousmediator, produced by tumors, or by cells of the host. A more direct approach was required to demonstrate the existence of this agent. The isolation of cachectin and TNF, and the demonstration of their identity, led to widespread appreciation of the influence exerted by the immunesystem over the metabolic activities of manyhost tissues, and to the multifaceted response that may derive from the action of a single mediator.

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THE IDENTIFICATION CACHECTIN

AND ISOLATION

OF

Studies of cachectin originated in the mid-1970s, with the work of Cerami and Rouzer, who observed a marked hypertriglyceridemia in rabbits infected with T. brucei. This elevation in plasma triglyceride, which occurred despite the concomitant presence of anorexia and a profound wasting diathesis, was shownto result from a lipid clearing defect: a systemic deficiency of the enzymelipoprotein lipase (LPL)was present (7). Reasoning that aberrant lipid metabolism might reflect the action of immunefactors responsible for wasting, Cerami and co-workers devised a second model system to detect endogenousmediators capable of mediating LPLsuppression (8). Endotoxin-sensitive (C3H/HeN)mice, when challenged with LPS, were shownto exhibit a hypertriglyceridemic response similar to that observed in trypanosome-infectedrabbits. While endotoxinresistant (C3H/HeJ)animals did not exhibit this response following direct challenge with LPS, a hypertriglyceridemia caused by LPLsuppression could be elicited by infusion of post-endotoxin serum derived from the sensitive strain. Thus, a factor present in serum (and subsequently termed "cachectin") acted to suppress LPLexpression by fatty tissues throughout the recipient. Macrophages isolated from sensitive animals were shown to produce cachectin activity when stimulated with LPS in vitro (8). Cachectin was also capable of suppressing LPLexpression by cultured adipocytes (3T3Ll cells) (9). Cachectin was purified to homogeneity from cells of mouse macrophage line RAW264.7 and partially sequenced (10, l 1). Active at picomolar concentrations, cachectin was secreted in large quantities by macrophagesof this line, as well as by thioglycolate-elicited peritoneal macrophages.Indeed, it comprised 1-2%of the total secretory product of activated cells (10). Correspondingly, large quantities of the protein (milligrams per kilogram, body mass) were later found to produced in vivo after LPSchallenge (12, 13). However,cachectin was not produced by unstimulated cultures (10, 14). Active, radiolabelled preparations of the hormonewere used to detect and characterize a high-affinity receptor for cachectin, which was found to be present on cells of diverse lineage (10). Wheninjected into mice an intravenous route (12), cachectin was cleared from the blood with half-life of 6-7 minutes. Clearance was effected by binding to the tissue receptor. The N-terminal sequence of cachectin was soon found to be strongly homologous to the sequence determined for humantumor necrosis factor (TNF) (11, 15, 16). TNF,isolated by a very different approach, was

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sequently shownto be identical to cachectin by serologic and molecular cloning studies, as well as by direct comparisonof biological activities (11, 16, 17).

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THE HISTORY OF TUMORNECROSIS FACTOR (TNF) While many tumors appear to provoke an inflammatory response, the capacity of the immunesystem to exercise "immunesurveillance" has been challenged by the observation that profound immunodeficienciesof genetic origin are not associated with marked enhancement of tumor formation. However,it is clear that, under certain circumstances, immunemechanisms may dramatically hinder tumor progression. One of the most dramatic examples of immune-mediated tumor regression is the hemorrhagic necrosis of tumors observed in humans or animals in the course of an intercurrent bacterial infection, particularly one involving one-gramnegative organisms. William Coley, impressed by the hemorrhagic necrosis of certain tumors observed in patients with streptococcal (erysipelas) infections, attempted to reproduce this phenomenonby injection of live or killed bacterial organisms and of filtrates derived from their culture (18, 19). As result of the toxicity inherent in administration of bacterial products to human cancer patients, this chemotherapeutic approach was eventually discredited. However, interest remained in the phenomenonof bacterially induced tumor necrosis, and other workers attempted to isolate the bacterial product responsible for it, in the hope that the active factor might be separated from the toxic materials present in crude preparations. Shear and his colleagues succeeded in purifying a factor derived from gram-negative organisms which they termed the "bacterial polysaccharide;" this substance, nowknownas lipopolysaccharide, was very effective at inducing tumor necrosis (20-25). However, it was also amongthe most toxic constituents of the culture from which it was derived. Parallel studies (26) indicated that hemorrhagic necrosis might reflect generalized vascular collapse and that it was histologically similar to changes observed in tumors infarcted by ischemia. The work of Coley & Shear, motivated by the wish to develop antineoplastic agents with low inherent toxicity, thus appeared to have reached an impasse. Later work (27) rekindled interest in the effects of LPS,since it became evident that hemorrhagic necrosis might be attributable to the production of an endogenous mediator released in response to LPS, rather than to LPSitself. Shockserum derived from endotoxin-treated mice was shownto

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induce hemorrhagic necrosis of tumors implanted in the skin of untreated recipients. The factor responsible was not endotoxin itself, but a heatlabile material, presumedto be a protein. This observation might have been forgotten, were it not for the fact that a similar experiment (28) revealed much the same phenomenon; serum derived from mice treated with Bacillus Calmette-Guerin (BCG)prior to injection of endotoxin was shown to contain a necrotizing factor capable of acting upon tumors implanted in the skin of recipient mice. The same factor appeared to have a cytolytic effect on several transformed cell lines in vitro (28-31). Dubbed "tumor necrosis factor", the LPS-induced mediator became the object of enormousbasic and clinical interest. TNFwas purified to homogeneity, partially sequenced (l 5), and cloned (16, 32, 33) by several groups of investigators.

CACHECTIN/TNF STRUCTURE: HOMOLOGYTO LYMPHOTOXIN Cachectin/TNF has a subunit size of 17 kd. In humans, the molecule is nonglycosylated; in certain other species (notably the mouse), glycosylation occurs on a single N-linked site in the mature protein, but the sugar moietyis not essential for biological activity. Each monomer consists largely of beta-pleated sheet structure (34), and three monomerscombine noncovalently to form the active hormone (35, 36). Cachectin/TNF may be exposed to chaotropic agents such as urea, SDS, or guanidinium hydrochloride, and renatured with recovery of as muchas 50%of the initial biological activity. This renaturability perhaps reflects the limited number of internal disulfide bonds (one per monomer)required for maintenance of structure. Cachectin/TNFis initially synthesized as a prohormonecontaining an amino-terminal peptide of varying length, depending upon species. The propeptide segment of the molecule is highly conserved. Approximately 86%of the 79 amino acids preceding the mature hormone in the mouse are identical to the 76 amino acids preceding the mature hormone in humans. By contrast, the mature hormone (156 amino acids in mouse and 157 aminoacids in humans)is conserved to the extent of 79%(17, 37, 38). The function of the propeptide remains unclear. Recent data suggest that it may be essential for the secretion of cachectin/TNF, which may occur through cleavage of the molecule at the cell surface (39). While the existence of a "membraneassociated" form of cachectin/TNF has been reported (39, 40), the physiologic significance of this molecule remains be determined. It would appear that the prohormone is biologically inactive, and its ability to form trimers has yet to be established.

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It is also possible that the propeptide mayhave biological activities of its own, distinct from those of the mature polypeptide. The exact cleavage site(s) recognized in the generation of the mature cachectin/TNFmolecule have not been determined; however, proteolysis occurs at a minimumof one internal site, approximately 10 amino acids proximal to the beginning of the mature hormone(B. Beutler, unpublished data). Several regions of the cachectin/TNF molecule are highly conserved amongspecies. One such region spans amino acids 115 through 130; a second area that is generally conserved is near the C-terminus. Indeed, it has been reported that deletion of the C-terminal aminoacid (an invariant leucine) from cachectin/TNFablates biological activity. These regions are also conserved in lymphotoxin (41), a second polypeptide hormonederived from a closely linked gene, which displays approximately 30%homology to cachcctin/TNF at the amino acid level. The cachectin/TNF and lymphotoxin genes are separated by only about 1100 base pairs. Lymphotoxinlies 5’ to cachectin/TNF, and both genes reside within the major histocompatability complex (chromosome6 in humans, and chromosome 17 in the mouse) (42-45). Within the complex of the mouse, cachectin/TNF and lymphotoxin are located approximately 70 kilobases proximal to the D-locus (46). Despite its relatively limited sequence homology to cachectin/TNF, lymphotoxinexerts a range of biological activities that are, both in vivo and in vitro, highly concordant with those of cachectin/TNF. Initially identified as a presumptive mediator of cell-mediated hypersensitivity (4750), lymphotoxin was shown to cause hemorrhagic necrosis of tumors in vivo, and to bring about the lysis of certain transformed cell lines. The two hormones share a commoncell surface receptor, binding to it with comparable affinity (51). However, the tissue of origin and of stimuli required for elaboration of these proteins are quite diffcrent. Crystals of both cachectin/TNF and lymphotoxin have been produced, and preliminary structural data from work performed on the former have recently been published (36, 52). It is reasonable to expect that highresolution models of both molecules will be forthcoming. PRODUCTION LYMPHOTOXIN: AND KINETICS

OF CACHECTIN AND SOURCES, STIMULI,

Once it was believed that cachectin/TNF was strictly a product of mononuclear phagocytic cells, and that lymphotoxin was derived only from lymphocytes. This initial picture has been somewhatcomplicated by the

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observation that lymphocytes are also capable of producing cachectin/TNFwhenexposedto such stimuli as phorbol ester, in conjunction with a calcium ionophore (e.g. A23187)(53). However,no reports lymphotoxinproduction by macrophageshave been made. Cachectin/TNF is also reportedly produced(in very small quantities) by natural killer (NK)cells (54, 55) and appears to comprisethe "colony inhibiting activity" of these cells. Transformed (L-929fibrosarcoma)cells, rendered resistant to lymphotoxinby continuousexposureto the hormone, are said to producesmall amountsof cachectin/TNFconstitutively (56), and certain tumors derived from humansources are also reported to be capable of spontaneous TNFproduction (57). REGULATION EXPRESSION

OF CACHECTIN/TNF

GENE

As previously mentioned,cachectin/TNFis one of the major products of activated macrophages.So far as is known,it is not producedby macrophages under normalcircumstancesand does not exist in a stored form. It is synthesizedde novofollowingactivation and is efficiently exported fromthe cell. Indeed, as described below, its productionand release may have deleterious effects uponthe organism,and therefore, its synthesis must be tightly governed. Cachectin/TNF biosynthesisis controlled at multiple levels (58-61). response to LPS, cachectin/TNFgene transcription (which is detectable in resting macrophages) is accelerated approximatelythree-fold; however, cachectin/TNFmRNA levels rise within the cell by a factor of 50 to 100, and cachectin/TNFprotein secretion, whichis undetectable in quiescent cultures, rises by a factor of about 10,000. Under some circumstances, cachectin/TNF mRNA may be detected within cells in the absence of hormoneproduction or release. This is most readily observed in macrophagesderived from C3H/HeJ(endotoxin unresponsive) mice, or in dexamethasone-treatedmacrophagesthat are subsequentlystimulated with LPS.To a large extent, therefore, cachectin/TNFbiosynthesisappearsto be controlled at a translational level. Theposttranscriptional control ofcachectin/TNFgeneexpression seems to dependpartly uponsequences that reside within the 3’-untranslated segmentof the molecule.Caputet al (17) noted that the 3’-untranslated region of cachectin/TNFmRNA contains a long U + A-exclusive region, bearing overlapping and repeating octameric elements (UUAUUUAU). This sequence was found to be conserved in toto between humanand murine forms of the cDNA.Moreover, many inflammatory cytokine

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mRNAs, as well as mRNAs encoding certain proto-oncogenes, were found to havesimilar 3’-untranslated sequences. It wassoon determinedthat one such sequence,derived fromthe human GM-CSF gene, wheninserted into a rabbit/%globingene in the 3’-untranslated region, wouldcause markedinstability of the modifiedmRNA (62). Subsequently,the existence of a selective nucleolytic activity capableof hydrolyzing mRNA bearing multiple copies of the cachectin/TNFUUAUUUAU sequence was demonstrated (63). Recently, it has becomeclear that the instability of mRNA molecules bearing UUAUUUAU-rich sequences is a specific example of a more general principal. It was noted that the dinucleotide UpAis unusually susceptible to hydrolysis by cytoplasmicribonucleases whenpresent in a single-stranded form (64). The markedsusceptibility of UpAto ribonucleases has led to a scarcity of UpAin most mRNA molecules, save those which, like cachectin/TNF mRNA, conserve the dinucleotide to encourageinstability (64). The propensity for strand breakage at UpA may, indeed, have been responsible for the adoption of UAAand UAG as the two universal "stop" codons,for whichno anticodonexists, since in primordial biosystems, UpAmayhave been entirely absent from RNA (64). In addition to its ability to confer instability, the UUAUUUAU-rich sequence of GM-CSF acted to confer superinducibility (62). Moreover, the UUAUUUAU-rich sequence ofinterferon-fl whenlinked to a reporter genesuppressestranslation in Xenopusoocytes andin reticulocyte lysates, but not in wheatgermtranslation systems(65, 66). Suppressionof translation, in these instances, doesnot appearrelated to instability. Theseobservationswouldsuggestthat specific proteins withinthe ceil, in additionto ribonuclease, must interact with such UA-richsequences, perhaps altering the access of the mRNA to translation. Further work is required to characterizesuch factors and to understandhowtheir activity is governed. Recently, certain endogenousfactors have been shownto influence the production and action of cachectin/TNF.Among these, the effect of glucocorticoid hormoneson cachectin/TNF mRNA translation, and on cachectin/TNF genetranscription, bears special mention,since this action of corticosteroids maylargely explain their markedprotective effect against lipopolysaccharide toxicity. Dexamethasone has been shownto diminish the rate of cachectin/TNFgene transcription, and to exert an evenmoremarkedeffect on hormonebiosynthesis at a post transcriptional level: in the presence of dcxamethasone, cachectin/TNF mRNA is not effectively translated. However,glucocorticoidsare only capableof inhibiting cachectin/TNFbiosynthesis if administered prior to LPS;their effect is a preemptiveone (58). If administeredfollowingLPSchallenge,

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cachectin/TNFbiosynthesis proceeds unimpeded.This fact mayexplain the limited utility of glucocorticoidsin the treatmentof septic shockas it occursin a clinical setting.

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RECEPTOR

AND POSTRECEPTOR

EFFECTS

Oncesecreted, cachectin/TNFinteracts with a plasmamembrane receptor that is widelyrepresented on mammalian cells and tissues (12, 67). As direct result of hormone-receptorinteraction, one or morebiological effects maybe triggered within the target tissue. Approximately one-third of transformedcell lines surveyed(67) werekilled or growth-inhibited cachectin/TNF.Onthe other hand, certain fibroblast lines (67, 68) are actually growth-stimulated by cachectin/TNF. Someof the effects of cachectin/TNFare manifested rapidly (69, 70), whereasmost take hours to develop. The nature of the cachectin/TNFreceptor, from whichall of these cellular effects emanate,remainsquite poorly understood,as does the nature of the signal evokeduponbinding of the hormone. Recentstudies suggest that the intact cachectin/TNF receptor is a protein with a molecularweightof approximately300 kd (71), possibly consisting of dissimilar subunits(72). Thebindingsubunitof the receptor, established by cross-linking studies, has a molecularsize of approximately 75 kd (72-76). A glycosyl componentis suggested by experiments in which certain lectins have beenshownto prevent the biological action of cachectin/TNF on its target cells, without impedinghormonebinding (77). Moreover, dissociation betweenbinding and exertion of biological effect has been suggestedby studies in whichcachectin/TNFderived from different mammalian species has been shownto bind to the receptor found on tissues fromoneof the specieswith similar affinity, yet trigger an effect (cytolysis) of different magnitude(71). Whencachectin/TNFbinds to its receptor, it does so with an affinity constant of approximately 3 x 109 (10). Variable numbersof cachectin/TNFreceptors are observedon different tissues; however,receptor numberandaffinity do not correlate with cytotoxic effect. Considerableeffort has been devotedto the elucidation of the postreceptor mechanismof action of cachectin/TNF. Sometime ago, it was noted that lymphotoxin-mediated cell injury was associated with fragmentation of genomicDNA,occurring well before cell lysis (78, 79). This finding promptedspeculation that DNA fragmentationmightplay an essential role in the destructionof target tumorceils. Morerecently, it was shownthat agents inhibiting ADP-ribosyltransferase activity are capable of blockingthe cytotoxic effect of cachectin/TNF,at least in sometarget

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cells (80). This wouldsuggest that ADP-ribosylation, induced by DNA fragmentation,mightendin destruction of the cell. However, the proximal messageleading to these events remains unknown.

