Annual Review of Immunology Volume 18 2000

Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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813 829

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Annu. Rev. Immunol. 2000. 18:1–17 Copyright q 2000 by Annual Reviews. All rights reserved

DISCOVERING THE ROLE OF THE MAJOR HISTOCOMPATIBILITY COMPLEX IN THE IMMUNE RESPONSE Hugh O. McDevitt Departments of Microbiology and Immunology and Medicine, Stanford University School of Medicine, Stanford, California 94305; e-mail: [email protected]

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Key Words immunogenetics, class II MHC, autoimmunity, genetic susceptibility, polymorphism Abstract The discovery that genes in the major histocompatibility complex (MHC) play an important role in the immune response depended on the chance interaction of several unrelated events. The first, and most important, was the decision by Michael Sela to synthesize a series of branched, multichain, synthetic polypeptides based on a backbone of poly-l-lysine. The prototype compound, (T,G)-A–L, was tipped with short random sequences of tyrosine and glutamic acid. This resulted in a restricted range of antigenic determinants composed of only two or three amino acids with a variable length—ideal for binding to the peptide binding groove of MHC class II molecules. The second was the decision by John Humphrey to immunize various strains of rabbits with this synthetic polypeptide. Two of these rabbit strains showed very large quantitative differences in antibody response to (T,G)-A–L. In transferring this system to inbred mouse strains, the third bit of good fortune was the availability at the National Institute of Medical Research, in Mill Hill (London), of the CBA (H2k) and C57 (H2b) strains. The H2b haplotype is the only one mediating a uniform high antibody response to (T,G)-A–L. The fourth critical ingredient was the availability of numerous congenic and H2 recombinant inbred strains of mice produced earlier by Snell, Stimpfling, Shreffler, and Klein. A search for congenic pairs of mice expressing the responder and nonresponder H2 haplotypes on the same background revealed that these strains responded as a function of their H2 haplotype, not of their inbred background. Extensive studies in a variety of inbred strains carrying recombinant H2 haplotypes, as well as a four-point linkage cross, mapped immune response to (T,G)A–L within the murine MHC, between the K and Ss loci. The demonstration that stimulation in the mixed lymphocyte reaction (MLR) mapped to the same region quickly led to attempts to produce antisera in congenic H2 recombinant strain combinations. These antisera identified I-region associated (Ia) antigens. Immunoprecipitation and blocking studies showed that the gene products controlling specific immune responses, the mixed lymphocyte reaction, and the structure of Ia antigens were one and the same—now designated as the I-A MHC class II molecules. These antisera and inbred strains enabled Unanue to demonstrate the peptide binding function of class II MHC molecules. 0732–0582/00/0410–0001$14.00

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BEGINNINGS—‘‘as the twig is bent—’’ Chance plays a greater part in our lives than most of us are willing to admit. My father was born in Cincinnati, Ohio, in 1888, into a first generation Irish Catholic family with some pretensions, largely on my grandmother’s part, to have made the transition from shanty Irish to ‘‘lace curtain’’ Irish. My grandfather, who ran a large drayage business in Cincinnati, was a convivial Irishman devoted to trotting horses, the Democratic party, and Irish camaraderie. He could, however, be a stern parent. After graduating from St. Xavier High School, my father enrolled as a freshman at St. Xavier University in Cincinnati. Quite early in his college career, he encountered a Jesuit priest who stated flatly that Darwin’s theory of evolution was wrong. This led to a heated discussion, a threat of corporal punishment by the priest, and forcible physical resistance by my father. Naturally enough for those times, the incident resulted in his expulsion from the University. My grandfather’s response was to conclude that my father’s chance for higher education was at an end. Through friends in the Democratic party (which was then in power in Cincinnati), he found my father a position as a rodman on a surveying gang, laying out the north part of Vine Street. During his year in that position, my father noticed the sad and empty expressions on many of the workers on the surveying team, and the way they looked forward to a lunch box containing a thermos filled with whiskey-laced coffee. The experience stimulated him to look about for other opportunities for further education. At that time, it was possible to enroll in medical school directly out of high school, and accordingly (with his mother’s support), he enrolled in the University of Cincinnati College of Medicine in 1907, graduating in 1911. In medical school, he was exposed to Ehrlich’s side chain theory, which at the time had attracted wide attention and interest (1). Much later, working as a urologist treating syphilis and other venereal diseases, he became acquainted with the uses of arsphenamine and 606 to treat syphilis, and with Ehrlich’s concept of finding ‘‘magic bullet’’ drugs, which would selectively bind to ‘‘side chains’’ on the spirochete and destroy it, while failing to bind to ‘‘side chains’’ on host tissues. As a young child of 8 or 9, I would often sit on our front porch with my father, watching late afternoon summer thunderstorms pass over our small village just outside of Cincinnati. During those discussions, my father would sometimes talk of his tremendous admiration for Ehrlich, the side chain theory, and the many drugs he developed for the treatment of syphilis. He was particularly amused by the fact that Ehrlich allegedly flunked his histology course in medical school but went on to develop many tissue-specific stains and to become an outstanding research scientist. When I asked my father to explain Ehrlich’s side chain theory, he would answer that that was very difficult, but I would understand it once I was in medical school. (Having failed to force my two older brothers to pursue science in high school, or to apply themselves to premedical studies, he switched

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to furthering his desire to have one of his sons become a physician by indirection. He succeeded in making medicine and research seem like a wonderful career.) These early influences were reinforced by my father’s announcement, while I was in the third grade, that I no longer had to attend church on Sunday (a duty that held little appeal for me). Instead, I was invited to accompany him into the city as he made his rounds at various hospitals, following which we would pick up a quart of ice cream for Sunday dinner at the city’s leading candy and confectionery store. In the summer time, this routine was sometimes changed, and my mother and several of my four older siblings would join us for a steak lunch at Mecklenburg’s German Beer Garden. Mecklenburg’s had a large outdoor dining area covered with a beautiful grape arbor. To me, it seemed like an enchanted garden. The result of all this was that I came to view a career in medicine as fascinating, fun, and certainly worth the effort. During my undergraduate years at Stanford, two experiences made a possible career in research both more appealing and more concrete. The first was a course in evolution taught by Richard Goldschmidt, a visiting professor from the University of California at Berkeley. Goldschmidt had developed a theory of evolution that he termed macroevolution (2, 3). He presented his theory at the end of a fascinating series of lectures reviewing the evidence then available in support of Darwin’s original theory. The second experience was the opportunity to do independent research in genetics under the supervision of Raymond Barratt in the department of biology. The Stanford Department of Biology in the 1940s and 1950s had become prominent due to the work of Beadle and Tatum, who used UV radiation-induced mutants of Neurospora to map out basic biosynthetic pathways. My project was to map the adenineless locus in the first Neurospora linkage group, using a four-point mapping cross. The results were inconclusive because the loci chosen were too far away from the adenineless locus. However, the techniques and concepts were identical to those used to map the Ir-1 gene (controlling the immune response to a synthetic polypeptide antigen), after linkage between that gene and the major histocompatibility complex had been established (see below). These two experiences strengthened an interest in research, but research itself was put aside for several years during medical school, internship, residency, and service in the army, which was then required by the doctor’s draft law and the Berry plan.

THE FATE OF ANTIGEN AND GENETIC CONTROL OF THE IMMUNE RESPONSE Following two years of service as an ‘‘obligated volunteer’’ in the Army Medical Corps, I spent two years learning the fundamentals of immunology under the supervision of Albert Coons, who had invented and perfected the fluorescent

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antibody technique (4). Looking back now, it requires a major shift in mental perspective to recall how very rudimentary our knowledge of immunology and the mechanics of the immune response was in 1959, at the start of this first postdoctoral fellowship. A 1960 paper by John Humphrey in the Proceedings of the first Antibody Workshop (5) caught my attention. In this paper, Humphrey proposed to test the instructive theory of antibody formation, originally put forward by Pauling and Haurowitz (6). This theory in its simplest form postulated that the polypeptide comprising the antibody molecule was folded around the antigen as a template in the antibody producing cell. Coons and others had shown that antibody producing plasma cells contained and secreted large numbers of antibody molecules. If the instructive theory was correct, this would require the presence of appreciable amounts of antigen in the antibody producing cell. Humphrey proposed to utilize a synthetic polypeptide antigen developed by Michael Sela (7) to obtain a radioactive polypeptide with high specific activity. The antigen, (T,G)-A-L, is a branched multichain synthetic polypeptide with a backbone of poly-L-lysine, side chains of poly-D,L-alanine, and short, random sequences of tyrosine and glutamic acid at the ends of the side chains. Humphrey predicted that by synthesizing this peptide with dehydroalanine, and then reducing the double bonds with tritium gas, it should be possible to detect a very small number of (T,G)-A-L molecules in a single antibody-producing cell (Figure 1). If no antigen were to be found in an antibody-producing cell, it followed that antibody structure must be a function of primary amino acid sequence and that the selective theory of antibody formation (8–10) must be correct. It occurred to me that it might be possible to combine immunofluorescence with autoradiography to detect radioactive (T,G)-A-L in specific antibody-producing plasma cells.

Figure 1 A schematic diagram of the structural pattern of the multichain synthetic polypeptide antigens used in this study. The numbers designate different preparations of each polypeptide.

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The proposal appealed to John Humphrey, and he accepted me for a two-year postdoctoral fellowship in his laboratory at the National Institute for Medical Research at Mill Hill, outside of London. In carrying out these studies, I was fortunate to enjoy the supervision of Brigitte Askonas as well as John Humphrey. Both taught me a great deal about scientific rigor. We found that there was no detectable antigen in specific antibody producing cells, but that is a separate story (11). After settling in at Mill Hill, my first assignment was to produce a reliable, high-titered antiserum specific for (T,G)-A-L. John Humphrey had already begun this task by immunizing several rabbits of the Sandylop strain with (T, G)-A-L in complete Freund’s adjuvant. However, the antibody response to this polypeptide antigen was almost undetectable. John wrote to Michael Sela saying that (T,G)-A-L appeared to be a poor antigen. Michael replied, with some heat, that it was a perfectly good antigen in his rabbits at the Weizmann Institute for Science in Rehovot, Israel. This remark stimulated John Humphrey to test other strains of rabbits, and accordingly, he had already immunized three strains of rabbits that were not inbred but did breed true for coat color, size, and a few other characteristics. The three strains were the original Sandylop strain, the Dutch strain, and the Himalayan (or Californian) strain. (This was the first of several instances in this experimental saga in which a good idea combined with exceptionally good luck to point the way to the next step.) After some false starts, I decided to do antigen-antibody precipitin curves on the sera from the three strains of rabbits. The results were quite striking. Himalayans uniformly produced 1–1.2 milligrams of antibody/ml of serum following immunization with (T,G)-A-L. Sandylops, on the other hand, produced only 30–50 micrograms of antibody, while Dutch rabbits were variable and produced antibody titers intermediate between these two extremes. The possibility that there might be inherited differences in the ability to produce antibody, or to make an immune response to a specific antigen, was very exciting. I proposed to John Humphrey that we attempt to test this possibility by crossing Sandylops with Himalayans, producing F1s, and then backcrossing the F1s to the two parental strains. Although there was not sufficient animal space to carry out this experiment at Mill Hill, John Humphrey negotiated with Ashley Miles, the head of the Lister Institute, to let us use two large rabbit rooms at the Institute’s animal facilities in Elstree, not far from Mill Hill. When the crosses and backcrosses were complete, immunization of all of the different offspring with (T,G)-A-L showed a clear genetic effect, but with a very complex inheritance pattern. Since these rabbit strains were not inbred, this could have been due to genetic heterogeneity in the parental strains, or to the fact that the trait under examination was determined by multiple genes. The logical next step was to attempt to find the same genetic difference in ability to respond to (T,G)-A-L in inbred animals—the obvious choice being inbred mice. At that time, the only inbred mouse strains available at Mill Hill

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were CBA and (CBA 2 C57 BL/6) F1. Mice of the parental C57 BL/6 strain were only available through Avrion Mitchison, who very kindly provided several animals for immunization as well as for production of F1 and F1 2 parent backcross animals. This was a second stroke of luck. We now know that uniform high response to (T, G)-A-L is seen only in mice expressing the H2b haplotype, such as C57BL/6, while mice of the H2a,k,u,s,f,q haplotypes are low responders. Immunization of CBA, C57, the F1, and the two backcross populations with an optimal dose of (T,G)-A-L in complete Freund’s adjuvant revealed a clear dominant inheritance of ability to respond. CBAs were very low responders, C57s were high responders, and the F1 was intermediate. The two parental backcrosses were low and intermediate for F1 2 CBA and intermediate or high in the F1 2 C57 population (12). In discussing these results with John Humphrey and Michael Sela, I wondered if it might be possible to test the specificity of this genetic control of the immune response by immunizing the same strains with related synthetic polypeptide antigens that had a different amino acid composition. I was delighted to learn from Michael Sela that, as a part of his study on the nature of antigenicity, he had also synthesized the same type of branched synthetic polypeptide antigen in which tyrosine was replaced by either histidine or phenylalanine—to produce (H,G)-A-L and (Phe, G)-A-L. Immunization of these same strains of mice with (H,G)-A-L revealed exactly the opposite pattern—CBA mice were high responders, C57 mice were low responders, and the ability to respond was inherited as a dominant trait (7, 13). These results showed that the phenotype of this particular form of genetic control of the immune response was codominant and specific for the amino acid composition of the antigenic deterimants in the immunizing antigen. However, the mechanism of this genetic control, and the gene or genes, if this were to be a polygenic trait, were a complete mystery. The next several years were the ‘‘wilderness years,’’ devoted to testing various possibilities for the mechanism of this genetic control, combined with efforts to establish linkage of this genetic trait with known genes in the murine linkage map. Numerous mechanisms and linkages were tested, with mostly negative results. The metabolism and degradation of (T, G)-A-L was the same in CBA and C57, and no linkage could be established with immunoglobulin allotype, or with the isotype of specific anti-(T, G)-A-L antibody, coat color, or other easily scored genetic traits (14). In fact, there was no direct evidence to show that this difference in antibody response was due to a gene expressed in immunocompetent cells. It seemed equally possible that this genetic trait could be the indirect result of some gene not directly involved in antibody formation. Michael Edidin, then a postdoctoral fellow in Coons’s lab at Harvard, suggested that it might be possible to transfer the ability to respond from a high responder strain into a low responder strain by transplantation of high responder bone marrow into irradiated low responder recipients. Unfortunately, these experiments, utilizing either neonatal or adult recipients, gave variable and inconclusive results because of the frequent occurrence of graft vs. host disease and/or rejection of the bone marrow transplant.

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DISCOVERY OF LINKAGE WITH THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) In the fall of 1966, I took up a position as an assistant professor in the division of immunology in the Department of Medicine at Stanford University School of Medicine. I quickly became acquainted with Leonard and Leonore Herzenberg in the Department of Genetics. Our discussions covered a wide range of subjects, and I outlined to them how I hoped to show that this genetic control of a specific immune response was expressed in immunocompetent cells by transferring (CBA 2 C57) F1 spleen cells into irradiated CBA low-responder parental recipients. At that time, Len and Lee were also interacting frequently with Marvin Tyan at the Hunter’s Point naval radiation research laboratory. Marv Tyan joined in many of these discussions and offered to carry out the cell transfer experiments, since cell and bone marrow transplantation in mice was his main research interest. Accordingly, I supplied him with F1 and CBA mice to carry out the cell transfers. The results showed quite clearly that transfer of F1 responder spleen cells into irradiated low responder parental recipients transferred the ability to respond to (T,G)-A-L. This clearly established that this genetic control was transferable with immunocompetent cells, although it did not establish whether the gene(s) responsible was expressed exclusively in this cell population. While these experiments were going on, I continued to search for a means of establishing homozygous high responder lymphocyte populations in homozygous low responder recipients. As in the earlier experiments with Michael Edidin, this was difficult because CBA (H2K) and C57 (H2b) differ at the major histocompatibility complex. In one of these discussions, Lee Herzenberg mentioned that congenic strains were available from the Jackson Laboratories in Bar Harbor, Maine. These inbred congenic strains were homozygous for several different H2 haplotypes on several different inbred strain backgrounds. Thus, C3H mice were available with their original H2k MHC haplotype, or as C3H.SW mice, which are congenic with C3H except at the MHC, H2b. (See Table 1.) It was a simple matter to determine that C3H mice like CBAs were low responders, while C57 BL/10 (B10) mice (H2b) were high responders to (T,G)-A-L. Three sets of congenic strain pairs were ordered from the Jackson Laboratories (Table 1) and immunized TABLE 1 Strain

H-2 Type

C3H C3H.SW

k b

A A.BY

a b

B10 B10.BR

b k

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with (T,G)-A-L. This experiment was done simply to establish that both of the C3H strains and both the A strains were low responders, and that both the B10 strains were high responders to (T,G)-A-L. The results of this experiment were exactly the opposite of that predicted (15): C3H.SW, A.BY, and B10 were all high responders, while C3H, A, and B10.BR were all low responders. Thus, these three congenic pairs responded to (T,G)-AL as a function of their H2 haplotype and not as a function of their inbred strain background. These results clearly implied that genetic control of the immune response to (T,G)-A-L and (H,G)-A-L was linked to the major histocompatibility complex. This was quickly confirmed by formal linkage analysis of a cross between CBA and C57 mice. The offspring of a cross between F1 high responder and the CBA low responder parent showed a clear segregation into high responders and low responders, and high response segregated almost perfectly with the H2b haplotype (15). (Progeny testing of the few H2b low responders showed that their offspring were 50% high responders and that the first immunophenotyping was incorrect.) This result, presented at several meetings and seminars in the fall of 1967, stimulated great interest on the part of George Snell, and considerable skepticism on the part of some of the other workers in the H2 field. One criticism was to the effect that the gene had to be located somewhere on the murine linkage map, and it just happened to be linked to the MHC. Snell offered to help in any way he could, and he pointed out that Donald Shreffler, Jan Klein, and Jack Stimpfling (each of whom was more than generous with their mice and reagents) had produced a number of congenic mouse strains on the B10 and A backgrounds that were homozygous for crossover events within the murine MHC (see Figure 2).

THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX IN 1968 It was immediately apparent that mapping the gene or genes determining genetic control of this specific immune response offered the most direct means of ultimately identifying the gene or genes involved. Good fortune once again was at

Figure 2 The map of the murine MHC on chromosome 17. The only definitely established loci in the murine MHC in 1968 were the K and D loci encoding the serologically identified gene products (which are now known to be the two major class I MHC molecules), and the Ss locus, identified by a serological polymorphism in a serum protein, now known to be the fourth component of complement.

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hand, this time in the form of a large number of congenic strains of mice homozygous for crossover events known to have occurred either between the K and Ss loci or between the Ss and D loci. These various strain combinations are listed in Table 2. They had been produced over the preceding several years by Shreffler, Klein, and Stimpfling, with the goal of creating a series of crossover MHC haplotypes that could be used for further detailed mapping of the murine MHC. Two approaches were taken to determine the map position of the genetic control of the immune response to (T,G)-A-L and (H,G)-A-L. (Because this was the first genetic control of the immune response to be linked in the murine linkage map, it was given the gene designation Ir-1, for Immune response – 1.) The first approach utilized 11 inbred mouse strains and lines, which were homozygous for a known crossover event within the MHC. Nine of these H2 recombinants, with the position of the crossover event, are listed in Table 2. They are derived from crossovers between H2a and H2b, between H2k and H2d, between H2s and H2al, and between H2q and H2a. Immunization of these strains with (T,G)-A-L, (H,G)A-L, and (Phe,G)-A-L showed that 9 of the 11 strains responded as did the donor of the K locus end of the recombinant H2 chromosomes. This would map Ir-1 near or to the centromeric side of the K locus. However, two of these recombinants, H2tl and H2y, gave the opposite result; they responded as did the donor of the D locus end of the recombinant H2 chromosomes. Both of these latter two H2 recombinants arose from crossovers between the K and Ss loci. These results would be compatible either with the mapping of the Ir-1 gene within the MHC, between the K and Ss loci, or they could have been due to a double crossover event. However, the H2y recombinant was derived from a known single crossover event because of outside markers in the cross giving rise to this H2 recombinant. Thus, this result almost certainly mapped Ir-1 within the murine MHC, lying between the K and Ss loci. The results described above were confirmed by the second approach, utilizing a four-point mapping cross. This cross utilized a strain developed by Margaret TABLE 2 Strain

H2 Haplotype

B10.A (1R) B10.A (2R) B10.A (3R) B10.A (4R) B10.A (5R) H2al H2tl H2th AQR

h h i h i (not inbred) al (not inbred) tl (not inbred) th (not inbred) Y

Crossover at K-Ss

Crossover at Ss-D x x

x x x x x x x

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Green, carrying an H2q chromosome with additional markers approximately 10 centimorgans (brachyury) and 5 centimorgans (tufted) centromeric to the H2 complex. This mapping cross is diagrammed in Figure 3. Complete analysis of the offspring of this mapping cross required the testing of more than 1500 mice because the offspring of this cross were typed and confirmed by progeny testing. In 484 offspring, only 2 recombinants were detected. Both were confirmed by progeny testing, which showed that the crossover event had occurred within the H2 region itself. Thus, the two approaches were concordant in mapping Ir-1 within the murine MHC, between the K and Ss Loci (16) (Figure 3). The map position of the Ir-1 gene within the MHC suggested very strongly that the gene product, then completely unknown, might in some way be related to the known genes within the major histocompatibility complex. However, the striking antigenic specificity of the Ir-1 gene effect, combined with the recent demonstration by Mitchell and Miller (17, 18) that T cells had separate, distinct regulatory functions over B cells, raised the possibility, first put forward by Benacerraf, that the Ir-1 gene(s) might encode an antigen-specific receptor on T cells. I, and many others (see below), found this hypothesis very appealing, particularly because of the sharp discrimination of the Ir-1 genetic control, which could readily differentiate between (T,G)-A-L (H2b) and (Phe, G)-A-L. Mice carrying the H2q haplotype are capable of responding to (Phe, G)-A-L but do not respond to (T,G)-A-L, although the antibodies are quite cross-reactive. (At this point, a warning and a disclaimer about the accuracy of memory versus the written record is appropriate. For example, the description given above of the mapping of the Ir-1 gene is a reasonable statement of my memory of the process. In looking up the original papers on this subject, I was stunned to find a paper (19) presented at a 1969 symposium on immunogenetics of the H2 system, at Liblice, Czechoslovakia, in which I stated that the results to date indicated that Ir-1 was either within the H2 locus or near the left-hand margin and that ‘‘complete resolution may not be possible on the basis of the present experiments.’’ Continued analysis of that mapping cross ultimately produced the result described above, but I had little memory of my gloomy assessment of the progress of that experiment in late 1969. Later in this discussion, I mention one or two other instances in which my memory of the progression of thinking about these experiments and their implications does not completely fit the written record. I am now much more in agreement than I was previously with those historians of science who insist that it is only the written record of science that can be relied upon.)

FINDING THE IR-1 GENE PRODUCT In the spring of 1972, Maurice Landy enlisted my help in organizing the last of a series of five NIAID-supported symposia on immunology entitled ‘‘Genetic Control of Immune Responsiveness.’’ The symposium covered a wide variety of topics, with particular emphasis on MHC-linked genetic control of immune

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Figure 3 T is the short tail allele at the brachyury locus, while tf is the tufted phenotype allele at the tufted locus. These are linked to H2q and the mapping cross was carried out with an F1 between this linkage testing strain and the H2s haplotype from A.SW mice.

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responses, immunoglobulin allotype–linked immune response genes, and several related topics. In my memory, the most exciting new finding was the presentation by Fritz Bach and Jan Klein. These colleagues had noticed a 1971 study by Yunis & Amos (20) showing that ability to stimulate in a mixed lymphocyte reaction (MLR) was linked to HLA, but separable by recombination from the genes encoding HLA A and B in three families with one recombinant offspring in each family. Bach and Klein used mice carrying recombinant H2 haplotypes to search for the same phenomenon in the mouse. Their results showed that a gene or genes mapping in the same position as Ir-1 encoded structures on the surface of lymphocytes that were responsible for the strongest stimulation in the mixed lymphocyte culture reaction (MLR) (21). Since MLR stimulation involved a recognition by T cells of alloantigenic structures on other lymphocytes, the implication was clear that at least the structures responsible for stimulation in MLR were expressed on the surface of lymphocytes, and that it should therefore be possible to produce antisera to these molecules (later called Ia antigens) by immunizing one inbred strain with lymphocytes from another inbred strain that was genetically identical except for differences in the Ir-1 region of the MHC. (For example, A.TL and A.TH are two inbred strains with identical backgrounds and identical MHCs, except for Ir-1 and Ss). These MLR studies used many of the same H2 recombinant strains that had been used to map Ir-1 and raised the possibility that the Ir-1 gene product and the MLR stimulation gene product might be similar or identical gene products. In fact, a review of this study (21) again jogged my memory because I had forgotten that Bach and Klein had suggested that ‘‘it is possible that the Ir product is the T cell receptor and that it is this same molecule which can act as the stimulatory agent in MLC.’’ In any event, the identical map position of Ir-1 and MLR stimulation immediately stimulated several laboratories to attempt to produce antisera to the gene products of this region, by the method described above. There were two other very important findings presented at this conference. The first is the set of experiments by Gene Shearer and Edna Mozes using limiting dilution cell transfer techniques, which indicated that Ir-1 gene products are expressed on B cells. As is know well known, this ultimately proved to be correct, and my refusal to accept Shearer and Mozes’ results at face value, and my stubborn belief that Ir-1 encoded some form of T cell receptor, remain a source of embarrassment to this day. The Irish may be lucky, but they can also be stubborn (22). The other exciting new finding at this Brook Lodge conference was the report from Ira Green that guinea pig anti-MHC alloantisera inhibited antigen-induced proliferation of responder lymphocytes in an Ir gene–specific manner. Green also presented preliminary data which suggested that T cells could only respond to an Ir gene–controlled antigen when the antigen was presented by cells sharing the responder Ir genotype (22). These results are discussed further below. Immediately following the Brook Lodge conference in the fall of 1972, a number of laboratories undertook the production of antisera specific for I region

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gene products by immunizing in strain combinations, such as A.TL and A.TH, which differed only at the I region, or at the I region and the Ss locus. In my laboratory, these sera were produced by Beverly Deak and Gunter Hammerling. By the late spring of 1973, Gunter and Bev were finding that these antisera exhibited complement-mediated cytotoxicity primarily for B cells of the immunizing genotype. These results were contrary to the expectation of many of us, and Gunter and I had many extensive discussions about all of the proper controls that needed to be done. However, by the fall of 1973, the results showed beyond any doubt that anti-I region sera identified a polymorphic protein(s) that was expressed primarily on the surface of B cells and macrophages and was not expressed on T cells (23). Although both Shreffler and Klein’s initial results suggested that I region gene products were expressed on T cells, further testing, as well as the independent findings of David Sachs, confirmed that I region antigens were expressed primarily on B lymphocytes (see references in Reference 23). It is beyond the scope of this essay to describe the numerous experiments carried out over the next several years, which finally established beyond any doubt that the I region contains several genes encoding class II MHC molecules (also called Ia antigens) that are responsible for the observed genetic control of specific immune responses and for stimulation in the mixed lymphocyte culture reaction. Among the most notable findings in this regard were the results presented by Green at Brook Lodge (see above). These findings led to several studies by Shevach and Rosenthal (24, 25) showing that interaction between antigen-presenting macrophages and T lymphocytes occurred only when the two cell types were histocompatible for at least one MHC haplotype. When the antigen under study was also under Ir gene control, (responder 2 non-responder) F1, T cells could only respond when the antigen was presented on macrophages of responder genotype. Presentation of the antigen by nonresponder macrophages failed to stimulate F1 responder T cells (25). In a sense, these observations were ahead of their time. In retrospect, these observations, particularly the latter experiments (25), are a clear demonstration of the expression of Ir genes in macrophages and of MHC restriction on the ability of macrophages and T cells to interact. However, at the time these observations were made, the Ir gene products—the Ia antigens or class II MHC molecules—had not been described; little was known about the guinea pig MHC; and the necessary but complex terminology made it difficult to completely understand the now obvious implications of these experiments. An even earlier demonstration, using anti-H2 antisera, of the dependence of T-B cooperation upon products of the MHC was published by Kindred and Shreffler in 1972 (26). However, the antigenic system used was not one known to be under Ir gene control, and the full implication of this finding, as with the other findings noted above, was not widely appreciated. The clear evidence from my own and other laboratories that molecules encoded in the I region were expressed on B cells and macrophages was difficult to reconcile with an equally persuasive set of results, also from my laboratory, obtained

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by Katie Bechtol and John Freed using tetraparental embryo fusion chimeras (27), which indicated that Ir-1 was expressed in T cells and not in B cells. Resolution of this paradox came when Joan Press repeated these experiments using tetraparental bone marrow chimeras assayed by a plate-binding antibody assay (28). Antisera from both the tetraparental embryo fusion and bone marrow chimeras showed that almost all of the specific antibody was of responder allotype. Our resulting publication (28) was described by Jan Klein as the ‘‘Oops, we goofed’’ paper (29). The resolution of the tetraparental results, as well as findings from many other laboratories, finally established that the Ir-1 gene product, Ia antigens, and the stimulating molecules in the MLR are all one and the same entity—namely class II MHC molecules. However, the mechanism by which class II MHC molecules influenced the immune response to specific antigens remained a mystery for several more years. The solution to that mystery has required the contributions of a large number of laboratories, the cloning and sequencing of many class II MHC molecules by my own (30, 31) and many other laboratories, and most importantly, the demonstration by Unanue and colleagues that MHC class II molecules can bind a large number of different peptides 9 to 25 amino acids in length—which will undoubtedly soon be the subject of another essay in this series.

THE MHC AND DISEASE Long before class II MHC molecules were identified either in mouse or in human, it seemed reasonable to test the possibility that MHC genotype might play a role in susceptibility or resistance to infectious diseases or to autoimmune diseases. Because of my position in a Division of Immunology that was primarily concerned with rheumatologic diseases, and because of the striking autoimmunity manifested in systemic lupus erythematosus (SLE), it seemed reasonable to study the MHC genotypes in patients with this disease, compared to a normal control population. Fortune again smiled on this enterprise, because Stanford was also home at that time to Rose Payne and Julia and Walter Bodmer. Walter readily agreed to an exploratory study of HLA genotype in patients with SLE, and the study was accordingly carried out with the help of Carl Grumet. The results showed that patients with SLE had an increase in HLA B8 and B15 compared to the control population (32). These results stimulated us to publish a brief article describing the implications of these findings and suggesting that similar findings might be found in a number of autoimmune diseases (33). In the ensuing years, studies from many laboratories around the world revealed that susceptibility to most autoimmune diseases is determined in part by HLA genotype. Particularly strong associations between specific HLA haplotypes, and in some cases HLA sequence polymorphisms, and susceptibility to rheumatoid arthritis (34), Type I insulin-dependent diabetes mellitus (35, 36), and several other diseases were dis-

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covered. It is beyond the scope of this essay to describe these results in detail. It is now clear that most autoimmune diseases are polygenic traits in which MHC genotype, and in particular class II MHC genotype, can play a major role in some instances, or a relatively minor role in other instances. One of the most important challenges today is to understand the mechanism by which particular sequence polymorphisms mediate susceptibility or resistance to specific autoimmune diseases (37).

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ENDINGS—the twig is bent Preparing a reminiscence such as this has its good and bad sides. It has been wonderful fun to look up and reread some of the original studies that led to the discovery of linkage between genetic control of the ability to mount an antibody response to synthetic peptides and genes in the major histocompatibility complex. At the outset, the meaning of this genetic linkage was a mystery. Within a very few years, it became apparent that MHC genes play a major role in the development of the immune system and in the initiation of the cellular and humoral immune response. The progress that has been made in our understanding of the genetics, structure, and function of the MHC class I and class II molecules is awe-inspiring. All of us who have worked in this field over the years have been extremely fortunate to be a part of a series of major advances in our understanding of the immune system and of vertebrate biology. The bad side of preparing such a memoir lies in the similarity to preparing one’s own obituary. While it gives you an opportunity to put the best possible face on the matter, it does inspire some morbid thoughts—another notable trait of the Irish character. Fortunately, science in general, immunology in particular, and especially the study of the MHC continue to present us with mysteries and challenges that have yet to be met. The extraordinary degree of genetic polymorphism in MHC class I and class II genes, the many structurally similar genes with as yet undiscovered functions, the evolutionary origin of MHC genes, and the mechanisms by which MHC molecules determine disease susceptibility, and the ability to respond or not respond to particular peptides are all matters that have yet to be worked out in complete detail. And for many of these problems, the devil is in the details. One problem that continues to elude us is the understanding of the molecular mechanism by which MHC class I or class II sequence polymorphisms determine susceptibility to a wide variety of autoimmune diseases. That problem continues to tantalize, and there may yet be time to see it through to its solution. The twig, once bent, stays that way.

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LITERATURE CITED 1. Ehrlich P. 1900. The Croonian Lecture to the Royal Society. Proc. R. Soc. London 66:424 2. Goldschmidt R. 1940. The Material Basis of Evolution. New Haven, CT: Yale Univ. Press 3. Gould SJ. 1977. The return of hopeful monsters. Nat. Hist. 86:22–30 4. Coons AH, Creech HJ, Jones RN. 1941. Proc. Soc. Exp. Biol. Med. 47:200 5. Humphrey JH. 1960. Methods for detecting foreign antigen in cells, and their sensitivity. In Mechanisms of Antibody Formation. Proc. Symp., Prague, May 27–31, 1959. Prague: Czech. Acad. Sci. 6. Pauling L. 1940. J. Am. Chem. Soc. 62:2643 7. Sela M, Fuchs S, Arnon R. 1962. Studies on the chemical basis of the antigenicity of proteins 5. Synthesis and characterization of some multichain and linear polypeptides containing tyrosine. Biochem. J. 85:223 8. Jerne NK. 1955. The natural selection theory of antibody formation. Proc. Natl. Acad. Sci. USA 41:849 9. Talmage DW. 1959. Immunological specificity. Unique combinations of selected natural globulins provide an alternative to the classical concept. Science 129:1643 10. 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 11. McDevitt HO, Askonas BA, Humphrey JH, Schechter I, Sela M. 1966. The localization of antigen in relation to specific antibody-producing cells. I. Use of a synthetic polypeptide [(T,G)-A—L] labeled with iodine-125. Immunology 11(4): 337–51 12. McDevitt HO, Sela M. 1965. Genetic control of the antibody response. I. Demonstration of determinant-specific differ-

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ences in response to synthetic polypeptide antigens in two strains of inbred mice. J. Exp. Med. 122(3):517–31 McDevitt HO, Sela M. 1967. Genetic control of the antibody response. II. Further analysis of the specificity of determinant-specific control, and genetic analysis of the response to (H,G)-A–L in CBA and C57 mice. J. Exp. Med. 126(5):969–78 McDevitt HO. 1968. Genetic control of the antibody response. III. Qualitative and quantitative characterization of the antibody response to (T,G)-A–L in CBA and C57 mice. J. Immunol. 100(3):485– 92 McDevitt HO, Tyan ML. 1968. Genetic control of the antibody response in inbred mice: transfer of response by spleen cells and linkage to the major histocompatibility [H-2] locus. J. Exp. Med. 128(1):1–11 McDevitt HO, Deak BD, Shreffler DC, Klein J, Stimpfling JH, et al. 1972. Genetic control of the immune response. Mapping of the Ir-1 locus. J. Exp. Med. 135(6):1259–78 Miller JFAP, Mitchell GF. 1968. Cell to cell interaction in the immune response. I. Hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocites. J. Exp. Med. 128:801–20 Mitchell GF, Miller JFAP. 1968. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J. Exp. Med. 128:821–37 McDevitt HO, Shreffler DC, Snell GD, Stimpfling JH. 1970. Genetic control of the antibody response: genetic mapping studies of the linkage between the H-2 and Ir-l loci. In Immunogenetics of the H2 System, Proc. Symp., Liblice, Czech.,

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1970, ed. M Vojtiskova, A Lengerova, pp. 69–75. Basel, Switzerland: Karger Yunis EJ, Amos DB. 1971. Three closely linked genetic systems relevant to transplantation. Proc. Natl. Acad. Sci. USA 68:3031–35 Bach FH, Widmer MB, Bach ML, Klein J. 1972. Serologially defined and lymphocyte-defined components of the major histocompatibility complex in the mouse. J. Exp. Med. 136:1430–44 McDevitt HO, Landy M. 1972. Genetic Control of Immune Responsiveness. New York/London: Academic Hammerling GJ, Deak BD, Mauve G, Hammerling U, McDevitt HO. 1974. Blymphocyte alloantigens controlled by the I-region of the major histocompatibility complex in mice. Immunogenetics 1(1):68–81 Rosenthal AS, Shevach EM. 1973. Function of macroophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes. J. Exp. Med. 138:1194–1212 Shevach EM, Rosenthal AS. 1973. Function of macroophages in antigen recognition by guinea pig T lymphocytes. II. Role of the macrophage in the regulation of genetic control of the immune response. J. Exp. Med. 138:1213–29 Kindred B, Shereffler DC. 1972. H-2 dependence of cooperation between T and B cells in vivo. J. Immunol. 109:940 Bechtol KB, Freed JH, Herzenberg LA, McDevitt HO. 1974. Genetic control of the antibody response to polyL(Tyr,Glu)-poly-D,L-Ala—Lys in C3H in equilibrium with CWB tetraparental mice. J. Exp. Med. 140(6):1660–75 Press JL, McDevitt HO. 1977. Allotypespecific analysis of anti-(Tyr,Glu)-AlaLys antibodies produced by Ir-1A high

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and low responder chimeric mice. J. Exp. Med. 146(6): 1815–20 Klein J. 1986. Natural History of the Major Histocompatibility Complex. New York: Wiley. p. 542 Mathis DJ, Benoist CO, Williams VE II, Kanter MR, McDevitt HO. 1983. The murine Ea immune response gene. Cell 32(3):745–54 Benoist CO, Mathis DJ, Kanter MR, Williams VE II, McDevitt HO. 1983. Regions of allelic hypervariability in the murine Aa immune response gene. Cell 34(1):169–77 Grumet FC, Coukell A, Bodmer JG, Bodmer WF, McDevitt HO. 1971. Histocompatibility (HLA) antigens associated with systemic lupus erythematosus. A possible genetic predisposition to disease. N. Engl. J. Med. 285(4):193–96 McDevitt HO, Bodmer WF. 1972. Histocompatibility antigens, immune responsiveness and susceptibility to disease. Am. J. Med. 52(1):1–8 Nepom GT, Erlich H. 1991. MHC class II molecules and autoimmunity. Annu. Rev. Immunol. 9:493–525 Acha-Orbea H, McDevitt HO. 1987. The first external domain of the non-obese diabetic mouse classII I-Ab chain is unique. Proc. Natl. Acad. Sci. USA 84(8):2435–39 Todd JA, Bell JI, McDevitt HO. 1987. HLA-DQb gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 329(6140): 599–604 Chao CC, Sytwu HK, Chen EL, Toma J, McDevitt HO. 1999. The role of MHC class II molecules in susceptibility to type I diabetes—identification of peptide epitopes, and characterization of the T cell repertoire. Proc. Natl. Acad. Sci. USA 96:9299–9304

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Annu. Rev. Immunol. 2000. 18:19–51 Copyright q by Annual Reviews. All rights reserved

RECEPTOR SELECTION IN B AND T LYMPHOCYTES David Nemazee Department of Immunology, Scripps Research Institute, La Jolla, California 92037; e-mail: [email protected]

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Key Words immunoglobulin genes, T cell receptor, immune tolerance, receptor editing, gene rearrangement Abstract The process of clonal selection is a central feature of the immune system, but immune specificity is also regulated by receptor selection, in which the fate of a lymphocyte’s antigen receptor is uncoupled from that of the cell itself. Whereas clonal selection controls cell death or survival in response to antigen receptor signaling, receptor selection regulates the process of V(D)J recombination, which can alter or fix antigen receptor specificity. Receptor selection is carried out in both T and B cells and can occur at different stages of lymphocyte differentiation, in which it plays a key role in allelic exclusion, positive selection, receptor editing, and the diversification of the antigen receptor repertoire. Thus, the immune system takes advantage of its control of V(D)J recombination to modify antigen receptors in such a way that self/non-self discrimination is enhanced. New information about receptor editing in T cells and B-1 B cells is also discussed.

INTRODUCTION Lymphocytes often express a single antigen receptor despite their genetic potential to simultaneously express many. This fact has focused interest on how expression of multiple receptors is limited. It has recently come to light that cells bearing antigen receptors may actively attempt to express new ones, which can lead to expression of multiple receptors, inactivation of previously expressed receptors, or replacement of old receptors with new ones—a process called receptor editing. Receptor editing occurs through ongoing or renewed antigen receptor gene rearrangement. In this overview, we discuss features of both T cell receptor (TCR) and immunoglobulin (Ig) loci that promote secondary receptor gene rearrangements, and we review the evidence for receptor editing in B and T cells. Receptor editing has recently been the subject of several brief reviews (1–4).

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SECONDARY REARRANGEMENTS V(D)J recombinase rearranges and ligates together dispersed variable (V), diversity (D), and joining (J) minigene elements and brings the newly assembled V(D)J exon in proximity to the constant (C) exons of these genes (see accompanying article by Schatz, ‘‘Evolution and Mechanisms of VDJ Recombination,’’ pp. 495– 97). An important feature of some antigen receptor genes is the ability to undergo so-called secondary, or nested, rearrangements, which can salvage the functionality of loci with primary out-of-frame rearrangements and favor receptor editing, i.e., the modification of already functional genes. This process is affected by several factors including the number of gene segments in the locus, their organization along the DNA, and the organization and types of recombination signal sequence (RSSs) adjacent to the gene segments in question. Some typical organizations of receptor genes and their influence on secondary rearrangements are shown schematically in Figures 1 and 2. As we discuss below with respect to the well-studied mouse and human systems, some antigen receptor gene loci appear to be particularly specialized to carry out secondary rearrangements, whereas other loci may disfavor them. The organizations of mouse and human antigen receptor gene loci are shown schematically in Figure 3.

Loci That Favor Secondary Rearrangements Ig-j Rearrangements in the mouse involve initial joining of one of ;140 Vj elements to one of four functional Jj elements (5) (Figure 3A) (6–12); a similar organization is seen in humans, who have about 66 Vj elements and 5 Jjs (13) (Figure 3B) (14). The j locus lacks D gene segments; consequently, upon primary VjJj joining on one allele, secondary rearrangements between remaining upstream Vjs and downstream Jjs can occur in a single step (shown schematically in Figure 1B). In the mouse, Jj1 and Jj2 rearrangements are preferred (15), which retains downstream Js available for secondary rearrangement (16). Furthermore, because many Vj genes are placed in a transcriptional orientation opposite to the Jj elements, j loci often rearrange by inversion, retaining thereby the entire repertoire of Vjs for subsequent rearrangements (shown schematically in Figure 2B, bottom). When the V gene segments are in the same transcriptional orientation as the segments to which they rearrange, deletional rearrangements excise intervening DNA, which is permanently lost from the chromosome (Figure 2B, top). Analysis of the Ig-j loci in mouse or human B cell lines has shown that a single allele can undergo two or more successive V-J recombinations (16–23). Isolation of the excised circular DNA from sequences intervening in the V/J recombination revealed that they often contained independent V/J joins, indicating that secondary j rearrangements are common in the mouse splenic B cells analyzed (24, 25). B cells of mice lacking the j-locus on one chromosome have increased usage of downstream Jjs on the remaining allele, consistent with dis-

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Figure 1 Role of gene organization in facilitating or inhibiting receptor editing. V, D, and J coding elements are flanked by recombination signal sequences carrying 12-bp spacers (open triangles) or 23-bp spacers (black triangles) that constrain the range of possible rearrangements in cis. Recombinase joins elements with 12-bp spacers to those with 23 bp. Because of their overall organization, loci vary in their abilities to support receptor editing type rearrangements. (A) Cartoon of one type of gene organization similar to mouse and human Ig-H loci (see Figure 3 for details). The presence of D elements along with V genes in the same transcriptional orientation as the J/C cluster forces deletional rearrangements. Primary VDJ assembly cannot be replaced by recombination using conventional signal sequences. (B) In contrast, in loci without D elements, sequential rearrangements are often possible. In this example, a primary (10) V4-to-J join is replaced by a subsequent secondary (20) rearrangement between V2 and the downstream J. Such secondary rearrangement permits the replacement of potentially functional V4-to-J joins, i.e., receptor editing. This type of organization is also seen in mammalian TCRa loci.

placement of out-of-frame VJ joins by nested rearrangement (23). Thus, j locus structure permits replacement reactions, allowing receptor editing. TCRa At the TCRa locus, any one of about 100–200 Va segments can be joined to one of 50 Ja elements (Figure 3A, B) (26–29). A primary VJ rearrangement may yield an out-of-frame join that can be replaced by subsequent rearrangement of an upstream Va segment with one of the remaining downstream Ja loci (30). T cells may undergo many nested Va-Ja recombinations (31, 32). In addition, thymocytes rearranging TCRa loci retain excised DNA circles containing the TCRd locus, usually in a rearranged form (30, 33). Since these episomal DNAs are lost with cell proliferation (30, 34, 35), TCRa rearrangements

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Figure 2 Gene organizations that inhibit or facilitate receptor editing. (A) Cluster type receptor gene organization is used in many lower vertebrates and is retained in certain mammalian receptor gene loci, such as mouse Ig-k. Rearrangements occur within clusters but not between adjoining clusters, preventing editing and potentially posing problems for isotype exclusion. (B) Inversional rearrangements are dictated by gene orientation. Variable gene segments in inverted transcriptional orientations relative to J/C clusters are indicated by upside-down Vs. Such elements join though inversion rather than excision of intervening DNA. Hypothetical V3 and V4 elements must undergo deletion during primary rearrangement to Js, whereas V1 and V2 elements rearrange by inversion. Note that subsequent secondary rearrangement can again occur through either deletion or inversion, but inversional rearrangements retain more V genes and change the orientations of V elements intervening the break points.

functionally exclude TCRd expression. Furthermore, TCRa rearrangements favor initial use of Ja’s at the 58 end of the locus (36), possibly suggesting progressive use over time of 58-to-38 Ja’s. The TCRa locus organization constrains rearrangement to occur by deletion rather than inversion (27) (shown schematically in Figure 1B) (28, 29).

Loci That Disfavor Secondary Rearrangements Ig-H In contrast to the Ig-j and TCRa loci, some loci are so constructed that they tend to suppress secondary rearrangements, at least through conventional recombination signal sequences. VH genes are arranged in the same transcriptional orientation as the D and J elements (37, 38), forcing VDJ assembly at the Ig-H locus to occur by deletion of the intervening DNA (38) (Figure 3). As a consequence, correction of nonfunctional VDJ rearrangements by secondary rearrangement is prevented because no more D regions are available for recombination to upstream VHs. While unrecombined upstream VHs and downstream JHs are typically retained, they both have recombination signal sequences carrying

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Figure 3 Antigen receptor loci of (A) mouse and (B) human. Note that Ig-j, TCR-a, TCR-d, and TCR-b have structures that are compatible with secondary, replacement rearrangements in both mouse and human. Conventional V(D)J recombination disfavors receptor editing at the Ig-H locus of mouse or human because of the 12/23 rule and the arrangement of VH elements in the same transcriptional orientation as the JH/CH cluster. In the mouse Ig-k and TCR-c loci, functionally rearranged genes cannot efficiently be altered by secondary rearrangements because of their cluster type organization, whereas editing is possible in the human versions of these loci provided that the 38 most J’s are not initially used. In the TCR-d locus of both mouse and human, TCR-a rearrangements exclude TCR-d expression.

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23bp spacers, and therefore they cannot be joined together by recombinase (see Figure 1A). Despite this, secondary rearrangements can sometimes occur on the Ig-H locus. Early in mouse B cell development, secondary D-to-J joining can replace primary DH-to-JH rearrangements by deletion or inversion (39), but this apparently does not occur in humans (40). Furthermore, VH elements can sometimes undergo secondary recombination to preformed VDJ elements through VH replacement by targeting noncanonical, but conserved, RSS-like sequences that are present in the 38 end of the rearranged VH coding sequence (41). VH replacement is discussed in a later section. Ig-L k Loci The lambda locus in the laboratory mouse is composed of two linked miniclusters spanning about 200 kb (42–47). Vk2, Vkx/Jk2 Ck2 lies upstream of Vk1/Jk3-Ck3, Jk1–Ck1, but most rearrangements occur between V and J regions of the same cluster (Figure 3A). All rearrangements involve DNA deletions. The recombined Vk1-Jk1-Ck1 gene is the most common type of k rearrangement (48). Thus, combinatorial diversity is extremely limited in the mouse k locus, and receptor editing is precluded (shown schematically in Figure 2A). The human k-locus has an array of ;70 Vk genes upstream of seven JkCk clusters; each J-C cluster has a single J, and only three of the clusters are functional (Figure 3B) (49). Unlike in the mouse k-locus, this organization allows nested recombination and receptor editing (50).

Suppressive Rearrangements RS/kde An additional mechanism facilitated by receptor gene organization is the ability to preclude function of certain loci by nested rearrangements that can themselves have no functional product. One important example of a rearranging, but noncoding, gene element that illustrates this mechanism is the RS/kde element (‘‘recombining sequence’’ in mouse, ‘‘kappa deleting element’’ in human) (51). This element inactivates the Ig-j locus by deletional recombination (52). RS is found ;25 kb downstream of the mouse Cj exon (53) (Figure 3A). (RS should not be confused with the RSS, recombination signal sequence, carried by all rearranging receptor genes). The kde is in a similar position in the human j-locus (54) (Figure 3B). Both RS and kde contain an RSS sequence with a 23-bp spacer that can recombine through a V(D)J recombinase–dependent process either to unrearranged Vjs or to noncanonical RSS sites in the Jj-Cj intron (25, 51, 55). RS/kde rearrangements occur in ;75% of mouse B cells that express k L-chain (56) and in an even higher fraction of human k-producing cells (13, 57). RS rearrangements also occur on the second allele of approximately 12% of mouse j-expressing cells (58). Because the RS/kde encodes no protein, these joins are nonfunctional (55, 59). It appears that the RS/kde elements have no other purpose than to inactivate j-genes, many of which were previously functional (57). Since RS rearrangement appears concurrently with Ig-k locus recombination, they may

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be predicted to clear the way for k expression (59, 60). Thus, j locus structure favors replacement reactions that allow receptor editing.

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wJa /drec A similar process of deletional recombination can occur in the TCRd locus, where the wJa element joins to drec, eliminating the entire TCRd locus that is embedded in the TCRa locus (Figure 3A,B) (61, 62). This type of nonprotein coding recombination is reported to occur in ;70% of human T cells that go on to express TCRa (63). Like the wJa element, RS and kde have homology to J regions (64), but unlike most antigen receptor pseudogenes, they retain consensus RSSs (51, 52, 55), implying evolutionary conservation of rearrangement function.

QUASI-ORDERED REARRANGEMENTS IN DEVELOPMENT B Cells Rearrangements generating antigen receptor genes generally occur in a particular temporal sequence regulated by control of gene accessibility. In mouse B cell development, H-chain genes are assembled first. PreB cells bearing in-frame, functional IgM H-chains (l-chains) are selected for clonal expansion and differentiation, whereas cells lacking l-chains are eventually targeted for elimination (65). Cells lacking l-chains often attempt rearrangements on the second allele, which, if functional, can rescue the cells. This selection process is a major developmental checkpoint that involves the testing of l-chain based on its ability to associate with surrogate L-chain components k5 (66) and VpreB (67) and to mediate transmembrane signals through associated Ig-a/b signal transducers (68– 71). Only after a proliferative burst of approximately six cell divisions (72) do cells exit cell cycle; then recombination activity is redirected to the Ig L-chain loci generating sIgM` B cells (73). Gene Assembly and Lineage Commitment in T Cells In abT cell development, TCR loci are similarly rearranged in a temporal sequence, beginning with DJ rearrangements at the TCRb loci, followed by Vb-to-DJ rearrangements, a proliferative burst of those cells expressing functional b-chains, and subsequent redirection of recombinase to the TCRa loci (74–77). In a further parallel to B cell development, testing of the b-chain in T cell development involves protein association with a surrogate chain, pTa (78), and signaling through a preTCR complex (79). Thymocytes lacking b-chains, pTa, or molecules involved in preTCR signaling fail to develop further (80–82). Enforced expression of b-chain transgenes blocks endogenous b-chain gene assembly (83) and promotes development to TCRa gene rearrangement and expression (84).

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Expression of a complete a/bTCR is clearly insufficient for RAG downregulation (31, 32, 85, 86). a/b T cells are subject to the process of positive selection (87), in which cells with a receptor biased to weak MHC self-reactivity are selected to advance in development. This maturation is coincident with the cessation of TCRa rearrangement (88, 89). RAG downregulation can be experimentally induced in thymocytes by antibody cross-linking of the TCR (90). The abundance of V genes and J genes in the TCRa locus allows cells to undergo multiple rearrangement attempts, which may be necessary for efficient positive selection (31, 32, 86). T cell development in the thymus is further complicated by two factors. First, common precursors can give rise to either cd or ab T cells, a situation that is regulated in part by the functionality of rearrangements. And, although the antigen receptors of these cells use different protein chains, the TCRd locus is embedded in the TCRa locus (Figure 3) and is physically excised in the process of TCRa rearrangements (27, 29, 91). The d and c loci rearrange prior to a and b loci (92, 93), and an intact cd receptor likely promotes development in much the same way as b/pTa heterodimers. Lineage determination is also under control of the Notch pathway (94). Likewise, it has been proposed that cd- or b/pTa-mediated signals may affect the Notch pathway itself (95). Regardless of the details of ab vs. cd lineage commitment, it is of interest to observe patterns common to the organization and rearrangement of different antigen receptor genes. First, in all lymphocytes, one chain partner gene is preferentially rearranged and expressed before the other. Two waves of RAG gene expression correspond to the points in development in which each locus is recombined (96, 97). After expression of the first chain, cells are selected for proliferative expansion (78, 96). The initially expressed Ig-H, TCR-b, and TCR-d loci invariably include D elements, whereas loci rearranging later lack D regions (Figure 3). Absence of D regions facilitates receptor editing (Figure 1) but limits potential receptor gene diversity, suggesting that receptor editing may be an important selective force in evolution. The initially rearranging chains are each encoded in a single locus, as is the TCR-a locus, which should theoretically limit dual expression. However, in humans and mice the Ig-L chain is encoded by two or three loci, respectively, each with two alleles that rearrange independently of one another, compounding the problem of allelic exclusion with one of isotypic exclusion.

ALLELIC EXCLUSION The vast majority of mammalian B cells bear a single antibody heavy and light chain (98–103), despite the ability to produce two, one encoded by each allele. In the mouse, most cells express a single light chain (104, 105), despite the ability to express six (2 alleles 2 3 isotype loci: j, k2/kx, k1/k3). As discussed below, allelic and isotypic exclusion are ensured by a variety of active processes. The

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evolutionary selective forces that make allelic exclusion desirable are not particularly clear. It has been argued that B cell monospecificity is important for both immune self-tolerance and efficient antibody effector function. In T lymphocytes, several mechanisms ensure that cd receptors are excluded from abT cells and vice versa, despite the ability of both types of receptor to rearrange in the same cells (95). Furthermore, in abT cells the lack of allelic exclusion (allelic inclusion) of TCRb chains is rare. On the other hand, in these same cells coexpression of two TCRa chains is common (86, 106, 107). Allelic inclusion is also observed in TCRd (108) and TCRc (109). Why exclusion should be strict in some situations but not in others is unclear. The presumed risk of autoimmunity that may be caused by relaxed allelic exclusion remains to be proved.

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Mechanisms of Allelic Exclusion Stochastic Factors Several mechanisms in B cells have been proposed to explain the phenomena of allelic and isotypic exclusion (e.g., expression of only Ig-j or Ig-k, but not both). One major mechanism is inherent to the recombination machinery itself. Because recombination introduces small deletions and insertions at the coding join, about two thirds of rearrangements are out of frame. In some cases, the V, D, or J elements themselves may harbor stop codons, or such codons may be created in the process of recombination. In theory, these stochastic mechanisms alone reduce allelic inclusion of a single locus to less than 20%. This frequency drops further if a time limit is imposed on the rearrangement process, and further still if rearrangement is intrinsically preferred in one allele over another. Some evidence suggests both of these factors play a role in L-chain allelic and isotypic exclusion. In the mouse j-light chain locus, DNA demethylation and concomitant accessibility to recombination is, at least initially, monoallelic (110), though this is clearly not a strict rule since about half of all B cells have rearrangements on both j alleles (104). Furthermore, most j-expressing cells lack k rearrangements, whereas virtually all k-producing cells bear j rearrangements, usually on both alleles (13, 56, 111–113). During B cell development in the bone marrow, k-bearing B cells emerge 24 h later than j` B cells (114), and analysis of k-producing hybridomas revealed that most k` cells had rearranged a single k-gene, suggesting that k gene recombination is both inefficient and time limited (57). While these stochastic factors reduce allelic and isotypic inclusion, they are complemented by more active regulatory processes. Selective Factors A second mechanism that could enforce allelic exclusion in mature cells is counter selection against double producing cells. As originally conceived, excess H-chain expression was thought to be toxic to cells (115). Evidence suggesting this possibility came from analysis of myeloma cells, in which L-chain loss is toxic if H-chains are produced. While this toxicity explanation is not supported by experiments in which double H-chain expressing B cells were generated by transgenesis in vivo (116), it is nevertheless possible that

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double producers are counterselected for reasons other than toxicity. If random H/L pairs are frequently self-reactive, as has been predicted on a priori grounds (117), cells bearing two different receptors would be much more likely to be autoreactive than cells with a single H/L pair. Consequently, counterselection of double producers may occur as a consequence of tolerance induction. This might explain the curious finding that in k5-deficient mice, which manifest poor allelic exclusion of H-chains in the preB compartment (118), allelic exclusion is restored in peripheral B cells (66).

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Feedback Suppression of Recombination Ig-H While selective forces obviously play a major role in lymphocyte biology in general, a considerable body of evidence argues that instructive mechanisms have a dominant role in establishing allelic exclusion. In preB cells, functional l-chains actively block further H-chain rearrangements (66, 119–121). This inhibition occurs through assembly of the surrogate L-chain components k5 (120, 122) and VpreB (67) with the membrane-bound form of l-chain (121, 123–125), followed by signaling via the associated Ig-a/b complex (125–127). Forced expression of a heavy chain by transgenesis substantially blocks VDJ assembly on the H-chain locus (119, 123, 128, 129). Disruption of the preB cell receptor (preBCR) signaling complex inhibits both B cell development and allelic exclusion at this stage (103, 118). H-chains that fail to associate with surrogate Lchain, such as certain chains that include VH81X sequences, are essentially absent from the peripheral B cell pool (72, 130, 131). In normal B cells, many cells have incomplete, DJ rearrangements on the nonfunctional H-chain gene allele, suggesting that the rate of V-to-DJ recombination is slow compared to the ability of the cell to perceive functional H-chain assembly in a preBCR complex (132, 133). In addition, in the mouse, otherwise nonfunctional Dl chains generated from partial D-J rearrangements that involve D reading frame 2 can terminate recombination and block further development (65, 134, 135). Overall, there is compelling evidence for active feedback regulation of H-chain gene recombination that contributes to allelic exclusion. TCRb An analogous feedback process in thymocytes regulates allelic exclusion of the TCRb locus. Pairing of TCRb chain with the invariant preTCRa chain generates a signaling complex with CD3 that signals cessation of V(D)J recombination and developmental progression (78, 79, 136, 137). Enforced expression of a b-chain transgene (Tg) blocks endogenous rearrangements and promotes developmental progression (83, 138).