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ACTIONS OF CACHECTIN TISSUE TARGETS

AND LYMPHOTOXIN:

Theability of cachectin/TNFand lymphotoxinto modulatecellular function, both in vivo and in vitro, has beenintensively studied. Theindependent action of these hormoneshas been examined, as well as their behaviorin concert with related proinflammatorypolypeptides, including interferon-gamma,interleukin-l, and interleukin-2. Manydifferentiated cell lines havebeenstudied, as havemanyphysiologicparameters. Specific examplesto the contrary notwithstanding(81), it maygenerally be stated that the principal actions of cachectin/TNFand lymphotoxin are catabolic and inflammatory;in manyinstances, the effects of the hormoneon isolated tissues and cells reflect the responsesof the entire organism. Below,we summarizethe biological actions of cachectin/TNF as established in a numberof experimentalsystemsdealing with specific cell andtissue types. Thephysiologicresponseto cachectin/TNF,observed in living animals following administration of the hormone,are covered subsequently. Vascular Endothelium Theshock-promotingeffects of cachectin/TNF,and its ability to induce hemorrhagicnecrosis of certain tumorsin vivo, appear to dependlargely uponits vascular effects. Cachectin/TNF downregulatesendothelial cell expression of thrombomodulin and causes the elaboration of a procoagulant activity (82) as well as the release of IL-1 fromendothelial cells vitro (83, 84). IL-1, in turn, exerts effects on endothelialcells substantially similar to those of cachectin/TNF itself (85-91). Cachectin/TNF also prompts the adhesion of neutrophils to vascular endotheliumthrough effects on eachof the cellular participants (69, 85, 92). Theinitial phase of neutropenia observed following LPSchallenge, and the margination and transudation of neutrophils that occur during local inflammatory processes, maythus dependuponcachectin/TNF.Among its other effects on vascular endothelial cells, cachectin/TNF is growthinhibitory (93, 94) and causes morphologicchanges(93, 95) including the reorganization vascular endothelial monolayers(95) such that the cells flatten, overlap, rearrangeactin filaments, and lose stainable fibronectin whentreated with the hormone.Synergy between interferon-gamma and cachectin/TNF in causing these changes has been noted (95). Cachectin/TNF modulatesthe

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expression of various surface antigens on endothelial cells (85, 86, 96, 97). It induces endothelial phospholipase A2 activity (98). Tissue-type plasminogen activator is suppressed, and type-1 plasminogen activator inhibitor is induced in endothelial cells following exposure to cachectin/TNF (90). Cachectin/TNF and IL-1 have both been reported to sensitize vascular endothelial cells to lysis by circulating antibodies obtained from patients with Kawasaki syndrome. Conceivably, this might predispose to the vascular complications associated with this disease (88). Hematopoietic

Elements

NEU~ROPHILS The importance ofneutrophils in the pathogenesis ofendotoxic shock is widely accepted (99, 100), and considerable significance may be attached to the fact that cachectin/TNFappears to be a major mediator of neutrophil adhesion and activation. Cachectin/TNFprompts neutrophil adhesion by a direct effect (69, 101,102) that is maximalwithin 5 minutes of contact with the hormone and does not appear to require protein synthesis. In vivo, this may lead to their margination and transudation from the intravascular space, and thus, to tissue injury. In addition to enhancing neutrophil adhesion to endothelial surfaces, cachectin/TNF stimulates enhanced phagocytosis of latex beads (101), enhanced production of superoxide anion (103), release of lysozyme and hydrogen peroxide (102), and degranulation (102). Neutrophils also enhanced microbicidal activity when stimulated by cachectin/TNF (104). Cachectin/TNF has recently been shown to modulate the neutrophil response to F-met-leu-phe receptor activation (105, 106). In addition, cachectin/TNF increases the expression of complementreceptor on human neutrophils and maythereby contribute to localization of these cells within an inflammatory focus (107). In the opinion of some, cachectin/TNF triggers the emigration of neutrophils into skin, perhaps by a direct chemotactic effect (108, 109). The chemotactic activity of cachectin/ TNFhas been disputed by other workers (110), who nonetheless note that cachectin/TNF appears to inhibit neutrophil migration. EOSINO~’HILS Cachectin/TNF has been shown to enhance the toxicity of eosinophils to schistosomula in vitro (111); it wouldappear that cachectin/TNF is the principal factor produced by U-937 cells responsible for this bioactivity (112). Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)also enhances the cytotoxicity of eosinophils for schistosomula (113); the mechanismby which the cytotoxic response is augmentedremains to be determined, as does the significance of the phenomenon in vivo. However, these observations support the

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concept that cachectin/TNFmayhavea beneficial role to play in chronic parasitic infections (114-116).

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MONOCYTE/MACROPHAGESA large

number of investigators have coneluded that the macrophage,in one or more of its manyforms, is the principal source of cachectin/TNF(61, 117-132)producedin response manytypes of invasive stimuli, LPSbeing the most potent of these. Less work, to date, has concerned the effects of cachectin/TNF on monocyte/macrophagesthemselves, although it is clear that these cells bear receptors for cachectin/TNF(133), and internalize cachectin/TNFonce is boundto them. Cachectin/TNF has been reported to exert a chemotactic effect on monocytes,just as it does on neutrophils (108). Macrophage activation appears to cause a markedenhancementof LPLsecretion by macrophages;however, this enhancementdoes not appear to be mediated by cachectin/TNF(134). Cachectin/TNFaugmentsthe production of GMCSFby a variety of cell types and thus mayaffect macrophagefunction indirectly (135). Cachectin/TNF also inducesthe differentiation of certain myeloidcell lines in vitro (136)andhas beenshownto stimulatethe release of IL-1 and PGE2production from resting macrophages(137). Like eosinophils, macrophagesare capable of killing schistosomula whenactivated by cachectin/TNFor lymphotoxin, and macrophagesare synergistically activated to achievethis effect by coincubationwith interferon-gamma (138). Cachectin/TNF, alone or in combinationwith IL-2, has been shownto activate macrophages,allowing themto kill Mycobacterium aviurnorganisms(139). Todate, no clear data haveemergedto support the conceptthat cachectin/TNF is capable of inducing cachectin/TNFbiosynthesis by primary macrophages.The function of cachectin/TNFas it concerns macrophages themselvesis still rather poorly understood, and muchadditional work remainsto be donebefore this issue can be fully resolved. LYMPHOCYTnS Overa decade ago, the ability of post-endotoxin serumto promoteantigen-directed B-cell activation wasreported and presumptively ascribed to the action of tumornecrosis factor (140, 141). In addition, crude preparations of cachectin/TNFwere shownto direct the maturation of thymocytesin vitro (142). Similar crude preparations of cachectin/TNF were also found to be cytotoxic to humanlymphocytes (143) as was lymphotoxin(144, 145). Morerecently, the effects of cachectin/TNFon T and B lymphocytes havebeen studied in greater detail. Cachectin/TNF induces the expression of additional cachectin/TNFreceptors on primary cultures of T lymphocytes (146) and also increases the expression of HLA-DR antigen and high affinity IL-2 receptor. Thus, cachectin/TNF-treatedT cells showan

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B~OLOGV OF CACnEc’rr~/a~rF 637 enhancedproliferative response to IL-2. Cachectin/TNFalso enhances IL-2-dependent production of interferon-gamma. Other workers (147) havesuggested that lymphocyte-fibroblastadhesionis inducedby interferon-gamma and cachectin/TNF.Onthe other hand, B-cell proliferation anddifferentiation, occurringin responseto the B-cell activator pokeweed mitogen, are inhibited by cachectin/TNF048). Finally, cachectin/TNF, lymphotoxin,and various interferons have been shownto enhancethe T cell-mediatedresponseto antigenic challengethrougha direct effect on T cells 049). Thesignificanceof these observations,whichderivefromin vitro studies, remainsto be demonstratedat the level of the organism.However,it would appear likely that cachectin/TNFis an importantmodulatorof B-cell and T-cell function and that it mayact as an autocrine factor capable of influencingthe progressionof certain hematopoieticmalignancies(57). Very recent work has implicated cachectin/TNFand lymphotoxinas essential participants in mixedlymphocyte reactions (150). Thesedata, well as other studies pointing to the involvementof cachectin/TNFas a reactant in the early phase of graft-vs-host disease (151), suggest that the hormonedoes indeed affect T-cell function, with importantsystemic consequences. .Adipose Tissue Cachectin/TNF,by the namecachectin, was first identified as a hormone capable of modulatingthe metabolicactivities of adipocytes (7-11, 14, 152-154).It wasnoted to cause suppressionof lipoprotein lipase, acetyl CoAcarboxylase, and fatty acid synthetase whenapplied as a crude factor, andto activate the release of glycerolfromdifferentiatedfat cells, presumablyindicating an effect on the hormone-sensitivelipase (155). Subsequently, workwith purified recombinantcachectin/TNFconfirmed that the hormonedid, indeed, mediate most of these changes(156-160). Cachectin/TNFwas also shownto inhibit the morphological differentiation of adipocytes(154) and, whenchronically administeredto these cells, to inducemorphologicalde-differentiation. It maybe supposedthat these effects on lipid metabolismcould serve an adaptive function in an acute, self-limitedillness, by favoringthe mobilizationof energystores for use in the immuneresponse. Muscle Reflectingits role as a presumptivemediatorof wastingin chronicdisease, cachectin/TNFwasregarded as a potential mediatorof protein catabolism in musclecells, and as a mediatorof the diminishedtransmembrane potential frequently observedin shock. Indeed, cachectin/TNFhas beenshown

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to lowertransmembrane potential in isolated skeletal musclepreparations (70). Whileit mayalso be involved in the breakdown of skeletal muscle protein, no direct effect of this type has beendemonstrated(161, 162). Recently, it was shownthat cachectin/TNFmodulates LPLin cardiac muscle(163, 164). At high concentrations, cachectin/TNFhas also been foundto stimulate glucose uptake by L-6 musclecells in vitro; the significance of this "insulin-like" effect (165)remainsto be determined.

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Liver and Gastrointestinal

Tract

Onlyrecently, the toxic effects of cachectin/TNF on hepatocyteshavebeen examined. However,for sometime, it has been apparent that galactosaminehepatotoxicity is a function of lymphoreticular cells in mice (166). Thus, a factor of hematopoietic origin must ultimately mediate the necrotizing effect of this compound.Direct measurements (167) have revealed that recombinant cachectin/TNF depresses cytochromeP450dependent drug metabolism in mice. Manyaspects of the acute-phase response--including diminishedproduction of albuminand enhancedproductionof fibrinogen, ~l-acid glycoprotein, and cysteine protease inhibitor-seem, at least in part, to involve action of cachectin/TNFand IL-1, acting in concert (168-171).Total hepatic protein synthesis is suppressed by cachectin/TNF(172), and in rats, chronic administration of cachectin/TNFhas beenshownto cause liver injury that is discernableat a lightmicroscopiclevel (173). Theliver is the first reticuloendothelial organ to be exposedto blood draining enteric structures, and its burdenof endotoxin;the capacity of the liver to producecachectin/TNF,and its function as a locally active hormone,remainto be fully determined(40). Approximately10%of an injected dose of cachectin/TNFlocalizes in the gastrointestinal tract (12). Systemicadministration of cachectin/TNF to experimentalanimalsleads to bowelnecrosis, concentratedin the region of the cecum(174, 175). This effect of the hormone maytrigger the release of additional LPSfrom the bowel, leading to "irreversible endotoxin shock"(176). It is believed that the necrotizing effect of cachectin/TNF maybe mediatedby platelet activating factor (177), whichthe hormone knownto induce. Ultimately, the cause of necrosis maybe vascular, as witnessedin the case of susceptible tumors. Cachectin/TNFalso appears to delay gastric emptying(175) and may, in part, produceanorexiathroughits effects on gastrointestinal structures. Central Nervous System Cachectin/TNFcrosses the blood-brain barrier in vanishingly small amounts(12). However,it is knownthat central productionof the hormone

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may occur 078, 179). High levels of cachectin/TNF have been measured in the cerebrospinalfluid of patients with bacterial, but not viral, meningitis (179), and hormoneconcentrations mayprove to be of prognostic import-

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ance.

Acting within the central nervous system, cachectin/TNF is capable of inducing a pyrogenic response (162, 180), apparently through a direct effect on hypothalamic neurons (which produce enhanced quantities of PGE-2 in response to cachectin/TNF) and through induction of IL-1 release. Cachectin/TNFalso causes anorexia, which may, in part, have a central basis (172, 181-183). Cachectin/TNFhas also been shownto stimulate thermogenesisby a central effect (184). Recently, it has been reported that ~ melanocyte-stimulating hormone is capable of blocking some of the central effects of both cachectin/TNF and IL-1, including the thermoregulatory effect (185). In the wake reports that IL-1 stimulates adrenocorticotropic hormonesecretion in a negative feedback loop (186), related observations have been made with cachectin/TNF (187), which also appears to induce the secretion of prolactin (187). Considerable thought has been addressed to the proposal that cachectin/TNF, or the related hormonelymphotoxin, may be involved in chronic inflammatory central nervous disorders, such as multiple sclerosis. In experimental allergic encephalomyelitis, T lymphocyte clones capable of producing lymphotoxin also appear to be capable of causing the most severe neurologic lesions (M. Broome-Powell, personal communication). Further work is required to establish a cause-and-effect relationship, and to understand howglial injury is actually mediated. Adrenal Cachectin/TNF has been noted, in vivo, to cause adrenal hemorrhage (174). It has also been noted that adrenalectomy sensitizes mice to the lethal effect of both IL-1 and cachectin/TNF (188). Since the inhibitory effect of glucocorticoid hormoneson production of cachectin/TNF is well established (58), it wouldseem likely that tonic steroid production may exert a physiologic restraint both on the production of cachectin/TNF, and on its action in vivo. Cortisol levels are markedlyincreased following infusion of cachectin/TNFin toxic doses (189); this mayreflect an indirect responseto stress or a direct effect of the hormoneeither at a central level, or uponcells of the adrenal gland itself. Skin Approximately 30% of an intravenously administered dose of cachectin/TNF may be recovered from skin (12). Interestingly, the phenomenon

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of hemorrhagicnecrosis is also principally observed in tumors with a dermalblood supply. Thedermalvasculature, whichin any case is subject to a high degree of autoregulation, maythus be quite responsive to the effects of cachectin/TNF.Both cachectin/TNFand lymphotoxinhave been shownto induce dermal inflammation(109, 190-192), and it is believed that cachectin/TNF,together with IL-1, maycontribute to the localized Shwartzmanphenomenon. Humandermal fibroblasts (193) have been shownto secrete collagenase and PGE-2in response to cachectin/TNF. Other fibroblast-derived cells (FS4 cells) are knownto undergoa proliferative response followingcontact with the hormone. Macrophagesare stimulated to produce cachectin/TNFby contact with advancedglycosylation endproductsthat characteristically accumulatein mesenchymal tissues (194). Thus, certain chronic inflammatoryand regenative processes responsible for the remodelling of normaltissues may dependuponthe action of the hormone. Bone and Cartilage Avariety of inflammatory joint diseases mightpossiblyoccur as the result of elaboration of cachectin/TNF,IL-1, or related hormones.Cachectin/ TNFhas been shownto stimulate the resorption of proteoglycan in cartilage (195) and to stimulate bone resorption (196) in vitro. Proteoglycanand bone synthesis are also inhibited in the respective assay systems. Cachectin/TNFand lymphotoxinappear to synergize with IL-1 in their ability to promptboneresorption (197), althoughthe latter hormone, acting in isolation, is a far morepotent activator of osteoclasts than either of the former. Osteoclast-like cells of the line C3-E1(198) are stimulatedby cachectin/TNF to produce macrophagecolony stimulating factor (M-CSF)and PGE-2.Therole played by these secondarymediators in bone resorption remains to be fully quantitated. Transforminggrowth factor-r, which antagonizesthe effects of cachectin/TNFin manyother systems, also has the ability to stimulate bone resorption and PGE-2productionin cultured mousecalvarium (199). Theclinical significance of lymphotoxinas an osteoclast activator has best been demonstratedby the studies of Mundyand his colleagues (200) whohave shownthat humanmyelomacells mayproduce lymphotoxinin an autonomousmanner, and that monoclonalantibody directed against lymphotoxininhibits the bone-resorbing potential of myelomacells in vitro. Thus, at least someof the destructive effects of myeloma maybe directly attributable to the elaboration of lymphotoxin..