Ig-L-Chain Allelic Exclusion That Ig-L chain loci must similarly be subject to feedback suppression was predicted on a priori grounds, but the evidence for feedback suppression in light chain gene rearrangements is much less compelling or clear than the evidence for

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H-chain or TCR-b allelic exclusion. B cells from normal animals manifest substantial allelic exclusion (102), but sensitive techniques are able to identify double producing cells above background (139–141). In contrast to the ability of H-chain transgenic constructs to suppress H-chain rearrangements, enforced expression of L-chain transgenes led to mixed results. Initial experiments by Storb and colleagues using the MOPC 21 j-chain Tg were consistent with a strict feedback regulation model, as hybridomas generated from these mice appeared to lack endogenous j expression when H-chain was also expressed (142). Curiously, in this study many hybridomas lacked H-chain expression but expressed endogenous j-chains (142). It was later found that k-chain-producing hybridomas from these mice not only coexpressed the j Tg but also often rearranged and expressed endogenous j-loci (139). Some other transgenic mouse lines apparently failed to exclude endogenous rearrangements because of insufficient protein expression (119), but this quantitative effect could not explain how in the MOPC 21 mice endogenous rearrangements were excluded in some B cells but not in others. Similarly, in MOPC-167 j-gene transgenic mice, endogenous j expression was suppressed in only a subset of B cells (123). It is unlikely, but not formally excluded, that the incomplete allelic exclusion observed in these j-transgenic mouse experiments was solely the result of defective Tg expression. Certain conventional j transgenic mice manifest excellent allelic exclusion in a substantial proportion of cells (143, 144). Several lines of evidence suggest that in some cells even normal L-chain gene expression is not sufficient to suppress further L-chain gene rearrangement. Several sIg ` B cell tumor lines and long-term IL-7-dependent bone marrow B cell lines express new receptor chains through secondary rearrangements (22, 145– 149). In an analysis of episomal DNA present in mouse spleen cells, Harada & Yamagishi found that of 16 clones containing VjJj joins excised by nested j rearrangements, 5 were in-frame and theoretically functional (24), suggesting that in these B cells the natural genes were frequently incapable of suppressing further rearrangements. Perhaps more convincingly, in mice in which functional VjJjgenes were targeted to the natural locus, endogenous j rearrangements were inhibited in some, but not all, B cells (144, 150). Again, only a subset of cells, which varied in frequency depending upon the variable region gene used, appeared subject to feedback suppression. A criticism of these experiments is that even the targeted j-genes may be aberrantly expressed owing to the premature juxtaposition of a Vj promoter near the Cj locus. This could conceivably target recombination to the j locus abnormally early in development, perhaps in cells that lack H-chains and that therefore are unable to perceive the presence of light chain by BCR signaling. Conversely, if DNA accessibility in the j-locus is stochastically controlled (110), the targeted gene may at first be transcriptionally silent in some cells that make the other allele accessible. The targeted allele might then be coexpressed at a later time, resulting in allelic inclusion of a significant fraction of cells, an unexpected but well-documented, property of targeted Lchain mice (144, 150, 151).

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In contrast to these results, it appears that when expressed as transgenes, certain H/L pairs are extremely efficient at suppressing endogenous L- and H-chain protein expression and RAG expression in the bone marrow (152), suggesting that BCR specificity plays a critical role in development. One interpretation of these results is that, like T cells, B cells are positively selected by self-antigens that downregulate RAG expression. Some studies with cell lines are consistent with this idea (145, 146). However, if antigen-specific positive selection was associated with cessation of V(D)J recombination in B cells, then allelic inclusion would be predicted to occur frequently among normal B cells in vivo (as is the case for TCRa exclusion in T cells). On the contrary, as we discuss below, there is substantial evidence from the study of autoantibody transgenic mice that negative selection, i.e. tolerance mediated by encounter with self-antigens, blocks developmental progression and prevents recombinase downregulation. These results can be reconciled with the above-mentioned results by taking into account the influence of immune tolerance on feedback suppression of recombination.

RECEPTOR EDITING Receptor Editing Monitored In Vivo in Transgenic Models of Immune Tolerance The fact that H`L antibody transgenes could be used to generate mice in which most B cells had a defined specificity (119) stimulated studies analyzing immune tolerance in B and T cells (153). Transgenic mouse models using autoantibodies to HEL, MHC class I alloantigens, DNA, erythrocytes, and other antigens have been useful in defining a number of ways that self-reactivity is controlled (154). In transgenic autoantibody models in which developing B cells were confronted with antigen in a multivalent form, autoreactive cells were absent from the peripheral lymphoid system, but a population of cells carrying a low level of receptor was present in the bone marrow (155–157). Two lines of evidence suggested that receptor editing, i.e. autoantigen-induced secondary Ig gene rearrangements, might be a mechanism of immune tolerance in these models. In mice transgenic for the 3–83 antibody (anti-H-2Kk,b), B cells were numerous and they almost exclusively expressed the Tg-encoded antibody as a consequence of feedback suppression of recombination (158). But when cognate antigen was introduced by breeding the 3–83 transgenes to the appropriate MHC background, spleen and lymph node B cells were reduced in number, and those cells that remained lacked self-reactivity. B cells in the peripheral lymphoid organs retained surface expression of the 3–83 H-chain, but not the 3–83 L-chain, and an extremely high percentage of cells expressed endogenous k-chain. In the bone marrows of antigen-expressing mice, transgenic sIg ` B cells expressed high levels of recombinase mRNA and manifested rearrangements at the endogenous L-chain loci. Based on these findings, it was suggested that autoantigen binding

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by immature bone marrow B cells could reinduce or prolong L-chain gene rearrangements, allowing a cell to alter specificity and escape death (158). It was further postulated that the ability to undergo receptor editing was limited to early stages of B cell development because 3–83 mice that expressed cognate antigen only in the periphery, under control of a liver-specific Kb Tg, underwent profound B cell deletion without appreciable receptor editing. Independent evidence for receptor editing was obtained in transgenic mice carrying H ` L genes of dsDNA antibody 3H9 (159). Spleen cells of H ` L Tg mice and hybridomas generated from them were analyzed for antibody specificity and endogenous rearrangements. While dsDNA-specific B cells were lacking from the hybridoma sample, indicating that specific tolerance was induced, splenic B cell numbers were surprisingly normal, particularly in older mice. Splenic B cells retained transgenic H-chain expression on the cell surface but lacked 3H9 L-chain expression as detected with specific anti-idiotypic antibody. The hybridomas expressed endogenous L-chains that apparently altered B cell specificity and extinguished dsDNA binding. Suppression of dsDNA specificity was reversible in one hybridoma, when the endogenous j was lost, suggesting that binding of the transgenic H-chain with the endogenous L-chain outcompeted transgenic L-chain. In addition to this evidence for ‘‘phenotypic’’ allelic exclusion, among these hybrids an excessive use of endogenous Vj12/13 genes associated with downstream Jjs was observed. In control mice transgenic for just the 3H9 L-chain, only 6/25 hybrids excluded endogenous L-chain expression, but the endogenous L-chains that were expressed had the typical distribution of Vj and J usage. Radic et al (160) and Prak et al (23) extended this work to test for toleranceinduced receptor editing in 3H9 H-chain-only Tg mice. This test was possible because 3H9 H-chain binds DNA in association with diverse L-chain partners (161), but spleen hybridomas from 3H9 H-chain Tg mice lack reactivity to dsDNA (160, 162). Hybridoma analysis again revealed a strikingly limited usage of Vj genes in splenic B cells with excess use of Vj12/13 and skewing to downstream Jj’s, particularly Jj5. These studies also documented that in many cells multiple rearrangement attempts were made on a single chromosome. Assuming that additional attempts were required to replace j-chains that conferred autoreactivity, these results provide an explanation for skewing of Jj usage. This would predict that many VJ joins displaced by secondary rearrangements were in-frame and functional, an assumption that was not directly tested. An independent study by Eilat and colleagues of a different set of VH11 anti-DNA H-chain mice yielded similar results (163). Since in these studies no L-chain Tg was present, the later stages of B cell development should have been relatively normal. Furthermore, because simple apoptosis of autoreactive cells from an initially random population would be unlikely to account for the observed skewing in Jj usage, these results suggested that autoreactive cells were rescued by ongoing Igj recombination.

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Analysis of Receptor Editing in Gene Targeted Antibody Gene Mice VH Replacement Gene targeted autoantibody Tg mice were generated to more closely mimic the natural genomic context and to test for VH replacement in vivo. In theory, gene targeting to the natural locus should facilitate editing because secondary rearrangements on the targeted allele, should they occur, can silence or modify antibody expression. In conventional Tg mice, the suppression of Tg expression is highly inefficient because transgenes are typically inserted on chromosomes without access to potential rearrangement partners. A complicating feature of mice with targeted H-chain loci is that they retain upstream D regions, which are normally absent after VDJ assembly. These dangling Ds can rearrange to remaining J elements of the constructs or to the heptamers embedded in the targeted VH region. Early studies (164) failed to identify use of the conserved heptamer embedded in the expressed VH, but destructive rearrangements caused by D joining to other heptamer-like sites in the gene were observed. Another study (165) found frequent editing by D-regions, but also found VH-to-VDJ replacement events that rescued H-chain function, a result predicted from prior work with cell lines (41, 166). This and subsequent results from studies of other H-chain targeted mice (116, 163, 167, 168) have verified that such replacement events at the Ig-H locus occur at detectable frequency in these animals, indicating that nested rearrangements alter specificity in vivo. But there is debate about the frequency, stage specificity, and tolerance inducibility of the VH replacement reaction (169). In vitro analyses indicate that the embedded heptamer is extremely inefficient at targeting V(D)J recombination relative to conventional RSSs (170). When VH-to-VDJ replacements have been observed in mouse B cells, they almost always include additional ‘‘N’’ nucleotides derived from TdT activity, indicating that most VH replacements occur at the proB stage, prior to L-chain gene rearrangements (116, 165, 167). This in turn suggests that tolerance signaling through intact surface immunoglobulin (Ig) does not drive the VH-to-VDJ replacement response. Editing at the j-Locus The possibility that editing on the H- or L-chain loci could be tolerance induced was more directly tested in models of H`L autoantibody gene targeted mice. Chen and colleagues analyzed mice coexpressing heavy and light chain targeted constructs encoding the 3H9H/Vj4 anti-dsDNA antibody or the 3H9 H-chain with a different targeted L-chain (Vj8) that conferred reactivity to ssDNA (152). The 3H9/Vj4 dsDNA reactive combination was subject to extensive j-chain editing in the absence of editing on the H-chain locus. Ninety-eight percent of cells retained the expression of the 3H9 H-chain, and the vast majority of these failed to express the other H-chain allele, but at the L-chain locus extensive rearrangements occurred on both the targeted and untargeted jalleles. Only 4% of hybridomas lacked detectable secondary j-rearrangements. L-chain editing occurred by inversional rearrangements in 57% of the hybrid-

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omas, deletional type rearrangement events in 28% of the cells, and inclusional rearrangements (i.e. on the opposite allele with an unaffected targeted allele) in only 11% of cells. In the 3H9H/Vj8 mice, B cells bearing this receptor are often seen in the periphery, in an anergic state (162). But suggestive evidence of receptor editing was also observed in the 3H9H/Vj8 B cells because in both conventional and 3H9H/Vj8 gene-targeted mice a subset of B cell hybridomas showed exaggerated usage of Vj12/13 along with skewed Jj usage on the untargeted allele (152). (In the targeted 3H9H/Vj8 mice the Vj8 gene was joined to Jj5, providing no additional functional rearrangement possibilities on that allele.) These results suggested that tolerance-induced receptor editing was focused on the L-chain loci and could be induced by both strong and weak toleragens, dsDNA and ssDNA, respectively. In a second study, targeted j- and H-chain gene mice were generated encoding the 3–83 antibody, specific for self-MHC molecules H-2Kk and H-2Kb, and light chain editing in B cells was monitored (151). Quantitative southern blotting assessed the extent of recombination in the j-loci and indicated that in the autoreactive combination at least 30% of targeted alleles and 59% of wild-type alleles were rearranged, while in B cells of j-only mice, these percentages were 10% and 27%, respectively. As a control, the 3–83 j mice were bred with H-chain gene mice of an irrelevant, nonautoreactive specificity. (Unfortunately, in this study, it was not possible to compare 3–83 H`L targeted mice with and without antigen because the targeted H-chain gene was linked to a cross-reactive antigen locus contributed by the embryonic stem cell strain–129.) Most control cells appeared to retain expression of the targeted L-chain, whereas at least 85% of the autoreactive B cells lost expression of the autoreactive specificity on the cell surface. RS-type recombinations inactivating the j-loci were 60-fold more prevalent in the autoreactive context than in the presence of an innocuous receptor. In the bone marrows of mice with an innocuous H`L receptor, small preB cells were absent owing to accelerated developmental progression. However, in mice with autoreactive receptors that manifested receptor editing at the DNA level, this compartment was very large, suggesting that receptor editing specifically caused a retrograde step in development from sIg` to sIg1 stages. This is as one might predict from the high frequency of nonfunctional secondary rearrangements associated with inactivation of the targeted j-gene. In both this study and that of Chen et al (152), it appeared that receptor editing could allow a rather efficient rescue of previously autoreactive cells because peripheral B cell numbers were relatively normal.

Central B Cell Tolerance Is Associated with Developmental Block It has been known for a long time that B cell production can be suppressed by anti-IgM treatment (171). Early transgenic mouse studies on B cell tolerance to membrane MHC molecules indicated that self-reactive B cells were absent from

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the peripheral lymphoid organs, but large numbers of newly formed autoreactive cells bearing a low density of sIg were retained in the bone marrow (155, 156). Because these data were superficially consistent with the clonal selection hypothesis and classical studies on B cell tolerance (172), they were interpreted to mean that autoreactive cells were eliminated at the preB-to-B cell transition. It now appears most likely that autoreactivity initially prevents B cell developmental progression and promotes receptor editing that allows some cells to change their specificities. While cells failing to alter their receptors eventually die, this tempo of cell death is essentially identical to the rate of turnover of preB cells that fail to generate any receptor at all. Thus, a cell death program succeeds a receptor editing phase. An early indication that this might occur was provided in studies performed in the 1970s, in which bone marrow B cells were challenged with anti-IgM antibodies (173, 174). In these cultures, immature B cells rapidly lost sIg but were not reduced in number, as assessed by quantitation with anti-MHC class II antibodies; after removal of the anti-Ig stimulus, only a small subset reexpressed sIg. In contrast, mature splenic B cells rapidly reexpressed sIg after similar treatment. This ‘‘irreversible receptor modulation,’’ as it was called at the time, was probably the first evidence for editing, which is predicted to frequently lead to nonfunctional secondary rearrangements. More recently, in experiments in which highly purified bone marrow B cells, rather than unpurified bone marrow preparations, were challenged with anti-IgM antibodies, this stimulus was found to result in apoptosis (172, 175, 176). A possible resolution of the discrepancy between the two types of study is that the presence of bone marrow accessory cells may be required for the editing response (177). Experiments studying antigen-induced apoptosis in immature B cells of autoantibody transgenic mice are consistent with a model in which cell death is slow and delayed. Culture of anti-HEL Tg bone marrow B cells with membrane-bound HEL blocked developmental progression at the fraction E (immature B cell) stage but failed to cause rapid cell death (178). The block was reversible upon antigen removal during the first 1–2 days, but at later times B cell death occurred. Enforced expression of Bcl2 in immature autoreactive B cells was shown to further prolong survival at this developmental stage (178) but was unable to relieve the developmental block. The receptor editing model provided a rationale for this delayed and reversible death program in immature B cells. In short-term cultures of 3–83 (anti-MHC) B cells, anti-BCR antibodies failed to accelerate B cell death during the initial 48 h of culture but stimulated secondary L-chain gene rearrangements in a considerable proportion of cells, estimated to be from 25% to 50% of all cells (179). This same study reported that anti-BCR treatment could also induce receptor editing in normal, nontransgenic bone marrow B cells. In another model system, immature bone marrow B cells of 3–83 Tg mice were expanded in IL-7 and challenged with antigen. Under these conditions, autoantigen blocked developmental progression and promoted receptor editing but did not appreciably accelerate cell death (180, 181). Consistent with this idea, Bcl-2 overexpression did not allow autoreactive receptor-bearing B cells to escape from the bone marrow

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but appeared to improve the efficiency of their escape by receptor editing, probably by prolonging the time window of secondary rearrangement (182). In an independent study, Bcl-2 overexpressing immature B cells spontaneously switched from j to k L-chain expression in vitro (146). Somewhat analogous results were obtained in Bcl-xl-overexpressing anti-HEL/mHEL double Tg mice, in which central tolerance was apparently disrupted, yielding enhanced peripheralization of autoreactive B cells with an anergic phenotype along with enhanced receptor editing (183). Curiously, however, the same Bcl-xl Tg had minimal effects in the 3–83 model of central tolerance (J Lang, D Nemazee, unpublished data). Overall, these data suggest that receptor editing is associated with a developmental checkpoint. The notion that through secondary recombination autoreactive cells can overcome a developmental block implies that inhibition of secondary recombination in autoantibody Tg mice should prevent B cells from maturing to populate the periphery. This idea was directly tested in both the anti-H-2Kk,b and anti-DNA models by breeding Ig-H`L transgenes to a RAG-1- or RAG-2-deficient background (184, 185). B cell development did not advance past the late bone marrow stages, and B cells did not populate the peripheral lymphoid organs. The developmentally arrested RAG-deficient autoreactive cells underwent apoptosis in situ (185). In contrast, on an antigen-free (H-2d), RAG-deficient background anti-H2Kk,b B cells did develop and populated the spleen. These studies are consistent with a model in which developmental arrest is coupled to receptor editing, and cells that fail to appropriately modify their receptors die.

Locus Specificity of B Cell Receptor Editing Several lines of evidence suggest that tolerance-induced receptor editing in immature B cells is focused primarily on the Ig-L chain loci, although some editing at the H-chain locus may also occur. One indication of this is that most autoantibody Tg mice exhibit excellent H-chain exclusion, but poor L-chain allelic exclusion (152, 158, 159). Other evidence has been gleaned from cell culture models of receptor editing. Bone marrow B cells of 3–83 Tg mice cultured in IL-7 maintain excellent H and L-chain exclusion in the absence of cognate antigen, but jrearrangements could be induced in over two thirds of cells upon anti-BCR treatment (181). In these same cells, VDJ assembly on the H-chain locus was not detectable by a sensitive PCR assay. However, as discussed above, conventional H-chain transgenic mice do not allow an assessment of VH replacement events, and when targeted H-chain autoantibody mice have been analyzed, VH-to-VDJ replacement occurred infrequently and usually included N-region additions indicative of recombination at the proB stage prior to sIgM expression (152).

Developmental Stage Specificity of Receptor Editing Immature bone marrow B cells sensitive to tolerance-induced receptor editing rapidly lose this sensitivity as they mature, and that stage is succeeded by an apoptosis-sensitive one. These cells can be distinguished by a series of surface

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markers (186) and by the ability of the latter cells to migrate to the spleen (187). More detailed analysis has been facilitated by the ability to selectively expand receptor editing–sensitive B cells from bone marrow using IL-7 and to follow their differentiation after IL-7 withdrawal (146, 188). In these cultures, IL-7 withdrawal is required for recombinase expression, possibly because cell cycle events in proliferating cells suppress recombinase expression (189). RAG-2 protein is selectively degraded in cycling cells (189). Upon IL-7 withdrawal, cells express increasing amounts of sIg, and the ability to respond to antigen challenge by editing decreases and apoptosis sensitivity increases concomitantly. Since more mature cells presumably also must retain the option to fix their receptors and to participate in the immune response, the inability of these cells to be induced to receptor editing by antigen alone makes sense. Is there a role for receptor editing in receptor diversification? Besides providing an elegant self-tolerance mechanism, receptor editing may have an additional biological benefit. V(D)J recombination is often far from random because differences exist within sets of variable and joining segments in their recombination signal sequences, promoter regions, and proximity to other cis-acting elements that affect the efficiency of recombination. As a result, overrepresentation of certain genes is common and perhaps inevitable. Secondary and higher order rearrangements may play an important role in promoting a higher degree of randomness in V and J gene usage than would occur if each locus underwent only a single recombination.

PERIPHERAL EDITING Receptor Revision Even mature B cells, it was shown recently, can undergo receptor editing during the immune response in germinal centers (190–193). The first indications of this were that RAG mRNA and protein were inducibly expressed in mature B cells under certain culture conditions or upon a germinal center immune reaction (190, 194). Histological analysis of spleens and lymph nodes of immunized mice indicated that the cell type expressing RAG in vivo was the centrocyte (190, 195). The centrocyte is a nondividing cell that has recently proliferated in the germinal center reaction and is intimately contacted and selected by the follicular dendritic cell network (196). Concerted treatment of splenic B cells with CD40 agonist and IL-4, agents that probably mimic T cell help and induce heavy chain class switching, could rapidly induce upregulation of RAG genes (193, 195, 197). Combined treatment with bacterial lipopolysaccharides and IL-4 had a similar effect (190, 195). Later studies suggested that IL-7 could substitute for IL-4 and was probably the critical cytokine in vivo because antibodies to IL-7R, though they permitted germinal center formation, prevented RAG expression in centrocytes (197).

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RAG expression in mature B cells was strongly associated with B cell death both in vivo and in vitro and was often present in cells that were engulfed by macrophages (190, 195). In histological sections of germinal centers, RAG protein expression appeared to be localized to the cytoplasm rather than the nucleus (190, 194). RAG gene products induced in vivo and in vitro were nevertheless shown to cause double-stranded DNA breaks adjacent to recombination signal sequences (191) and new L-chain protein expression (192, 193, 198). Curiously, the cells undergoing these recombination reactions and receptor alterations did not manifest a loss of surface Ig expression (192), which would be predicted to be a common occurrence, suggesting rapid death of sIg- cells in these cultures. Perhaps most intriguingly, the cell fractions that expressed recombinase also expressed other markers characteristic of B cell precursors, including surrogate L-chain components (199), IL-7R (197), the surface marker GL-7 (194), and, in human germinal center cells, TdT (199). The reexpression of surrogate L-chain components is particularly interesting because of the likelihood that many editing events would initially silence L-chain production, perhaps requiring surrogate Lchain to temporarily pair with H-chain. Expression of surrogate L-chain components is also observed in cycling, centroblast cells (199). These unexpected similarities between bone marrow and germinal center cells, first pointed out by Han, Kelsoe, and colleagues on the basis of RAG expression (194), suggest that many elements of B cell development are recapitulated during the immune response. However, in the regulation of recombinase expression, there are critical differences as well. In the bone marrow microenvironment, immature B cells require only BCR ligation to undergo receptor editing, whereas different stimuli drive receptor editing in germinal center cells, and BCR stimulation under such conditions actively blocks the recombinase response (198, 199). In one study, in vivo RAG expression appeared to be present in a subset of germinal center centrocytes distinguishable by reduced CD45 levels and nonoptimal receptor gene usage for cognate immunogen (191). It is therefore unlikely that immune tolerance induces V(D)J recombination in germinal center cells. Instead, the data are most consistent with a role for receptor editing in the diversification of the receptors of antigenreactive cells. However, as we discuss below, this interpretation also presents problems. Very recently, data has been presented suggesting that receptor revision may occur concurrently with somatic hypermutation. Weigert and colleagues have identified a clone of anti-dsDNA reactive cells captured as hybridomas from MRL/lpr mice harboring targeted L-chain and H-chain genes encoding anti-DNA (F Brard, M Shannon, EL Park, S Litwin, M Weigert, in press). In this clone the L-chain Tg was heavily mutated, and all clone members shared a lethal stop mutation. The clone expressed a second L-chain on the other kappa allele that had acquired fewer mutations. Assuming that the new j-gene rearrangement appeared some time after the initiation of somatic mutation, these results imply that revision can occur in cells that undergo point hypermutation and that editing

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may be specifically stimulated by the loss of BCR expression. In a second study, de Wildt et al (200) identified among 365 human primary IgG` clones a pair of cells expressing an identical hypermutated H chain gene and two different expressed L-chain genes, which were presumably rearranged after these mutations. Since the L-chain genes themselves showed evidence of somatic mutation, hypermutation and receptor editing can apparently occur concurrently in these cells. A second recent study argues that receptor revision can occur in mouse B-1 cells (201), which are not thought to take part in germinal center reactions. IgM`B220low peritoneal cells, particularly those from the autoimmune-prone NZB strain, express RAG mRNA and possess double-strand breaks at Jj RSSs. In analysis of mice carrying functional replacements of IgH and IgL variable genes, B-1 cells lost idiotypic determinants, indicative of receptor editing at the protein level. The function of such modification is unclear and, as in the case of germinal center B cells, has important implications for the generation of autoantibodies. Locus Specificity of Receptor Revision Several studies documented renewed recombination of L-chain genes in mature B cells (191–193, 197, 199, 201), and a single study provided possible evidence of rearrangements at the IgH locus (192). This latter result was obtained in a VDJ replacement mouse in which both nonphysiological D-to-VDJ joins and potentially physiological V-to-VDJ replacement reactions are possible. Thus, either this experimental system may represent the ideal model to reveal physiologically relevant receptor revision on the IgHlocus, or it may allow recombination events on the IgH-locus that rarely occur in normal cells. It will be important to determine if receptor revision can occur at the H-loci through V-to-VDJ replacements.

RECEPTOR EDITING IN T CELLS As discussed earlier, TCRa allelic exclusion essentially fails to occur because of ongoing rearrangements in cells prior to positive selection. This raises the question of whether an editing mechanism may play a role in thymocyte negative selection. Wang et al (202) established a targeted replacement mouse using a rearranged TCRa VJ gene from a pigeon cytochrome c (PCC)–specific T cell hybridoma inserted in the 58 region of the germ-line J locus. This gene was expressed in most double positive cells but was lost, presumably through nested rearrangements, in all but ;3% of peripheral T cells. Retention of gene expression was greatly improved when a TCRb Tg encoding the original hybridoma was coexpressed on a positively selecting background but was not improved on a nonselecting background. Most importantly, in the presence of PCC, negative selection was not associated with loss of thymic cellularity, in contrast to what was seen with conventional anti-PCC TCR transgenic mice, and nonautoreactive

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cells populated the periphery. While these results failed to prove that autoreactivity promoted further rearrangements, the possibility is not excluded, and this model should allow this question to be further tested. It is equally possible that the autoreactive cells in this system were positively selected and subsequently lost by apoptosis, but the compartment was filled with competing cells that had lost expression of the targeted TCRa gene prior to antigen encounter. Perhaps more remarkable is the recent suggestion that receptor editing can occur during peripheral T cell tolerance (203). In this experiment, a TCRb-chain (Vb5) Tg mouse was constructed and tolerance to the endogenous mouse mammary tumor virus superantigen MTV-8 was assessed. In antigen-expressing mice most Vb5` cells were deleted, and the remainder were functionally inactivated. However, over time, a Vb51 population emerged. It was concluded that in a subset of the tolerant cells, extra-thymic V(D)J recombination occurred, altering and replacing their receptors because RAG expression and doublestranded breaks adjacent to TCR Db2 and Ja50 were detected in a subset of peripheral T cells. Furthermore, the appearance of Vb51 cells, but not tolerance, was dependent on the presence of B cells, suggesting that a B:T interaction, such as a germinal center reaction, might be required for this process. A potential inconsistency between the data and this model was that the RAG-expression was detected in Vb51 rather than Vb5` cells. These points will no doubt be addressed in the near future as experimental systems are designed with these questions in mind (202). Finally, it should be noted that the structure of the TCRb loci is consistent with receptor editing, as active genes using the 58-most D/J/Cb cluster can in theory be subsequently eliminated and replaced by V-to-DJ rearrangements to the 38 D/J/Cb cluster on the same chromosome (Figure 3). In addition, the spacers of their RSS elements are theoretically compatible with direct Vb-to-Jb rearrangement, but this has never been observed.

CONCLUSION The ability of immune cells to turn V(D)J recombinase on and off, or to redirect its activity to new gene loci, appears to regulate lymphocyte specificity in a novel way distinct from, and complementary to, the control of cellular growth and survival. This regulation of recombinase is largely controlled by signaling through the antigen receptor itself and constitutes a feedback mechanism that allows cells to ‘‘repair’’ receptor genes whose products are dysfunctional, or of inappropriate specificity, including those with too high or too low affinity for self-antigens. Receptor editing facilitates immune self-tolerance during B cell development and also permits developing thymocytes to screen multiple TCRs for potential positive selection. Mature B lymphocytes (and possibly also T cells) may re-express recombinase upon foreign antigenic stimulation, possibly allowing more rapid clonal evolution to high-affinity reactivity. The organization of antigen receptor genes appears to facilitate these processes, often at the cost of limiting potential

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receptor gene diversity and lymphocyte monospecificity. The full extent to which these receptor selection processes normally occur, or benefit the organism, remains to be elucidated. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health and the Arthritis Foundation. The author thanks Anne Feeney, Norman Klinman, Martin Weigert, and members of the laboratory for their comments on the manuscript, and Kathy Offerding for secretarial assistance.

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Note Added in Proof Since the submission of the manuscript, the cloning by Zachau’s group of the murine j-locus has made extraordinary progress. For information on this fastmoving area and associated recent publications, the reader is referred to their web site: http://www.med.uni-muenchen.de/biochemie/zachau/kappa,htm. Visit the Annual Reviews home page at www.AnnualReviews.org.

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Annual Review of Immunology Volume 18, 2000

CONTENTS

Annu. Rev. Immunol. 2000.18:19-51. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Annu. Rev. Immunol. 2000. 18:53–81 Copyright q 2000 by Annual Reviews. All rights reserved

MOLECULAR BASIS OF CELIAC DISEASE Ludvig M. Sollid Institute of Immunology, Rikshospitalet, University of Oslo, N-0027 Oslo, Norway; e-mail: [email protected]

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Key Words celiac disease, gluten, T cells, HLA, DQ2, peptides Abstract Celiac disease (CD) is an intestinal disorder with multifactorial etiology. HLA and non-HLA genes together with gluten and possibly additional environmental factors are involved in disease development. Evidence suggests that CD4` T cells are central in controlling an immune response to gluten that causes the immunopathology, but the actual mechanisms responsible for the tissue damage are as yet only partly characterized. CD provides a good model for HLA-associated diseases, and insight into the mechanism of this disease may well shed light on oral tolerance in humans. The primary HLA association in the majority of CD patients is with DQ2 and in the minority of patients with DQ8. Gluten-reactive T cells can be isolated from small intestinal biopsies of celiac patients but not of non-celiac controls. DQ2 or DQ8, but not other HLA molecules carried by patients, are the predominant restriction elements for these T cells. Lesion-derived T cells predominantly recognize deamidated gluten peptides. A number of distinct T cell epitopes within gluten exist. DQ2 and DQ8 bind the epitopes so that the glutamic acid residues created by deamidation are accommodated in pockets that have a preference for negatively charged side chains. Evidence indicates that deamidation in vivo is mediated by the enzyme tissue transglutaminase (tTG). Notably, tTG can also cross-link glutamine residues of peptides to lysine residues in other proteins including tTG itself. This may result in the formation of complexes of gluten-tTG. These complexes may permit gluten-reactive T cells to provide help to tTG-specific B cells by a mechanism of intramolecular help, thereby explaining the occurrence of gluten-dependent tTG autoantibodies that is a characteristic feature of active CD.

INTRODUCTION Celiac disease (CD), or gluten sensitive enteropathy, is a condition in which ingested wheat gluten or related proteins from rye and barley are not tolerated (1). CD, like type 1 diabetes, rheumatoid arthritis, and multiple sclerosis, has a chronic nature where particular HLA alleles are overrepresented among the patients (2). Commonly these disorders are multifactorial; HLA genes and other genes together with environmental factors are involved in disease development. The expression of CD is strictly dependent on dietary exposure to gluten and

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similar cereal proteins (1). Patients go into complete remission when they are put on a gluten-free diet, and they relapse when gluten is reintroduced into the diet. CD is in this respect unique among the chronic inflammatory HLA- associated diseases in that a critical environmental factor has been identified. CD is primarily a disease of caucasians (1). It is most frequently recognized among Europeans, although there is an increasing awareness of this disorder in the United States. CD commonly presents in early childhood with classic symptoms including chronic diarrhea, abdominal distension, and failure to thrive (3). The general condition of these children is severely impaired. The disease may also present later in life with symptoms that tend to be more vague and include anemia, fatigue, weight loss, diarrhea, constipation, and neurological symptoms (4). CD patients on a gluten-containing diet have increased levels of serum antibodies to a variety of antigens, including gluten and the autoantigen tissue transglutaminase (tTG) (5, 6). The presence of antibodies to gluten and tTG is strictly dependent on dietary exposure to gluten. Testing of serum antibodies to gluten and tissue tTG is utilized to predict CD, and this provides a great aid in clinical practice (5, 7, 8). The final diagnosis of CD, nevertheless, rests on the demonstration of typical mucosal pathology by histological examination of small intestinal biopsies. The reported prevalence of disease with overt symptoms varies enormously in the populations of Europe and North America. Assessment of the prevalence by biopsy examination of individuals identified by antibody screening has however demonstrated surprisingly similar prevalence rates of about 1:200 to 1:400 throughout Europe and North America (9). Many of the patients identified in these studies have no symptoms or only mild symptoms that are often associated with decreased psychophysical well-being and anemia (4, 9). The clinical expression of CD is probably influenced by environmental factors. In Sweden an ‘‘epidemic’’ of CD in children under the age of two years produced a dramatic fourfold increase in incidence rates in the period 1985–1987 and a similar rapid decline in the incidence rates from 1995–1997 (10). These changes in incidence concur with changes in infant feeding practices and suggest that the amount and timing of the gluten introduction (perhaps in conjuction with the breast feeding duration) is important for precipitation of the disease in children (10). Whether the pattern of gluten feeding in infants affects only the age of onset of the disease or whether it ultimately changes the overall population prevalence is still an open question. Current treatment of CD is a lifelong exclusion of gluten from the diet. Poor diet compliance by patients and undiscovered disease are associated with complications including increased risk of anemia, infertility, osteoporosis, and intestinal lymphoma (4). Notably, untreated CD is associated with increased mortality. Research into the molecular basis of the disease has already lead to improved diagnosis, and it is hoped this research will lead to better treatment in the future.

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THE CELIAC LESION The lesion in CD is localized in the proximal part of the small intestine. Villous atrophy, crypt cell hyperplasia, lymphocytic infiltration of the epithelium, and increased density of various leukocytes in the lamina propria characterize the classic textbook type of lesion (1). These alterations represent one end of a spectrum of mucosal pathology that Marsh (11) has classified into three stages: the infiltrative, the hyperplastic, and the destructive lesions. The infiltrative lesion is characterized by infiltration of small nonmitotic lymphocytes in the villous epithelium without any other sign of mucosal pathology. The hyperplastic lesion is similar to the infiltrate lesion but in addition has hypertrophic crypts whose epithelium may be infiltrated by lymphocytes. The destructive lesion is synonymous to the classic lesion described in textbooks. Oral challenge experiments with gluten have demonstrated that these stages are dynamically related (12). The existence of a spectrum of pathological stages in CD is interesting when considering the polygenic nature of CD. In the NOD mouse model of autoimmune diabetes, where at least 14 different loci are involved in the control of the disease, nearly all NOD mice develop insulitis, but many animals do not go on to develop diabetes (13). Notably fewer susceptibility genes are required to produce insulitis than diabetes (13). It is conceivable that in CD different susceptibility genes contribute at different stages to the development of the end-stage disease. The pathological alterations and the type of cellular infiltrates found in the classical, flat-destructive lesion are well characterized, and the major features are summarized in the following.

Enterocytes In CD there is an increased loss of epithelial cells and increased proliferation of epithelial cells in the crypts. Both these factors have been used to explain the villus atrophy found in CD (14, 15). It is not clear whether the two phenomena are causally linked, and if so, which of them is primary or secondary. The increased epithelial cell loss probably reflects increased apoptosis of enterocytes (16), whereas the increased enterocyte proliferation appears to be due to an increased production of keratinocyte growth factor (KGF) by stromal cells (17). Several molecules with immune function are known to have an altered expression in CD. There is an increased epithelial expression of HLA class II molecules with strong expression of DR and DP molecules, but with little or no expression of DQ molecules (18, 19). The expression of the polymeric Ig receptor is also upregulated (20). Notably, this enhanced expression of the polymeric Ig receptor is accompanied by increased transport of IgA and IgM into the gut lumen (21).

Intraepithelial Lymphocytes Three major lineages of intraepithelial lymphocytes (IELs) occur in the normal human small intestine; the most prominent is the TCRab` CD8`CD41 popu-

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lation, while the TCRab` CD81CD4` population and the TCRcd` CD81CD41 population are also present. Both the TCRab` CD8`CD41 and the TCRcd` CD81CD41 populations are expanded in CD. In contrast to the TCRab` CD8` IELs that return to normal when gluten is removed from the diet, the TCRcd` IELs appear to remain at an elevated level (22). However, IELs of both the TCRab` CD8` and TCRcd` lineages express the Ki67 proliferation marker, suggesting intraepithelial proliferation of both populations in CD (23). Interestingly, the majority of TCRcd` IELs express the Vd1 TCR variable region (24, 25). Spies and co-workers have demonstrated that cd T cells expressing this variable region recognize MICA and MICB molecules (26)— molecules that are mainly expressed by intestinal epithelial cells (27). Activated human IELs are able to produce a number of cytokines including IFN-c, IL-2, IL-8 and TNF-a and are known to have a lytic potential (28). Furthermore, in CD, but not in giardiasis, the IELs stain positive for granzyme B and TiA (a marker characteristic for cytotoxic lymphocytes), indicating that some IELs in the celiac lesion may be activated cytotoxic T cells (29).

Lamina Propria Leukocytes A marked infiltration of TCRab` T cells appears in the lamina propria in the active lesion. These T cells are mostly CD4` and carry a memory phenotype (CD45RO`) (30). Notably, an increased percentage of these lamina propria T cells express the CD25 (IL2R a-chain) activation marker but lack the Ki67 marker associated with proliferation (23). Thus, gluten appears to induce a nonproliferative activation of CD4` lamina propria T cells. This fits well with the results of several studies reporting increased cytokine production by T cells in the lamina propria (31–33). There seems to be a particular increase in cells producing IFNc, whereas no increase appears in cells producing IL-4 or IL-10 (33, 34). mRNA for IFN-c has been found to be increased more than 1000-fold in untreated disease related to a small increase in the message for IL-2, IL-4, IL-6, and TNF-a (33). Furthermore, the IFN-c mRNA level of biopsies of treated patients has been demonstrated to reach that of untreated patients by in vitro stimulation with gluten (33). Altogether, these results are consistent with the conception that glutenreactive T cells in the lamina propria have a cytokine profile dominated by production of IFN-c. A characteristic of the CD lesion is an accumulation of IgA-, IgM-, and IgGproducing plasma cells (35). The specificities of the antibodies produced by these cells have been only partly characterized; however, in vitro culture of biopsies has demonstrated that antibodies to gliadin (36) and endomysium (i.e. tTG) (37) are produced. Just beneath the epithelium in the normal mucosa a high number of macrophage/dendritic-like cells stain positive for CD68 (38). It is conceivable that these cells are involved in sampling of luminal antigens. The expression of the HLA

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class II, ICAM-1, and CD25 molecules is increased in these macrophage/ dendritic-like cells, suggesting that they are activated in the disease state (18, 23, 39).

The Extracellular Matrix

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In the normal small intestine extracellular matrix formation (ECM) by stromal cells balances ECM degradation mediated by matrix metalloproteinases (MMPs). Increased ECM degradation has been suggested to play a role in the villous atrophy of CD. This is supported by the demonstration of a decreased ratio of cells expressing collagen I and tissue inhibitor of metalloproteinases (TIMP)-1 mRNA to those expressing matrix metalloproteinase (MMP)-1 and -3 mRNA in untreated CD (40). Expression of MMP-1 and MMP-3 mRNA is mainly localized to subepithelial fibroblasts and macrophages. It is likely that the increased expression of metalloproteinases is related to activation of mucosal T cells (see later).

THE GENETICS OF CELIAC DISEASE A high prevalence rate (10%) among first degree relatives of CD patients indicates a strong genetic influence on susceptibility to develop CD (41). Familial clustering can be expressed as the ratio of the prevalence in relatives of affected individuals over the prevalence within the population as a whole (42). The ratio ks based on the sibpair risk is the most commonly used. If this ratio is close to 1, then there is no evidence for genetic factors in susceptibility. In contrast, the ks value for CD is estimated to be 30–60 (42, 43), which is high compared with other multifactorial disorders like rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. The strong genetic influence in CD is further supported by a high concordance rate of 70% in monozygotic twins (44). The sibship aggregation attributable to HLA (ks HLA) is estimated to be 2.3–5.5 (42, 43). Using these estimates and assuming a multiplicative model of disease predisposing genes, the overall importance of non-HLA genes has been calculated to be greater than that of HLA genes (42, 43). However, attempts to map predisposing genes by linkage analysis have, with the exception of the HLA, failed to reveal unambiguous candidate genes or chromosomal regions (45–48). This suggests that each of the yetunmapped predisposing CD genes has only a minor genetic influence. Indications for susceptibility regions at 5qter and 11qter are weak (47). As with other polygenic inflammatory diseases, little is known about the non-HLA susceptibility genes. Conceivably, however, the gene products of many of these genes have immune-related functions. In the case of CD, the HLA genes (see later) and the non-HLA genes shape the immune response to gluten so that immunopathology is produced in the small intestine. Relevant to this are the recent reports that the CTLA-4/CD28 gene region contains a CD susceptibility gene (49, 50), although this finding is not consistent in all populations (51).

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HLA GENES IN CELIAC DISEASE CD was first found to be associated with the HLA class I molecule B8 (52, 53). Later stronger associations were found to the HLA class II molecules DR3 and DQ2 (54–56). The genes encoding DR3 and DQ2 are in strong linkage disequilibrium, and DR3 and DQ2 are both contained within the B8-DR3-DQ2 or the B18-DR3-DQ2 extended haplotypes. The B8-DR3-DQ2 and the B18-DR3-DQ2 haplotypes are both associated with CD (57, 58). This is significant, as these two haplotypes are conspicuously dissimilar in the regions outside the DR-DQ region. CD is also associated with DR7 (59, 60), but this association is seen almost only when DR7 occurs together with DR3 or DR5 (61, 62). This is unlike the susceptibility associated with DR3, which is seen irrespective of the accompanying DR allele. In studies to date, most CD patients have been shown to carry either the DR3-DQ2 haplotype or are DR5-DQ7/DR7-DQ2 heterozygous. Evidently, CD patients with these DR-DQ combinations share the genetic information conferring CD susceptibility (63) (see Figure 1, bottom part). The DQA1*0501 and DQB1*0201 alleles of the DR3-DQ2 haplotype (64, 65) are also found when

Figure 1 Patients with CD who are DR3 or DR5/DR7 heterozygous express the same HLA-DQ2 molecule, HLA-DQ(a1*0501, b1*02). The DQA1*0501 and DQB1*02 genes are located in cis (on the same chromosome) in DR3 individuals, whereas they are located in trans (on opposite chromosomes) in DR5/DR7 heterozygous individuals.

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the DR5-DQ7 and DR7-DQ2 haplotypes are combined. The DR5-DQ7 haplotype carries the DQA1*0501 and DQB1*0301 alleles (66), and the DR7-DQ2 haplotype carries the DQA1*0201 and DQB1*0202 alleles (67, 68). Notably, the DQB1*0201 and DQB1*0202 alleles are identical except for the codon of residue 135 located in the membrane proximal domain of the DQb chain (69). Recombination (crossing over) seems to be an important mechanism for the generation of HLA haplotypes (70). Accumulating evidence suggests that the DR3-DQ2, DR7-DQ2, and the DR5-DQ7 haplotypes have a close evolutionary relationship. Based on microsatellite analysis, fragments of DNA flanking the DQA1 gene of the DR3-DQ2 haplotype have been identified on the DR5-DQ7 haplotype, and fragments of DNA flanking the DQB1 gene of the DR3-DQ2 haplotype have been identified on the DR7-DQ2 haplotype (71, 72). Thus, the genetic information in the DQ subregion of the DR3-DQ2 haplotype is reestablished in DR5-DQ7/ DR7-DQ2 heterozygotes, although the sequence information is split between two chromosomes. It can be argued that susceptibility for CD depends on an interaction between at least two genes on the DR3-DQ2 haplotype that are reunited in DR5-DQ7/ DR7-DQ2 heterozygous individuals. Theoretically this gene interaction could involve any HLA-linked genes in the DQ region. However, complete sequencing of an 86-kb genomic fragment spanning the DQ subregion of the DR3-DQ2 haplotype failed to identify genes other than the DQA1 and the DQB1 genes in this region (73). Furthermore, the DQA1 and DQB1 are very good candidates because their products interact by forming a class II heterodimer and because they are situated close to the putative recombination site. This evolutionary consideration together with the fact that most CD patients share a particular pair of DQA1 and DQB1 genes located either in cis or in trans are strong arguments that the DQA1*0501 and DQB1*0201 alleles jointly confer susceptibility to CD by coding for the DQ(a1*0501, b1*02) heterodimer (Figure 1, top part). In most populations studied, 90% or more of the CD patients carry the DQ(a1*0501, b1*02) heterodimer, compared to 20%–30% in healthy controls (63). The fraction of patients in different populations that encode this DQ heterodimer by genes in cis or in trans position depends on the haplotype frequencies of DR3-DQ2, DR5-DQ7, and DR7-DQ2 haplotypes in the given populations (74). In a few patients the DQ(a1*0501, b1*02) heterodimer may be found to be encoded in cis position by haplotypes other than DR3-DQ2 or in trans position by individuals being heterozygous for combinations other than DR5-DQ7/DR7DQ2 (63). There is no increase of the DQ(a1*0501, b1*0301) or DQ(a1*0201, b1*02) heterodimers alone in CD demonstrating that susceptibility is dependent on both the DQa and DQb chains in the DQ(a1*0501, b1*02) heterodimer. Many studies have reported a particular increased risk for CD among individuals who are DR3-DQ2 homozygous and DR3-DQ2/DR7-DQ2 heterozygous (for references, see 63). This could be explained by a gene dosage effect of the DQB1*02 allele possibly caused by an increased expression of the DQ(a1*0501, b1*0201) heterodimer in such individuals (75). A gene dosage effect of DQB1*02

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could also provide an explanation of the high degree of HLA haplotype identity observed among affected siblings (74, 76). Depending on the populations studied, about 2%–10% of CD patients do not carry the DQ(a1*0501, b1*02) heterodimer. The great majority of these patients carry different subtypes of DR4. The genetic determinant responsible for the HLA association in these individuals is likely to be different from that of the DQ(a1*0501, b1*02)-expressing individuals. To unequivocally identify the responsible molecule encoded by the DR4 haplotype by a genetic approach is, however, difficult. Notably, there is a clear skewing in the representation of the DR4-DQ8 vs the DR4-DQ7 haplotype among these patients (77–79). This implies that DQ8, i.e. DQ(a1*0301, b1*0302), is most probably the molecule responsible for susceptibility. An opposing view is that the susceptibility is mediated by the DR53, i.e. DR(a*, b4*0101), molecule that is carried on most of the DR4, DR7, and DR9 haplotypes (80). The majority of DQ(a1*0501, b1*02)-negative patients would fit into this category. Importantly, however, this model does not account for the observed skewing of the DR4-DQ8 vs. the DR4-DQ7 haplotypes. Moreover, DQ(a1*0501, b1*02)-negative CD patients who carry the DRB1*0701DQB1*03032 haplotype exist (79), and this haplotype is reported to carry a non-expressed null allele at the DRB4 locus (81). Further studies including typing for the DRB4 null allele are needed to clarify the role of DR53 as a susceptibility molecule in CD. Genes located in the HLA gene complex other than DQ might also contribute to CD susceptibility. Associations to particular DP alleles have been reported in different populations, but many of these associations can be explained by linkage disequilibrium between the involved DP allele(s) and the DQA1*0501 and DQB1*02 alleles (for further discussion, see 63). Moreover, no independent associations to alleles at the TAP1 and TAP2 loci have been found (82–84). Several studies have consistently indicated that DQA1*0501/DQB1*02-positive individuals carrying the DR5/DR7 genotype have a higher risk to develop disease than do those of the DR3/DRX genotype (X ? DR7 and DR3) (84–86). Furthermore, it has been indicated that the risk of the DR3/DR7 genotype is higher than that of the DR3/DR3 genotype (84, 86), although this is not a consistent finding (75, 87). This has led to the suggestion that a gene on the DR7-DQ2 haplotype confers an additive effect to that of the DQA1*0501/DQB1*02 genes (86). To note, a locus with a protective allele of the DR3-DQ2 haplotype would produce the same effect. Studies of Irish CD patients have indicated an additional predisposing role of TNF genes, an association independent of DQ2 that has been demonstrated using a microsatellite polymorphism situated near the TNF genes (88). Moreover, a polymorphism of the TNF-a gene promoter has been demonstrated to be a component of the DR3-DQ2 haplotype (89). A Finnish study failed to reproduce the finding of a DQ2-independent association of the TNF microsattelites (90). These discrepant results may relate to population differences. Recently, an allele of a locus (D6S2223) that is located 2, 5 Mb telomeric to the HLA-F locus was found by Lie et al (91) to be less frequent among DR3-

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DQ2 homozygous CD patients compared to DR3-DQ2 homozygous controls. The same allele of the D6S2223 locus was also found to be underrepresented among DR3-DQ2 homozygous type 1 diabetes patients, and it was transmitted less often than expected from DR3-DQ2 homozygous parents to diabetic siblings (92). These findings suggest that a gene(s) in the vicinity of D6S2223 is involved in the pathogenesis of both CD and type 1 diabetes. In addition, the MIC-A and MIC-B genes are interesting candidate susceptibility genes in CD, as the MIC molecules are ligands for TCRcd T cells. The MIC genes are located near the HLA-B locus, and the MIC-A*008 (5.1) allele is in strong positive linkage disequilibrium with HLA-B8 (93, 94). This allele is particularly interesting since it bears a frameshift and a premature stop codon in exon 5 (95) that might affect the expression of the molecule. Taken together, available data strongly suggest that susceptibility to develop CD is primarily associated to two conventional peptide-presenting DQ molecules: i.e. DQ(a1*0501, b1*02) (4DQ2) or to a lesser extent DQ(a1*03, b1*0302) (4DQ8). An issue still to be clarified is whether there are additional molecules encoded by unidentified genes in the HLA gene complex that also contribute to the genetic predisposition for CD. However, any effect of these additional genes is likely to be moderate. A key question for the understanding of the molecular basis for CD is therefore to define the functional role of the DQ2 and DQ8 molecules.

PEPTIDE BINDING MOTIF OF DISEASE-ASSOCIATED DQ MOLECULES Peptides binding to DQ2 have anchor residues in the relative positions P1, P4, P6, P7, and P9 (96–100). This is the same spacing as previously found for DR molecules, suggesting that DQ2 bound peptides adopt to a conformation similar to that of peptides bound to DR molecules. The peptide-binding motif of DQ2 illustrated in Figure 2 is quite different from other class II–binding motifs that have been identified (101). Notably, the preference for negatively charged residues for the three anchor positions in the middle seems to be unique for DQ2. The binding motif of DQ8 is different from that of DQ2, but DQ8 also displays a preference for binding negatively charged residues at several positions (i.e. P1, P4, and P9) (102, 103). Hence, both the DQ2 and DQ8 molecules share a preference for negatively charged residues at some of their anchor positions. The peptide-binding motif of DQ2, i.e. DQ(a1*0501, b1*02), is different from the motifs of the closely related DQ(a1*0501, b1*0301) and DQ(a1*0201, b1*02) molecules (96, 99, 104), which do not confer susceptibility to CD (see above). The binding motif of the DQ(a1*0501, b1*0301) molecule is clearly different from that of DQ(a1*0501, b1*02) with differences at the P4, P7 and P9 pockets (96), whereas the differences between DQ(a1*0501, b1*02) and

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Figure 2 Schematic depiction of the peptide binding groove of HLA-DQ2 (i.e. HLADQ(a1*0501, b1*02) with the peptide-binding motif displayed with the one letter code for the amino acids. A bound peptide and a TCR recognizing the peptide/HLA complex are also indicated. This motif description is based on a compilation of results from references 96–100.

DQ(a1*0201, b1*02) are more subtle (96, 99). The molecules have similar binding motifs with the most apparent difference being an additional anchor residue at P3 for DQ(a1*0201, b1*02) (99, 105).

PREFERENTIAL PRESENTATION OF GLUTEN-DERIVED PEPTIDES BY DISEASE-ASSOCIATED HLA MOLECULES TO INTESTINAL T CELLS The DQ2 and DQ8 molecules could confer susceptibility to CD by presenting disease-related peptides in the target organ or alternatively by shaping the T cell repertoire during T cell development in the thymus. This issue has been addressed by studies of T cells derived from the celiac lesion. Stimulation of small intestinal biopsy specimens with a peptic/tryptic digest of gluten induces rapid activation (i.e. expression of CD25, the IL-2 receptor a-chain) of the T cells in the lamina propria of CD patients, but not of non-CD control subjects (106). Gluten-reactive T cells can be isolated and propagated from intestinal biopsies of CD patients but

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not from non-CD controls (107–109). These T cells are CD4` and use the ab TCR. Importantly, T cells isolated from biopsy specimens of patients carrying the DR3-DQ2 haplotype typically recognize gluten fragments presented by the DQ2 molecule rather than the other HLA molecules carried by the patients (107). Both DR3-DQ2-positive and DR5-DQ7/DR7-DQ2-positive antigen-presenting cells (i.e. carrying the DQA1*0501 and DQB1*02 genes in cis or in trans position) are able to present the gluten antigen to these T cells (107, 110). Likewise, T cells isolated from small intestinal biopsies of DQ2-negative, DR4-DQ8-positive patients predominantly recognize gluten-derived peptides when presented by the DQ8 molecule (111). It is notable that no DR(a, b1*01)-restricted intestinal T cells specific for gluten have been reported supporting a role of DQ8 rather than the DR(a, b1*01) molecule in conferring susceptibility to CD. Taken together, these results allude to presentation of gluten peptides in the small intestine as the mechanism by which DQ2 and DQ8 confer susceptibility to CD. A thymic effect of the same DQ molecules on the TCR repertoire selection is, however, not excluded by these results. The DQ2 and DQ8 molecules are not preferential antigen-presenting molecules in the intestinal mucosa irrespective of antigen. T cells specific for astrovirus (a common gastroenteritis virus) are predominantly DR restricted (109), which suggests that the peculiar HLA restriction pattern of the gliadin-specific T cells of the intestine must be related to the antigen. Interestingly, gluten-specific T cells can also be found in the peripheral blood (112). These T cells are restricted either by DR, DP, or DQ molecules, and they do not therefore display the the predominant DQ2 or DQ8 restriction observed for gluten-specific T cells from the intestinal mucosa (112). One explanation for this could be that the majority of gluten-specific T cells of peripheral blood recognize epitopes different from those recognized by T cells of the small intestine. Studies of lamina propria T cells in situ have, as mentioned above, indicated that gluten reactive T cells have a cytokine profile dominated by IFN-c. This notion is sustained by the characterization of gut-derived DQ2 and DQ8-restricted gluten-specific T cell clones. These T cells uniformly secrete IFN-c at high concentrations, and some produce IL-4, IL-5, IL-6, IL-10, TNF-a, or TGF-b in addition (113).

T CELL RECOGNITION OF DEAMIDATED GLUTEN PEPTIDES Wheat gluten is a mixture of numerous proteins grouped into the gliadin and glutenin fractions. These proteins serve as a source of nitrogen and carbon for the growing seedling during germination. A vast sequence heterogeneity among gliadin and glutenin proteins probably reflects that these proteins have been subjected to few structural constraints during evolution. Generally, gluten proteins

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contain a large percentage of proline and glutamine residues, while many other amino acids, including glutamic and aspartic acid, are unusually scarce. Feeding experiments have demonstrated that the gliadin fraction can precipitate CD (114), whereas the role of glutenins is still inconclusive. Proteins of the gliadin fraction can be subdivided according to their sequence into the a-, c-, and x-gliadins (115). A large number of different gliadins exist within each of these gliadin families. Estimates suggest that as many as 50 to 150 different a-gliadin genes may be present in a single wheat cultivar (116). For the work of identifying peptide fragments recognized by T cells, the complexity of this antigen presents a big challenge. Initially it was difficult to reconcile the DQ2 (and DQ8) binding motifs with presentation of gluten peptides because gluten proteins have an unusual scarcity of negatively charged residues. A clue to help explain this paradox came from the observation that the stimulatory capacity of gliadin preparations for gliadinspecific intestinal T cells was significantly enhanced following treatment at high temperatures and low pH (117). These conditions are known to cause nonspecific deamidation of glutamines to glutamic acid and may thus convert gliadin from a protein with very few peptides with the potential to bind to DQ2/DQ8 into one with many such. An important and general role for deamidation of gluten for T cell recognition was sustained by analysis of the response pattern of a panel of polyclonal, gliadin-specific T cell lines derived from biopsies (118). All the lines responded poorly to a gliadin antigen prepared under conditions of minimal deamidation (chymotrypsin-digestion), compared to the same antigen when further heat-treated in an acidic environment. The characterization of gluten epitopes recognized by intestinal T cells has extended the knowledge about the importance of deamidation for their T cell recognition. So far five unique epitopes of gluten that are recognized by gut T cells have been identified; three restricted by DQ2 (118, 119) (Table 1 and Figure 3) and two restricted by DQ8 (120, 121) (Table 1). The three DQ2-restricted peptides, one from c-gliadin and two from a-gliadins (DQ2-c-gliadin-I, DQ2-agliadin-I and DQ2-a-gliadin-II), fail to stimulate T cells in their native form but are potent antigens when a single glutamine residue is exchanged with glutamic

TABLE 1 Epitope DQ2-c-I-gliadin DQ2-a-I-gliadin DQ2-a-II-gliadin DQ8-a-I-gliadin DQ8-I-glutenin

Derived from protein

Presentation element

Recognized by patients

Reference

c-gliadin a-gliadin a-gliadin a-gliadin Glutenin

DQ2 DQ2 DQ2 DQ8 DQ8

Infrequently Frequently Frequently Frequently? Not known

(118) (119) (119) (120) (121)

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Figure 3 A cartoon showing the amino acid sequence (one letter code) and binding of the three known gluten epitopes recognized by HLA-DQ2 restricted intestinal T cells of CD patients. All three epitopes contain a glutamic acid (E) residue that has been converted from glutamine (Q) by deamidation. The glutamic acid residues formed by deamidation improve the binding affinity and are critical for T cell recognition in all three epitopes. Notably, glutamic acid residues are accommodated in the P4 pocket for the DQ2-a-IIgliadin epitope (119), in the P6 pocket for the DQ2-a-I-gliadin epitope (119), and in the P7 pocket for the DQ2- c-I-gliadin epitope (118). In some a-gliadins, the DQ2-a-I-gliadin and DQ2-a-II-gliadin epitopes are part of the same fragment, and it is the very same glutamine that is modified by tTG in both epitopes.

acid in certain positions. The recognition of one of the DQ8-restricted peptides from a-gliadins (DQ8-a-gliadin-I) is augmented by introduction of negatively charged residues (122), whereas this is not seen for another DQ8-restricted peptide of glutenin (DQ8-glutenin-I) (121). These data demonstrate that most, but not all, gluten-specific intestinal T cells from CD patients recognize gluten proteins only after they have undergone deamidation. Moreover, the results with the glutenin epitope demonstrate that intestinal T cells can recognize gluten proteins other than gliadins (121). This raises the question of whether glutenins are also able to precipitate the disease.