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ACTIONS OF CACHECTIN/TNF LYMPHOTOXIN: PHYSIOLOGY

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AND

The importance of cachectin/TNF as a mediator of shock was suggested long before this monokinewas purified or cloned (118, 201-204). At the same time, there existed a strong suspicion that the same hormone must confer a protective effect (114, 115, 125, 205-209). Suchspeculations could be subjected to test only after purification of the hormonewas achieved. The identity of cachectin and TNF(l l) underscored the possibility that single polypeptide hormonemight fulfil both beneficial and deleterious functions and might mediate many, if not all, of the biological effects of lipopolysaccharide. Passive immunizationstudies (210) revealed that the secretion of cachectin/TNF was, indeed, an important element in the pathogenesis of endotoxicity. Subsequentwork, (21 l, 212), in which monoclonalreagents were employedand other species examined, has supported this view. Cacbectin/TNF has been shown to produce a shock syndrome when administered in pure form to rats and dogs (174, 189). The hormone induces hypotension, tachypnea, metabolic acidosis, an initial phase of hyperglycemia followed by hypoglycemia, hemoconcentration, and multiple end-organ damagevery similar to that seen in endotoxin-poisoned animals. Such changes, witnessed following acute administration of cachectin/TNF, were later reported by other workers as well (175, 213-216). Lethality resulting from bolus administration of cachectin/TNF was, in the short run, usually attributable to respiratory arrest, which occurred as a result of acute interstitial pneumonitis(174). Whensmaller doses of the hormonewere administered, cecal necrosis was often observed, occasionally with perforation of the gastrointestinal tract, leading to acute intraabdominal infection. Also supporting the notion that cachectin/TNF is an important mediator of endotoxicity is the finding that agents capable of sensitizing animals to the lethal effect of LPS (e.g. galactosamine and actinomycin) also appear to sensitize to cachectin/TNF (214, 217); moreover, adrenalectomy, which greatly diminishes the mean lethal dose of LPS in many species, also predisposes to the toxicity of cachectin/TNF(188). Interestingly, chronic administration of the hormonein smaller doses led to a syndromeentirely distinct from that observed following injection of large quantities of cachectin/TNF. Oliff and coworkers inoculated nude mice with neoplastic (Chinese hamster ovary) cells that had been genetically modified, causing them to secrete cachectin/TNF(218) continuously.

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The resulting tumors in these animals were small and nonmetastatic; however,the chronic elaboration of cachectin/TNFwasassociated with a profoundwasting diathesis, while control tumors (bearing an emptyvector) causedno weightloss. This finding strongly supportedthe original supposition that chronic exposureto the hormonemight lead to cachexia (6, 10). Thepathogenesisof wasting wasnot entirely clarified in these studies; however,it appearedthat anorexiawaslargely responsiblefor the weightloss observed. Continuousinfusion of cachectin/TNFhas also been shownto cause hepatictoxicity (173), to suppressfoodintake andhepaticprotein synthesis (171,172, 181,183),and to cause anemia(219) in animals. Someeffects cachectin/TNFappear to be mediatedcentrally, as noted above. Appetite suppression maybe amongthese. In addition, cachectin/TNFhas well defined effects on thermogenesis(184, 220) that are both central and peripheral, and that maycontribute to the dissipation of energyobserved duringacute and chronic infectious disorders. CACHECTIN/TNF

IN DISEASE

Since it was demonstrated that cachectin/TNFcould play an important role in the pathogenesisof endotoxicshock (174, 210) and cachexia(218), a numberof attempts have been madeto measure the hormonein biological fluids derived from humanand animal subjects suffering from a variety of infectious and neoplastic diseases. Conflicting results have arisen in these studies, in large part becauseof the noveltyof the available assays. Cachectin/TNFwas demonstratedin serum derived from patients with proven meningococcal septicemia, and high levels correlated with a negative outcome(221). Induction of cachectin/TNFalso occurred mouselung tissue during experimental infection with Legionella pneumophila(222); however,these authors concluded(223) that the hormone exerts a protective effect in this disease. Cachectin/TNF wasmeasuredin a variety of parasitic infections (224) in humans;however,in this study, high percentage of normal controls also displayed elevated hormone concentrations in their serum. Humanpatients with acquired immunodeficiency syndrome(225) also display elevated serumconcentrations of cachectin/TNF.Whilelevels are markedlycorrelated with the stage of the disease, it remainsto be determinedwhetherthe inducing stimulus is the humanimmunodeficiency virus itself or the infectious complications that characterize AIDS.It has beensuggestedthat cerebral malaria might depend, in large part, uponthe elaboration of cachectin/TNF(202, 203). Recentexperimentalwork(226, 227) has supported the view that cachectin/TNF is an essential mediator of murine cerebral malaria. Separate

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studies (151) indicate that cachectin/TNFis involved in the mediation graft-vs-host disease in mice. The potent abortifacient effect of LPScaused some suspicion that cachectin/TNF might cause premature delivery, such as that witnessed during intrauterine infection. Recent work suggests that macrophage-like cells of the placental decidua are capable of producing abundant quantities of cachectin/TNF, and that cachectin/TNF inhibits the growthof cells of the amnion, possibly leading to rupture of the fetal membranes(228). Indeed, high levels of cachectin/TNF can be detected amniotic fluid derived from patients with intrauterine infections (L. Casey, personal communication). Cachectin/TNF has been assayed in serum derived from a large number of humancancer patients and detected in a substantial number of those cases (229). Conflicting data have been reported by a second laboratory (230), in which cachectin/TNFwas absent from the serum of 19 individuals with cachexia related to cancer. It remains to be fully established whether the hormoneactually participates in most cases of unexplained wasting, and if so, whether it is produced intermittently or whether undetectable hormone concentrations are adequate to cause wasting if chronically present.

CLINICAL USES OF CACHECTIN/TNF: THE FUTURE Successful treatment of disease consists, in large part, in the restoration of homeostasis. To the endocrinologist, this implies the judicious administration or antagonism of specific hormones. For example, an insulindependent diabetic maybe effectively treated by insulin replacement therapy; a patient with Graves’ disease may be helped by pharmacological measuresthat inhibit thyroxine release. Onlyrarely can disease be alleviated by administration of hormones that were never deficient to begin with. The use ofcytokines as antineoplastic agents provides one such instance. The rationale for administration of immunomodulatoryagents to cancer patients is quite slender. Manycytokines, like cachectin/TNF, .exhibit toxicities that prove dose-limiting long before a therapeutic goal is achieved. Our understanding of the role for which cachectin/TNF evolved is too shallow to allow a precise assessment of conditions under which administration or removal of the hormonewould prove to be of benefit. It is clear that the hormonecan be lethal whenover-produced;it is equally clear that it did not evolve to producea lethal effect. Cachectin/TNF,like the inflammatory response of which it is a part, is

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a mixed blessing. Perhaps in understanding its physiolo~c function, we may better understand the purpose of inflammation as a whole and gain insight into the circumstances under which "cachectin/TNF deficiency" and "cachectin/TNF excess" obtain. Then, a rational and specific therapeutic approach to diseases that involve the production of this hormone mayat least be at hand.

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ACKNOWLEDGMENTS

Wewish to thank Ms. Betsy Layton for her secretarial assistance in the preparation of the manuscript. This investigation was supported also by NIH Research Grant CA-4552.

Literature Cited 1. Michalek, S. M., Moore, R. N., McGhee, J. R., Rosenstreich, D. L., Mergenhagen, S. E. 1980. The primary role of lymphoreticular cells in the mediation of host responses to bacterial endotoxin. J. Infect. Dis. 141:55~53 2. Vogel, S. N., Moore, R. N., Sipe, J. D., Rosenstrelch, D. L. 1980. BCGinduced enhancement of endotoxin sensitivity in C3H/HeJmice. I. In vivo studies, J. Immunol. 124:2004-2009 3. Ha, D. K., Gardner, I. D., Lawton, J. W. 1983. Characterization of macrophage function in Mycobacterium lepraemurium-infected mice: sensitivity of mice to endotoxin and release of mediators and lysosomal enzymes after endotoxin treatment. Parasite Immunol. 5:513-26 4. Wood,P. R., Clark, I. A. 1984. Macrophages from Babesia and malaria infected mice are primed for monokine release. Parasite Immunol. 6:30~17 5. Haranaka, K., Satomi, N., Sakurai, A., Haranaka, R. 1984. Role of fast stimulating agents in the production of tumor necrosis factor. Cancer lmmunol. Immunother. 18:87-90 6. Cerami, A., Ikeda, Y., Le Trang, N., Hotez, P. J., Beutler, B. 1985. Weight loss associated with an endotoxininduced mediator from peritoneal macrophages: the role of cachectin (tumor necrosis factor). Irnmunol. Lett. 11:173-77 7. Rouzer, C. A., Cerami, A. 1980. Hypertriglyceridemia associated with Trypanosoma brucei brucei infection in rabbits: role of defective triglyceride removal. Mol. Biochem. Parasitol. 2: 31-38

8. Kawakami, M., Cerami, A. 1981. Studies of endotoxin-induced decrease in lipoprotein lipase activity. J. Exp. Med. 154:631-39 9. Kawakami, M., Pekala, P. H., Lane, M. D., Cerami, A. 1982. Lipoprotein lipase suppression in 3T3-LI cells by an endotoxin-induced mediator from exudate cells. Proc. Natl. Acad. Sci. USA 79:912-16 10. Beutler, B., Mahoney,J., Le Trang, N., Pekala, P., Cerami, A. 1985. Purification of cachectin, a lipoprotein lipasesuppressing hormonesecreted by endotoxin-induced RAW 264.7 cells. J. Exp, Med. 161:984-95 11. Beutler, B., Greenwald, D., Hulmes,J. D., Chang, M., Pan, Y.-C. E., Mathison, J., Ulevitch, R., Cerami, A. 1985. Identity of tumour necrosis factor and the macrophage-secretedtZactor cachectin. Nature 316:552-54 12. Beutler, B., Milsark, I. W., Cerami, A. 1985. Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. J. Immunol. 135: 3972-77 13. Abe, S., Gatanaga, T., Yamazaki, M., Soma, G., Mizuno, D. 1985. Purification of rabbit tumornecrosis factor. FEBS Lett. 180:203-6 14. Mahoney,J. R. Jr., Beutler, B. A., Le Trang, N., Vine, W., Ikeda, Y., Kawakami, M., Cerami, A. 1985. Lipopolysaccharide-treated RAW264.7 cells produce a mediator which inhibits lipoprotein lipase in 3T3-L1cells. J. ImtnunoL 134:1673-75 15, Aggarwal, B. B., Kohr, W. J., Hass, P. E., Moffat, B., Spencer, S. A., Henzel, W. J., Bringman, T. S., Nedwin, G. E.,

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THE T-CELL RECEPTOR REPERTOIRE AND AUTOIMMUNE DISEASES Vipin Leroy

Kumar, Dwight H. Kono, James L. Urban, Hood

Division of Biology 147-75, California Institute Pasadena, California 91125

and

of Technology,

INTRODUCTION The ability to distinguish "self" from "non-self" is central to the normal function of the immunesystem. An essential condition for self-nonself discrimination is the immunological tolerance to self components. The mechanismsunderlying the development and maintenance of self-tolerance are thought to involve one or more of the following: (i) "clonal deletion" or "clonal anergy" of relevant effector cells (l-5), (ii) active regulation through T-suppressor cells (6-10), (iii) regulation through idiotypic works (11-14), and (iv) sequestering of self-antigens (15, 16). If the anisms regulating self-tolerance break down, an excessive immune responseagainst a self-antigen sets in, sometimesresulting in a pathological condition or an autoimmunedisease. Autoimmunediseases affect at least 2% of the US population with a myriad of presentations including rheumatoid arthritis, insulin-dependent diabetes, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, and pemphigusvuigaris. For manyof these diseases, the events which result in end organ damage are known; however, little is known about how an autoimmuneresponse is activated and perpetuated. Most of the early studies focused on B cells and autoantibodies (17-25). With the recent advent of new methodologies for studying T cells, there is now compellingevidence that helper T cells play a pivotal role in the activation of the autoimmuneresponse (26-30). This article reviews the role played by T lymphocytesin the effector phase of autoimmunediseases. In particu657 0732-0582/89/0410-0657502.00

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lar, wediscuss the molecularbasis for the T-cell recognition of a selfantigen, whichinitiates an autoimmune response leading to experimental allergic encephalomyelitis(EAE),and the implications that these observations have for the treatment of autoimmune diseases in general.

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STRUCTURAL OF ANTIGEN

BASIS

FOR T-CELL

RECOGNITION

In general, T cells respondto a protein after it has undergonea process of denaturation and/or degradation and has been cleaved into peptide fragmentswhichare presented in association with products of the major histocompatibility complex(MHC)on the surface of antigen presenting cells (31-35). Therefore, T-cell recognition of antigen requires a trimolecular complexconsisting of a class-I or class-II MHC molecule, a peptide antigen, andthe T cell antigen receptor (Figure 1). HelperT cells generally recognizeantigen in association with class-II proteins, and most of the cytotoxic T cells recognizeantigen in the context of class-I MHC products. The concept of a trimolecular complexis important for the study of autoimmunitybecause it provides a structural frameworkfor analyzing

T-Cell

I

T-Cell Receptor

MHC molecular complex

Antigen-presenting cell (APE;) TRIMOLECULAR COMPLEX: Figure 1 A diagrammatic

representation

of the trimolecular

T-cell

activation

complex

comprised of the T-cell receptor molecule, antigen and MHCmolecule. V~, Va, C~, and C a represent variable and constant regions, respectively, of a and fl chains of the T-cell receptor. a l, a2, a3 and f12 represent the three ~ chain regions and f12 microglobulin (or fl chain domain for class II) of the class I MHCcomplex.

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each of the three interacting components individually. Theoutcomeof this interaction is decisive in discriminatingbetweenself-tolerance, immunity, and autoimmunity.In addition, therapy directed successfully at any one component of this complexwouldbe relatively specific. Several factors influence antigen recognition by T cells, affecting one of the three componentsof the trimolecular complex.To define these interactions, it is necessaryto understandthe structural characteristics of each component.

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MHCMolecule The MHC clags-I and class-II proteins are madeup of two polypeptide chains, a heavychain and fi2-microglobulinfor the formerand a pair of ~ andfl chainsfor the latter. Crystallographicstudies haveshownthat two alpha helixes of a class-I moleculeforma cleft that serves as the site of peptide binding to the MHC molecule(36-38). Thelength of this groove, approximately20 aminoacids long, is consistent with the observedlength of peptides recognizedby T cells. Similar structural characteristics for bindingof an antigen to the class-II moleculehaverecently beenproposed (38). Details of binding are discussedelsewhere(3941). In general, overlappingpeptides comprisingan entire protein have beenanalyzedfor reactivity, different MHC moleculeshavebeenfoundto "present" different peptide determinantsto T cells (38, 39, 42). TheMHC class-I and class-II molecules influence T-cell recognition of antigen by (i) determiningthe repertoire of peptides that can be presented to the TCR,and (ii) determiningTCRrepertoire by participating in negative and positive selection of T cells as they emigratethroughthe thymus (42-47). MHC molecules mayalso potentially present autoantigens to T cells if these MHC moleculesare "aberrantly" expressedin tissues that normallydo not express MHC antigens (reviewedin 48).

T Cell Antigen Receptorand Its Repertoire Theantigen-specific T cell receptor is a disulfide-linked heterodimeric transmembraneglycoprotein comprisedof an ~ and a//chain (49-52) and clonally distributed on T cells in association with the CD3complex(53). Thesepolypeptidesare encodedin the germlineby various dispersed gene segmentscomprisingvariable (V), diversity (D, for the // chain gene), joining (J), and constant (C) genesegments.Functionalg and/~genes formedduring T-cell developmentby DNA rearrangementsthat generate V-(D)-Jgenes, whichare then joined to a C-regiongene segmentby RNA splicing, followingtranscription (reviewedin 54, 55). Diversity in the T-cell receptor repertoire is generatedby three basic mechanisms:randomutilization of a large numberof germline V, D, and J genesegments;junctional variation generatedbetweenthe genesegments

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during the joining process; and the combinatorialassociation of a and fl chains. T cell receptor genes do not undergosomatic mutation, possibly thus avoidingthe potential for generatingautoreactivespecificities outside the selective environmentof the thymus. Thea-chain germlinegene repertoire in mice and humansis thought to comprisemorethan 100 different V-genesegmentsand 50--100 different J-genesegments(54, 55). Thepredictedt-chain generepertoire consists about 75 V-gene segments in the humanand approximately 30 V-gene segmentsin mice whichrearrange to approximately15 J-gene segmentsin both (54, 55). Basedon the sequencedata of TCRgenes, there exists a region of enormousjunctional diversity in whichit appears that all 20 aminoacids maybe found at each residue. Basedon the calculated number of junctional sequences,the germlinepotential and combinatorialassociations, there is the potential for generating2.9 × 1022different receptors. If one considers that perhapsonly 1%of T cells actually leave the thymus becauseof the requirementfor positive or negative selection, 1.9 × 102° potential TCRsare possible fromthe germlinerepertoire in a single mouse

(56). TheavailableT-cell repertoire is determinedby at least four factors: (i) the inherited germlinegenesegmentrepertoire; (ii) preferential rearrangementof certain T cell receptor V-genesubfamilies;(iii) the positive and negative selection in the thymus;and (iv) the fact that, as a result peripheral tolerance induction or idiotype interactions, all the expressed receptors will not be available for antigenic recognition. An important point to consideris that the T-cell repertoire is constantlychangingas new T cells emigrate fromthe thymus. Antigen and the Structure of a T-Cell Epitope In contrast to immunoglobulins whichrecognizethe tertiary structure of molecules,the antigenic determinantthat T lymphocytesrecognizeis not the intact protein moleculebut rather a small peptide fragment(57-59). It appears only those pcptidcs that can bind to a MHC moleculewill be recognizedby T cells. Onthe basis of in vitro bindingstudies, it has been suggestedthat a single MHC moleculeshould be able to bind a variety of peptideson the cell surface(60-62).Severalgeneralpropertiesof peptides, recently determined,appear to makethemmorelikely to be recognizedby T cells. Althoughnot alwaysthe case, peptides that forman amphipathic alpha helix (63, 64) and peptides that contain the motif of a glycine or charged group followed by 2-3 hydrophobicresidues and then a hydrophilic ami’noacid appear to be immunodominant epitopes whenthe whole moleculeis used for the priming (65, 66). It remainsto be determined whetherthese properties are importantfor the actual binding to the MHC

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or T cell receptor molecule or for deciding the most likely peptide to be produced by antigen processing. The minimal length of a peptide that can be recognized by T cells is about 7 aminoacids long. For a particular MHChaplotype, a single protein molecule generally has several non-overlapping peptide determinants that can bind to the MHCmolecule. Along each of these MHC-binding peptide determinants there can be multiple different T-cell epitopes. The individual aminoadds along a peptide that bind to the MHCmolecule may be interspersed or even the same as some of the amino acids required for TCRbinding (67, 68). The various T-cell determinants and epitopes from a single protein are not recognizedwith equal efficiency, and there appears to be a hierarchy in the ability of any of these epitopes to be recognizedby T cells (42). The primary sequence and possibly the structure of the protein antigen itself likely contribute to this "immunodominance" &certain peptide fragments by determining the preferred sites of proteolytic cleavage during antigen processing, the relative affinities of the peptides to the MHC molecule, and competitive binding between two peptides to the same MHCmolecule (69). Antigenic similarity or "molecular mimicry" between antigenic determinants of infectious agents--e.g, virus, bacteria or parasites, and host self determinants--has been implicated as a primary causal event in the induction of autoimmunity(70, 71). Shared epitopes between a viral protein and myelin basic protein encephalitogenic determinant have been shown to cause experimental autoimmune encephalomyelitis (72; V. Kumar, unpublished data). Similarly, cross-reactivity between a Mycobacterium tuberculosis immunogen and a self componentof cartilage implicates molecular mimicryin the triggering of adjuvant arthritis in rats (73). Recently, T lymphocytesfrom rheumatoid arthritis patients have also been isolated that recognize a Mycobacteriumtuberculosis epitope that crossreacts with a determinant within cartilage (74). Identification of the selfantigen will be extremely important for understanding the pathogenesis of human autoimmune diseases.