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DEAMIDATION IN VIVO IS LIKELY TO BE ENZYMATICALLY MEDIATED BY TISSUE TRANSGLUTAMINASE The deamidation of gliadin may take place in the acidic environment in the stomach (118). Alternatively, it can be mediated by the enzyme tissue transglutaminase (tTG) as demonstrated by Molberg et al (123) and later also by van de Wal et al (122). tTG is expressed in many different tissues and organs; in the small intestine it is expressed just beneath the epithelium in the gut wall (123). Notably the activity of tTG is elevated in the small intestinal mucosa of CD patients in both the active disease phase and in remission (123a). The enzyme is present both intracellularly and extracellulary, and in the extracellular environment tTG plays a role in extracellular matrix assembly, cell adhesion, and wound healing (124). The calcium-dependent transglutaminase activity of tTG catalyzes selective crosslinking or deamidation of protein-bound glutamine residues (125). Notably, tTG is the same protein that Dieterich et al found to be a major focus of the autoantibody response in CD (6). In contrast to the nonenzymatically mediated deamidation that results in a near random deamidation of the often numerous glutamine residues in gliadin peptides, tTG appears to carry out an ordered deamidation of some few specific glutamines (123). For all the three DQ2-restricted gliadin epitopes recognized by gut T cells and the DQ8-a-gliadin-I epitope, the residues critical for T cell recognition are all specifically targeted by tTG (119, 122, 123). Interestingly, the deamidation of glutamines that are not targeted by tTG (e.g. by acid treatment) can be deleterious for T cell recognition (105, 122). Additional evidence for a role of tTG comes from experiments where T cell lines have been established from biopsies challenged with a minimally deamidated gliadin antigen (chymotrypsin-digested) and then tested for recognition of this antigen or the same antigen treated with tTG (Ø Molberg, S McAdam, KEA Lundin, C Kristiansen, K Kett, EH Arentz-Hansen, LM Sollid, manuscript in preparation). In 14 out of 15 patients, the T cell lines responded better to the antigen that had been subjected to treatment with tTG. Similarly, T cell lines established from two DQ2` patients by stimulating biopsies with a chymotrypsin-digested recombinant a-gliadin were found to recognize synthetic peptides representing the DQ2a-gliadin-I and DQ2-a-gliadin-II epitopes, but not the corresponding nondeamidated peptides (Ø Molberg, S McAdam, KEA Lundin, C Kristiansen, EH Arentz-Hansen, K Kett, LM Sollid, manuscript in preparation). Taken together, these results indicate that deamidation in vivo is mediated by tTG. It is intriguing to hypothesize that tTG plays a central role in the selection of gliadin T cell epitopes. Credence to this idea comes from the observation that the intestinal T cell response to a-gliadin in adults is focused on a single deamidated glutamine (in the related DQ2-a-gliadin-I and DQ2-a-gliadin-II epitopes) that is targeted by tTG (119). Knowledge of the substrate recognition sites of tTG should allow further testing of this hypothesis. Unfortunately, the available information

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on sequences targeted by tTG is not presently sufficient to establish the overall substrate specificity of the enzyme.

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HIERARCHIES OF GLUTEN T CELL EPITOPES? The existence of multiple epitopes in gluten that are recognized by small intestinal T cells of CD patients raises several interesting questions: Are only some of the epitopes pathogenic and thereby relevant to explain the HLA association? Are responses toward some of the epitopes generated during the early phases of disease development, while the responses to others are a result of epitope spreading? Are different epitopes recognized by distinct groups of patients (e.g. children vs. adults)? Are some epitopes more relevant to disease as responses to them are found in the majority of the patients or because there is a higher precursor frequency of T cells in the lesion specific for these epitopes? The answers to most of these questions must await further investigations. At present we know that for the DQ2-a-gliadin-I and DQ2-a-gliadin-II epitopes, intestinal T cell reactivity is found in most if not all adult DQ2` patients (119), whereas for the DQ2-cgliadin-I epitope, intestinal T cell reactivity is found in only a minority of DQ2` patients (118). Less is known about the DQ8-restricted epitopes because few DQ8-positive patients have been tested so far. However, the DQ8-a-gliadin-I appears to be frequently recognized (120). What causes the variance in responsiveness to the different epitopes and whether this reflects qualitative or quantitative differences between the patients are presently unclear. Epitope spreading (126) may be a mechanism relevant to CD that could explain the existence of several gluten epitopes. In experimental autoimmune encephalomyelitis where epitope spreading occurs, along with spreading of new antigenic epitopes there is also a ‘‘spreading’’ of MHC class II molecules involved in epitope presentation (127, 128). The strict restriction of DQ2 and DQ8 as presentation elements for gluten-reactive T cells of the disease lesion clearly deviates from the picture found in experimental autoimmune encephalomyelitis and may suggest that other mechanisms are operating. Further studies are clearly needed to sort out this question. The mapping of epitopes of gluten proteins recognized by intestinal T cells is incomplete; the actual number of distinct epitopes is currently a matter of speculation. However, recent results from testing intestinal T cells of Norwegian adults against a panel of recombinant a-gliadins suggest that the number of epitopes might be more limited than initially thought (119). From the sequences represented in a panel of full-length recombinant a-gliadins, there seems to be only a single immunodominant fragment that contains the two related epitopes DQ2-agliadin-I and DQ2-a-gliadin-II. The disease relevance of epitopes defined using peripheral blood T cells must also be questioned because peripheral blood T cell gliadin epitopes do not appear to be limited in their presentation by DQ2 or DQ8 (112) nor to be enhanced by

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treatment with tTG (123). Furthermore, a DQ2-restricted epitope of a-gliadin, which was defined by peripheral blood T cells of a CD patient (129) and which induces mucosal changes in peptide feeding experiments (130), fails to be recognized by gluten-reactive polyclonal intestinal T cell lines from six patients even after tTG treatment (119).

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GLUTEN-SPECIFIC T CELLS MAY PROVIDE HELP FOR AUTOANTIBODY PRODUCTION The IgG and IgA serum antibodies to tTG (also termed anti-endomysial antibodies) are a hallmark of CD, and detection of serum IgA tTG-antibodies is utilized to predict the disease (7, 8). As the B cells producing the tTG antibodies have undergone an isotype switch, it is likely that these are T cell-dependent antibody responses. This poses a problem since the existence of T cells recognizing tTG is doubtful. tTG is expressed ubiquitously in the human body, and staining with sera from untreated CD patients indicates that the antigen is also expressed in fetal thymus (131). Most likely, T cells reactive with tTG are therefore deleted by negative selection in thymus, and if they should exist they would likely have induced serious systemic autoimmunity. Interestingly, gluten seems to drive the antibody production, as the presence of tTG antibodies is strictly dependent on dietary gluten exposure (8). This raises the possibility that gluten-reactive T cells provide help for tTG-specific B cells by a mechanism of intramolecular help (132) analogous to the hapten-carrier system (133). As mentioned earlier, an important physiological role of tTG is the catalysis of isopeptide bond formation between glutamine and lysine residues (125). Indeed, it is the substitution of water rather than lysine in this reaction that results in deamidation. In vitro treatment of gliadin fragments with tTG leads to some gliadin fragments becoming covalently attached to tTG by autocatalysis (6, 123). tTG-specific B cells may selectively bind and internalize gliadin-tTG complexes via specific surface immunoglobulins. The gliadin fragment may finally be processed and presented by DQ2 or DQ8 to the gliadin-specific T cells, thereby providing cognate help for B cell maturation, isotype switching, and antibody secretion. This model can explain why tTG antibody levels in CD are dependent on the presence of gliadin in the diet because its removal will also abolish the T cell help needed for antibody production. Autocatalysis by tTG should be more likely to occur when the concentration of other amine donors (lysine containing proteins/primary amines) is low. In fact, deamidation is also likely to happen when the amount of primary amines is low or absent (125). Formation of gliadin-tTG complexes and deamidation of gliadin may thus reflect an altered microenvironment in the gut mucosa. The unusual ability of gliadins to act as excellent amine acceptor substrates for tTG may result in a local depletion of lysine/polyamines, and the altered microenvironment may hence be established in situations where increased levels of gluten proteins get access to the subepithelial area.

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PERTURBED ORAL TOLERANCE TO GLUTEN IN CELIAC DISEASE? Although the concept of oral tolerance is not as firmly established in humans as it is in rodents, it is clearly necessary that mechanisms that allow for tolerance to soluble food antigens exist in humans (134). In keeping with this thinking, oral tolerance to gluten in patients with CD either is not established properly or is broken. A deeper understanding of this issue should shed new light on the mechanism behind oral tolerance in humans. Given the preferential intestinal T cell response to deamidated gluten fragments in CD patients, it is conceivable that deamidation is central to the perturbation of the oral tolerance. Deamidation increases the binding affinity of gliadin peptides for DQ2 from poor but significant binders to epitopes with reasonable, but by no means exceptional, affinity (118, 119). The moderate binding affinity of these epitopes concurs with the finding that they do not carry optimal anchors in all the anchor positions. It is interesting that the modified glutamine residues for the three defined DQ2-restricted gliadin epitopes recognized by intestinal T cells occupy different pockets within DQ2 (Figure 3). This suggests that the altered affinity of the gliadin peptides for DQ2 is a critical factor involved in loss of tolerance rather than recognition of a single ‘‘pathogenic’’ motif that binds to DQ2 (119). Concurrent with the increase in affinity for DQ2 caused by deamidation of the gliadin peptide is a change in conformation of the gliadin/DQ2 complex. This is apparent by the failure of the T cells to recognize the unmodified peptides even at higher concentrations that should compensate for their lower affinity for DQ2. However, the simple modification of glutamine residues that act as major T cell receptor contact residues appears not to be sufficient to break tolerance as none of the modified glutamines are found in such positions (105, 119). Gliadin fragments containing two glutamine residues targeted by tTG may well be deamidated and cross-linked to other proteins that contain lysine. Conditions may exist in the gut, where T cell epitopes are both created and trapped locally by tTG, that prevent the epitopes from being presented by antigen-presenting cells that induce tolerance in the gut. Alternatively, it may prevent these epitopes from spreading systemically, a factor thought to be important in the establishment of oral tolerance (135). In this regard it is interesting that the motif targeted by tTG and shared in the DQ2-a-gliadin-I and DQ2-a-gliadin-II epitopes is repeated within many of the a-gliadins (119).

MECHANISMS INVOLVED IN FORMATION OF THE CELIAC LESION The evidence discussed above provides strong evidence that CD4` TCRab` T cells in the lamina propria are central for controlling the immune response to gluten that produces the immunopathology of CD. The knowledge of the events

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downstream of T cell activation is, however, still incomplete. The characterization of mechanisms operating in the model of human fetal gut explant cultures, where activation of T cells induces villous atrophy and hyperplasia of the crypts, has provided interesting clues and indicated some major pathways (136). However, knowing how the immune system usually utilizes a multitude of effector mechanisms for fighting its opponents, it is reasonable to believe that multiple effector mechanisms may well be involved in the creation of the celiac lesion. Adding to the complexity, recent in vitro organ culture studies have indicated that gluten exerts additional immune relevant effects independent of T cell activation (137, 138). Some of these effects have rapid kinetics, and conceivably the direct effects of gluten may facilitate subsequent T cell responses. Cytokines produced by lamina propria CD4` T cells may be involved in the increased crypt cell proliferation and the increased loss of epithelial cells. IFN-c induces macrophages to produce TNF-a. TNF-a activates stromal cells to produce KGF, and KGF causes epithelial proliferation and crypt cell hyperplasia (17). IFN-c and TNF-a can jointly have a direct cytotoxic effect on intestinal epithelial cells (139). It is also conceivable that IELs and in particular cd T cells play a role in the epithelial cell destruction by recognizing MIC molecules induced by stress (26). Alterations of the extracellular matrix can also distort the epithelial arrangement, as the extracellular matrix provides the scaffold on which the epithelium lies. Enterocytes adhere to basement membrane through extracellular matrix receptors so that modification or loss of the basement membrane can result in enterocyte shedding. Evidence for increased extracellular matrix degeneration in CD exists, and this degeneration may be important for the mucosal transformation found in CD (40). The increased production of metalloproteinases by subepithelial fibroblasts and macrophages is likely to be directly or indirectly induced by cytokines that are released from activated T cells. Do the autoantibodies play a role in the pathogenesis of CD, or are they just an epiphenomenon? The significant increase in prevalence of CD among IgAdeficient individuals (1) speaks against a role of the antibodies. However, most CD patients also have elevated levels of serum IgG endomysial (i.e. tTG) antibodies (5), and little is known about the antibodies found locally in the mucosa of IgA-deficient CD patients. Interestingly, the endomysial (i.e. tTG) antibodies can, as suggested by Ma¨ki and coworkers (140), be involved in the disease development by blocking interactions between mesenchymal cells and epithelial cells during the migration of epithelial cells and fibroblasts from the crypts to the tips of the villi. tTG is necessary for activation of transforming growth factor-b (TGFb) (141). Indirect inhibition of TGF-b activation by anti-tTG antibodies can be envisaged to have broad effects as TGF-b is known to affect the differentiation of the intestinal epithelium (140), to stimulate extracellular matrix formation (142), and to regulate the function of many immune competent cells within the gut microenvironment (143). In addition, tTG has been demonstrated to be involved in attachment of fibroblasts to the extracellular matrix (144), suggesting

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that the autoantibodies could also be involved in lesion formation by perturbing important contacts between fibroblasts and extracellular matrix components. The tTG antibodies may in addition modulate the deamidating activity of tTG in either an inhibiting or a promoting fashion (145). Further research is clearly needed to establish whether and how the tTG antibodies play a role in CD pathogenesis.

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HLA ASSOCIATION WITH DISEASE: LESSONS TO BE LEARNED FROM CD Strong evidence suggests that the primary HLA association in CD is to the classical peptide presenting HLA molecules DQ2 and DQ8. These HLA molecules predispose to disease by presenting gluten peptides to CD4` T cells in the affected organ, although an effect mediated by shaping of the T cell repertoire in the thymus cannot yet be excluded. This has clear relevance for studies of other HLAassociated diseases where the identity of the HLA molecules involved are less well defined and where the triggering antigens have not been identified. The DQ2 and DQ8 molecules bind gluten peptides that after specific deamidation become good peptide ligands for DQ2 and DQ8. Exactly why no other class II molecules are able to present gluten peptides in the gut that result in disease is not yet fully understood. Likely related is that peptides that become deamidated in the gut mucosa are particularly effective in inducing a pathologic immune response, and that the DQ2 and DQ8 molecules are especially suited to bind deamidated peptides. The DQ(a1*0501, b1*0301) and DQ(a1*0201, b1*0202) molecules which are related to the predisposing DQ(a1*0501, b1*0201) molecule and which do not predispose to CD have different binding motifs, although the binding motif of DQ(a1*0201, b1*0202) is very similar. Interestingly, DQ(a1*0501, b1*0201) and DQ(a1*0201, b1*0202) expressing B lymphoblastoid cell lines exhibit abilities to present the DQ2-a-gliadin-II epitope that differ according to when the epitope is incorporated into a complex antigen that requires processing as compared with the peptide that is processing independent (105). This might suggest that factors involved in processing and peptide binding act differentially for loading of the gliadin peptides to two DQ molecules and that this is relevant for explaining the HLA association. Modification of self-proteins analogous to gliadin in CD would create epitopes recognized as nonself. This could be a more general mechanism for breaking of immunological tolerance and precipitation of autoimmune disease. Perhaps as many as 50% to 90% of the proteins in the human body are posttranslationally modified (P Roepstorff, personal communication), and the degree and type of modification are likely to be altered in an inflamed microenvironment. Epitopes harboring a posttranslational modification may go unreported, as the standard use of recombinant proteins and synthetic peptides for the characterization of T cell epitopes means that most in vivo modified epitopes would escape detection. This class of epitopes should not be overlooked, and it will be important to devise

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strategies that will identify modified T cell epitopes of potential autoantigens so that their role in autoimmune disease can be clarified. Another important point illustrated from the studies of CD is how a foreign antigen drives autoantibody production. For most autoimmune diseases, autoantigens have been defined by use of the autoantibodies. It is often inferred that T cells must exist that are reactive with the autoantigen because the antibodies are of the IgG or IgA isotypes whose formation is dependent on T cell help. In some cases this assumption may turn out to be unjustified. It is in my opinion appropriate to intensify the search for unknown foreign agents that might be hosted by the human body (virus, bacteria, etc) and that are capable, after combining with a self-protein, of providing help for autoimmune responses similar to that found in CD. This review illustrates that the molecular basis of CD is complex. Given the multifactorial etiology of the disease with involvement of several genes and environmental factors, this is not unexpected. Despite the recent advances in understanding of critical steps in disease development, there is much still to be learned about the disease. Several predisposing genes are yet to be identified. Given the difficulty in defining susceptibility genes with modest effects, a combined functional and genetic approch will be required for their identification. The full understanding of multifactorial inflammatory diseases is surely a formidable challenge for scientists. Compared with the other diseases of this nature, however, CD stands out as a disease for which it should be easier to decipher both the actions of the predisposing gene products and how they interact with other gene products and environmental factors. In this situation it is justified to call for intensified research on CD as this can serve as an illuminator for the other multifactorial inflammatory diseases. ACKNOWLEDGMENTS I thank Knut E. A. Lundin, Stephen N. McAdam, Øyvind Molberg, and Frode Vartdal for critically reading the manuscript. I also thank members of the ‘‘celiac disease research group’’ at my Institute for stimulating discussions and Erik Thorsby for continous support. The author’s research is supported by grants from the Research Council of Norway and the European Commission (BMH4-CT98– 3087). Visit the Annual Reviews home page at www.AnnualReviews.org.

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Triggered human mucosal T cells release tumour necrosis factor- alpha and interferon-gamma which kill human colonic epithelial cells. Clin. Exp. Immunol. 83:79–84 Halttunen T, Ma¨ki M. 1999. Serum immunoglobulin A from patients with celiac disease inhibits human T84 intestinal crypt epithelial cell differentiation. Gastroenterology 116:566–72 Nunes I, Gleizes PE, Metz CN, Rifkin DB. 1997. Latent transforming growth factor-b binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-b. J. Cell Biol. 136:1151–63 Bonewald LF. 1999. Regulation and regulatory activities of transforming growth factor ß. Crit. Rev. Eukaryo. Gene Expr. 9:33–44 Letterio JJ, Roberts AB. 1998. Regulation of immune responses by TGF-b. Annu. Rev. Immunol. 16:137–61 Verderio E, Nicholas B, Gross S, Griffin M. 1998. Regulated expression of tissue transglutaminase in Swiss 3T3 fibroblasts: effects on the processing of fibronectin, cell attachment, and cell death. Exp. Cell Res. 239:119–38 Sollid LM, Scott H. 1998. New tool to predict celiac disease on its way to the clinics. Gastroenterology 115:1584–86

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Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:83–111 Copyright q 2000 by Annual Reviews. All rights reserved

POPULATION BIOLOGY OF LYMPHOCYTES: The Flight for Survival Antonio A. Freitas1 and Benedita Rocha2 1

Lymphocyte Population Biology Unit, URA CNRS 1961, Institut Pasteur, 25 Rue du Dr. Roux, 75015 Paris, France; e-mail: [email protected] 2 INSERM U345, Institut Necker, 156 Rue Vaugirard, 75015 Paris, France; e-mail: [email protected]

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Key Words lymphocyte survival and life spans, lymphocyte competition, lymphocyte homeostasis, naive and memory lymphocytes Abstract In this essay we suggest that the primary goal of the cells of the immune system is to ensure their own growth and survival. In adults, in steady-state conditions, the number and distribution of lymphocyte populations is under homeostatic control. New lymphocytes that are continuously produced in primary and secondary lymphoid organs must compete with resident cells for survival. We discuss recent findings supporting lymphocyte survival as a continuous active process and implicating cognate receptor engagement as fundamental survival signals for both T and B lymphocytes. The conflict of survival interests between different cell types gives rise to a pattern of interactions that mimics the behavior of complex ecological systems. In their flight for survival and in response to competition, lymphocytes use different survival signals within different ecological niches during cell differentiation. This is the case for T and B lymphocytes and also for naive and memory/activated T and B cells. We discuss how niche differentiation allows the co-existence of different cell types and guarantees both repertoire diversity and efficient immune responses.

INTRODUCTION Life started when self-replicating molecules arose (1). The first unicellular organisms emerged when these molecules developed the capacity to control their immediate surroundings to ensure their own survival and replication. With time, single-cell individuals evolved to give rise to multicellular organisms. In these complex individuals, each cell is still imprinted with the same primordial program for self-replication and survival (2). A hierarchical organization is, however, established in which the survival and the rate of replication of the different cell types are restrained and their number controlled (3). The individual cells of the immune system follow the same program, but they can survive only within the limited constraints imposed by the host. In adult mice the total number of lymphocytes remains constant and shows a ‘‘return tendency, 0732–0582/00/0410–0083$14.00

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due to a density dependent process to approach a stationary distribution of population densities’’ (4), usually referred to as homeostasis. T and B lymphocytes are, however, produced continuously in either the primary lymphoid organs or by peripheral cell division; it follows that each newly formed lymphocyte can persist only if another resident lymphocyte dies. In an immune system in which the total number of cells is limited, cell survival can no longer be a passive phenomenon; it is rather a continuous and active process (5) in which each lymphocyte must compete with other lymphocytes (6). It can be said that lymphocytes follow the Red Queen Hypothesis—‘‘it takes all the running you can do to keep in the same place’’ (5, 7).

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Naive T Cells Maintenance of naive T cells in peripheral lymphoid organs requires continuous T cell receptor (TCR) engagement by major histocompatibility complex (MHC) molecules. This was established for both CD4` and CD8` lymphocytes using various experimental approaches. In the case of naive CD8` T cells, their capacity to survive was initially studied after adoptive transfer into mice expressing different MHC class I alleles. Monoclonal naive CD8` cells from TCR transgenic (TCR-Tg) mice, restricted to MHC class I H-2Db, survived without dividing when transferred to mice lacking H-2Kb but expressing normal levels of H-2Db (8). In mice expressing lower levels of H2Db, only a fraction of the transferred cells survived—this fraction was proportional to the level of H-2Db expression. In H-2Kb`Db1 host mice the TCR-Tg cells rapidly disappeared (8). The requirements for the peripheral survival of this naive CD8` T cell clone mimic, therefore, the requirements for positive selection of the same clone in the thymus (9); these requirements include the continuous ligation of the TCR by MHC and that the number of selected cells is related to the level of MHC class I expression (10). The existing experimental evidence indicates that the results obtained with this TCR-Tg clone apply to naive CD8` T cell populations in general. Naive monoclonal CD8` T cells, expressing a TCRTg specific for the gp33–41 peptide of LCMV and restricted to H-2Db (11), also persist after transfer into CD3e-/-H-2Kb1Db` hosts but these cells disappear by one week after injection into CD3e-/-H-2Kb`Db1 hosts (N Legrand, AA Freitas, unpublished). When wild-type thymic epithelium was grafted into MHC class I– deficient mice, mature CD8` T cell differentiation in the thymus was restored, but naive CD8` T cells produced in the thymus were unable to survive at the periphery (12). Naive CD4` T cells also require allele-specific MHC class II interactions to survive (13, 14). Monoclonal TCR-Tg CD4` T cells, restricted to I-Ad, survive after transfer into I-Ad` recipients but disappear after transfer into I-Ad1Ab` recip-

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ients. Persistence of non-Tg CD4` polyclonal populations also depends on interactions with MHC class II. Using retroviral vectors, class II expression was transiently induced in the thymus of class II–deficient mice, allowing cohorts of CD4` T cells to be generated and exported into class II1 peripheral pools (15). Alternatively, MHC class II` fetal thymus grafts were implanted into class II– deficient hosts (16). In both cases, permanent CD4` survival was dependent on the expression of class II at the periphery, but the exported CD4` T cells took 100 days to disappear in the absence of class II. Although these results demonstrate that permanent mature CD4` T cell engraftment requires TCR/MHC class II interactions, in the absence of these interactions polyclonal CD4` populations appeared to survive much longer than CD8` T cells. This relatively prolonged survival could be due to the presence of activated cells; as most persistent survivor cells were of an activated phenotype (16), these cells (when compared to naive T cells) could be less dependent on MHC for survival (see below). It was also likely that within a heterogeneous polyclonal T cell population, some clones could interact with MHC class I- or class II–like molecules and thus would be able to survive longer in absence of bona fide TCR/class II interactions (17). Alternatively the prolonged CD4` survival could be due to the presence of MHC class II–bearing dendritic cells (DC) exported by the class II` thymus (18). When a class II` thymus depleted of all BM-derived class I` cells was grafted into class II1 mice, the CD4` T cells generated in the thymus did not survive at the periphery (18). When the transplanted class II–deficient hosts expressed a transgene of the same MHC class II molecule on the peripheral DC, the CD4` cells exported from the thymus survived in the periphery (18). In this case CD4` T cells were shown to be in close contact with transgenic class II` DC, suggesting that this direct interaction plays a key role in CD4` survival. When these hosts expressed at the periphery a class II transgene of a different haplotype from the thymus class II, the majority of the naive CD4` T cells generated and exported from the thymus did not survive (T Brocker, personal communication). These findings indicate that polyclonal naive CD4` T cell populations require TCR/MHCrestricted interactions for peripheral survival. TCR/MHC class II interactions are also required for the expansion of CD4` T cells in T cell–deficient hosts (19). Thymus positive selection requires the simultaneous recognition of MHC and peptide molecules presented by the MHC. The role of such peptide recognition in peripheral T cell survival was also investigated by comparing the fate of mature T cells in environments of restricted peptide complexity. In H-2Ma-deficient mice, the level of expression of MHC class II is not modified, but the loading of peptides into the MHC class II molecules is impaired. Most of the I-A molecules are occupied by the invariant chain peptide CLIP (20). This limited peptide display affects thymus selection, and the peripheral T cells from these mice have a restricted repertoire diversity (20). Polyclonal CD4` T cells and TCR-Tg cells restricted to I-Ab do not survive when transferred either to H-2Ma–deficient mice, to mice expressing a different MHC class II allotype, or to MHC class II–deficient hosts (14). The similar behavior of peripheral T cells in environments of restricted

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peptide diversity or MHC deficiency establishes that peptide recognition is essential for T cell survival. The peptides involved are probably similar to those mediating thymus positive selection. Thus, CD4` T cells from H-2Ma–deficient mice that were selected by peptides expressed by the H-2Ma–deficient thymus survived when transferred into H-2Ma–deficient peripheral pools but not when transferred into MHC class II–deficient hosts (14). CD4` T cells selected by a single MHC class II/peptide complex survived without dividing in mice expressing the same complex at the periphery (21). These results demonstrate that, like thymus positive selection, peptide recognition plays a role in peripheral T cell survival. They also suggest that the peptides involved in thymus positive selection and peripheral survival may be similar. It thus appears that naive mature T cells, following emigration from the thymus, must continuously recognize MHC/peptide complexes in order to survive in the periphery. Like thymus positive selection, this TCR ligation event (a) is MHC restricted, (b) involves recognition of similar self-peptides, (c) maintains bcl-2 levels of expression, since when TCR/MHC interactions are discontinued bcl-2 levels decrease (13), and (d) does not induce extensive cell division because the vast majority of naive peripheral T cells appear to survive as resting T cells. CD441 TCR-Tg or wild-type CD8` cells do not divide (8, 22, 23). Naive CD4` T cells either do not divide or they cycle very slowly (21, 24). In spite of these similarities, peripheral survival and thymus positive selection signals probably do not overlap. Positive selection is mainly determined by interactions between T cells and MHC/peptide complexes expressed by the thymus epithelial cells, a cell type absent at the periphery. At the periphery, a close interaction between surviving CD4` T cells with class II` DC was identified (18). It is not known if survival of naive CD8` T cells also needs interactions with particular cell types. Besides, thymus positive selection and naive T cell survival also seem to differ in lymphokine requirements, as suggested by studies on H-Y antigen-specific TCR-Tg mice, deficient for the common c chain of the IL-2 receptor (cc-mice). The cc-deficient mice have severe defects of thymus T cell differentiation. Because productive TCR rearrangements are rare, immature precursors survive poorly, most dying before TCR gene rearrangement. These abnormalities of thymus differentiation can be partially overcome when the cc-mice are crossed into an ab-TCR-Tg background (25). Although producing CD8` mature T cells in the thymus, these mice still lack peripheral CD8` T cells. These results suggest that peripheral T cell survival is not solely dependent on signals mediated by the TCR; it may also depend on signals transmitted by lymphokine receptors. The lymphokine/s required for naive T cell survival were not yet identified.