Factors Affectin9 T-Cell Reactivity to Self-Antiyens In order for an autoantigen to elicit an immuneresponse in an individual, not only must the autoantigen give rise to peptides that can bind to classII MHCmolecules, but also the individual must have T cells beating receptors able to recognize that particular combination of peptide and MHC (75). This would imply that what is a disease-causing antigen in one individual maynot induce disease in another individual with a different MHC(MHCpolymorphism) or T cell receptor repertoire (T cell receptor polymorphism).Similarly, polymorphismof a self-antigen itself mayalter

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the degree ofT-cell activation. Since a correlation betweenimmunogenicity of an antigen and Ia binding has been documented,an immuneresponse for a given antigen mayvary in different individuals dependingupon differences in the binding affinity of that antigen to the MHC molecule. Avariety of cells (e.g. astrocytes,thyrocytes,andislet cells) other than macrophagesand B cells have been shownto express MHC molecules underpathological conditions (76-79). Theaberrant MHC expressionon the cell surface maybe importantin presenting autoantigensin target tissues (Figure 3). It is also possible that differential processingof a protein different antigen presenting cells maygenerate distinct peptide products whichin turn wouldalter the repertoire of expressed epitopes on the surface of these cells (80, 81). Finally, immune responseto an autoantigen mayalso be affected by an anti-idiotypic or suppressorcell network(82, 83) that further skewsthe available T-cell repertoire and therefore maybe importantin determininga disease-causingepitope in that individual. EXPERIMENTAL ANIMAL AUTOIMMUNE DISEASES

MODELS

FOR

The etiopathogenesis of autoimmune diseases in humanscannot be easily studied. Thereforeappropriateanimalmodelsoffer the best hopeof understanding the underlying mechanisms of autoimmunity.Becauseof the ease with whichgenetic and other manipulative studies can be performedin experimentalanimals,they are exceptionallyvaluabletools for identifying genes and loci involved in the pathogenesisof autoimmunity. Certain experimental animal modelsof autoimmunedisease have demonstrated that helper T cells are the primarymediatorsof autoimmunity (Table 1). T-cell involvementin autoimmune diseases has been inferred fromthe followingfindings: (i) T-cell lines or clones wereable to transfer diseasein naiverecipients (28, 29, 84-86);(ii) eliminationof helper T subset with anti-L3T4antibodies in vivo resulted in abrogationof disease (26, 27, 87, 88); and (iii) immunization with attenuated antigen-specific helper T cells protected against subsequentattempts to induce active disease in experimentalanimals(29, 86, 89, 90). Althoughthe relationship of these experimental modelsto humandisease is unproven, the information gained from the study of the recognition of self-antigens and maintenance of tolerance in these experimental animals will be fundamental to the understanding of autoimmunephenomenain general.

ExperimentalAlleryic Encephalomyelitis( EAE) EAEis currently the best characterized modelof an antigen-specific, T cell-mediated autoimmunedisease. EAEis an autoimmunedemyelinating

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disease caused by an immuneresponse to myelin basic protein (MBP),and it serves as an animal modelfor multiple sclerosis (MS)(91, 92). Although the etiology of MSis not known,the principles derived from this experimental model may provide a better understanding of the role that the immunesystem plays in MS. EAEin mice clearly is a helper T cell-mediated disease. Studies involving T-cell subset depletions (88), transfer of disease with helper T cells into naive syngeneic mice (93-95) or nude mice (96), and treatment of with anti-L3T4 all support the hypothesis that EAEis caused directly by helper T cells. This model provides a unique opportunity to study the effects of intervention directed against a specific componentof the trimolecular complex, without inadvertently affecting other effector cells of the immunesystem as would be seen with a B-cell mediated disease. The trimolecular interactions in EAEhave been studied in detail. Early studies showedthat the MHC determined in part the susceptibility of mice to develop EAE(97-100). Proteolytic fragments of MBPwere found cause EAEonly in certain strains of mice (Figure 2). H-2u and H-2k mice develop EAEwhen challenged with the 1-37 N-terminal fragment, and H-2s and H-2q mice develop EAEwhen challenged with the C-terminal 89-169 residues of guinea pig MBP(101). Subsequent studies using synthetic peptides have characterized the peptide determinants causing EAE in H-2u mice and H-2~ mice (102-104). Study of the SJL/J encephalitogenie T-cell epitope, using overlappingand truncated synthetic peptides, has identified multiple encephalitogenic T-cell epitopes along what appears to be a single peptide determinant (102; Figure 2B). Interestingly, minor peptide epitopes appear also to have the capacity to elicit an encephalitogenie response, despite the fact that following immunizationwith the native moleculeno T-cell proliferative response can be detected against these determinants (MBPpeptide residues 68-91, 87-98, and truncated peptides). The severity of EAEappears to be greater with the immunodominant T-cell epitopes, comparedwith the minor determinants; in one instance a minor determinant, MBPpeptide residues 68-91, did not induce EAE. The presence of MBP-reactive T lymphocytes and induction of EAEby other CNSproteins (195-107) are consistent with the concept of the absence of clonal deletion of T cells to "protected" or "sequestered" self antigens (108). The basis for this maybe that only antigens present within the thymusduring T-cell maturation lead to clonal deletion of T cells (109, 110). However,the lack of response to these sequestered antigens maynot be due only to the protected location of these antigens; the lack mayalso be due to an anti-idiotypic or suppressor network. The fact that minor peptide determinants can cause EAEhas implications for the eventual detection of autoantigens in nonantigen-induced,

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A.

37

mouse MBP+ Act MOUSE STRAINS

87

168

ENCEPHALITOGENtC MBP PEPTIDES 31--50

PL and BIO.PL (H-2U) Ac s) SJL(H-2 A/J

(H.2

k)

Ac

--

.37

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BIO.T((~R)(H-2q)

87

168

Bo MBP PEPTIDES 68

PROLIFERATION

80

90

100

EAE

110

LPQKSQHGRTQDENPWHFFKNIVTPRTPPPSQGKGRGLSLSRFSWG

Figure

2 T-cell

and encephalitogenic

determinants

of

murine

+ +++

+++

++

++

++

++

+

+

+

+

+

NT

+ +

NT +

myelin

basic

protein.

A.

Synthetic andproteolytic peptides of MBP which bothstimulate T-cellresponses andinduce EAE in PL/J Truncated including

or B10.PL

peptides

of

minor T-cell

(H-2U), the

SJL/J

SJL/J T-cell

epitopes

(H-2S),

A/J (H-2k),

determinant

which can also

induce

and B10.T(6R)

demonstrate

multiple

q)

mice . B.

T-cell

epitopes

disease.

spontaneousautoimmune disease. Since autoimmunity maybe due either to mimicryof a non-self antigen(e.g. an infectious agent homologous to a self-antigen)or to presentation of a minorself-determinant, detectionof the immune responsedirected against a minordeterminantmayonly be possiblethroughthe use of overlappingsyntheticpeptides. Recentadvancesin our understanding of the molecularbiology of the T cell receptorshavemadeit possible to studythe receptorsof THcells mediatingEAE in great detail. TheT cell receptorsof THcells mediating EAE haverecentlybeenidentified in twomousestrains of the H-2u haplotype, B10.PLandPL/J (110-112). Themajorityof encephalitogenic cells from these mice recognize an immunodominant N-terminal MBP nonapeptide (1-9 NAc)in association with the I-Au moleculeof the MHC. TheV~andV~genes of B10.PLhelper T cells whichare specific for the 1-9 NAcpeptide andrestricted by the I-Au moleculehaverecently been

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sequenced and found to be highly restricted (110, 111). Out of 33 clonally distinct THcells analyzed, 58%were found to utilize the same V~2.3gene segment and the remaining 42%the V~4.2 gene segment. All the TH cells utilized the same J~39 gene segment. For the V~ genes, 79%employedthe same V~8.2gene segment joined to the J~2.6 gene segment, and the remaining 21%employedthe V~ 13 and J¢2.2 gene segments. All of these receptors possess nearly identical antigen-binding sites, as determinedby fine specificity analysis using synthetic N-terminal analogues of MBP.Thus, there is a striking restriction of the T cell receptor repertoire directed against the immunodominantMBPepitope 1-9 NAcin B10.PL mice. A similar restriction exists in the repertoire of PL/J mice recognizing this epitope, although differences exist in gene segmentusage between the two strains. In an analysis of eight THclones mediating EAEin PL/J mice, Acha-Orbeaand coworkers (111) found that all clones share the V~4.3gene segment, and seven out of eight share the Va8.2 gene segment. All eight clones exhibited the same antigen fine specificity, determined by using a series of 9 N-terminal MBPpeptides differing from the parent 1-9 NAc peptide by single alanine substitutions. This pattern of fine specificity is identical with that for encephalitogenic B10.PLTHcells. Whereas the Va8.2 gene segment is exactly the same segment used by the majority of B10.PLand PL/J encephalitogenic THcells, the V~ gene segments are not the same--e.g. V~4.2 and V~4.3, respectively. PL/J Tn ceils also fail to express the V~2.3gene segmentutilized by the majority of B10.PLcells, and the alternate Va gene segmentexpressed by these cells is Va4 rather than the alternate Val 3 gene segmentexpressed by B10.PLcells. Except for the Ja2.6 gene segment, which is used by 79% of B10.PL fl chains and 50%of PL/J fl chains, T cell receptors of the two strains also employdifferent J~ and Ja elements. These differences in gene segment usage mayrelate to polymorphicdifferences in T cell receptor genes that exist betweenthe two strains. The critical point is that amongindividuals with the same MHC haplotype, differences in germline or expressed T cell receptor genes maydramatically influence the nature of T cell receptor restriction to a given autoantigen. Alternatively, the use of certain ¥ gene segmentsmaybe so critical to the response to particular autoantigens that they will be invariantly expressed in individuals expressing the same MHC molecules. Although the presence of an MBP-specific TCRis necessary for causing disease, it alone is not sufficient. Otherfactors are also required for T cells uto be encephalitogenic. A majority of the MBP(1-9 NAc)specific, I-A restricted, helper T cell clones, bearing different T cell receptors (different V, D, J gene segments), induce EAEwhen transferred into syngeneic animals (110-112). However,T cells with the same antigen-fine specificity

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andidentical T cell receptors (identical ¥, D, J genesegments)havebeen shown to differ in their ability to causedisease(111). This differencein the biological activity of these T lymphocytes can not be correlated with either the cell-surface receptor concentrationor lymphokine production,as these lymphocytesproduce varying amountsof interleukin 2 (IL-2) and lymphotoxin (LT) in vitro (113; N. H. Ruddle, personal communication). Whyis it that someclones using identical receptors producedifferent amountsof lymphotoxinand havediffering abilities to transfer disease? Whetherthese differences are dueto the artifacts generatedas a result of in vitro propagation,e.g. loss of homing receptor, is not dear at present. T CELL RECEPTOR REPERTOIRE IN OTHER ANIMAL MODELS OF AUTOIMMUNE DISEASES The primary role of T lymphocytesin the developmentof autoimmunity has beenwell established. Preliminarystudies in susceptible animalssuggest a possiblecorrelation betweenthe usageof a particular T cell receptor variable (V) gene segment and autoimmunepathology in someof the experimental models of autoimmunediseases. Recent genetic studies in hybrids between susceptible (SWR,H-2q) b) and resistant (B10, H-2 mousestrains has shownthat one of the genetic determinantsof collagen arthritis (CA)mayrelate to the presence of the Vp6gene segment susceptible mice(114; C. S. David, personal communication). Similarly, deletion of ~ 8.8 kb spanningCpl, D~2,and J~2 gene segmentsof the rchain of the T cell receptor in autoimmuneNZBor (NZB× NZW) miceand a significant skewingof the T-cell repertoire in favor of Va8 subfamilygenes usage in MRL-lpr/lprmicecorrelate with the development of a lupus-like autoimmune disease in these mousestrains (115-118).The role these findings play in the pathogenesisof murinelupus has not been clarified. Interestingly, NOD (non-obesediabetic) mice, whichnormally do not express I-E molecules,developinsulin-dependentdiabetes mellitus (IDDM)(119, 120). However,expression of I-E in transgenic NOD prevents the developmentof autoirnmunedisease (121). The mechanism leading to the preventionof IDDM in these miceis not dear, although it seemsreasonable to speculate that the autoimmune I-E reactive T cell clones (utilizing Vo17and others) maybe involved in autoimmunity,and their deletion mayprotect these mice from IDDM. T-CELL INVOLVEMENT DISEASES IN HUMANS

IN

AUTOIMMUNE

Although evidence for a primary involvementof T lymphocytesin the initiation and perpetuation of humanautoimmunediseases has not been

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compelling,the bulk of the experimentaldata in humanpatients suggests a role for abnormalimmuneregulation and T-cell mediationin autoimmune pathology.Thefollowingstudies support T cells as an importantmediator of autoimmune reactions. Abnormalmixed lymphocytereaction (AMLR) and the delineation the T-cell imbalances,usingmonoclonal antibodiesagainst the cell surface antigens on T cells, in a numberof immunologicallymediateddiseases demonstrateda possible deficiencyof cytotoxic (Tc) or abnormalityin the TH/Tcratios in the peripheral blood of affected individuals (122-129). Whetherabnormalhelper or cytotoxic T cell activity is the primarycause or result of the immune reactions has not beenestablished. Infiltration of both cytotoxic and helper T cells has been found in autoimmunetarget organs (130, 131). Auto-antigen-specific helper T cell lines have been isolated fromaffected individuals (132-134).Whetherthese cells are the primarycause of autoimmune pathologyremainsto be clarified. An"aberrant" expression of MHCclass-II antigens has been demonstrated in target organs,e.g. endocrinecells of the pancreasin IDDM (76), thyroid epithelial cells in autoimmune thyroiditis (77), andbile duct epithelial cells in primary biliary cirrhosis (135). AlthoughMHC expressiondoes not seemto be the primaryinitiating event it mayexacerbate or perpetuate the disease by presentingself-antigensto the activatedT-cells infiltrating the target organ. Althoughautoimmune reactions seemto play an important role in many autoimmune diseases, the precise immunologicalmechanismsinvolved are presently unclear. In general, autoimmunedisorders are complexand multifactorial in etiology. Genetic, immunologic,hormonal, environmental, and possible viral factors mayall play a role in the pathogenesis of autoimmune diseases in humans.Possible events leading to autoimmune pathologyhavebeen outlined (Figure 3). Abnormal triggering of helper cells appears to be the commonpathway leading to tissue damageby various mechanisms such as delayed-typehypersensitivity (DTH)and cellmediatedor antibody-mediatedcytotoxicity.