Memory T Cells Memory T cells express at the cell surface a vast array of receptors (lymphokine receptors, costimulatory and adhesion molecules) that are absent or expressed at low levels in naive cells (8, 26–28). The expression of these receptors will likely

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modify the interactions of memory cells with their environment in a yet unidentified way. Changes in overall receptor expression may contribute to modify the T cell avidity for antigen and/or MHC and to lower the thresholds of survival and activation (28–33). Moreover, some of these receptors may be able to transmit activation signals and induce cell division independently of TCR engagement. The requirements for the survival and division of memory T cells are not yet completely characterized. Current studies, however, indicate that they differ from those of naive T cells, suggesting that lymphocytes are ‘‘able to tune their threshold of activation in response to recurrent signals’’ (34). Memory CD8` T cells expressing the H-Y-specific TCR-Tg, restricted to MHC H-2Db class I, survive and expand in the absence of either H-Y male antigen or the MHC-restricting element in female H-2Kb`Db1, but they disappear in mice lacking both b2microglobulin and H-2Db (presumably expressing few MHC class I molecules) (8). The same anti-H-Y TCR-Tg cells stimulated in vitro by the cognate peptide were transferred to TAP-1` Rag-deficient or TAP-11 Rag-deficient mice (35). All memory cells survived for at least 70 days in mice expressing TAP-1, while most Tg cells disappear two weeks after transfer into TAP-1-deficient recipients. To investigate if the behavior of this T cell clone can be extrapolated to CD8` memory T cell populations in general, we studied the survival of CD8`CD44` polyclonal memory cells from normal B6 mice. Since the MHC-restriction elements of this polyclonal population cannot be identified we followed their fate after transfer into syngeneic hosts lacking simultaneously b2-microglobulin, H2Db and H-2Kb MHC class I molecules. We found that one week after transfer 80% to 90% of the transferred memory T cells disappeared (B Rocha, AA Freitas, F Lemonnier, unpublished). Although long-term survival has yet to be studied, these results suggest that the vast majority of the memory CD8`CD44` polyclonal T cell populations still require some type of TCR/MHC class I interactions to survive. The requirements for the survival in vivo of memory CD4` T cells have not yet been investigated, but probably they also differ from those of naive CD4` T cells. In MHC class II–deficient mice, the rare CD4` T cells that are able to persist at the periphery bear a very activated phenotype. Cells with a similar phenotype also persist in class II and b2-microglobulin–deficient mice, which appear to recognize MHC-like molecules (17). Although memory T cell survival may require TCR-MHC-peptide interactions, in contrast to naive cells this recognition does not appear to be MHC restricted (8). It is difficult to envisage how such nonrestricted interactions would trigger memory T cells. Nonspecific signaling through the CD4/CD8 coreceptors might be involved, as Lck was found to be targeted to the CD8 coreceptor in LCMVspecific memory CD8` T cells (36). Alternatively a lower threshold of T cell activation would allow signaling by allo-MHC. It must be noted, however, that so far memory T cell survival has been studied only after transfer into irradiated hosts. In these mice, memory T cells (but not naive cells) divide extensively (37). Part of this proliferation may be due to mitogenic factors liberated after irradia-

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tion. The crossing of MHC-deficient mice into a CD3e-/- background will allow the study of memory T cell survival in intact hosts in the absence of possible artifacts induced by irradiation. Memory T cells also differ from naive cells in the state of activation associated with survival. While naive T cells persist mainly as long-lived resting cells, memory cell survival is accompanied by cell division (self-renewal). In normal mice a fraction of memory T cells are cycling and dividing in the absence of nominal antigen stimulation (8, 23, 32, 35, 37–42). The rate of memory T cell division can be enhanced by injection in vivo of lymphokines such as IL-2, IL-12, IL-15, and a-interferon (43). During immunization the increased production of growth factors also leads to the by-stander activation of memory T cells of unrelated specificity (31, 44). Memory T cell proliferation increases in conditions of T cell depletion. When mice are sublethally irradiated, residual activated T cells expand (37). T cell expansion is evident when T lymphocytes are transferred to T cell– deficient or irradiated hosts (38, 45). This expansion is at least partially responsible for the early increase in T cell numbers observed in treated AIDS patients (46) and in irradiated adults after BM transplantation (47). In these conditions peripheral mature T cells retain a considerable expansion potential that, in the absence of intentional immunization, allows a single CD4` T cell to generate up to 1015 daughter cells upon sequential transfers into successive T cell–depleted hosts (38). The finding that memory T cells divide in the absence of their cognate antigen raises several (as yet unanswered) questions. The first concerns the existence of long-lived resting memory T cells. It is possible that no memory T cell is actually in the Go phase of the cell cycle. Memory populations are likely not constituted by cells that cycle and cells that never divide. By studying the dilution of CFSE labeling in LCMV-specific memory T cells in steady-state conditions, all these antigen-specific memory T cells were shown to divide (K Murali-Khrishna, R Ahmed, in preparation). Results of studies of BrdU accumulation by memory CD8` T cells are also compatible with long cycle times and asynchronous division (23). If all memory cells are in some type of pre-activated state, this may favor their rapid and efficient engagement in secondary responses (H VeigaFernandes, U Walter, A McLean, B Rocha, submitted). The second question refers to the type of signals driving antigen-independent division. Survival and proliferation of memory cells may be driven by IL-15 (48) or by several other cytokines that are able to increase the rate of proliferation of memory T cells (43). It is probable, however, that some type of cognate TCR/MHC/peptide interactions may also be involved: CD4` cells selected by a single MHC/peptide complex do not divide after transfer to irradiated mice expressing the same complex, but they expand after transfer into normal mice (21). These results suggest that peptides responsible for thymus-positive selection and peripheral survival differ from those driving peripheral T cell division, the latter phenomenon involving some type of TCR ‘‘cross’’-reactivity.

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Finally the most important question concerns the functional activity induced by non-specific T cell stimulation. What are the functional differences between these naturally activated T cells and T cells triggered in the course of an immune response? Several (non-exclusive) possibilities may be considered. Low-affinity signals may (a) reduce the frequency of the cells engaged in different effector functions, or (b) reduce the efficiency of each different effector function. For example, production of cytokines after non-specific stimulation may be so low that they can only promote autocrine growth (and maintain antigen-independent division of memory T cells); they would be unable to act on neighboring cells. (c) Different effector functions may require different thresholds of activation. Some type of cytokine secretion (required for memory T cell growth) may be induced by weak interactions, while T cell cytotoxicity depends on a strong T cell activation. In a lymphocyte receiving multiple signals through several cell surface receptors, what will determine the strength of T cell activation? How lymphocytes integrate TCR-mediated signals with signals transmitted by other cell surface molecules is largely unknown. These signals may act independently, antigen-specific engagement being absolutely required to induce some effector functions. Alternatively some type of integration may take place: In this latter case, a non-specific TCR engagement associated with other intense stimulation through other receptors may also be able to induce strong T cell activation. The answers to these questions have major implications for our understanding of autoimmunity. Mature memory T cells are continuously triggered by nonspecific TCR/MHC/peptide engagement. Strong activation through alternative receptors may explain the emergency of auto-aggression in conditions of lymphokine imbalance (49, 50) or of deficient control of T cell activation and growth (51, 52). Secondly, situations in which T cell repertoires are restricted are now frequent. In AIDS or after irradiation or chemotherapy, the restoration of immunecompetence may be dependent on our capacity to induce low-affinity T cells to mediate effector functions.

B Cell Survival Survival of naive B cells in the peripheral pools also appears to involve interactions between the B cell receptor (BCR) and yet unidentified ligand(s). This was first suggested by experiments in which a transgenic BCR could be ablated by an inducible Cre-LoxP recombination event. After BCR ablation, B cells reportedly rapidly disappeared from the peripheral pools (53). This study, however, did not directly correlate BCR signaling and peripheral B cell survival, as BCR ablation also leads to the arrest of new B cell production in the BM (54). In the absence of the newly formed BM migrants, a significant fraction of the peripheral B cells is rapidly lost (55). In mice with a deletion mutation of the Iga cytoplasmic tail, early B cell development in the BM exhibited only a small impairment, but the generation of the peripheral B cell pool was severely reduced (56). The question remained as to whether the mere presence of a signaling complex, e.g. IgM-

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Iga-Igb or other, at the cell surface suffices to signal B cell survival, or if B cell survival requires ligand recognition. Other studies addressed directly the role of ligand-mediated recognition in peripheral B cell survival. B cells lacking the Vregion of the IgM receptor have a very short life-expectancy (57). The presence of the truncated membrane IgM transgene, lacking the V-region, provides constitutive signals that suffice to signal allelic exclusion and to promote pre-B cell development in the absence of the surrogate light chain k5 (58), but the transgene fails to support long-term survival of the Tg B cells (57). Differences in the antibody repertoires expressed by pre-B and peripheral B cells can also be invoked to suggest the involvement of ligand-mediated recognition in peripheral B cell positive selection and persistence (59–61). In contrast to T cells, in which TCR survival signaling seems to require the recognition of MHC class I or class II molecules, the nature of the ligand(s) that might be involved in B cell survival remains elusive. Analysis of VH-gene family expression has provided evidence for a very conserved pattern of VH-gene family usage that is independent of the VH-gene number, is strain and tissue specific, and is tightly regulated during B cell differentiation (60). After B cell generation in the BM, naive B cell survival is associated with a strong peripheral selection of B cells expressing particular VH-gene families (61, 62). These observations suggest that the recognition interactions related to B cell survival may not require the involvement of the full antigen-binding site and might be exclusively VHmediated. It is possible that this type of ligand recognition may not lead to full cell activation and that low avidity interactions suffice to promote cell survival. It was recently shown that the BCR is capable of differential signaling and that B cell responses may differ depending upon the properties of the antigen. Thus, while some B cell responses were better correlated with antigen-BCR affinity than with receptor occupancy, others were only weakly dependent on antigen affinity (63). Memory B cells not only show phenotypic changes and express a hypermutated BCR of a different isotype with an increased avidity for antigen, they also show a higher rate of cell division and a lower threshold of activation, and they occupy a different habitat within the secondary lymphoid organs. Studying memory responses to both thymus-dependent and independent antigens and using different cell transfer systems has revealed that long-term persistence of B cell memory requires continuous cell division in the presence of antigen (64–66). Recent observations in mice in which an inducible Cre-loxP-mediated gene inversion (67) is used to change the specificity of the BCR contradict these results. In these experiments, after antigenic stimulation and the generation of memory B cells, memory cells survived even after expressing a new BCR unable to bind to the original activating antigen (M Maruyama, K Lam, K Rajewsky, personal communication). These findings suggest that antigen may be required only at early stages of cell activation and selection of high-affinity B cells in the germinal centers. Once these B cells acquire a memory phenotype, they may no longer require antigen recognition for long-term survival.

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In this context it is important to mention that the difference in the interactions required for the survival of B and T cells may explain the different rates of mutation of B and T cell receptors. Since T cell survival requires recognition of ubiquitous MHC molecules, somatic mutation of the TCR might always be disadvantageous as it may cause loss of MHC recognition and thus cell death. In contrast, somatic mutation may increase the BCR-ligand avidity and favor selection of high-affinity B cells which, compared to the initial population of nonmutated B cells, have a competitive survival advantage within the germinal center. In their flight for survival, B cells are still able to use mechanisms of receptor editing (68–70) and to change BCR specificity to escape cell death; they thus may gain a survival advantage over other rival cells. Lymphocyte survival is likely to involve multiple mechanisms. Different signals may engage different cell surface receptors using the same or differing survival pathways. Naive T cell survival depends on signals transmitted via lymphokine receptors (71), and it requires the expression of LKLF (72) or NFAT4 (73) transcription factors. The constitutive expression of the Epstein-Barr virus LMP2A protein in transgenic mice bypasses BCR signaling and allows the survival of receptor-less B cells in the peripheral pools (74). Downstream of the BCR signaling pathway, defects in CD45 (75), Btk (76), Syk (77), or NF-jB (78) or in the OBF-1 transcription factor (79) also affect peripheral B cell maintenance. Lymphocyte survival is also modified through the balance between different apoptotic and anti-apoptotic proteins (80–82). BCR signaling and increased levels of intracellular bcl-2 ensure lymphocyte survival through independent pathways (53, 57). The expression of bcl-2 may simply increase cell efficiency by lowering the threshold of resource requirements for survival.

HOMEOSTASIS AND COMPETITION Lymphocyte Production In adult mice the potential to produce new lymphocytes in the primary lymphoid organs or by peripheral cell division largely exceeds the number required to replenish the peripheral pools. Firstly, the number of immature T cell precursors in the thymus is not limiting. A normal-sized T cell pool can be generated in Rag2-deficient mice reconstituted with a mixture of 50% normal BM and 50% BM cells from a CD3e-/- deficient mice that are unable to generate T cells. The thymus production of mature T cells is also in excess as shown in mice in which thymus export is artificially reduced. Mice with reduced thymus export are Rag2-deficient hosts reconstituted with normal BM cells diluted in TCRa-deficient BM (that cannot generate mature T cells). In these mice, peripheral T cell numbers are maintained in spite of the lower production of mature T cells (A Almeida, AA Freitas, unpublished). Finally, T cells can also be produced by peripheral cell division. In a normal mouse,

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peripheral division is restricted. When peripheral T cells are transferred into a T cell–deficient host, the cells are capable of considerable expansion (38). Each lymphocyte could generate a progeny of about 1015 cells (38). In theory, a single T cell could be sufficient to repopulate the peripheral T cell pool of a mouse. Secondly, an increase in lymphocyte production is not directly reflected in the size of the peripheral T cell pool. Mice grafted with increasing numbers of thymuses do not show a proportional increase in the total number of naive peripheral T cells (83–85). All these results indicate that the total number of peripheral T cells is not necessarily determined by T cell production but is limited at the periphery. Mature B cells are produced in much higher numbers than those required to fill the peripheral B cell pool. The number of B cell precursors is not limiting, as shown in mice in which the B cell precursor number is artificially reduced. The number of pre–B cells can be reduced in Rag2-deficient mice, which were lethally irradiated and reconstituted with mixtures of normal BM cells diluted among incompetent BM cells from B cell–deficient (lMT) donors. In these chimeras the number of BM pre–B cells is proportional to the fraction of normal BM cells injected. Mice containing less than 25% of the normal number of pre–B cells had reduced peripheral B cell numbers. A normal-sized peripheral B cell pool, however, was generated in mice containing only 30% of the normal number of pre– B cells. These results demonstrate that about one third of the normal number of BM B cell precursors suffices to maintain the peripheral B cell pool size (86). A similar conclusion was reached after parabiosis between one normal and one or two B cell–deficient mice. In these circumstances B cell production was restricted to the BM of the normal mouse since no chimerism was detectable in the BM of the different partners. In mice triads each individual mouse had physiological B cell numbers, i.e. the B cell production of one mouse was sufficient to populate the peripheral pools of three mice (86). These results demonstrate that peripheral B cell numbers are not determined by the rates of BM B cell production but are limited at the periphery.

Demonstration of Lymphocyte Competition In an immune system where new lymphocytes are continuously produced in excess but their total numbers are kept constant, newly generated cells have to compete with other newly produced or resident cells to survive. Competition can be defined as ‘‘an interaction between two populations, in which, for each, the birth rates are depressed or the death rates increased by the presence of the other population’’ (87). There are two main established criteria accepted as evidence of competition among populations: 1. The presence of competitors should modify the equilibrium size of a population, and 2. their presence should alter the dynamics, e.g. the life span, of a population (88). The question of whether competition arises between B and T cells was addressed by comparing the development and the fate of TCR or BCR-Tg and non-Tg populations in several different lines of

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mouse BM chimeras (89, 90). It was found that (a) when injected alone, Tg and non-Tg cell populations show an identical behavior and generate peripheral pools of similar size and (b) when Tg and non-Tg cells are mixed in the same host, they initially accumulate at the same rate. However, after reaching steady-state numbers, a preferential selection of the non-Tg cells occurs at the periphery. These observations fulfil the first criterion for competition since they demonstrate that the presence of non-Tg populations modifies the number of the Tg cells (89, 90). In these experiments the life-expectancy of the Tg, T, or B cells varied according to the presence and the type of other competing cells (89–91). These latter findings fulfil the second main criterion as they prove that the presence of competitors alters the life span of a population. By considering each cell clone as a species, competition between Tg and nonTg cells represents an example of inter-specific competition. Competition may also occur between individuals of the same species (intra-specific competition) (87). Studies using monoclonal mice may allow determination of whether competition also occurs among cells of the same clone.

Types of Competition and Resources Competition may arise through different processes. In interference competition, populations may interact directly with each other, or one population can prevent a second population from occupying a habitat and from exploiting the resources in it. Thus, although interference competition may occur for a resource, it is ‘‘only loosely related to the resource level’’ (87). In exploitation competition, different populations have a common need for resources present in limited supply. In this case competition is directly related to level of resources available. We may define resource as any factor that can lead to increased cell survival or growth through at least some range of their availability (88). In a broad sense, a resource is any factor used by a cell and for that reason no longer available to other cells. Resources can be essential, complementary, substitutable, antagonistic, or even inhibitory (92). Ample evidence exists for the role of resources in lymphocyte competition. First, the kinetics of accumulation of Tg and non-Tg populations after reconstitution in different groups of mouse chimeras followed a density-dependent growth curve, which follows the Monod growth function, i.e. ‘‘it increases in a saturating manner with resource availability’’ (93). During the expansion phase of cell reconstitution, resources are abundant, and Tg and polyclonal populations accumulate at the same rate. When population growth reaches equilibrium, polyclonal populations become dominant, i.e. competition only occurs when resources are limiting (89, 90). Second, as discussed below, changes in resource level should alter the balance between populations. Administration of antigens to chimeras hosting different Tg cell populations favors dominance by the antigen-specific Tg cells (91, 94). Third, experiments demonstrating that the total numbers of peripheral T and B cells are not determined by rates of new cell production, but are

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limited at the periphery, represent further evidence for the existence of resource competition. In the immune system, many molecules can function as resources, e.g. antigen, MHC molecules, ligands for costimulatory and adhesion molecules, mitogens, interleukins, chemokines, hormones, and other growth factors, etc. Resources can be external to the immune system or be produced by the lymphocytes themselves. By producing their own resources lymphocytes also contribute to generate their own ecological space.

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Resource Availability Shapes Lymphocyte Populations Resource competition may also contribute to the regulation of the size of peripheral lymphocyte pools (94); if peripheral resources are plentiful, many more newly generated T and B cells are able to survive (86). Any manipulation of resources may modify lymphocyte populations. When resources are used by many cell types, changes in these common resources will modify overall cell numbers. Hormones and mitogens are examples of pleiotropic resources. The size of T and B cell pools is diminished in mice deficient in growth hormone and is increased in adrenalectomized, ovarectomized, castrated, pseudo-pregnant, and pregnant mice (6). In axenic mice, lymphocyte numbers are reduced and lymphocytes expressing activation or memory markers are rare or absent (B Rocha, unpublished). Normal-sized peripheral pools are rapidly restored as soon as the normal gut flora is reestablished or after its colonization by a single fungus or bacteria species. Activated lymphocytes and antigen-presenting cells (APC) are major producers of their own resources. Antigen stimulation increases resource availability by inducing APC and lymphocyte activation and the production of numerous cytokines. In this context, lymphocytes can increase in numbers as during the expansion phase of the immune response (95). Once antigen is eliminated, cytokine production decreases. Reduced resource levels are unable to maintain the same number of lymphocytes: Most will die during the contraction phase of the immune response (95). Antigen can also be considered as an example of a private resource, i.e., that used by a particular cell set. Changes in the levels of private resources modify the composition of lymphocyte populations, as when antigen injection to chimeras carrying mixtures of two different Tg B cells or of Tg and non-Tg B cells shifted the balance between populations to favor the antigen-specific Tg B cells (91, 94). In conditions of resource competition, one would also expect changes in morphology of a population in response to the presence of competitors: a process known as character displacement (96). The IgM secreted into the serum by a population of normal B cells exhibits different binding patterns according to the presence or absence of a population of Tg B cells (94). This implies that the presence of the Tg B cells leads to changes in the selection of the non-Tg B cell clones, a process that at a population level may be considered to mimic character displacement.

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The Competition Exclusion/Diversity Paradox

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According to the competition exclusion principle, competition should lead to the progressive dominance of a limited number of clones and consequently to the exclusion of most clones (87). In this context lymphocyte competition would be incompatible with maintenance of diversity. This traditional model of population dynamics assumes that all cells interact equally well with each other in small closed environments. The more recent metapopulation approach takes into account difference in the age and structure of the populations, their migration capacity, the heterogeneity and patchwork distribution of fragment habitats, and their change over time (4, 97). This approach explains the co-existence of multiple potentially competing species within larger areas of space. According to this theory, there are several mechanisms through which the immune system can solve the apparent competition-diversity paradox and select preferentially for diversity. 1. Continuous cell production. The continuous generation of naive cells ensures the permanent availability of new cell specificities. The contribution of new cell production to the maintenance of diversity can be evaluated by comparing lymphocyte repertoires in young and old individuals. With age the progressive decrease in new lymphocyte production both in the thymus and in the bonemarrow generally correlates with an increased frequency of pauciclonal repertoires. 2. Terminal differentiation. Control of cell proliferation by terminal differentiation prevents unlimited expansion and dominance (34). This is the case of B cell terminal differentiation into Ig-secreting plasma cells. 3. The redundancy of resources. The capacity to use multiple alternative factors for survival and proliferation may provide a critical advantage for cells competing for limiting resources. Indeed, B cells from a LPS-reactive mouse strain have a clear competitive advantage when compared to B cells from a non-LPS reactive strain (89). 4. Diversification in resource usage. The use of different resources by different populations of lymphocytes will allow the co-existence of different cell types. Cell differentiation is associated with the acquisition of receptors for different chemokines and growth factors or by regulation of homing receptors. These parameters contribute to create a heterogeneity of habitats that favors coexistence of diverse, potentially competing cell populations (4). Migration ensures the distribution of cells from source to the periphery and between the different peripheral environments (98): Cell survival is associated with the adaptation to new ecological niches.

The Ecological Niche The variety of resources and the heterogeneity of anatomical structures in the lymphoid organs permits that different lymphocyte types may find an ecological

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niche to survive. The inability of the cell to migrate into the niche may compromise survival; exclusion of B cells from follicular niches in the secondary lymphoid organs has been claimed to result in cell death (99). Niches may also play a role in homeostasis and in the control of cell numbers by forcing the B cells to migrate into the primary follicles (100) where they must compete for trophic factors present in limited supply. Lymphocytes may help to define their appropriate niches. Thus, differentiation of epithelial cells in the thymus and the organization of the thymus cortex and medulla cellular networks depend on the presence of thymocytes (101); the maturation of the splenic follicular dendritic cell networks and the organization of B cell follicles depend on the expression of LTa and b and TNF by B lymphocytes (102–104). Germinal centers that develop in the B cell follicles during T cell–dependent antibody responses are one of the best-characterized ecological niches in immunology (105–107). Germinal centers are oligoclonal; the B cells that give rise to germinal centers are initially activated outside follicles; on the average three B cells colonize each follicle. Massive clonal expansion of the initial founder cells, driven by antigen held by follicular dendritic cells, prevents colonization of the germinal center by B cells specific for a second unrelated antigen. Expansion of the B cells is accompanied by BCR hypermutation. Competition among the proliferating B cells, based on their ability to interact with antigen held on FDC, will lead to the preferential survival of the cells capable of high-affinity recognition and to the death by apoptosis of other cells. Germinal centers play therefore a critical role in the maturation of immune responses by selecting cells with high avidity for antigen binding. They seem to have evolved to provide the appropriate niche for selecting B cells that attempt to gain competitive advantage for survival by somatic hypermutation. Indeed, somatic hypermutation is present in philogeny (108) well before the development of the affinity maturation of immune responses (109). Affinity maturation of the immune response, however, is only present in species capable of germinal center formation. When heterogeneous populations occupy heterogeneous habitats, the probability of extinction of a species is higher for the rare populations (97). The same rules may apply to the immune system. In mice, arrest of new B cell production leads to preferential extinction of B cells expressing rare V genes (61). The probability of extinction of a population is also related to the area of habitat occupied. Destruction of wide areas of habitat can be predicted to be more damaging for the survival of the most frequently found species with a wider distribution than for some rare species that commonly occupies restricted niches and may therefore escape catastrophic events. Large structural changes in secondary lymphoid organs (e.g. sub-lethal irradiation) affect predominantly dominant B cell clonal responses (110).

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NICHE DIFFERENTIATION

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Different Types of Lymphocytes Occupy Different Niches Different lymphocyte subsets likely use different resources. In adult mice the size of T and B lymphocyte populations is independently regulated. The number of mature B cells is similar in normal mice or in mice that lack T cells (111). In mice that lack B cells, the number of T cells is similar to that of normal mice (54). B and T cells also occupy different locations in the secondary lymphoid organs (112, 113). These observations suggest that B and T cells belong to different guilds, i.e. they exploit different resources. Among the T cell subsets the number of ab and cd T cells is also independently regulated (114): In the absence of ab T cells, the number of cd T cells does not increase significantly and vice versa. Within ab T lymphocytes, the number of CD4` and CD8` T cells is, however, coregulated (38). In the absence of either of the two CD4` or CD8` T cell subsets, cell loss can be compensated by the remaining cellular subset, and the total number of ab T cells remains similar to that of normal mice (115–117), suggesting that CD4` and CD8` T cells may partially share common resources.

Naive and Memory/Activated Cells Belong to Different Niches In response to the presence of competitors, a population may modify its survival requirements and adapt to a new ecological niche. During lymphocyte development, cells bearing the same clonal specificity but at different stages of differentiation (immature vs. mature; naive vs. memory; Th1 vs. Th2) either modify their use of resources or interact differently with the same resources. The variety and the patchy distribution of resources in different habitats will cause the adaptive lymphocyte environment to change according to time and tissue localization. Lymphocytes engage in niche differentiation as is the case for naive and activated/memory populations of cells. The peripheral pool of CD8` T cells was divided in two compartments (naive and memory) of equivalent size that are regulated by independent homeostatic mechanisms. Mice manipulated to contain only naive CD8` T cells, in spite of available space, have only half of the total number of CD8` T cells. Similarly, in mice engineered to contain only memory CD8` T cells, the total number of CD8` T cells is also reduced by half (37, 118). To reconstitute a normal-sized compartment, both naive and memory T cells are required. The existence of independent niches for naive and memory T cells ensures the coexistence of both cell types; this fact has major functional implications. Thus, the continuous thymus output of naive T cells cannot lead to the extinction of previously generated memory responses as exported naive thymus migrants do not replace resident memory T cells (119). Survival of naive T cells relies on the nature and number

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of other competing naive T cells (90, 119). Conversely, accumulation of memory cells will not lead to the contraction of the naive T cell pool; successive antigen encounters will not necessarily result in pauciclonal repertoires, as the diverse naive repertoire will remain. Survival of resident memory T cells will rather be determined by the expansion of newly generated memory T cell populations. Upon the sequential immunization of mice with different types of virus (120), response to a second antigen leads to the reduction of the residual memory to a first antigen. It is not yet known if CD4` T cells follow the same strategy, but preliminary results suggest that this may be the case (A Almeida, AA Freitas, unpublished). In the case of B lymphocyte populations, the numbers of resting and activated IgM-secreting cells are also independently regulated (86, 121), as shown in mice engineered to contain different numbers of B cells. In mice with very low numbers, a significant fraction of the B cells shows an activated phenotype, and the number of IgM-secreting cells and serum IgM levels are as in normal mice (86, 121, 122). Mature resting B cells only accumulate once the activated pool is replenished (121). These results suggest that the immune system is organized to first ensure the maintenance of normal levels of natural antibodies (innate immunity), which constitutes an initial barrier of protection, while keeping a reserve of diversity in the resting B cell compartment that allows responses to new antigenic stimuli. The independent homeostatic regulation of resting and activated/ memory lymphocyte compartments implies the existence of a hierarchical organization of the immune system.