T CELL RECEPTOR GENE POLYMORPHISM HUMAN AUTOIMMUNE DISEASES

IN

Recently, serological and/or DNA markers for genes that confer susceptibility to autoimmune diseases, includingrheumatoidarthritis, insulindependentdiabetes mellitus, pemphigus,and multiple sclerosis, havebeen identified for the class-I and class-II moleculesof the HLA system(136, 137). Manyindividuals, however,with disease-associated class-II alleles

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o/ --.F’,,.=

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do not develop autoimmune disease. Furthermore, the low concordance rates for certain autoimmunediseases in HLAidentical siblings (138, 139) and the higher concordance rate in genetically identical monozygotictwins (comparedto dizygotic twins) indicate either that other genes contribute to disease susceptibility or that interaction with the environment is a necessary prerequisite. Since the histopathology of the lesions in these diseases demonstrates infiltration with T cells, and since the T-cell repertoire is biased against the self-MHC,it is quite possible that the biological events by which MHC alleles produce disease susceptibility are mediated by the T cell receptors selected for by the MHC.Also, since the T-cell repertoire is dependent on the available T cell receptor V, D, and J gene segments, analysis of genetic elements at least possibly in linkage dis- . equilibrium with these genes could be of importance. Polymorphismsin the germline V genes are likely to affect the repertoire of T cell receptors and, thus, antigen recognition, e.g. specific V gene segmentusage for a defined antigen (111,140-142). Restriction fragment length polymorphisms (RFLPs) of the C and V gene segments encoding ~ and fl chain of the T cell receptor have recently been described (143-146). In view of the fundamental importance of the ternary complex (TCR, MHC,and antigen) in the immuneresponse, there are reports of the possible association of these RFLPswith various autoimmune diseases including multiple sclerosis (147, 148), myasthenia gravis (149), membranousnephropathy (150), Graves’ disease (151), and insulin dependentdiabetes mellitus (152, 153). Although inconclusive, these studies suggest that disease susceptibility genes may be located in the region of the TCR~ and fl gene complex. More detailed studies with multiplex families are needed to draw any definitive conclusions regarding disease susceptibility and T cell receptor V-genepolymorphisms.Since the available T cell repertoire depends upon the process of thymic selection and the anti-idiotypic or suppressor cell network in the periphery, RFLPstudies alone may not identify a skewed, expressed T cell receptor repertoire in a susceptible individual, unless one can demonstrate germline deletions of someof the V-region gene segments or that the RFLPcorrelates with changes in either the coding or 5’ non-codingregulatory regions. Therefore, novel strategies, other than RFLPstudies need to be designed to correlate the available expressed T cell receptor repertoire with disease susceptibility in humans. TREATMENT

OF

AUTOIMMUNE

DISEASES

The therapeutic approaches to autoimmunediseases in the past have been very generalized and nonspecific, e.g. the use of immunomodulatingor

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immunosuppressiveagents like cyclosporine A, cyclophosphamide, corticosteroids, sex hormones,and total lymphoid irradiation (154-156). Similarly, antisera or monoclonal antibodies directed against MHC molecules and/or T-cell marker (e.g. L3T4) were used to suppress or reverse autoimmunediseases in experimental animals (87, 88, 157-160). Preventive immunoablationof only specific disease-causing helper T cell subset with a T cell receptor V region specific monoclonalantibody F23.1 (161) has recently been demonstrated (110, 111). In vivo treatment BI0.PL or (BI0.PL x SJL) FI with anti-V~8 monoclonal antibody depletes specific encephalitogenicVt~8.2+ helper T cells and results in the prevention and reversal of peptide-induced experimental allergic encephalomyelitis (110, 111). Therefore, this approach allows the elimination of specific cell responses linked to the pathogenesis of an autoimmunecondition with minimal impairment of the overall host immuneresponse. A potential complication to this approach in humansinvolves the allelic heterogeneity in MHC and T cell receptor genes exhibited by different individuals. Since different mousestrains (PL/J and SJL/J) demonstrate different encephalitogenic MBPepitopes and employ different T cell receptor genes for their encephalitogenic THcells, it is presumedthat for humanseach different MHChaplotype may employ different subsets of T cell receptors in the induction of a particular autoimmunedisease. Thus, for a given disease a correlation in the T cell receptor usage with the MHChaplotypes may be necessary before one can identify appropriate sets of T cells that can be ablated. Similarly, the anti-clonotypic antibody approach may also be tried because of the remarkablesimilarity seen in the fine specificity of the helper T cells utilizing four different T cell receptor V genes. This shared T cell receptor specificity could also perhaps be a specific target for experimental approaches utilizing peptide fragments as an antagonist and tolerogen to induce therapeutic suppression of the disease. Althoughthe existence of suppressor cells or a regulatory T-cell network has been a matter of debate (162-164), recent studies on the suppression or prevention of autoimmunedisease (such as EAEand thyroiditis) (29, 86, 89, 90; V. Kumar,unpublished data) after vaccination with attenuated T lymphocytes suggest a regulatory role for an anti-idiotypic T-cell network. Suppressor or anti-idiotypic T-cell clones (CD4+ and CD8+) or lines have recently been isolated from rats recovered from EAEor immunized with attenuated helper T cell encephalitogenic clones (165-167). These anti-idiotypic T-cell clones or lines are specific in that they only recognize and suppress MBP-specifichelper T cell clones in vitro, and they prevented EAEinduction when transferred into naive animals. While the exact mechanismof resistance in vivo by the anti-idiotypic network or the suppressor cells remains unknown,it does suggest that immunenetwork

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interactions, if they exist, could be exploited in the future to control autoimmunedisease.

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FUTURE

PERSPECTIVE

The major obstacle in studying human autoimmune diseases has been the inability to identify the target self-antigens. Since specific immune intervention can only be achieved after analyzing the T-cell repertoire directed against the specific autoantigen, both the identification of the antigen and isolation of the specific T-cell clones should give rise to ways of designing therapeutic approaches for various autoimmunediseases. Similarly, identification of clonal T-cell population in the damagedorgans or lesions and characterization of their T-cell receptors might permit the analysis of prevalence of distinct T-cell receptor idiotypes in patients relative to MHCmatched controls or siblings. Concurrently, studies of the T cell receptor repertoire, antigen processing and recognition by lymphocytes, lymphokineproduction, and the structural details of the trimolecular complexwill be of immenseimportance for the eventual understanding of the mechanism of autoimmunity and pathogenesis of the disease process. Specific immune-ablation of T cells responsible for the induction of a particular disease or anti-idiotypic suppressionof the disease causing T cells, may be considered the beginning of new ways to control autoimmunity by exploiting one of the most important features of the immunesystemi.e. specificity. Finally, a fundamental question is: how can we find the original, presumably pauciclonal, T-cell clones which initiate an autoimmuneresponse in humans? Recently, a functional humanimmunesystem has been reconstituted in SCID(severe combined immunological deficiency) mice with the transfer of either the bone marrowcells or fetal thymus, fetal liver, and lymph nodes from humans(168, 169). This will be an ideal system search for humanT lymphocytes capable of initiating autoimmunedisease and, even more important, to study potential use of therapies to delete specific T-cell subsets. It will also be a useful assay system to identify the antigen which initiates autoimmunedisease. Clearly the physician’s approach to the therapy of autoimmunedisease will change in fundamental ways in the next 10 years. ACKNOWLEDGMENTS

The literature search for this article was completedin August, 1988. This work was supported by the National Institutes of Health. Vipin Kumaris a postdoctoral fellow of the National Multiple Sclerosis Society, USA.

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D. H. Konois a recipient of the Arthritis Investigator Awardand J. L. Urban is supported by a Leukemia Society of America Fellowship. We thank C. S. David, N. H. Ruddle, T. Shinohara, A. Theofilopoulos for communicatingtheir results prior to the publication. Wealso thank Steve Beall, Tim Hunkapiller, and Dennis Zaller for critical reading of the manuscript, and Cathy Elkins, Connie Katz, Rita Grable, and Bertha Jones for help in preparing the manuscript.

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Polymorphicmarkers of humanT-cell receptor alpha and beta genes. Family studies and comparisonof frequencies in healthy individuals and patients with multiple sclerosis and myasthenia gravis. Hum.Immunol.22:111-21 148. Beall, S. S., Concannon, P., Charmley, P., McFarland,H. F., Gatti, R. A., Hood,L. E., McFarlin,D. E., Biddison, W.E. 1988. The germline repertoire of T-cell receptor/3-chaingenes in patients with multiplesclerosis. J. Neuroimmunol. In press 149. EdvardSmith, C. I., Borgonovo,L., Carlsson, B., Hammarstrom, L., Rabbitts, T. H. 1987. Molecularprobingof disease susceptibility genes in myasthenia gravis patients: Ananalysis of T-cell receptor and HLAclass II genes using restriction fragmentlength polymorphism.Ann. N.Y. Acad. Sci. 505: 388-97 150. Demaine, A. G., Vaughan, R. W., Taube, D. H., Welsh, K. I. 1988. Association of membranousnephropathy with T-cell receptor constant fl chain and immunoglobulin heavy chain switch region polymorphisms. lmmunogenetics27:19-23 151. Wcctman,A. P., So, A. K., Roe, C., Walport,M.J., Foroni, L. 1987.T-cell receptor c~ chain V region polymorphism linked to primary autoimmunehypothyroidism but not Graves’ disease. Hum.Immunol.20: 16773 152. Millward,B. A., Welsh,K. I., Leslie, R. D. G., Pyke, D. A., Demaine, A. G. 1987. T-cell receptor beta chain polymorphismsare associated with insulin-dependentdiabetes. Clin. Exp. Immunol. 70:152-57 153. Hoover,M. L., Capra, D. J. 1987. The T-cell receptor and autoimmunediseases. Mol. Biol. Med.4:123-32 154. Schindler, R. 1986. Cyclosporin in Autoimmune Disease. Berlin: SpringerVcrlag 155. Thomson,A. W., Webster,L. M. 1988. Theinfluence of cyclosporinA on cellmediatedimmunity. Clin. Exp. Immunol. 71:369-76 156. Talal, N. 1986. Newtherapeutic approaches to autoimmunedisease. SpringerSemin.lmmunopathol. 9: 105-16 157. Steinman, L., Rosenbaum, J. T., Sriram, S., McDevitt,H. O. 1981. In vivo effects of antibodies to immune responsegeneproducts. II. Prevention of experimental allergic encephalomyelitis. Proc. Natl. Acad. Sci. USA 78:7111-14

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senblatt, R. B. 1988. A newmodelof autoimmunedisease. Experimental autoimmune uveoretinitis in mice with twodifferent retinal antigens. J. lmmunol. 140:1490-95 185. McAllister,C. G., Wiggert,B., Chader, G. J., Kuwabara,T., Gery, I. 1987. Uveotogenicpotential of lymphocytes sensitized to interphotoreceptorretinoid-bindingprotein. J. lmmunol.138: 1416-20 186. Shinohara,T., Donoso,L., Truda, M., Yamaki,K., Singh, V. K. 1988. S-antigen: structure, function and experimental autoimmuneuveitis (EAU). Pro#. Ret. Res. 8:1-16 187. Bach,J.-F. 1988. Mechanisms of autoimmunityin insulin-dependent diabetes mellitus. Clin. Exp. Immunol.70: I-8 188. Bendelac,A., Carnaud,C., Boitard, C., Bach,J. F. 1987. Syngeneictransfer of autoimmunediabetes from diabetic NOD mice to healthy neonatal require+ andLyt2+ T cells. mentfor both L3T4 J. Exp. Med.166:823-32 189. Koike,T., Itoh, Y., Ishii, T., Ito, I., Takabayashi, K., Masuyama, N., Tomioka,H., Yoshida, S. 1987. Preventive effect of monoclonalanti-L3T4 antibody on developmentof diabetes in NODmice. Diabetes 36:539 190. Andrews, B. S., Eisenberg,R. A., Theofilopoulos, A. N., Izui, S., Wilson,C. B., McConahey, P. J., Murphy,E. D., Roths, J. B. Dixon,F. J. 1978. Spontaneous murinelupus-like syndromes. Clinical and immunopathological manifestations in several strains. J. Exp. Med. 148:1198-1215 191. Morse,H. C., Davidson,W.F., Yetter, R. A., Murphy,E., Roths, J. B., Coffman, R. L. 1982. Abnormalities inducedby the mutantgenelpr: expansion of a unique lymphocytesubset. J. Immunol. 129:2612-15 192. Theofilopoulos, A. N., Dixon, F. J.

1985. Murinemodelsof systemic lupus erythematosus.Adv. Immunol.37: 26990 193. Sekigawa,I., Ishida, Y., Hirose, S., Sato, H., Shirai, T. 1986.Cellularbasis of in vitro anti-DNAproduction: evidence for T-cell dependenceof IgGclass anti-DNAantibody synthesis in the (NZB× NZW) F 1 hybrid. J. Immunol. 136:1247-52 194. Ando, D. G., Sercarz, E. E., Hahn, B. H. 1987. Mechanismsof T- and Bcell collaborationin the in vitro production of anti-DNAantibodies in the NZB/NZW F~ murine SLE model. J. Immuno.138:3185-90 195. Mihara, M., Ohsugi, Y., Saito, K., Miyai, T., Togashi, M., Ono, S., Murakami,S., Dobashi,K., Hirayama, F., Hamaoka,T. 1988. Immunologic abnormality in NZB/NZWF~ mice: thymus-independentoccurrence of Bcell abnormalityand requirementof T cells in the development of autoimmunedisease, as evidenced by an analysis of the athymic nude individuals. J. Immunol.141:85-90 196. Roths, J. B., Murphy,E. D., Either, E. M. 1984. A new mutation, gld, that produceslymphoproliferationand autoimmunity in C3H/HeJmice. J. Exp. Med. 159:1-20 197. Davidson,W.F., Dumont,F. J., Bedigian, H. G., Fowlties,B. J., Morse,H. III. 1986. Phenotypic,functional and molecular genetic comparisons of abnormal lymphoid cells of C3Hlpr/lrp and C3H-gld/gldmice.J. Immunol. 136:4075-84 198. Mikecz,K., Giant, T. T., Poole, A. R. 1987. Immunity to cartilage proteoglycans in BALB/cmice with progressive polyarthritis and ankylosing spondylitis induced by injection of humancartilage proteoglycan.Arthritic Rheum.30:306~18

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T-CELL RECOGNITION OF MINOR LYMPHOCYTE STIMULATING(MI s) GENE 1PRODUCTS Ryo Abe and Richard

J.

Hodes

Experimental ImmunologyBranch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 INTRODUCTION The immunesystem has evolved with a highly diverse antigen-specific repertoire capable of recognizing an extraordinarily large universe of foreign antigens. As a corollary of this wide diversity, the precursor frequencyof T cells specific for any given foreign antigen is generally very low, so low, in fact, that proliferative responses of naive T-cell populations to these antigens are undetectable. However, prominent exceptions to this generalization have been identified. In multiple species, cell surface alloantigens encoded by genes of the major histocompatibility complex (MHC) are recognized by T cells at a high precursor frequency and are capable of eliciting a strong proliferative mixedlymphocyteresponse by unprimedTcell populations (1, 2). In the mouse, a second set of determinants, the minor lymphocyte stimulatory (Mls) determinants, also induce strong proliferative responses by naive T cells (3-5). The finding that T cells are reactive to MHCand to Mls determinants at extraordinarily high precursor frequencies (1, 2, 6, 7) has generated interest in understanding the biologic significance of these two systems. A great deal of information has been accumulated concerning the structure and function of MHCgenes and their products, and the biologic importance of MHC-encoded determinants has been extensively documented(reviewed in 8). In contrast, although Mls gene products were described as lymphocyte ~ The USGovernmenthas the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

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activating determinants at almost the same time as MHCgene products (9, 10), the nature of Mls genes and their products remains largely unknown.No antibodies specific for Mls products have been identified, and the biochemistry of these products is completely uncharacterized. The existence of Mls products is in fact defined at this time solely by the response of T lymphocytes. Recently, significant developments in several areas have added to our understanding of the Mls system. A series of experiments has led to a revised understanding of the genetics of the Mls system (11-17; review in 18), and to the identification of distinct Mls determinants. In addition, it has been established that the reactivity of T cells to certain Mls gene products is highly associated with the expression of selected T cell receptor (TCR)Vfl segments by these T cells (19-22), and that mature T cells bearing these Vfl gene segmentsare deleted in mice expressing the corresponding Mls type. It nowappears that the study of the Mls system may provide important insights into the general mechanismsof T-cell recognition and activation, and into the role of non-MHC products in the selection of the T-cell repertoire. This review discusses the nature of the Mls system with an emphasis upon recent findings that have heightened interest in this problematicarea.

MLs DETERMINANTS AND GENETICS Initial Characterization of the Mls System Whenresponding T cells of one inbred strain or individual are cultured in vitro with a genetically dissimilar population of stimulator cells, T-cell proliferation results, a response termed the mixed lymphocyte reaction (MLR).The determinants expressed on stimulator cells that play an essential role in triggering the proliferation of responding lymphocytes were initially termed lymphocyte activatin 9 determinants (LAD)(23). In murine system, where a panel of inbred and congenic mouse strains was available, it was first shownthat genes controlling LADare encodedwithin H-2, the mouse MHC(9). However, it was also observed that strong MLRscould be generated between strains which shared the same H-2 haplotype but which differed in their non-H-2 backgrounds (10). These non-H-2 MLRswere first postulated to represent cumulative responses to multiple minor histocompatibility loci (24, 25). However,whenFestenstein performed genetic analysis of non-MHCmixed lymphocyte reactions, he obtained results which led him to propose that these responses were induced by the product of a single dominant gene, the M locus, later designated as the minor lymphocyte stimulatory or Mls locus (reviewed in 3). The initial evidence suggesting the existence of the Mls locus camefrom

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OF MLS GENE PRODUCTS

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the observation that coculturing of lymphocytes from MHC-identical BALB/cand DBA/2mice caused a strong MLR(10). A backcross analysis demonstrated that approximately 50% of the offspring of a BALB/cx (BALB/c× DBA/2)F1breeding were stimulatory to BALB/clymphocytes, and this led to the conclusion that the reactivity in MLRbetween BALB/c and DBA/2was determined by antigen(s) encoded or regulated by a single genetic region (3). Using similar strategies to study MLRsbetween H-2 identical strains, Festenstein proposed that the Mls locus was composedof four alleles, a, b, c, and d. Each allele was presumedto encode polymorphic determinants with widely differing stimulatory capacities: Mlsa (expressed by strains including AKR/J, DBA/2, DBA/1), and Mlsd (CBA/J) are strongly stimulatory; Mlsc (A/J, C3H/HeJ)is weak; and Mls~ (C57BL/10, C57BL/6, C57L, BALB/c,CBA/H)is nonstimulatory (3, 4). The genetics of Mls expression were further explored by Festenstein, whoused segregation analysis to demonstrate that the locus encoding Mls~ is unlinked to H-2 (3). Later, this Mls locus was mapped to chromosome1 using recombinant inbred (RI) strains established between AKR/J(Mls°) and C57L (Mls b) (the AKXLRI series), or between B6 (Mls ~) and DBA/2 (MlsO(the BXDRI series) (26). It should be noted that no formal analysis wascarried out at the time of this initial characterization to test the allelic relationship of the genes encoding Mls~, Mlsc, and Mlsd. Morerecently, a fifth allele designated Mls~ has been proposed (27). Pattern

of MLRs Elicited

by Mls Gene Products

In general, MLRselicited by MHC disparities are bidirectional: T cells from one strain can respond to those from any other strain, and inactivated (irradiated or mitomycin C-treated) lymphocytes from one strain can stimulate those from any other strain. In contrast, Mls-specific proliferation in MLRs is strikingly different in this aspect. The pattern of Tcell response to MHC-compatible,Mls-disparate stimulators amongthe H-2k strains AKR/J(Mls~), B10.BR (Mlsb), C3H/HeJ (MlsO and CBA/J (Mlsd) is shownschematically in Figure 1. Mlsb T cells respond to each of the three other Mls types, Mls~, Mls~, and Mlsd; but Mls~ stimulator cells do not elicit responses by T cells of any other Mls type. Mlsd T cells do not respond to Mls~, Mlsb, or Mls~, but Mlsd stimulator cells induce responses by T cells of each of these Mls types. Mutual or bidirectional stimulation is seen in only one combination, between Mls° and Mls*. Anotherstriking characteristic of the Mls-specific MLR is the quantitative variation in T-cell responses to different Mls types. For example, responses ofMls~ T cells to Mls° and Mlsd are generally muchstronger than responses to MlsC; and Mlsc T cells respond muchmore strongly to Mlsd stimulators than do Mls~ T cells.

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AKR/d ] a )(MIs

/ CBA/J]

b }(Mls

(Mlsd)

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/

Responsepattern of T ceils to Mls disparate cells,

Controversies Concerning the Mls System

Polymorphism

and Allelism

of

Although the stimulatory pattern shown in Figure 1 has been observed independently by several laboratories (3, 11, 14, 16, 28, 29), some investigators have described different patterns. For example, the weak stimulatory ability of Mlsc is not always detectable, and Mlsa and Mlsa are highly cross-reactive and even antigenically indistinguishable in certain conditions (30-32). Furthermore, in secondary MLRs,Peck et al found an unexpected pattern of cross-reactivities by T-cell blasts which had been generated in MLRsbetween H-2 compatible, Mls-disparate strains (33). For example, B10.BR(H-2k, Mlsb) T cells selected by initial stimulation with CBA/J splenocytes responded not only to CBA/J but also to AKR/J (H-2k, Mlsa), C3H/HeJ(H-2k, MlsC), DBA/2(H-2d, Mlsa) and C57BL/6 (H-2b, Mlsb. Molnar-Kimber& Sprent also demonstrated that bulk cultures of C3H/HeJ(Mls~) lymph node cells cultured with CBA/J(H-2k, Mlsd) or AKR/Cum(H-2k, Mlsa) responded equally to stimulators from either strain (34). Therefore, it has been suggested by someinvestigators that the Mls locus may encode nonpolymorphic cell surface determinants and that different strengths of stimulatory effect mayreflect only quantitative difference in expression of the samedeterminant by strains differing in Mls type (35). Similarly, Molnar-Kimber& Sprent have suggested that the Mls locus has only two alleles: the aid allele causing strong MLRand the null b allele (32). However,in contrast, Click et al have reported that Mlsc cells stimulated Mls~ responding T cells almost as well as Mls~ cells stimulated Mlsc T cells, and that even Mlsb could stimulate Mls~ and MlsdTcells (36). From these experimental results, they proposed that the products of the different Mls alleles consist of commonframeworkdeterminants and variable determinants specific for each allele (36).

Annual Reviews T-CELL RECOGNITIONOF MLS GENEPRODUCTS687

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Recent

Analysis

of Polymorphism

Among Mls Determinants

A more definitive analysis of the presence or absence of polymorphism amongMls determinants required the availability of monospecific typing reagents. Since, as is discussed below, there are no serologic reagents available for the study of Mls, the use ofT-cell clones specific for putatively different Mls products has played a critical role in recent analysis of this system. A number of laboratories have succeeded in generating cloned T-cell populations specific for Mls~ determinants by positive selection in vitro through stimulation with M/s~-expressing cells and by subsequent limiting dilution cloning. The determination that such clones are indeed specific for Mlsa has generally been based upon the patterns of clone responses to BXDor AKXL RI strains of previously established Mls type (12, 29, 37, 38). A number of T-cell clones with established Mlsa specificity were found to exhibit consistent and strong cross-reactivity to Mlsd (CBA/J) stimulators (12, 29, 32, 37, 39), suggesting that Mlsa and Mlsd maynot be independentor distinct sets of determinants (32). These findings challenged the originally proposed polymorphismof the Mls system. A systematic attempt to analyze Mls polymorphism by clonal analysis was recently carried out by Abe and his colleagues (reviewed in 40). B10.BR(H-2k, Mlsb) T-cell clones were generated by repeated stimulation in vitro with H-2 identical, Mls-disparate stimulators, AKR/J(Mls~), C3H/HeJ(MlsC), and CBA/J(Mlsd), and by subsequent cloning. The MI~ specificity of AKR/J-specific clones was confirmed using BXDRI strains. Establishing the Mlsc specificity of C3H/HeJ-specific clones was more complicated, since unlike Mls~, the MlsC gene has not yet been formally mapped.Therefore, Mls~ specificities of T-cell clones were determined by comparison of their pattern of proliferative responses to the primary anti-Mls ~ responses of unprimed T cells to different stimulators. These comparisons were carried out using stimulators from standard inbred strains, BXHRI strains [established from B6 (Mls~) and C3H/HeJ(MlsC)], and (AKR/J x C3H/HeJ)F~ x AKR/J [(Mls ~ x MlsC)F~ x Mls~] backcross mice. Since the response patterns of unprimed heterogeneous AKR/J (Mlsa) T cells and C3H/HeJreactive cloned T cells to these stimulators were identical, it was concludedthat C3H/HeJ-reactiveclones were in fact Mist-specific. Once the Mls~ and Mls~ specificities of these clones were established, the response patterns of these clones to Mls-disparate stimulators were studied to clarify the polymorphism of Mls determinants. Several findings emergedfrom these studies: (i) No-M/s~-specific clones reacted to Mlsc, and no MlsC-specific clones reacted to Mlsa, demonstrating that MI~ or Mls~ determinants recognized by T cells are antigenically

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688

ABE& I-lODES

distinct (29). (ii) All M/sa-specific and Mist-specific clones responded as strongly to Mlsa (CBA/J)stimulators as to their primary stimulators. Thus, Mlsd cells coexpress Mlsa and Mls~ determinants (11, 29). (iii) Clones generated by stimulation with CBA/J(Mlsa) stimulators fell into two categories based upon their cross-reactive responses to AKR/J(Mls") and C3H/HeJ(MlsC). One group was reactive to AKR/Jand another to C3H/HeJ; none were reactive to both; all AKR/Jcross-reactivity was shownto be Mls~-specific, and all C3H/HeJreactivity was MlsC-specific (11). These results were consistent with the conclusion that Mlsa cells express both MI~ and Mlsc determinants. A similar interpretation was proposed by Ryan et al based upon the pattern of primary MLRs(14). The fact that there was no clearcut primary MLR between (AKR/J× C3H/HeJ)F~(Mls~/~) T cells and CBA/J(Ml, 3) s timulators ( 11, 14, 16) further suggested that Mls~ cells maynot express any unique Mls determinants, and that Mls~ may not be an independent Mls type but only the sum of Mls" and Mlsc determinants. Anti-Mls a responses would thus appear to be composedof clonally distinct anti-Mls ~ and Mls~ specific responses (11). The availability of Mls~-specific and MlsC-specific T-cell clones also permitted the direct assessment of the expression of Mls~ or Mlsc determinants on multiple strains. In addition, recently accumulated knowledge about characteristics of Mls-specific T-cell responses (discussed in detail below) improved the sensitivity of Mls-specific primary MLRs.Under these conditions, a reappraisal of the Mls typing of inbred strains has been carried out (13, 15, 17; R. Abe, unpublisheddata), and the results of such analysis are summarized in Table 1. Although it has been demonstrated that spleen cells from a numberof strains originally described as cnon-Mls in fact express Mls~ determinants, original phenotypic designations (3, 4) are used in the text of this review for the sake of simplicity. Analysis

of Allelism

in the Mls System

As noted above, the initial formulation of the Mls system proposed a single locus multiallelic system (3). However,this concept was not based upon c, any direct analyses of the allelic relationship of stimulatory Mls~, Mls and Mlsa determinants. Recently, formal segregation analyses were carried out to test these relationships (11, 12). Segregation analysis of the genes encoding Mls~ and Mlsc was carried out employing progeny of an (AKR/J × C3H/HeJ)F1 × B10.BR [(Mls~ x MlsC)F1× Mls~] breeding (l 1). Offspring were typed for expression of Mls~ and Mlsc using the responses of both cloned T cells and primary MLR.Approximately 50% of offspring were either Mls~ or Mlsc in phenotype. However, 50%either coexpressed both Mls~ and Mlsc deter-

Annual Reviews T-CELL RECOGNITION OF MLS GENE PRODUCTS Table 1 Strain distribution

689

of Mlsa and Mlsc determinants Expression of Mls determinants #

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Strain AKR/J BALB.K B10.BR C3H/HeJ C58/J CBA/J CBA/CaH CE/J HRS/J MA/MyJ MRL/MpJ RF/J A/J C57BL/6 LP/J BALB/c DBA/2 NZB LT/Ch SEC ST/6J D BA/1 SJL/J PL/J SM/J

H-2 genotype k k k k k k k k k k k k a b b d d d d d d q s u v

Original Mls + type

~ Mls

~ Mls

+ + + + + + --+ + + + + + + +

+ + + + + +* + + _+ +_ + + + + + + +

a b b c a d b ? ? ? ? ? c b ? b a a ? ? ? a c x a

÷ Ref. 4, 5. # Results are basedon Ref. 13, 15, 17, and R. Abeunpublisheddata. * __. = weakexpression.

minants or expressed neither. From these results, it was concluded that the genes encoding Mls~ in AKR/Jand Mlsc in C3H/HeJwere not allelic as had originally been proposed. In fact, the Mls" and Mls~ genes were neither allelic nor linked (11). Since it had been shown that the Mlsd strain CBA/Jcoexpressed both Mls" and Mlsc determinants, it was next asked whether there exists a single gene encoding an Mlsd product (that bears both Mls~-like and Mls~-like determinants) or whether the Mlsd phenotype reflects the independent expression by CBA/Jcells of two different genes, one of them encoding Mls~ and the other encoding Mls~ determinants (12). The results obtained from segregation analysis using (CBA/J x B10.BR)F~x B10.BR [(Mlsd x Mlsb) x Mlsb] progeny indicated that the Mls"-like and Mls~-like

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products expressed by CBA/Jwere in fact encoded by distinct and unlinked genes. These observations strongly suggested that Mlsd does not represent an independent Mls genotype and further challenged the originally proposed allelism of the Mls genetic system.

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New Genetic

Model of the Mls System

The recent findings summarizedabove suggest a significant alteration of the original model of Mls genetics. What were originally defined as Mlsspecific T-cell responses nowappear to consist of responses to determinants controlled by at least two independentgene loci (Table 2). One set of these determinants is controlled by a gene located on the Mls locus mappedto chromosome1, and this gene product is expressed on Mls~ and Mlsa cells. Another determinant, designated as Mlsc, is expressed on Mlsc- and dMls type cells; the gene(s) encoding these determinants have not been mapped and their genetic organization has yet to be clarified. Preliminary results obtained from segregation analysis suggest that multiple loci may be involved in Mlsc expression (R. Abe, unpublished data). A proposed model of the Mls system is shown in Table 2. Tentatively, the locus encoding Mls~ is designated as locus 1 and the locus (or loci) encoding Mlsc is designated as locus 2. Each locus has a stimulatory allele (Mls~ or c, Mls respectively) and an apparently nonstimulatory or null allele. No clear evidence to date indicates that structural polymorphismexists within either the Mls~ or the Mls~ system. The allele of the gene encoding Mlsa on chromosome1 was originally described as Mlsb (4, 26), and most (but not all; 36, 41) experimental systems, the Mls~ phenotype is "non-stimulatory" to other Mls types (5, 18, 31,42). Therefore it is possible that the allelic forms of Mls~ or Mls~ are genes which encode nonstimulatory products or that stimulatory genes are simply deleted. A prototype of this circumstance is provided by the failure of somemousestrains

Table 2 Mls genetics of prototypic strains MIs determinants encoded by

Strain B10.BR AKR/J C3H/HeJ CBA/J

Conventional Mls type b a c d

Locus 1 (Chromosome 1) Nonstimulatory a Nonstimulatory a

Locus 2 (Unknown) Nonstimulatory Nonstimulatory c c

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T-CELL RECOGNITION OF MLS GENE PRODUCTS

691

to express the MHC class-II E~ gene and the consequent failure to express a cell surface EctEfl molecule.In this instance, it is possible to generatea oneway MLRin which T cells from an E0~-non-expressing strain responded to stimulators from an E~-expressing strain, but no MLRcan be generated in the reverse direction (23). However, it should be noted that some investigators have concluded that Mls~ is not a null phenotype. Click et al reported that the product of Mlsb was easily detectable by Mls~ and dMls responding cells and therefore is not null (36). Waters et al reported that one T-cell clone recognized autologous determinants encoded by Mls~ (43). The reason for the discrepancy between these results and others is not certain. Another observation which suggested the existence of stimulatory Mlsb gene products was reported by Berumenet al (41). With use of the a which differs from BALB/cb) Mls congenic strain, BALB.D2-Mls (Mls a only in genes linked to Mls (44), it was found that T cells from BALB.D2Mls° mice which had previously been hyperimmunized to BALB/ccould be restimulated in vitro with BALB/cstimulators to proliferate and to becomecytolytic to BALB/ctargets (41). Howeverit is not clear whether this effect was caused by Mls~ per se or by some other minor antigen controlled by a gene closely linked to the Mls~ locus. PROPERTIES

OF MLS GENE

PRODUCTS

¯

Tissue Distribution Von Boehmer& Sprent first reported that B cells but not T cells are stimulatory for Mls responses (45). Subsequently, a number of investigators reported expression of Mls gene products on macrophages(46), Ia + splenic adherent cells and peritoneal cells or dendritic cells (47). Recently, several lines of evidence suggest that B cells are a major stimulator for Mls responses. For example, (i) Highly-purified populations B cells elicit strong Mls~ responses(48). (ii) Spleencells from micethat been selectively depleted of B cells by treatment with anti-IgM antibody are poor stimulators for anti-M/s ~ responses (S. R. Webb, personal communication). (iii) Anti-IgD antibody treatment in vivo activates B cells and concurrently increases the stimulatory ability of spleen cells for T-cell responses to both Mls~ and Mlsc (42, 49). Mls gene products are not detectable serologically, and the identification of these products is completely dependent upon the measurementof T-cell responses to Mls disparities. This limitation creates a potential difficulty in the determination of tissue distribution of Mls. For example, the stimulatory ability of a given cell population is not necessarily dependent upon only the expression of Mls determinants but may be influenced by other factors, such as the degree of Ia expressionor other aspects of overall