Lymphocyte Substitution From birth to adult life, lymphocyte pools increase in size and can accommodate newly generated lymphocytes. The situation differs once steady-state numbers are reached, and the ‘‘space’’ is full: Survival of newly generated lymphocytes will be determined by the rate of cell substitution. Substitution is conditioned by the rate of colonization (invasion) by new cells, but it is also determined by the rate of extinction of pre-established cells. To survive, a cell must migrate and find the appropriate niche. If the niche is empty, there is no obstacle to cell entering. If the niche is already colonized, the newly arriving cell is either able to out-compete and displace the established cell or it is prevented from entering the niche and dies. A population pre-established in the correct niche and with a low extinction rate benefits from the founder advantage ‘‘first come, first served.’’ It may resist replacement by a newly arrived cell. The most extreme case of substitution is succession, in which the vast majority of a resident population is replaced by a population of newly arriving cells. This situation probably occurs during ontogeny, as lymphocytes produced during the perinatal period are replaced by adult cells. Thus, T lymphocytes generated early in ontogeny perform poorly when compared to adult T cells (123). They proliferate poorly and secrete low levels of certain cytokines in response to in vitro

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anti-TCR stimulation. In vivo proliferation in response to anti-TCR or antigenic stimulation is also impaired (124, 125), owing both to an intrinsic lymphocyte defect and to deficient antigen-presenting cell function (125). These characteristics of the perinatal environment are believed to be the basis for its increased susceptibility to tolerance induction (126). Affinity maturation may be absent, as suggested by the extensive cross-reactivity of neonatal T cell repertoires (127). Similarly, neonatal B cell repertoires differ from those of adult mice. In neonatal mice the DH proximal VH7183 gene family is used at the same frequency by 30% of the immature pre-B cells in the BM and 30% of the peripheral B cells (60), while in adult mice B cells expressing VH-genes of the VH7183 gene family are strongly counter-selected at the pre-B to B cell transition (62). Comparing the binding specificities of B cell hybridomas revealed that the vast majority of the antibodies were multireactive in neonatal mice, (128), while in adult mice most of the antibodies were monospecific (129). These changes of B cell repertoires correlate with overall modifications of B cell dynamics (55, 130), suggesting that B cell selection is permissive in neonatal mice during the phase of expansion of lymphocyte numbers, while it becomes strict in adult mice when resources become scarce and competition is established. The replacement of both T and B cell neonatal cross-reactive repertoires suggests that in ontogeny lymphocyte populations go through a process of ecological succession in which generalist cells are replaced by specialists. Analysis of the processes of succession also may reveal other types of interactions between populations (97). By modifying the environment, the presence of an early population may prepare the site for colonization by a later cell type, a process known as facilitation. Examples of these should be looked for in the immune system. How does substitution work in adult mice? Studies of the substitution of resident naive T cells by recent thymus migrants gave contradictory results. In irradiated TCR-Tg mice injected with normal BM cells, substitution of the naive Tg CD8` T cells by newly generated naive non-Tg cells was age-independent; e.g. thymus migrants and resident cells appeared to have the same survival probability at the periphery (119). Opposite results were obtained in mice transplanted with increased numbers of fetal thymus grafts. These grafts initially exported fetal T cells of donor origin but were subsequently colonized by host BM cells. The study of the substitution of T cells of graft origin by T cells of host origin suggested that recent thymus migrants survived better than resident naive T cells (85). Finally, the study of long-term colonization and persistence of recent thymus migrants labeled in vivo with CFSE seems to suggest the resistance of resident cells to replacement by the newly arriving cells (131). Similar conclusions were made from parabiosis experiments (90). The reasons for these conflicting results no doubt derive from the complexity of the different experimental systems required to study substitution. All experimental approaches involved stressful

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conditions such as irradiation or surgery. Very diverse populations (Tg vs. polyclonal; fetal vs. adult) were compared. Finally, conclusions on substitution rates rely on the quantification of very low T cell numbers. The definite answer to this question requires results from new, as yet unavailable, experimental approaches. As naive and memory T cells belong to different niches (see section on Competition Exclusion/Diversity Paradox), naive recent thymus migrants do not dislodge resident memory T cells (119). Surprisingly, thymus migrants are able to dislodge resident tolerant T cells. Tolerant CD8` Tg cells can persist at high frequencies for prolonged periods of time in the absence of other CD8` T cells (132). Tolerant Tg CD8` T cells can be substituted by both thymus migrants (119) and memory CD8` T cells (B Rocha, unpublished). This replacement is such that a significant part of these cells disappear, but a few cells remain for prolonged time periods. The residual tolerant Tg T cells are unable to eliminate antigen but are not inert. They secrete IL-10 and c-IFN (133) and may also play a role in the control of immune responses by interference competition (134, 135). In the naive B cell pool, new B cells produced in the BM replace resident B cells continuously. Maintenance of the physiological size of the peripheral B cell pool requires a minimal continuous input of new cells because mouse chimeras with a 10-fold reduction of B cell production show diminished B cell numbers (86). Abrogation of the de novo B cell production leads to a rapid decrease in naive B cell numbers (55, 136). The excess of B cell production in normal physiological conditions suggests the existence of a high attrition rate at the periphery (55). Tolerant B cells from double-transgenic mice coexpressing hen egg lysozyme (HEL) and anti-HEL Ig-genes have a relatively short life span when compared to normal B cells (137). In contrast to naı¨ve B cells, naturally activated IgM-secreting B cells can resist replacement by newly arriving B cells. They can also prevent the entry of new emigrating B cells into the activated cell pool (121). This negative feedback regulation may be due to the secretion of inhibitory factors, e.g. Igs, IL-10, cIFN. Alternatively, the first established population may occupy a niche required for the selection and survival of incoming cells, preventing colonization (87). The ability of newly formed B cells to differentiate into IgM plasma cells is dependent on the nature and number of cells already present at the periphery. Early during development and expansion of the immune system, an initial pool of activated B cells is selected that may resist replacement. The same IgM-secreting cells are also found in axenic mice, suggesting their activation by self-antigens. Recent evidence suggests a possible role of self-antigen in the positive selection and activation of autoreactive B cells (138). Competition based on the BCR diversity and the antigenic environment heterogeneity eventually leads to the substitution of the initially selected population by new specificities formed in the BM. The question remains concerning the possible functional differences between these naturally activated B cells and the B cells triggered in the course of an immune response.

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MUTUALISM Besides competition, populations of cells also interact differently. One of the most important types of interactions is cooperation, also called mutualism. Through cooperation the survival and growth rate of one population is increased in the presence of a second population. During evolution eukaryotic cells represent the first example of mutualism (139). Multicellular organisms, where no cell survives on its own, are yet another example of mutualism. Multiple examples of mutualism are found in the immune system: T and B lymphocytes evolved mutualistic interactions; CD4` T cells help proliferation and differentiation of antigen-specific B cells. Reciprocally, antigen presentation by B cells contributes to the expansion of antigen-specific T cells. CD4` T cells enhance cytolytic CD8` T cell responses. Similarly, T lymphocytes and antigenpresenting cells (APC) have evolved mutualistic interactions, essential for T cell survival and for triggering immune responses. Dendritic and other antigen-presenting cells may therefore play a keystone role in the establishment of lymphocyte communities. Predator-prey interactions may also shape lymphocyte populations. In chimeras reconstituted with mixed populations of BM cells from male and female donors, the immediate injection of TCR Tg CD8` T cells specific for the HY male antigen leads to the elimination of all cells of male origin, followed by an increase in the number of cells from female progeny (132). In fact, in this example, the fate of the two BM cell types mimics the dynamic behavior of two competing populations—apparent competition (87).

SELECTION OF REPERTOIRES: TOLERANCE AND AUTOIMMUNITY In an immune system where the total number of cells is limited, lymphocyte repertoires will be shaped by the differential ability of lymphocytes to survive. Lymphocyte survival relies on cell/ligand interactions, the availability of other resources, and the nature and number of competing rivals. In their continuous flight for survival, lymphocytes must acquire a selective advantage over their competitors. They must adapt to the immediate environment by modifying their survival thresholds (140) and or changing their requirements through differentiation. At different life stages, lymphocytes will require different signals to survive. These factors impose a continuous selective pressure throughout the lymphocyte life history. Initially, T and B cell repertoires are shaped in primary lymphoid organs at early stages of T and B cell differentiation (141, 142). In the thymus, selection is determined by the avidity of cognate receptor/ligand interactions; by the presence of growth and differentiation factors; by the size of the selecting niche (143);

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by the number of competitors present at the different stages/compartments of lymphocyte differentiation (90, 144). Immature B cell precursors are particularly sensitive to deletion and receptor-editing mechanisms by low-affinity interactions (145), and the outcome of the ligand/B cell interaction may be determined by the degree of receptor engagement (146). Selection of T and B cells continues at the periphery (147, 148). After activation, lymphocytes may follow a pre-established hierarchical program and differentiate first to effector functions, then to memory, to anergy and/or terminal differentiation (149). The duration of antigen persistence may be fundamental to trigger sequentially these stochastic processes (29, 150) and thus to determine the class of immune responses. Lymphocyte selection and adaptation also require co-evolution involving several different cell lines. Different subclasses of lymphocytes and antigenpresenting cells represent patterns of co-evolved interactions. These patterns allow the emergence of the holistic properties of the immune system. The global properties of cellular communities by far exceed the simple properties of the individual cells. The multiple interactions between all differing cells suffice to explain the immune system’s development and functions. The context of homeostasis, survival, and competition excludes any manicheism as a realistic explanation of the global behavior of the immune system. In contrast to the original postulate of the clonal selection theory (151), lymphocyte selection through competition occurs even in absence of exogenous antigens. The idiotypic network of variable-to-variable region interactions is not essential (152). A closed autistic self-referential immune system (153) is impossible. The immune system’s functions cannot be directed by self/non-self (151) or sense/non-sense (154) discrimination or by the detection of danger (155). Lymphocyte activation, rather than being determined by the ability of the cells to discriminate between one or two signals (156), will likely be triggered by the capacity of the cell to integrate different exogenous stimuli. Indeed, both TCR and BCR are capable of differential signaling according to the properties of the antigen and the environmental conditions of stimulation (63, 132). In a complex system where a vast number of different new cell specificities are continuously generated in both the central and the peripheral compartments, the presence of self-reactive cells is unavoidable. Besides the described mechanisms of tolerance induction that work at a single cell level, the global properties of the system may also contribute to avoid autoaggression. One of these properties is niche segregation. When a lymphocyte encounters an activating ligand in the correct niche and in the presence of plenty of resources, it will respond and expand proportionally. This is the case for infectious agents presented to lymphocytes in the appropriate niche (in the secondary lymphoid organs) and in the presence of an excess of new resources provided by the primary inflammatory reaction. In contrast, encounters with a self-ligand frequently occur outside lymphoid organs, where deficient antigen presentation and limiting resources may be major factors to restrict clonal expansion and autoaggression.

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The growth of the potentially harmful cells will be controlled by lymphocyte competition. In these circumstances, lymphocyte diversity associated with polyclonal stimulation may help prevent the emergence of large clone sizes, clonal dominance, and immune-pathology. Conversely, pauciclonal repertoires will determine higher fluctuations in clonal frequencies, which may increase the probability for the expansion and fixation of clones that in the absence of competition will become irreversibly dominant and induce pathology. This is supported by the inverse correlation between the world incidence of autoimmune diseases and the frequency of infectious diseases. The prevalence of autoimmune pathology is higher in developed countries with low population densities and very cold climates that do not favor spread of bacteria, parasites and viruses. Prevalence is lower in highly populated areas of third world countries (157). In this context polyclonal activation may have a therapeutic role in autoimmune disease, while immune suppression as a side effect may reinforce the already established selfreactive clonal drift. It may be more appropriate to refer to ‘‘horror monoclonicus’’ rather than ‘‘horror autotoxicus’’; in the survival/competition model the question is no longer ‘‘how large should the repertoire be to discriminate self and non-self?’’ (158, 159), but rather ‘‘what is the minimum size of the repertoire that protects against and avoids autoimmune pathology?’’ In their flight for survival lymphocytes may be caught in a Prisoner’s Dilemma game (160–162): They either cooperate, i.e. protect the host with strong immune responses, and continue to survive, or they defect, i.e. fail to protect or even destroy the host with uncontrolled responses, and so eventually die. ACKNOWLEDGMENTS We thank our colleagues F Agenes, A Almeida, S Guillaume, N Legrand, M Rosado, C Tanchot and H Veiga-Fernandes for continuous discussion. We thank Drs. T Brocker, P Ahmed and U Rajewsky for allowing us to quote their unpublished work. This work was supported by the Institute Pasteur, INSERM, CNRS, ANRS, ARC, DRET, Sidaction and MRT, France. Visit the Annual Reviews home page at www.AnnualReviews.org.

LITERATURE CITED 1. Miller SL, Orgel LE. 1974. The Origins of Life on Earth. Englewood Cliffs, NJ: Prentice-Hall 2. Dawkins R. 1989. The Selfish Gene. Oxford, UK: Oxford Univ. Press 3. Buss LW. 1987. The Evolution of Individuality. Princeton, NJ: Princeton Univ. Press

4. Hanski I. 1999. Metapopulation Ecology. Oxford, UK: Oxford Univ. Press 5. Freitas AA, Rocha B. 1997. Lymphocyte survival: a red queen hypothesis. Science 277:1950 6. Freitas AA, Rocha B. 1993. Lymphocyte lifespans: homeostasis, selection and competition. Immunol. Today. 14:25–29

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Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:113–142 Copyright q 2000 by Annual Reviews. All rights reserved

NONCLASSICAL MHC CLASS II MOLECULES Christopher Alfonso and Lars Karlsson The R.W. Johnson Pharmaceutical Research Institute, 3210 Merryfield Row, San Diego, California 92121; e-mail: [email protected]

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Key Words antigen processing, HLA-DM, HLA-DO Abstract Major histocompatibility complex (MHC) class II molecules are cell surface proteins that present peptides to CD4` T cells. In addition to these wellcharacterized molecules, two other class II–like proteins are produced from the class II region of the MHC, HLA-DM (DM) and HLA-DO (DO) (called H2-M, or H2-DM and H2-O in the mouse). The function of DM is well established; it promotes peptide loading of class II molecules in the endosomal/lysosomal system by catalyzing the release of CLIP peptides (derived from the class II–associated invariant chain) in exchange for more stably binding peptides. While DM is present in all class II– expressing antigen presenting cells, DO is expressed mainly in B cells. In this cell type the majority of DM molecules are not present as free heterodimers but are instead associated with DO in tight heterotetrameric complexes. The association with DM is essential for the intracellular transport of DO, and the two molecules remain associated in the endosomal system. DO can clearly modify the peptide exchange activity of DM both in vitro and in vivo, but the physiological relevance of this interaction is still only partly understood.

INTRODUCTION The recognition of antigen-presenting major histocompatibility complex (MHC) class II molecules by CD4` T cells is a crucial component of the defense against pathogens. MHC class II expression is normally restricted to a subset of antigen presenting cells–dendritic cells, macrophages, B cells, and thymic epithelium–but a number of other cell types can be induced to express class II molecules after stimulation with cytokines, in particular interferon-c. The class II molecules at the cell surface are heterotrimeric complexes consisting of two transmembrane glycoprotein chains (a and b) that form a binding scaffold for the third component, a peptide of 11–20 amino acids. The peptides are derived from proteins of endogenous or exogenous origin that have been degraded in the endosomal or lysosomal system of the presenting cell (1). Class II molecules, like other transmembrane proteins, are translocated into the endoplasmic reticulum (ER) after synthesis, where they associate with a third protein, the invariant chain (Ii). This molecule is a type II transmembrane protein 0732–0582/00/0410–0113$14.00

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that serves as a class II–specific chaperone that promotes the exit of class II-Ii complexes from the ER and prevents class II molecules from binding peptides and unfolded proteins in the ER and in the secretory pathway (2). A targeting motif in the cytoplasmic tail of Ii directs the complexes from the secretory pathway into the endosomal system, where the Ii is rapidly degraded and shed from the class II molecules. However, an Ii fragment, called CLIP, which occupies the peptide binding groove of the class II molecule, is in most cases not spontaneously released (3). The CLIP fragment serves as a substitute peptide that protects the class II binding pocket from collapsing both during intracellular transport and after Ii degradation in the endosomal system. Binding of antigenic peptides, generated from endocytosed proteins, requires an empty yet open binding site, and therefore CLIP has to be released while the open binding site needs to be stabilized to allow the binding of other peptides. DM has been well documented to mediate both of these functions, thus promoting the binding of antigenic peptides. After acquiring peptides, the class II molecules are transported to the cell surface via routes that are largely unknown. The general importance of DM and its mouse equivalent, H2-M, in the processing of antigens for CD4` T cells is now well established through a number of studies using mutant cell lines, isolated protein components, and genetically modified mice. Similar techniques have been used to study DO (and H2-O), but though it is clear that DO changes the function of DM, the physiological relevance of the DM-DO association is still being unraveled. In this review we present an overview of the current knowledge of the two nonclassical class II molecules, including their function and their influence on antigen presentation.

GENES AND TISSUE DISTRIBUTION The human MHC (also called HLA-D—human leukocyte antigen) is localized on the short arm of chromosome 6, while the equivalent mouse region (called H2) is localized on chromosome 17. The large interest in MHC-encoded molecules resulted in the class II genes (as well as the class I genes which are also located in this region) being among the earliest mammalian genes to be cloned and fully sequenced. Analysis by serology and of their ability to stimulate allogeneic T cells had revealed the existence of the three classical human class II molecules, DR, DQ, and DP, and the genes encoding these molecules had all been identified and characterized by the mid-1980s. In addition to these genes, however, two other genes encoding proteins similar to class II a and b chains, respectively, were found by cross-hybridization. These genes were called DZA (4, 5) (later renamed DNA and finally DOA) and DOB (6, 7), respectively, but no protein products were detected at the time. In the mouse, two b chain genes, Ab2 (7, 8) (homologous to DOB and later renamed Ob) and Eb2 (9, 10), were found, in addition to the genes encoding the two classical class II molecules, H2-A and H2-E. The number of class II genes exceeded what had been expected from

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functional data, and it seemed likely that all or most of the genes in the class II region had been located. This assumption turned out to be wrong, and a number of genes encoding proteins involved in antigen processing for MHC class I molecules, as well as the genes encoding DM, have since been mapped to the class II region in both human and mouse (11). The majority of the human and mouse class II regions have now been sequenced and a number of other new genes have been identified (12), but since none of these have structural homology to class II genes they are not discussed here.

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HLA-DM and H2-M The genes encoding DM and H2-M are quite dissimilar from other class II genes (including the DO and H2-O genes) (13, 14), and they were identified in a systematic search for genes in the class II region, rather than through crosshybridization (15). The overall amino acid identity between the DM chains and the corresponding a and b chains of the other human class II loci is only about 30%, while the DP, DQ, DR, and DO chains are about 60% identical when compared to each other (13, 14). Indeed, the b2 domain of DMb is almost as related to class I molecules as to class II molecules, suggesting that the DM/H2-M genes have evolved along different lines from other class II molecules. The DM a and b chain genes are located close to each other on the centromeric side of the TAP and Lmp genes (which are part of the peptide-generating machinery for class I molecules) (16). The human DM locus consists of one a chain gene, DMA, located on the centromeric side of a single b chain gene, DMB (13, 17). The mouse H2-M locus has a similar organization but contains two b chain genes, Mb1 and Mb2 (18, 19). The two b chain genes show a very high degree of identity– 96% in the coding regions, 93% if the entire genomic sequence is compared (19)–and they are likely to represent a recent gene duplication since other species including rats have only one Mb gene (20). Interestingly, the majority of amino acid differences are located in the b1 region (19, 21, 22), i.e. the region corresponding to the peptide-binding region of classical class II molecules, but it is unknown whether the presence of one or the other chain influences the efficiency or specificity of H2-M-mediated peptide exchange. It is somewhat unclear whether Mb1 and Mb2 are equally well expressed in vivo (21–23), but H2-M molecules consisting of either ab1 or ab2 heterodimers appear to be functional (21, 24, 25), although no systematic study comparing the two molecules has been published. Classical class II molecules display an extraordinary degree of polymorphism concentrated to the amino acids that contribute to the peptide binding site. In contrast, analysis of the DM and H2-M genes has revealed limited polymorphism where the minor differences that do occur appear to be spread throughout the coding regions (19, 21–23, 26, 27). A number of studies have investigated potential links between different DM haplotypes and autoimmune disease, but clear correlations have not yet been found (28–32). No functional comparisons have

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been made between DM or H2-M molecules of different haplotypes, but it appears likely that the peptide exchange activity will not be seriously influenced by the polymorphisms. DM and H2-M appear to be expressed in all class II–expressing cells, although the level of transcription is generally lower than that of conventional class II molecules. Interferon-c, which increases the expression of class II genes, also increases the expression of the DM and H2-M genes (13, 14). The promoter regions of these genes share the characteristic motifs (referred to as S, X, X2, and Y boxes) (17, 19) that are present in other class II genes; the transcriptional activators CIITA and RFX-5, which control class II transcription, are also necessary for efficient transcription of the DM and H2-M genes (33, 34). However, while class II expression appears to be totally dependent on these transcription factors, a low basal level of H2-M expression seems to persist both in mice deficient for CIITA (35) and in mice deficient for RFX-5 (36), suggesting that differences in transcriptional control do exist. This notion is also reinforced by the isolation of mutant cell lines that appear to have normal expression of class II molecules, but where the DM genes are not transcribed normally due to an unknown genetic defect mapping to chromosome 6p (37). Genes encoding DM-like proteins have been characterized in a number of other species (20, 38, 39) beside mouse and human; although the gene products have not been analyzed, they are likely to be the functional equivalents of DM.

HLA-DO and H2-O In contrast to the DM genes, the genes encoding DO a and b are as similar to the classical class II genes as these are to each other, and as a consequence of this similarity both genes were found using low stringency cross-hybridization (4–7). Although they were initially proposed to form a pair (5), several pieces of evidence suggested that this was not the case. First, in contrast to other class II a and b chain genes that are located pairwise, close to each other, the two DO genes are located quite far from each other (200 kb), with a number of other genes located in between the two. Second, while the transcription of the a chain appeared to be similar to that of other class II genes (although the level of transcription is at least tenfold lower— 4), the transcription of the b chain was distinctly different (6). Thus, DOb cannot be induced by interferon-c in most cell types (although exceptions to this finding have been reported— 40), and its expression is independent of CIITA (41). The transcription from the DOB promoter is also distinctly weaker than the transcription from conventional class II genes, and one study even concluded that the gene was transcriptionally silent (42). The fact that DOa and DOb actually are expressed and do associate with each other was only shown after the identification of the equivalent mouse molecule, H2-O (43). The H2-O b chain, initially called Ab2 (44), was discovered due to its homology to other b chains, but the H2-O a chain was mapped and cloned based on its association with H2-Ob at the protein level (45). The H2-O

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chains are both transcribed independently of CIITA, and H2-O is not upregulated by interferon-c (35, 46). In contrast, IL-4 appears to result in a moderate upregulation of H2-O protein expression in B cells (L Karlsson, unpublished data). H2-O and DO both have a more restricted tissue distribution than do other class II molecules, including DM (6, 43, 46–49). Thus, in peripheral tissues, H2O expression is largely restricted to B cells, while the vast majority of dendritic cells and macrophages do not express this molecule. The expression pattern of DO is less clear because this molecule has been reported to be expressed both by dendritic cells and by a melanoma cell line, as well as by B cells (49). The expression of DO by dendritic cells does not appear to be a general phenomenon, however (50; L Karlsson, unpublished), and it is possible that subsets of dendritic cells have different patterns of DO expression (A Vogt, H Kropshofer, personal communication). In the thymus, DO expression has been reported in both cortical and medullary epithelial cells (49), while immunohistochemistry showed H2-O expression to be most prominent on a subset of medullary epithelial cells (43, 47). Intriguingly, the thymic (but not the peripheral) H2-O staining was present also in mice lacking functional H2-Oa chains (51); (while this finding remains unexplained, analysis by in-situ hybridization has confirmed that mRNA for both H2-O chains are present in the thymic medulla (L Karlsson, unpublished data). The human DO chains have not been extensively analyzed for polymorphism, but the studies to date suggest that DO, like DM, is essentially nonpolymorphic (5, 7, 52, 53). This conclusion is also supported by sequence analysis of the mouse molecules where only minor differences could be found between different class II haplotypes (43, 54). The few allelic differences are spread throughout the open reading frames, suggesting that they are not likely to be functionally relevant.

H2-Eb2 The mouse class II region contains yet another class II b chain gene that may be classified as a nonclassical class II gene; Eb2. This gene, which does not appear to have any human homologue, is transcribed similarly to Ob (10) (i.e. it is expressed in B cells, but not macrophages, and is not inducible by interferon-c). No corresponding a chain gene has been identified, and it is unknown whether Eb2 is part of any functional protein.

INTRACELLULAR TRANSPORT AND SUBCELLULAR DISTRIBUTION The interaction between class II molecules and Ii, as well as the functional consequences of this interaction, have been very well characterized (reviewed in 2, 3, 55). Class II ab dimers rapidly associate with the Ii immediately after entry into the endoplasmic reticulum to form nonameric (abIi)3 complexes. The binding to Ii inhibits the class II molecules from associating with peptides in the endo-

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plasmic reticulum and in the secretory pathway, and maybe more importantly, it protects from the binding of nascent unfolded protein chains to class II molecules in the ER (56, 57). Invariant chain binding to class II molecules is largely mediated through the CLIP region of Ii (amino acids 81–104), which is thought to occupy the peptide binding groove of the class II molecule (58–61). In some class II haplotypes, the affinity to the CLIP peptide sequence is low, however (62), and other regions of Ii are important for the association with class II molecules (61, 63–65). Once assembled, the nonameric class II-Ii complexes are rapidly transported out of the ER into the secretory pathway. Two extensively characterized di-leucine-like targeting motifs in the cytoplasmic tail of the Ii direct the class IIIi complexes into the endosomal/lysosomal system (66–69), where Ii is removed and peptide loading of the class II molecules occurs (see below), before the assembled peptide-class II complexes are transported to the cell surface. It is presently unclear where class II-Ii complexes enter the endosomal system. A number of morphological and biochemical studies have suggested a direct path from the trans-Golgi network (TGN) to late endosomal compartments (70–72), but class II-Ii complexes have also been detected at the plasma membrane, suggesting that a fraction of newly synthesized class II molecules reach late endosomes or lysosomes via the cell surface or early endosomes (73–76). In the absence of Ii expression, class II molecules are largely retained in the ER due either to aggregation and functional inactivation or to binding of unfolded proteins, and only a minority of the synthesized molecules reach the plasma membrane (77–79). However, some of the class II molecules that do appear on the cell surface can acquire peptides after internalization and recycling back to the plasma membrane (80– 82). The cytoplasmic tails of H2-Ab, and of DR a and b have been shown to be able to mediate internalization of mature Ii-free class II molecules from the cell surface into recycling endosomes where peptide loading occurs under certain conditions (81, 82). Most peptide loading does not involve recycling class II molecules, however, but requires newly synthesized Ii-associated class II proteins (1, 83). The endosomal or lysosomal compartments where newly synthesized class II molecules acquire peptides have been the subject of intensive study in recent years. Morphological and sub-cellular fractionation techniques have been used to characterize and isolate compartments that are enriched in their class II content and that are likely to be the location where peptide loading occurs. Initial studies using electron microscopy suggested that the majority of class II molecules were localized in multilamellar structures with lysosomal characteristics, called MHC class II compartments (MIIC) (70, 84). A number of subsequent studies have described compartments with both endosomal and lysosomal characteristics as being important for peptide loading (85–89), and it has become apparent that celltype dependent differences (72, 90) as well as haplotype-dependent differences (91) can explain many if not all of the discrepancies seen in different experimental systems. The term MIIC has with time been expanded to include class II–

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containing multivesicular endosomal compartments as well as lysosomal compartments (72), but it remains uncertain whether class II–loading compartments are really specialized, or whether they are part of the normal endosomal/lysosomal system present in all cells. This question lies outside the scope of this review, however, and has been excellently reviewed elsewhere (1). After peptide acquisition, the mature class II–peptide complexes are transferred to the cell surface where they persist for extended periods of time, displaying the antigenic peptides to CD4` T cells.

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Intracellular Transport of DM/H2-M The DMA and Ma genes encode mature proteins of about 26 kDa, but the addition of two carbohydrate residues increases the actual molecular weight to 33–35 kDa. The mature core proteins encoded by the DMB and Mb genes have a predicted size of 27 kDa, and while this is the actual size of Mb2, DMb (and probably Mb1) are modified by the addition of one carbohydrate moiety to have an actual size of 30–31 kDa (21, 92). Newly synthesized DM/H2-M chains assemble in the ER after synthesis to form heterodimers, but unlike newly synthesized conventional class II molecules that rapidly associate with the Ii, physical association between DM and Ii has been difficult to show (92). In contrast, complexes of H2M and Ii have been co-immunoprecipitated after solubilization in digitonin (a mild detergent that tends to preserve weak protein-protein interactions) or after chemical cross-linking, but the significance of this interaction is uncertain (93). Assembly and exit from the ER of either H2-M or DM clearly do not require association with the Ii, since expression of the two DM (or H2-M) chains into a number of different cell lines that do not express Ii results in efficient assembly and intracellular transport of apparently functional DM heterodimers (21, 93–95). While class II molecules transiently pass through the endosomal system before reaching the cell surface, mature DM and H2-M molecules are permanent residents of the endosomal/lysosomal system (21, 96). Early immuno-electron microscopy studies showed that the majority of DM was located in multivesicular or multilamellar late endosomal or lysosomal structures (MIICs) (96, 97), but subcellular fractionation experiments in combination with further electron microscopy have shown that DM is present throughout the endosomal system (72, 98). In addition, small amounts of DM can be detected at the plasma membrane (99; A Vogt, H Kropshofer, personal communication). Both DM and H2-M are directed to (and probably maintained in) the endosomal system by a tyrosinebased targeting motif (amino acids YTPL) located in the cytoplasmic tail of the b chain of both molecules (93, 94, 100). Transfection experiments in HeLa or NRK cells (neither of which express endogenous DM or Ii) have shown that removal or mutation of the essential tyrosine or leucine residues in the motif results in disrupted sorting of DM/H2-M and accumulation of the mutated

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molecules at the cell surface. Somewhat surprisingly, considering the apparently low affinity between H2-M and Ii, co-expression with full length (but not targeting-deficient) Ii could rescue both the endosomal delivery and the peptide exchange function of the mutated H2-M, suggesting that the targeting motif in the H2-M tail is not essential for the localization of H2-M to endosomal/lysosomal structures in cells where Ii is present (93). Similar Ii-mediated rescue of DM function in cells transfected with targeting-deficient DM has also been reported, suggesting that DM does indeed associate with Ii to some extent (101), although this association may be weaker than the association between Ii and H2-M. How DM/H2-M reaches endosomal compartments has not been extensively studied, but transfection of a dominant negative form of clathrin that disrupts the formation of clathrin-coated vesicles suggests that DM and Ii-class II complexes may transit from the trans-Golgi network to the endosomal system in distinct vesicular compartments (102). Once in the endosomal system, DM and class II molecules must come in contact with each other in the same intracellular compartments, but it is not entirely clear whether the sorting signals in Ii and DM/ H2-M direct their respective cargoes to the same or to different compartments. One report has suggested that Ii–class II complexes are initially delivered to DMfree organelles where Ii is degraded before they are transported into DM-containing, peptide-loading compartments (103). Most available data seem to suggest, however, that Ii, class II, and DM are present in overlapping compartments (98, 104–106). The protease sensitivity of Ii complicates accurate detection in experimental systems, and the absence of detectable Ii does not necessarily mean that Ii was not responsible for the delivery of class II molecules to a particular compartment before its degradation. It is not known whether mature peptide-containing class II molecules (destined for delivery to the cell surface) are sorted away from DM and immature Iiassociated class II molecules in the endosomal system, or whether all the different molecules are deposited at the plasma membrane together, followed by rapid endocytosis of DM and possible residual Ii. Direct fusion of multivesicular class II–containing endosomes with the plasma membrane has been shown to occur (107), resulting in both the delivery of class II molecules to the plasma membrane and the secretion of the internal vesicles (called exosomes) into the surrounding medium (107, 108), but it is not clear if the majority of class II molecules are delivered to the plasma membrane by this mechanism. The vesicles have been reported to be enriched for class II molecules as well as for CD86 (B7.2) and several tetraspan molecules (CD37, CD53, CD 63, CD81 and CD82), while DM (and Lamp-1 and -2) were largely absent from the vesicles and instead concentrated on the surrounding endosomal membrane, suggesting that in this situation at least, DM and class II molecules may be sorted prior to delivery to the cell surface (109). Interestingly, several of the tetraspan molecules have been shown to associate with class II molecules as well as with DM and DO, but the significance of this finding is unclear (110).