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T-cell stimulatory capacity. The ability of mitomycinC-treated, but not irradiated B cells to stimulate an Mls-specific responseefficiently illustrates the importance of such considerations (48). For this reason, the generally accepted notion that T cells do not express Mls determinants maynot be strictly correct, since resting murine T cells do not express Ia and are generally not good APC.In fact, it has been observed that T cells from an Mls~ strain stimulate M/s~-specific T-cell clones in the presence of ~ Mls APC,presumably reflecting presentation of T cell~ierived Mlsa-product by the Mlsb APC(R. Abe, unpublished data). The issue of tissue distribution of Mls gene products thus remains controversial. Attempts

at the Serologic

Identification

of Mls Determinants

It might be expected that allogeneic determinants which are recognized by and whichstimulate a high proportion ofT cells could also stimulate B cells to produce specific antibody. Initial attempts to makeantibody against the Mls gene product used immunization of BALB/c(H-2d, Mlsb) mice with DBA/2(H-2d, Mls~) cells (50). The sera from these immunizedmice were found to react with a subpopulation of DBA/2lymphocytes, but the gene controlling the determinants recognized by these sera segregated independently from the gene encoding Mls~ determinants, and the sera failed to block a BALB/canti-DBA/2 MLR(50). Later, Tonkonogy Winnreported that the gene locus encoding the alloantigen designated LyM-1, defined by a C3Hanti-CBA/J antiserum, was linked to the Mls locus (51). Subsequently, Dickler et al reported that antisera from the same strain combination inhibited the binding of Ig complexes to B-cell Fc receptors and that determinants recognized by these antisera were controlled by a gene closely linked to Mls locus (52). Morerecently, close genetic linkage was reported between the Mls locus and the gent cncoding the alloantigens Lyl7 (53) and Lyre20 (54), which were independently identified by alloantiserum or monoclonal antibody. Subsequently, it was shown that anti-LyM-l, anti-LylT, and anti-Lym20 antibodies in fact are all specific for polymorphicdeterminants on the Fcy receptor of mouse B cells and monocytes (55, 56). Based on the strong linkage between the locus encoding Fcy receptor and the Mls locus, as well as the apparently similar tissue distributions of these two gene products, it has been suggested that the Mls gene product is the Fcy receptor (57). However, recombinations have now been demonstrated between the genes encoding Mls~ and the Fcy receptor, in studies in which the Fcy receptor has been identified either by serological analysis (58) or by restriction fragment length polymorphism(59). It therefore appears that the Mlsa gene is distinct from the gene encoding the Fcy receptor. The demonstration by Nicolas et al that Fc receptor expression can be dissociated from ~ Mls

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T-CELL

RECOGNITION

OF MLS GENRE PRODUCTS

693

stimulatory ability in a cloned cell line is also consistent with this conclusion (60). It is not clear whyit has not yet been possible to generate or identify antibody specific for Mls gene products, despite the fact that these products seem to be extraordinarily strong antigenic determinants for T cells. One possibility is that antibody can be (and perhaps has been) generated, but that because of the absence of an appropriate assay system, the existence of this antibody has not yet been identified. For example, if the cells expressing Mls gene products were a sufficiently small proportion of the cell populations used to assay antibody reactivity, cytotoxic tests or staining experiments would not allow antibody detection. Recently, cloned cell lines have been identified which express Mls determinants capable of stimulating T cells (60, R. Abe, unpublished data). The availability such lines mayassist in the generation and identification of Mls-specific antibodies. The failure to identify Mls-specific antibodies by assays which involve the blocking of Mls-specific T-cell proliferation maybe explained by the nature of T-cell recognition of Mls determinants. If Mls-specific T cells recognize Mls gene products as peptide antigens presented by APC, then the failure of "anti-Mls" antibodies to block Mls-specific T-cell responses is not surprising, since in general, antibodies against conventional foreign antigens do not block antigen-specific T-cell responses. Twoobservations suggesting this concept have been reported: Berumenet al showedthat cell-free culture supernatant of peritoneal adherent cells from Mls~ strains elicited a proliferative response of H-2 compatible bMls T cells (61). Taking advantage of the strong influence of MHCgene products on stimulator cells for Mlsa-specific T-cell responses (discussed in detail below), DeKruyffdemonstrated that although neither Mls~ cells of a nonpermissive H-2q hapiotype nor Mls~ kcells of a permissive H-2 haplotype were able to stimulate Mls~-reactive T-cell clones, a mixture of these two stimulator populations did stimulate cloned T cells (62). The same phenomenonhas been observed in responses of Mist-specific cloned T cells (R. Abe, unpublished data). These findings indicate that Mls reactive T cells can recognize Mls gene products contributed by one cell in association with MHC products expressed on another cell, and they raise the possibility that an Mls product can be processed and presented in association with la by APC,muchas has been shownfor defined peptide antigens. A second explanation for the failure to identify Mls-specific antibody is that Mls gene products in fact do not stimulate B cells to makeantibody. Several mechanismsmight underlie such B-cell unresponsiveness: (i) cells specific for Mls mightbe deleted in the process of tolerization to selfantigens. This might occur if, as earlier proposed by Katz & Janeway, all

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mouse strains express nonpolymorphic cell surface Mls antigens, and if different Mls types represent only quantitative differences in the expression of these gene products (35). (ii) There mayexist a suppressive mechanism which interferes with the antibody response to Mls gene products. It has been reported that preimmunization ofMls~ mice with strongly stimulatory Mls~ or Mlsd cells results in the suppression of either Mls-specific responses or nonspecific T-cell responses (61, 63). It has also been shownthat preimmunizationof mice with Mls~ or Mlsd cells protects against lethal graftvs-host (GVH) reactions caused by either H-2- or non-H-2-incompatibilities (64) and that similar preimmunization prevents in vivo generation of either allogeneic or antigen-specific CTL(65, 66). These downregulatory phenomenaare thought to be mediated by suppressor T cells (63, 64, 66). If such suppressor T cells are routinely generated by immunization of mice with cells expressing strongly stimulatory Mls determinants, these suppressor T cells mayinterfere with the efficient B-cell responses to Mls determinants. (iii) Mls genes maynot encode any surface structure but instead mayencode non-cell-surface molecules which influence the overall efficiency of interaction betweenAPCand T cells. If this were the case, attempts to generate and detect antibodies against Mls products might be extremely difficult. In such an event, it is possible that molecular biologic approaches involving the isolation and expression of putative Mls genes will be necessary for an eventual characterization of the Mls gene product. Functional

Properties

of Mls Products

Although Mls determinants share with MHCgene products the capacity to induce strong proliferative responses by unprimedT cells, Mls products appear to differ from conventional MHC products in a number of in vivo and in vitro functional properties. IN VIVOEFFECTS In early studies, it was shownthat skin grafts from Mlsincompatible backcross mice were rejected by parental mice in marginally but not significantly shorter time than grafts from Mls-compatible backcross mice (67). Since the strains used in these experiments differed numerousnon-MHC genes, it was difficult to evaluate the role ofMls gene ~, was made products per se. Later, an Mls congenic strain, BALB.D2-M/s a ~ by introducing Mls of DBA/2origin into the Mls strain BALB/c(44). The results obtained from experiments using these Mls congenic strains indicated that Mls gene products mayserve as transplantation antigens (41). Mls~, which is nonstimulatory in primary MLR,induced more rapid graft rejection [100% rejection, mean rejection time (MRT)37.5 days] than did Mls~ (33% rejection on day 200), which is strongly stimulatory

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T-CELL RECOGNITION OF MLS GENE PRODUCTS

695

in primary MLR (41). The possibility remains, however,that the genes responsiblefor this skin graft rejection are not the Mlsgenesbut are closely linked minorhistocompatibility genes. Althoughthe in vivo GVH reaction is thought in general to closely parallel the in vitro MLR (8), Mls determinantsappear unable to induce lethal GVHreaction or splenomegaly(68, 69). To the contrary, preimmunizationwith Mls~ or Mlsd cells wasfound to result in protection from the lethal GVH caused by other non-H-2or H-2 incompatibilities (64). Similar immunosuppressive effects inducedby preimmunizationwith strongly stimulatoryMls~ or Mlsd cells havealso beenobservedin several other systems. It has been reported by a numberof groups, using combinations of MHC-identical,Mls-disparateinbred strains (3) as well Mls congenicstrains (41, 61) that preimmunizationof Mlsb strains with Mls~ or Mlsd diminishesthe subsequentin vitro proliferative responsesof T cells from recipient mice to Mls~ or/and Mls~ gene products. In vivo suppressiveeffects inducedby preinjection with Mls incompatiblespleen cells were also observedin the generation of CTLagainst allogeneic MHC or "self+X" determinants (65, 66). Recently, Chow& Battisto analyzed the mechanismof this down-regulationof CTLgeneration and suggested that it is mediatedby suppressorT cells, macrophages, and their factors (66). rN VITRO EFFECTS WhileMls gene products are strong stimulatory signals for proliferation in naive T-cell populations, it has generally not been possible to generateCTLspecific for Mlsproductsby primarysensitization in vitro (70, 71). Oneinterpretation of this phenomenon is that Mls gene products can not be CTLtargets. However,evidence supporting the idea that Mls products can serve as CTLtargets has been reported. Macphail et al observed strong primary BALB/c(H-2d, Mls~) d, anti-DBA/2(H-2 Mlsa) CTLresponsesunder limiting dilution conditions but not at higher concentrationsof respondingT cells (72). Theyinterpreted this observation as indicating that these cultures inducednot only CTLbut also suppressor T cells that abrogate the generation of CTL(72). In contrast to primary CTLresponses, it is well knownthat hyperimmunization allows the generation of CTLagainst multiple minorantigens (73). Therefore,the identification of Mls specificity of CTLwhichhavebeen generatedby repeated stimulation in combinationsinvolvingmultiple genetic differences is complicated. However,using the Mls congenicstrain combinationdescribed ~, Berumenet al detected strong antiabove, BALB/cand BALB.D2-MIs a mice hyperimmunized with Misb CTLactivity from BALB.D2-MIs BALB/c (41). Rapidrejection of skin grafts in this combinationparalleled CTLgeneration. Mls~-specific CTLresponses by cells from hyperimmunizedmice have not yet been reported.

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Twosets of cytotoxic T-cell clones with possible specificity for stimula÷ (74) and another tory Mls gene products have been reported. One is CD8 is CD4÷CD8(75). Braciale & Braciale reported that H-2d restricted ÷ CTLclones are stimulated to proliferate influenza virus-specific CD8 with uninfected CBA/J(Mlsd), DBA/2(Mlsa), and NZB(Mls~) splenocytes in the presence of IL-2 (74). Althoughthese clones failed to kill Mlsa or Mlsd target cells, it could be argued that the Con A-activated lymphoblast targets used in their experiments might not be appropriate, since Ia molecules which are not expressed on these cells are knownto be important for Mls + recognition (see above). Katz et al presented evidence that CD4 Mlsa-specific clones have bystander killing effects presumably mediated via the lymphokine production induced by stimulation with Mls gene products (75). In the presence of AKR/J(Mls~) but not B10.BRb) (Mls stimulator cells, these clones kill certain tumortargets, although the biological meaningof this bystander killing is as yet obscure. Katz et al also demonstrated that, when mitomycin C-treated, the same Mls~ specific Tcell ,clones stimulated B cells of Mls~ or Mlsd but not Mls~ origin to proliferate and differentiate into antibody secreting cells (75). Theseresults indicate that Mls gene products expressed on B cells mayplay a role in T helper cell-dependent B-cell activation. Both MHCand Mls products can be recognized by murine T cells as allogeneic (non-self) determinants. In addition, syngeneic MHCdeterminants play a critical role as "restricting elements" in T-cell recognition of conventional foreign antigens. The absence of a similar role for syngeneic (self) Mls determinants appears to be a major difference between the functional roles played by MHC and Mls products in T-cell activation. However,Waters et al have reported a single instance of apparent T-cell recognition of antigen in association with syngeneic Mlsb determinants (43). T-CELL

RECOGNITION

OF

MLS

DETERMINANTS

The most prominent property of the Mls system is the extraordinarily high precursor frequency ofT cells reactive to Mls gene products (6, 7). Janeway et al first demonstratedthat the precursor frequency of T cells reactive to Mls is as high as or higher than the frequency of T cells reactive to allogeneic MHCproducts, with 1/300 normal splenic T cells capable of proliferating in response to Mls~ determinants (6). Later, Miller &Stutman ~ observed that 1/62 to 1/120 splenocytes secrete IL-2 in response to Mls gene products (7). The high precursor frequency of T cells recognizing Mls determinants has generated a great deal of speculation about the mechanismof T-cell recognition of Mls gene products.

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The Role of MHCGene Products Responses

in Mls-specific

697

T-cell

In general, non-MHC alloantigens are recognized by T cells in the context of self-MHCgene products, just as are nominal antigens. To elucidate the mechanismof T-cell recognition of Mls products, the role of MHCgene products in Mls-specific T-cell responses has been extensively investigated. A strong influence of MHCon responses to Mls was first reported by Peck et al (76) who showedthat the secondary proliferative response heterogeneous BI0.A (H-2~, Mlsb) or B10.BR(H-2k, Mls~) T cells which had previously been stimulated in vitro with A/J (H-2a, Mlsc) or C3H/HeJ (H-2k, MlsC), respectively, was affected by the H-2 type of the Mlsc cells used in the secondary challenge (76). Using linear regression analysis primary Mlsc responses of F~ T cells, Gress et al similarly described that T cells recognize Mlsc in conjunction with MHC gene products (77). Later, it was shownthat the magnitude of responses of unprimed heterogeneous (49) and cloned T-cell populations (78) Mlsc determinants is str ongly dependent on the 1-1-2 haplotype, in particular on the presence or absence of I-E molecules expressed on stimulator ceils (49, 78). In Mlsa-specific responses, using BUdRand light suicide or F~ into parent radiation bone-marrow chimeras, Janeway et al suggested that primary Mls~-specific responses involve recognition of self-MHC gene products (6). In contrast, Molnar-Kimber & Sprent demonstrated that negative selection of T cells to Mls~ determinants on H-2-incompatible ceils removedT-cell reactivity to Mls"-bearing H-2-compatiblecells (34). They also showedthat T cells primed in vitro or in vivo to Mlsa on H-2compatible cells gave high secondary responses to Mlsa presented either on H-2-compatible or H-2-incompatible stimulator cells. From these data they concluded that T cells can recognize Mls~ in conjunction with not only self but also allogeneic H-2 determinants (34). This concept was confirmed by the fact that Mls"-specific T-cell clones respond to MHCincompatible Mls" stimulators (12, 29, 32, 37, 39). However,similar to the Mlsc responses, it has been observed that the magnitude of both primary and cloned T-cell responses to Mlsa is influenced by the stimulator cell MHChaplotypes (37, 38, 49, 79). For example, Mlsa cells expressing the a, k, or d H-2 types are usually highly stimulatory, whereasan H-2b strain b is intermediate (79), and the H-2q haplotype is low or such as AKR-H-2 nonstimulatory (38, 79). In addition, several laboratories have reported that primary and cloned T-cell responses to Mls gene products are inhibitable with both anti-I-A and anti-I-E antibodies (6, 27, 78, 80, 81). Based on these observations, it has been suggested that T cells recognize Mlsgene products in conjunction with Ia determinants which are expressed by many,but not all, mousestrains (27, 38, 39).

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Mls Recognition by Subsets of T Cells At present, it is generally accepted that Mls-reactive T cells belong to the +, CD8-subset (6). This concept is based largely on two observations: CD4 (i) In primary Mls-specific T-cell responses, deletion of CD8+ T cells has minimal effect on response (6), whereas adding anti-CD4 antibody completely inhibits T-cell responses (27). (ii) A high percentage, up to + helper 30%(R. Abe, unpublished observation) of antigen-specific CD4 T-cell clones are cross-reactive to Mls gene products. Only one example + CTLclone apparently specific for Mlsa has been reported (74). of a CD8 + cells are preferentially specific for class-II products (82), the Since CD4 + T cells in Mls recognition mayreflect predominant involvement of CD4 the demonstratedrole ofclass-II products in these responses. Alternatively the accessory function of CD4may play some other critical role in Mls reactivity.

Preferential Expression of T-Cell Receptor Vfl Segments by Mls-Reactive T Cells Direct involvement of the T-cell receptor (TCR)~/3 dimer in Mls recognition was first suggested by the blocking of anti-Mls responses with antibody specific for the TCRfl chain (27), although this observation could also be explained by direct negative signaling to the T cell by this antibody (83). The recent demonstration of strong .associations between the expression of certain TCRVfl segments by T cells and the Mls reactivities of these cells has provided convincing evidence for a critical role of the TCRin Mls recognition. Kappler et al found that almost 80%of the Vfl8.1 positive T-cell hybridomas which had been selected with antibody against Vfl8 products were reactive to MlsL In contrast, only 13%of Vfl8.2 positive T-cell hybridomas were Mls~-reactive (19). Similarly, MacDonaldet al reported that vitro selection of BALB/c(H-2a, Mls~) a, T cells with either DBA/2(H-2 ~ (H-2a, Mls~) stimulators resulted Mlsa) or Mls congenic BALB.D2.Mls in an increase in the proportion of Vfl6+ T cells in the population to 65-80%(20). In both cases, the strong relationship between Mls~ reactivity and T-cell receptor gene usage was reinforced by the finding that peripheral T cells expressing the Vfl8.1 or Vfl6 gene segmentsare specifically deleted or markedly decreased in Mlsa-expressing inbred strains (including RI strains and Mls-congenics), and in F1 hybrids of Mlsa and non-M/s~ strains (19, 20). The expression of specific Vfl segments has also been shownto correlate with T-cell reactivity to Mlsc. In the studies of Abeet al (21), it was first

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T-CELL

RECOGNITION

OF MLS GENE PRODUCTS

699

observed that there is a striking degree of cross-reactivity betweenthe set of T cells specific for Mlsc and the set of T cells specific for pigeon cytochromec (cyt c) in association with E~kEflk. Since it had previously been demonstratedthat cyt c-specific T cells use a relatively limited set of TCRgenes (84), the possibility was suggested that M/f-specific T cells might similarly use selected receptor gene products. A survey of Mlsspecific T-cell clones revealed complete concordance between Mlsc reactivity and expression of V]/3 as detected by Northern .analysis (21). Observations reported by Fry & Matis reinforced this conclusion. They found ~ that although E~kE/~-restricted cyt c reactive T cells derived from the Mls strains BI0.A and B10.A(5R) are knownto use V/~3 predominantly cytspecific T cells from Mlsc positive strains and their F1 hybrids do not use V/~3 for cyt c recognition. Furthermore, they showed that V//3 mRNA is absent in lectin-stimulated peripheral T cells from strains expressing Mlsc (85). Using a specific antibody against the V/~3 product, Pullen et al have also recently identified a reduction in the numberof V/~3+ peripheral T cells in several Mlsc positive strains (22). They also showedthat most the T-cell hybridomasgenerated on the basis of positive selection of V]~3 expression respondedto Mlsc positive spleen cells (22). This apparent linkage betweenthe use of certain V//’s and reactivity to Mlsa or Mlsc strongly suggested that the g// receptor is directly involved in the T-cell recognition of Mls gene products. The available findings in fact suggest that expression of a particular V/~ gene is sufficient to confer Mls reactivity on a T cell, independent of other segmentsin the expressed TCR/~and ~ chains (19-22). This relationship between V// expression and Mls reactivity provides an explanation for the high precursor frequency of T cells specific for Mls~ or Mlsc, since, for example, 6-20%(20) of cells in various Mls~ negative strains express V/~6 and 5-8% of T cells express V/~8.1 (19). However,it should be noted that T cells which express neither V/~6 nor V/~8.1 have been shownto be Mls~-reactive (19, 86). These results indicate that other V/~ segmentor V~, V/~ combinationsmight serve for Mlsqrecognition. Mechanism of T-Cell

Recognition

of Mls

Although it appears that the TCR~/~ dimer is somehow involved in Mls-specific responses, the actual mechanismof Mls recognition remains uncertain. At least two models could account for a role of the TCR dimer in T-cell responses to Mls determinants. In the first model, the TCR wouldinteract directly with an Mls ligand, possibly in the form of an Mls peptide associated with the class-II MHCmolecules which appear to be critically involved in Mls recognition. Kappler et al have proposed that

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Annual Reviews 700 ABE& HODES Mls~ products maybe peptides involved in positive selection during T-cell development and that these peptides are preferentially associated with a relatively conserved site on MHCmolecules (19). These Mls~/MHC structures might have enhanced affinity for T-cell receptors, either for certain V/3 segments alone or for other combinations of ~ and/3 chains, that otherwise have only a weakaffinity for unmodified MHC.In a second modelof Mls recognition, a second T-cell receptor, specific for Mls gene products, is postulated (86, 87). In this model, Mls products are not cell surface antigens recognized in a conventional manner by the ~/3 T-cell receptor. Instead, the Mls product interacts with its unique receptor on the T cell, independent of any interactions between the conventional TCR and its antigenic ligands. Webbet al have provided evidence for the existence of such a unique T-cell recognition structure for MlsL They generated T-cell hybridomas that havedual reactivities to self plus X and to Mls", or triple reactivities to self plus X, allogeneic H-2, and Mls" (86, 87). T,a, king advantage the chromosomalinstability of T-cell hybridomas, they have monitored changes in the functional specificities of subclones and have correlated these changes with TCRexpression. Somehybrids selectively lost MI~ reactivity but retained their other reactivities, whereasothers showedthe converse pattern. In contrast, no hybrids showed any segregation between their self plus X reactivity and allogeneic H-2 reactivity. These findings suggested that Mls" recognition mayinvolve a receptor distinct from that used in conventional antigen recognition. However,Webbet al also found that hybridoma variants that were unresponsive to Mlsa had lost their original TCR~ chain. Based on these results, they proposed that the Mls-reactive T-cell response is triggered by two sets of receptor-ligand interactions: one is the interaction of the TCRwith its ligand, presumably a relatively nonpolymorphic MHCgene product expressed on syngeneic as well as some MHC-allogeneiccells; and the other is the interaction between a unique Mls receptor which is expressed on all T cells and its nonpolymorphicligand, the Mls gene product (87). According to this proposal, the two receptor-ligand interactions could be additive in their effects uponthe overall interaction betweenthe T cell and a stimulator cell. If the TCRexpressed by a given T cell has an affinity for self Ia, for example, whichis insufficient to mediate T cell-stimulator cell interaction and T-cell activation, the concomitant binding of Mls by its receptor mayallow sufficient cell interaction and T-cell proliferation. The role of the Mls receptor in this T-cell activation might be confined to enhancing the overall avidity of T cell-stimulator cell binding, or might involve a direct signaling function whichcontributes to T-cell proliferation. In the context of such a model, Mls-reactive T cells would be those cells

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T-CELL RECOGNITION OF MLS GENE PRODUCTS

701

which express TCRs(such as VflS.1 or Vfl6) which have an intermediate (sub-threshold) affinity for self Ia, and Mls nonreactive T cells wouldbe those with lower affinity for self Ia. Janeway et al have reported that APCfrom Mlsa mice present antigen more efficiently than do APCfrom Mlsb mice in the activation of antigenspecific T cells, suggesting that the binding ofMls~ to its receptor enhances the cell interaction mediated by the TCRand its MHC/Ag ligand (88). more recent report by Needlemanet al failed to confirm these findings (89). A related model has been proposed by Hammerlinget al who suggest that the interaction of T cells with Mls products has a generalized downregulatory effect on T-cell activation (90). The recent demonstration that Mls~ and Mlsc products are distinct determinants (29), and that different Vfl associations are found for Mlsa-reactive (19, 20) and Mist-reactive T cells (21, 22), must be accounted for in these models of Mls recognition. Thus, models which hypothesize the expression of unique (non-a//) receptors for Mls mayimply that all T cells simultaneously express an ~/3 TCR dimer, an Mls~ receptor, and a distinct Mls~ receptor. Tolerance

in the Mls System

The first series of experiments that directly addressed the issue of T-cell tolerance to Mls gene products was reported by Macphail et al and showed that neonatal injection of Mlsb mice with Mlsd cells caused long-lasting loss of anti-Mlsd responses (91). Limiting dilution analysis indicated that tolerance was specific, because the precursor frequency of Mlsd-reactive T a, cell decreased almost 300-fold, while responses to Con A, allogeneic H-2 and self-Ia were not affected. This specific tolerance was not caused by suppression but by clonal deletion or inactivation. Hosonoet al observed that injection of neonatal Mlsb mice with Mlsb/" bone marrowcells resulted in profound suppression of the host-vs-graft reaction of Mlsb host to ~ Mls cells as measuredin a popliteal lymphnode swelling assay (92). The strong correlation betweenMls reactivity and the expression of certain Vfl genes provided a unique opportunity to investigate the process of tolerance induction to these self-antigens. Based upon the demonstrated correlation between expression of Mls~ and deletion of Vfl6+ T cells in a series of inbred strains (discussed in more detail below), MacDonaldet al studied the T cells from Mlsb mice that had been neonatally injected with ~ Mls cells (93). T cells from these mice were not reactive to Mls~ stimulators; concurrently, they showeda selective loss of Vfl6-positive cells. This result indicated that neonatally induced tolerance to allogeneic Mls~, as well as physiologic tolerance to self M/s~, is mediatedby clonal deletion of reactive T cells (or by specific inhibition of TCRexpression by these cells). Evidence suggesting that deletion of self Mls-reactive T cells occurred

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intrathymicallywasfirst presentedby Morrisseyet al (94) in experiments employingirradiation and bone marrowreconstitution of mice that had beenthymectomized and engrafted with allogeneic thymuses.T cells within the thymuswerenot tolerant to Mls alloantigens that were expressedonly extrathymically,but T cells weretolerant to the Mlsalloantigensexpressed on thymicelements. Intrathymic elimination of self-Mls-reactive T cells wasfurther demonstratedby the disappearanceof Vfl8.1 ÷ or Vfl6+ T-cell populations of "mature" thymocytes.Kappleret al showedthat Vfl8.1 is expressedequivalently on immature(low TCRdensity) thymocytesin both Mlsa positive and Mlsa negative strains, but that expressionof Vfl8.1 on mature (high TCRdensity) thymocytes is muchlower in Mlsa positive than in Mlsa negative strains (19). Thesefindings were interpreted indicate that tolerance to self Mls~ occursby the deletion of Mls~-reactive Tcells in the thymusat a point in differentiation prior to expressionof the "mature" T-cell phenotype. MacDonaldet al also demonstrated that Vfl6 expressingT cells weredeleted in the cortisone-resistant thymocyte populationsof Mls~ positive strains (20) and in Mls~ micewhichhad been renderedtolerant to Mls~ by neonatalinjection of Mls~ cells (93). + T cells are principally responAsdescribedabove,it appearsthat CD4 + and CD8 +T sible for Mls responses (6, 27). Nevertheless, both CD4 a cells expressingV/~8.1or V/36are deletedin Mls positive strains (19, 20). + as well as CD4 + T cells may Oneexplanationfor this finding is that CD8 a be able to recognize Mls under certain conditions such as those which occur in vivo duringthe inductionof self-tolerance. Self-tolerance to Mls - and could then involve the independent deletion of both CD4+CD8 + CD4-CD8 T cells. Alternatively, tolerance induction to self-Mls deter+ and minants mayoccur at the level of a common precursor of both CD4 + + + CD8 cells, e.g. the immatureCD4 CD8 population (20). It should noted that muchof the information which has been accumulated concerning tolerance to Mls" parallels closely the data obtainedby Marrack, Kappler,and their colleaguesin studies of V/317aexpressionandtolerance to class-II MHC determinants(95, 96). CONCLUDING

REMARKS

Recent studies of the Mls system have generated prominentadvancesin two areas. Thefirst of these has beenthe reappraisal of Mls determinants and of the genetics of the Mls system. It nowappears that there are at least two unlinked Mls loci whichencoderespectively the antigenically distinct Mls~ and Mlsc determinants. The independentexpressionof these two determinantsprovides a logical explanationfor the uniquepattern of Mls-specific MLRs observedbetweenstrains (see Figure 1). Thesefindings

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T-CELL RECOGNITION OF MLSGENEPRODUCTS 703 establish that determinants recognized by naive T cells at extremely high precursor frequencies are encoded in at least three genetic regions: the MHC,the locus encoding Mls~, and the locus encoding c. Mis The second area of intense recent activity has involved the identification of strong associations between T-cell specificity for Mlsa or Mlsc and the expression of specific TCRVfl segments(Table 3). In fact, it appears that expression of a given Vfl is sufficient to confer Mls reactivity on a T cell, independent of which other a and fl chain segments are expressed by that cell. This finding parallels that recently reported for T-cell recognition of the E~tEfl class-II MHCproduct (see Table 3) and stands very much contrast to T-cell recognition of conventional foreign antigens, in which multiple regions of the TCR~ and fl chains appear to contribute to TCR specificity. The high precursor frequency of T cells reactive to Mls~ or to Mls~ (as well as to E~Efldeterminants) maytherefore be explained, at least in part, by the adequacy of selective Vfl expression (which occurs at reasonably high precursor frequency) to determine Mls specificity. Althoughthese findings indicate a critical role of the TCR~fl dimer in the overall process of Mls recognition, it is unclear whether this receptor interacts directly with an Mls product or whether a unique Mls-specific receptor (distinct from the ~//dimer) exists. A great deal remains to be determined concerning the structural identity of the Mls products and the role they play in T-cell activation or in the overall selection of the antigen-specific T-cell repertoire. It has been speculated that Mls products may be peptides that associate with MHC molecules and play a critical role in positive as well as negative selection of T cells during intrathymic differentiation. It has also been suggested that the role of Mls is to enhance the interaction between T cells and stimulator (or target) cells by a mechanismthat may not involve any specific affinity of the ~fl TCRfor Mls. The final clarification of these important issues will require the isolation and structural characterization of Mls products, and studies of the interactions between these products and defined T-cell receptor structures. Table3 Preferentialexpression of T-cellreceptorVfl segments byalloreactiveT cells Reactivity of Tcell

Vfl segmentusage

References

aMls

Vfl8.1 Vfl6 Vfl3 Vfll7a Vfll1

19 20 21, 22 95 (O. Kanagawa, personalcommunication)

Mlsc F_aE/~

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Todate, no evidenceindicates the existence of Mls-equivalentgene productsin species other than the mouse.Therefore,one mustquestion whetherthe Mls phenomenon is an anomalyof the murinesystem, not of generalizedimportance in otherspecies. However, it shouldbe notedthat detectionof Mlsdeterminantsis at presentdependent uponthe existence of polymorphism in Mlsexpressionwithina species. Thatis, if all members of a species expressa givenMlsproduct,its existencewouldnot be detected by available assays, despite the fact that this productmightsubservea biologically importantfunction. Theexpressionof Mlscdeterminantsby the vast majorityof inbredmousestrains (see Table1) approaches this circumstance;in the absenceof at least one Mls~-negativestrain, the existence of this productwouldnot havebeenevident. Oncethe murine Mlsgenesandtheir productshavebeenidentified, alternativestrategies will allowa better assessmentof the role whichsimilar productsmayplay in otherspecies, includingthe human. ACKNOWLEDGMENTS

The authors wish to thank Drs. W. Biddison and J. J. Ryanfor their critical reviewsof this manuscript. Literature Cited 1. Wilson, D. B., Blythe, J. L., Nowell, P. C. 1968. Quantitative studies on the mixedlymphocytereaction. III. Kinetics of the response. J. Exp. Med. 128: 115781 2. Ryser, J. E., MacDonald, H. R. 1979. Limiting .dilution analysis of alloantigen-reactive lymphocytes. I. Comparison of precursor frequencies for proliferative and cytolytic responses. J. Immunol. 122:1691-96 3. Festenstein, H. 1973. Immunogenetic and biological aspects of in vitro lymphocyte allotransformation (MLR) the mouse. Transplant. Rev. 15:62-88 4. Festenstein, H. 1974. Pertinent features of .M locus determinants including revised nomenclature and strain distribution. Transplantation 18:555-57 5. Festenstein, H. 1976. The Mls system. Transplant. Rev. 8:339~,2 6. Janeway, C. A. Jr., Lerner, E. A., Jason, J. M., Jones, B. 1980. T lymphocytes responding to Mls-locus antigens are Lyt-l+,2 and I-A restricted. Immuno9enetics 10:481-97 7. Miller, R. A., Stutman, O. 1982. Enumeration of IL2-secreting helper T cells by limiting dilution analysis, and dem-

8.

9.

10.

I1.

12.

13.

onstration of unexpectedly high levels of IL2 production per responding cell. J. Immunol. 128:2258-64 Sachs, D. H. 1984. The major histocompatibility complex. In Fundamental Immunology, ed. W. E. Paul, pp. 30346. New York: Raven Dutton, R. W. 1966. Spleen cell proliferation in response to homologous antigens studied in confenic resistant strains of mice. J. Exp. Med.1966. 123: 665-71 Festenstein, H. 1966. Antigenic strength investigated by mixedcultures of allogeneic mouse spleen cells. Ann. N.Y. Acad. Sci. 129:567-72 Abe, R., Ryan, J. J., Hodes, R. J. 1987. Mls is not a single gene, allelic system. Different stimulatory Mls determinants are the products of at least two nonallelic, unlinked genes. J. Exp. Med. 166: 115(~55 Abe, R., Ryan, J. J., Hodes, R. J. 1987. Clonal analysis of the Mls system. A reappraisal of polymorphismand allelism a~nongMls~, Mls~, and Mlsd. J. Exp. Med. 165:1113-29 Abe, R., Hodes, R. J. 1988. The expression of Mlsc determinants on

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