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Intracellular Transport of DO/H2-O The proteins encoded by the DO and H2-O genes are structurally similar to other class II proteins, consisting of a chains of about 25 kDa and b chains of about 28 kDa. Addition of 2 and 1 carbohydrate residues, respectively, results in an increase of the actual molecular weight to 32–34 kDa for the mature a chains and 33–34 kDa for the mature b chains (43, 48). The relatively large size of the b chains (compared to other class II b chains) is mainly a reflection of the unusually long cytoplasmic tail, present in both mouse and human molecules. After translocation into the ER, the a and b chains assemble to form heterodimers, but the dimerization was found to be relatively inefficient in transfected HeLa cells expressing transfected DO a and b alone, and exit from the ER of the assembled DO dimers could not be detected (48). The inefficient intracellular transport of DO is reminiscent of the transport of class II molecules expressed in the absence of Ii, but in the case of DO, co-expression of Ii resulted in only marginally improved transport out of the ER, and the association between the two molecules was substoichiometric. In contrast, co-expression of the two DM chains resulted in formation of heterotetrameric DMDO complexes that could effectively exit the ER (48). Analysis of B cell lines confirms that the DM-DO interaction is not a transfection artifact but occurs also in cells that express DM and DO endogenously (41, 48, 111, 112). A similar association between H2-O and H2-M is also seen in mouse B cells (48, 51). Both DO and H2-O do bind Ii to some extent in B cells (43, 48, 49), but this interaction is clearly less important than the interaction with DM and H2-M. Thus, H2-O dimers are formed in H2-Ma-deficient mice to some extent, but in the absence of functional H2-M, the H2-O molecules are not transported out of the ER and are instead rapidly degraded (48). In B cells the DMDO complexes are transported to endosomal/lysosomal compartments, and at steady state DO, like DM, is mainly localized in lysosomes (41, 48, 111). Most of the lysosomal DO molecules are associated with DM, and indeed it is uncertain whether free DO exists at all outside of the ER (48, 112). It is conceivable that DMDO complexes dissociate in lysosomes to generate free DM and DO, either spontaneously or in response to cellular activation or intracellular signaling events, but this has not been demonstrated so far. Free DM is present in DO-expressing cells, but the majority of DM seems to be associated with DO, both during intracellular transport and in lysosomes (48, 112). It is not known whether DMDO complexes have different targeting information than DM alone, and it is conceivable that DM and DMDO may be sorted to different compartments of the endosomal/lysosomal system, although no available data support this hypothesis. The long cytoplasmic tail of DOb contains two potential endosomal targeting motifs, and a recent report has shown that the DO tail may direct a transfected DRa-DOb heterodimer to lysosomal compartments, even though this chimeric molecule was also present at the cell surface (113). The targeting motifs are not conserved in the H2-Ob tail, however, and further study

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will be necessary to determine whether the cytoplasmic tails of DO and H2-O are important for DO or DMDO localization and function.

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FUNCTIONAL ASPECTS OF DM By the late eighties, it was becoming apparent that antigen processing and peptide aquisition by class II molecules were occurring in endosomal or lysosomal compartments of antigen presenting cells, but possible molecular requirements for this process were not known. A number of studies had shown that functional peptide– class II complexes could be formed by incubating antigenic peptides with class II molecules, either on cells or using purified class II molecules. Although this process was much slower than peptide loading in vivo, it was not immediately apparent that specific molecules would be necessary for mediating peptide loading. The first direct demonstration that peptide loading must be a regulated process came from the analysis of a panel of mutant cell lines that were defective in their ability to present protein antigens to CD4` T cells, but that had normal expression of class II proteins and normal or improved ability to present exogenously added peptides (114). These cell lines had been selected based on their failure to be recognized by 16.23, an HLA-DR3-specific monoclonal antibody that recognizes a subset of peptide-DR3 complexes (115). The antigen presentation defect was not limited to DR3-restricted antigens but was extended also to DP- and DQrestricted antigens. Biochemical analysis showed that the class II molecules from the mutant cells were less resistant to incubation in low concentrations of SDS than the molecules from wild-type control cells, leading to the suggestion that the class II molecules from the mutant cells might not contain any bound peptides. Subsequent studies in these and other mutant cell lines with similar phenotypes showed that the class II molecules were actually not empty, but instead contained a limited set of peptides, mainly derived from the Ii (116, 117). These peptides, called CLIP (class II associated invariant chain peptides), are intermediates in the degradation of Ii also in normal class II–expressing cells, but while they are normally released and replaced by antigenic peptides (118), CLIP release is blocked in the mutant cell lines. Genetic analysis showed that the defect in the mutant cell lines mapped to the class II region of the MHC (119, 120), and further work revealed that the deficiency could be rescued by transfection of functional HLA-DM genes (24, 121). How a class II–related molecule like DM could promote the loading of antigenic peptides onto conventional class II molecules was not immediately obvious and led to a period of intense study by a number of groups aimed at revealing the molecular mechanism of DM function.

DM Catalyzes Peptide Exchange In Vitro DM does have structural similarity to peptide-binding class II molecules; one initial suggestion was that DM, like other class II molecules would be able to

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bind peptides and in particular CLIP peptides released from maturing class II molecules. The notion that DM might function as a ‘‘CLIP sink’’ does not appear to be correct, however, and later experiments have not been able to show any stable peptide association with DM (122, 123). This conclusion is further strengthened by the fact that the recently solved crystal structures of both DM and H2M show a very reduced binding pocket, which is too small to accommodate peptides (124, 125). However, experiments using purified DR and DM molecules, eiher isolated from B cell lines or produced as recombinant proteins, revealed that DM alone is able to facilitate the release of CLIP peptides from CLIP–class II complexes, while at the same time promoting the binding of antigenic peptides (122, 126–128). CLIP may be the most important substrate peptide in vivo, but the peptide releasing effect of DM in vitro is not limited to CLIP, and in fact other class II–associated peptides, as well as longer Ii-derived protein fragments, can also be exchanged for antigenic peptides by incubation with DM. In addition to increasing the release of peptides from class II molecules, DM also increases both the rate of peptide binding and the maximal level of peptide binding in vitro (122, 126, 127). This effect can partly be explained by the increased release of previously bound peptides. As more binding sites become available, more new peptides can be bound, but the increased binding does also appear to be due to a direct chaperone effect of DM resulting in the stabilization of empty class II molecules, thus allowing them to maintain the ability to bind peptides (123, 129). Peptidefree class II molecules appear to be very unstable, and prolonged incubation at acidic pH in the absence of suitable peptide or DM results in an irreversible loss of ability to bind peptides (123, 130–132). Further analysis has extended the initial peptide release experiments to suggest that the dissociation rates of all class II– peptide complexes are actually increased by DM. The rate of dissociation in the presence of DM has been shown to be directly proportional to the intrinsic rate of dissociation of the peptide from the class II molecule (i.e. the dissociation in the absence of DM) (133, 134), and although DM is not strictly an enzyme, DMmediated peptide release appears to follow general rules for enzyme kinetics (133, 135). The notion that DM functions catalytically also in vivo is supported both by the long half-life of DM (.24 hours) (92) and by the fact that the intracellular concentration of DM is lower than the concentration of DR. In B cell lysates the ratio between DR and DM has been estimated to be 20:1 (122), while the more relevant quantitation of DR and DM levels in endosomal/lysosomal loading compartments has suggested that DR is present in a four- to fivefold excess over DM (106, 123). It should be noted, however, that relatively minor differences in DM expression have been reported to influence the efficiency of peptide exchange in vivo, under certain conditions (136). It has not been experimentally determined how DM causes the release of class II–associated peptides, but a direct interaction between DM and the class II molecule appears to be required (122, 126, 127, 137). The strength of the interaction between DM and class II molecules is unclear; reliable measurements have not been published, but DM-DR complexes can be isolated from cell lysates after

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solubilization using acidic conditions and gentle detergents (123, 129, 138). Many of these complexes do not contain bound peptides (123, 129), but it is presently not clear whether the stable interaction between DM and DR requires that the class II molecules are empty or whether the lack of peptides is a consequence of the extraction procedure. DR molecules containing a mutant form of DRa that has an additional glycosylation site in the membrane-proximal a2 domain, as well as several other mutations mapping to the same lateral face of DR, are unable to interact productively with DM both in vivo and in vitro, suggesting that the interacting DM and DR molecules are positioned parallel to each other in the membrane (126, 137; E Mellins, personal communication). The face of DR that contains these mutations corresponds to the end of the peptide-binding groove, which holds the N-terminus of the peptide, and this is likely to be the face that interacts with DM (124). The crystal structures have been determined for a number of class II molecules, including DR3-CLIP, but there are no obvious structural differences between the CLIP-containing molecule and other more stable class II–peptide complexes that could give more specific indications for how DM would bind to DR (59, 139, 140). However, both class II molecules and DM change conformation upon acidification, and these altered conformations may favor the association between the two molecules (141–144). The binding of the peptide to the class II–binding pocket is unusual in that it largely depends on a network of hydrogen bonds formed between the backbone of the bound peptide and the side chains of the amino acid residues lining the peptide binding pocket (59, 139). In addition to this network of hydrogen bonds, ‘‘good’’ class II–binding peptides are characterized by the presence of one or more anchor residues that fit in suitable pockets of the binding groove as well as by the absence of residues that are incompatible with binding (128, 145, 146). A likely explanation for how DM increases the rate of peptide exchange is that interaction between DM and a class II molecule stabilizes (or induces) a conformational change in the class II molecule that to some extent disrupts the hydrogen bond network holding the peptide in place (59, 124). Such structural changes would not have to be very substantial– a relatively minor dislocation of the two a-helices that line the peptide-binding pocket would be sufficient to ‘‘unzip’’ the hydrogen bonds locking the peptide in place (124). In this situation, the intrinsic affinity of the peptide for the open binding pocket may determine whether the peptide will be released or retained. It is not known how DM is released from the class II molecules when peptide binding has occurred, or indeed whether peptide binding is required for the release of DM. One likely possibility is that binding of a ‘‘good’’ peptide in the binding pocket of the class II molecule induces a conformational change that is sufficient to counteract the effect of DM, resulting in its release from the class II molecule. Data showing that DM appears to associate more stably with empty class II molecules than with peptide-containing class II molecules support this hypothesis (123, 129, 135). Alternatively, DM may be released from the class II molecule independently of the quality of the bound peptide. In this scenario ‘‘good’’ peptides would be trapped in the binding pocket more often than ‘‘bad’’ peptides,

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simply because their dwell time in the open DM-stabilized binding pocket would be longer. Repeated cycles of association and dissociation between DM and the class II molecule would result in accumulation of ‘‘good’’ peptides also in this case, and the DM-class II interaction would be disrupted either by pH-induced conformational changes occurring during transfer from lysosomal compartments to the cell surface, or by the physical separation of the two molecules by differential sorting, either in the endosomal system or maybe even at the cell surface, depending on where class II and DM are actually sorted away from each other. This model for DM function is supported by data showing that class II molecules containing ‘‘good’’ stably bound peptides can also be found associated with DM (129, 138). The two models are not mutually exclusive, but whether one or the other (if any) is more correct will have to be determined experimentally. A schematic view of how DM functions is outlined in Figure 1 (see color insert).

DM/H2-M Function In Vivo The antigen presentation defects in human cells lacking DM are quite striking, and they are shared by all three types of classical class II molecules (DR, DP and DQ) expressed by these cells (114). However, when human cells transfected with mouse class II molecules were analyzed for their ability to present exogenous antigens, the defects were generally less prominent and appeared to be haplotypeas well as cell-type-dependent (147, 148). The relatively small differences seen between the presence or absence of DM indicated that certain class II haplotypes, or indeed all mouse class II molecules, were less dependent on DM activity than initially suggested. However, the generation of H2-M-deficient mice by three independent groups very convincingly showed that the dependence on DM (or H2-M) is not species-specific (149–151). The level of class II expression at the cell surface of APCs is normal in these mice, but the class II molecules (H2-Ab) have abnormal conformation, a finding explained by the fact that they are overwhelmingly associated with CLIP peptides. The fraction of class II molecules that bind CLIP is so high that an accurate estimate of the CLIP content is difficult to make, but analysis of H2-Ab-associated peptides suggests that .99% of the molecules are loaded with this peptide (A Rudensky, personal communication). In contrast to the mutant human cell lines, which can present many endogenously synthesized antigens (152), as well as antigens internalized by efficient receptormediated uptake (153), the H2-Ab molecules from the H2-M-deficient mice are essentially incapable of presenting antigens, whether expressed endogenously (95, 149, 154) or internalized either by fluid-phase (150, 151) or receptor-mediated endocytosis (C Alfonso, L Karlsson, unpublished data). In addition, the ability of APCs from mutant mice to present pre-processed exogenously added peptides is lower than the ability of APCs from wild-type littermates (95, 150, 151). In contrast, the capacity to raise responses to peptides in vivo is similar between mutant and wild-type mice (155). The almost total inability to present protein

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antigens by H2-Ab is a haplotype-, rather than a species-dependent effect, and substantial fractions of H2-Ak and H2-Ek/b molecules expressed in the H2-Mdeficient animals (through breeding with H2-Ak or H2-Eak transgenic animals) are occupied with peptides other than CLIP, although their CLIP content is also increased compared to the H2-Ak and H2-Ek/b molecules from wild-type controls (156). Despite the lower CLIP content, the ability to form SDS-stable complexes is decreased for the H2-Ak and H2-Ek/b molecules from the H2-M-deficient mice, as is their ability to present exogenously added protein antigens to T cell hybridomas. In contrast, the ability to present exogenously added peptides is normal. The differences in antigen presentation and SDS stability of class II molecules between transfected human DM-deficient cell lines and antigen-presenting cells from H2-M-deficient mice are significant and are at least partly an effect of species mismatching, since DM interacts poorly with at least one class II allele, H2-Ad (25, 157, 158). Differences in Ii processing may also account for some of the discrepancies, since a fraction of CLIP peptides eluted from human DR molecules have extended N-termini compared with CLIP peptides eluted from mouse class II molecules (151, 159). This N-terminal extension, which has been reported to promote self-release of CLIP from DR molecules (160, 161), may be present also on CLIP bound by mouse class II molecules expressed in human cell lines, thus explaining their relative DM independence. The general importance of H2-M for peptide loading and peptide editing also in the absence of Ii is evident in mice lacking expression of both Ii and H2-M. The antigen presenting cells from these mice have low levels of class II (i.e. H2Ab) expression, comparable with the class II expression of APCs from Ii singledeficient mice (95, 162–164). The cells can present exogenously added peptides almost as well as the APCs lacking only Ii, but they have drastically decreased ability to present endogenously synthesized self-peptides, suggesting that H2-M is required not only for the removal of CLIP peptide, but also for the formation of stable peptide-class II complexes (95). In the absence of Ii, the peptide-binding pocket of the class II molecules is likely to be filled with various unstably bound endogenous peptides or proteins, and H2-M may be required to exchange these for better fitting endogenous peptides. Thus, H2-M appears to have a chaperonelike effect in vivo, consistent with results obtained with purified molecules in vitro. The antigen presentation defects in H2-M-deficient mice have expanded the understanding of antigen processing, but an equally important aspect is their contribution to the understanding of thymic selection. Thus, CD4` T cell selection in H2-M-deficient mice (expressing H2-Ab) is moderately decreased (by 50% to 70%) when compared to wild-type littermates (149–151). The T cells selected appear to have grossly normal repertoires of T cell receptor expression, but closer analysis has revealed that several different T cell receptors cannot be positively selected in the H2-M-deficient mice (154, 155, 165), and the receptors that are selected have more limited diversity than receptors from H2-M-expressing mice (166). A large part of this cell population (70%–80%) (165) appears to be made

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up of cells that have not undergone normal negative selection (due to the absence of self-peptides other than CLIP in the thymic medulla, where negative selection occurs), and the cells react vigorously to APCs from MHC-matched wild-type littermates. Despite this reactivity, the CD4` T cells from the H2-M-deficient mice appear to have low affinity for wild-type H2-Ab molecules and are not capable of mediating skin transplant rejection or causing lethal graft-vs-host disease (167). Bone-marrow chimeras, where the bone-marrow-derived APCs are wild-type (and thus able to mediate negative selection in the thymus), have approximately 5% of normal CD4` T cells, and these cells are no longer reactive with wild-type APCs because the self-reactive T cells have been eliminated in the thymus (154, 155, 165). The remaining cells are polyclonal and can respond to antigen after immunization but are not as diverse as CD4` cells from wildtype littermates. It is presently not clear whether the positive selection occurring in the H2-M-deficient mice is mediated by CLIP-class II complexes or by the few other peptide-class II complexes that are likely to exist in the animals (154, 155, 165, 168). H2-M-deficient mice can raise antibody responses after immunization with protein antigens (155), but affinity maturation is not detectable (C Alfonso and L Karlsson, unpublished). Although the immune responses to pathogens have not been extensively studied, H2-M-deficient mice do survive infections with lymphatic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV) (155), but in contrast to both wild-type mice and Ii-deficient mice, they cannot clear infection with Leishmania major (164). In mice expressing transgenic H2-Ak complexes, the number of selected CD4` T cells is not significantly changed compared to the mice expressing H2-Ab alone, but in contrast, the expression of H2-Ek,b results in wild-type levels of CD4` T cells (156). The selection of CD4` T cells by H2-Ek,b molecules is not normal, however, and the CD4` cells from these mice show a high degree of reactivity to H2-M-expressing littermates in mixed lymphocyte reactions (C Surh, personal communication). Surprisingly, despite the fact that the H2-Ek,b molecules are partly loaded with non-CLIP peptides, APCs from these mice are unable to stimulate allogeneic CD4` T cells. Mice lacking both H2-M and Ii have very low numbers of CD4` cells (10%–20% of normal, compared to 20%–30% in Ii-only–deficient mice), demonstrating that presentation of H2-M-dependent endogenous antigens is important for efficient T cell selection in the thymus (95, 162–164). Interestingly, B cell maturation has also been reported to be defective in mice lacking H2-M (or H2-M and Ii) (163), in a way similar to what has been described for mice lacking Ii (169) or H2-Aba (170) (but not H2-Abb) (169, 170).

FUNCTIONAL STUDIES OF DO The finding that a large proportion of DM is associated with DO in B cells was quite unexpected, particularly since DM alone appeared to be able to mediate peptide exchange both in vitro and in cell lines where DO is not expressed.

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However, since the majority of DM is actually bound to DO in B cells, a function for DO as a modulator of DM activity was likely. At the molecular level, this interaction could lead to three different outcomes; increased DM function, decreased DM function, or a change in the specificity of which peptides are exchanged by DM. These three possibilities are not necessarily exclusive when molecular function and other factors such as intracellular location for peptide loading and mode of antigen uptake are considered, and data supporting all three possibilities have been presented (41, 51, 111, 112). Ultimately, however, the presence of DO is likely to result in improved presentation of antigens that are physiologically relevant to B cells, but since other antigen presenting cells are mostly not expressing DO, it is unlikely that expression of DO benefits antigen presentation in general. Some of the published data regarding the molecular functions of DO are conflicting, but more importantly, the physiologic function of DO is still only partly understood.

Analysis of DO Function In Vitro DM function can be reconstituted effectively in vitro using purified or semipurified preparations of DM; similar approaches have been used by several groups to study the function of DMDO complexes in vitro (41, 51, 111, 112). In a direct comparison, DMDO complexes are less capable of mediating peptide loading onto DR molecules than is DM alone, although the degree of inhibition appears to vary depending on the experimental system. DO by itself, in contrast, has no measurable effect on peptide exchange (51, 112). The inhibition of DM by association with DO is not absolute, and with time the number of peptide-loaded class II complexes actually reaches the number formed in the presence of DM alone (51, 112). More significantly, the influence of DO on DM-mediated peptide exchange function appears to be pH dependent, so that the inhibitory effect of DO is most noticeable at pH over 5.5, while the peptide loading efficiency at pH ,5.0 approaches the efficiency of DM in the absence of DO (51, 112; M van Ham, J Neefjes, personal communication). DMDO complexes, as well as DO alone, undergo a substantial reversible conformational change upon acidification, and this structural change might explain the increased exchange activity at the lower pH (51). DMDO complexes have been reported to associate with DR molecules in lysosomal lysates from B cell lines (110, 112), but it is unclear whether the increased peptide exchange activity at acidic pH is due to increased catalytic activity of the intact DMDO complex or to dissociation of the complexes into free active DM and DO. One report has used kinetic analysis to suggest that DMDO complexes bind more tightly to class II molecules than DM does, thus providing a more potent chaperone effect (112). A tighter interaction would potentially result in the binding of higher affinity peptides to the class II molecule in vivo, since the time that the class II molecule would be available for peptide exchange (and thus for sampling peptides) would be extended. The tighter binding is not well docu-

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mented experimentally, however, and since other data show that peptide release from class II molecules, as well as peptide binding, is decreased in the presence of DO (at pH 5.5) (51), it remains to be shown that the time for peptide exchange is really increased in the presence of DO. When the rate of peptide release from DR molecules is analyzed for different peptide-DR complexes in the presence of DMDO complexes, the off-rate for any given peptide is proportional to the offrate in the presence of DM alone (or to the release in the absence of either DM or DMDO), suggesting that the presence of DO does not directly change the quality of released peptides, only the kinetics of peptide release (C Alfonso, L Karlsson, unpublished data). However, the repertoire of bound peptides is also influenced by the on-rate of available peptides (which is concentration-dependent) and by the rate of destruction of the molecules involved, so at any particular point in time the peptides bound by class II molecules may be different in the presence of DM or DMDO complexes (112).

DO/H2-O Function In Vivo Transfection of DO (or H2-O) in cell lines expressing class II molecules, Ii and DM, but not endogenous DO, has for the most part shown an inhibitory effect of DO on the function of DM. Thus, transfection of DO into class II–expressing T cell (41) or melanoma cell (111) lines has been reported to result in increased levels of CLIP–class II complexes both at the cell surface and intracellularly. The decreased release of CLIP in these cells was also accompanied by a decreased ability to present exogenous antigens to CD4` T cell clones, although the extent of this decrease was small (111). In contrast, another study has shown that the level of CLIP–class II complexes was decreased in a DO-transfected DR4 expressing melanoma cell line, compared with the DO negative parent line (112). The reason for these discrepancies is not clear, but the melanoma transfectants express different class II haplotypes (DR3 and DR4, respectively), and this may possibly explain the varying results. It should be noted, however, that the H2-Ab molecules from H2-O-deficient mice have normal (i.e. very low) CLIP content (51), suggesting that CLIP levels may not be a totally relevant marker for DO function. In contrast, several lines of evidence show that the repertoire of class II–associated peptides is different depending on whether the cells express DO or not. Thus, analysis of DR-associated peptides eluted from DO-transfected cell lines shows that these are partly different from the peptides eluted from the DOnegative parent cell lines (111, 112; M van Ham, J Neefjes, personal communication). Although the majority of the eluted peptides (around 90%) are identical in the DO-positive and DO-negative types of cells, some peptide species are present exclusively in one cell line or the other. Further evidence that the peptide repertoires are quantitatively different comes from analysis of antigen presentation by B cells derived from H2-O-deficient mice (51). Thus, while several protein antigens were presented equally well to T cell hybridomas independently of whether the B cells expressed H2-O, the presentation of other antigens was influ-

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enced by the presence of this molecule. In general, presentation of antigens internalized by fluid phase uptake of the antigen was found to be as good or better when H2-O-deficient B cells were compared to B cells from wild-type controls. In contrast, presentation of antigens internalized by receptor-mediated antigen uptake (achieved through breeding with mice expressing transgenic membrane immunoglobulin receptors) was more effective when the antigen-presenting B cells expressed H2-O. Also in this case, however, the presentation of several analyzed epitopes was independent of whether H2-O was present or not. The differences in presentation are epitope- rather than antigen-dependent, since different epitopes derived from the same protein may display different dependence on H2-O (C Alfonso, L Karlsson, unpublished data). Thus, after receptormediated uptake, two out of three analyzed epitopes derived from ovalbumin are presented better by the wild-type B cells than by the H2-O-deficient B cells, while the third epitope is equally well presented by both types of cells.

Why Is DO/H2-O Expressed in B Cells, But Not in Most Other APCs? Both DO and H2-O have been reported to be expressed on thymic epithelium, suggesting that these molecules may influence the thymic selection of T cells. No functional data exist to support this suggestion, however, and since DO and H2O have been reported to be expressed in different parts of the thymus, it is not easy to predict a possible function. The fact that H2-Ob staining is intact also in mice lacking a functional H2-Oa gene also needs to be explained before a function can be attributed to H2-O in the thymus. DO expression has also been reported to occur in dendritic cells, and it is conceivable that expression of DO may be favorable under certain conditions or during certain maturation stages of these cells. However, most dendritic cells and macrophages clearly do not express DO, and thus, the physiological function of DO/H2-O is likely to be a modification to antigen presentation that is beneficial mainly to B cells, rather than to antigen presentation in general. The finding that the majority of DM is associated with DO in B cells, but not in other APCs, is one of the first defined molecular differences between different types of APCs and may explain some of the described differences in antigen presentation between B cells and dendritic cells (171, 172). The reason for the DO/H2-O expression in B cells is likely to be related to the specialized function of B cells; i.e. to present antigens internalized by the clonal Ig receptor in order to receive help from CD4` T cells. The unfolding and degradation of protein antigens to protein fragments and peptides suitable for binding to class II molecules require acidic pH as well as the action of endosomal or lysosomal proteases, but excessive proteolysis will also result in the destruction of potential peptide epitopes (reviewed in 1). Different proteins have different resistance to denaturation at acidic pH and to proteolysis, and thus antigenic epitopes are likely to be released at different locations of the endosomal/lysosomal system. The capture of particular class II–binding

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epitopes may be optimal in different parts of the endosomal system, depending on the epitope, as well as on the haplotype of the class II molecule. Cathepsin S, the main enzyme degrading Ii in B cells and dendritic cells, is active in a broader pH range (including neutral pH) than most other lysosomal enzymes (173, 174), and it is therefore possible that Ii degradation precedes the bulk of antigen degradation (but not necessarily the bulk of epitope liberation). Since DM (in the absence of DO) is also active in the pH range between 6.5 and 4.5 (122, 126, 127), DM-mediated capture of antigenic epitopes by class II molecules is likely to occur throughout the endosomal/lysosomal system and may initially involve larger peptide fragments than the 11–20 amino acids long peptides that can be isolated from mature class II molecules. Most available in vitro data show that DO modifies the function of DMmediated peptide exchange by limiting the pH range in which effective peptide exchange can occur. The decreased activity of DM in the pH range between 6.5 and 5.5 suggests that the intracellular localization for peptide exchange may be skewed toward more acidic late endosomal and lysosomal compartments in cells expressing DO. Recent data support this conclusion by showing that expression of DO increases the sensitivity of antigen presentation to treatment with bafilomycin A, an inhibitor of the vacuolar ATPase (M van Ham, J Neefjes, personal communication). Treatment with this drug (which increases the endosomal pH) resulted in the abolition of antigen presentation by DO expressing cells, while presentation by the DO negative parent cell line was only partly decreased. In the case of B cells, relevant antigens are internalized after binding to membrane immunoglobulin (175, 176), and since the interaction with antibodies is well known to stabilize protein domains (177–179), the degradation patterns of free and antibody-bound antigens are often different. The pH in the endosomal system is not sufficient to dissociate most high-affinity antigen-antibody interactions, and release of antigenic epitopes from high-affinity antigen-antibody complexes is likely to require lysosomal conditions, due to the protease resistance of the antibodies themselves. Under these conditions, decreased DM-mediated peptide exchange in the earlier parts of the endosomal system due to the association with DO would limit presentation of antigens internalized by low-affinity receptors or by fluid phase uptake, without seriously influencing the presentation of antigens internalized by high-affinity immunoglobulin receptors. With age, H2O-deficient mice get increased levels of serum IgG1 (51), a finding that may reflect increased presentation of antigens internalized by low-affinity receptors or by fluid phase uptake. The presence of DO may serve to increase the specificity of the T cell–B cell interaction by promoting the presentation of peptides internalized by high-affinity antigen receptors, thus helping to focus the immune response and possibly avoiding the activation of irrelevant or harmful T and B cells. Antigen presentation in dendritic cells/macrophages compared to B cells is outlined in Figure 2 (see color insert).

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CONCLUDING REMARKS

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The general outline of antigen processing in the class II system in now well established, and it is quite apparent that DM plays an essential role in this process, not only by removing CLIP from maturing class II molecules, but also by editing the repertoire of bound peptides. The editing effect of DM can partly explain why certain peptides from a given protein are presented to T cells while others are not, but unfortunately it is still impossible to predict such peptides. In contrast to the clear and well-defined role of DM, the function of DO appears to be more subtle and is still being elucidated. Although our knowledge of antigen presentation and the components involved in this process is continually increasing, a complete picture of how antigens are processed will probably require a deeper understanding of how the endosomal/lysosomal system functions in general, and how this system is modified in different cell types. ACKNOWLEDGMENTS We thank our colleagues who have shared their preprints and unpublished data with us. We also thank A Brunmark for critically reading the manuscript and the rest of our colleagues at RWJPRI for their help and support. Visit the Annual Reviews home page at www.AnnualReviews.org.

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175. Rock KL, Benacerraf B, Abbas AK. 1984. Antigen presentation by haptenspecific B lymphocytes. I. Role of surface immunoglobulin receptors. J. Exp. Med. 160:1102–13 176. Lanzavecchia A. 1985. Antigen-specific interaction between T and B cells. Nature 314:537–39 177. Accolla RS, Cina R, Montesoro E, Celada F. 1981. Antibody-mediated activation of genetically defective Escherichia coli beta-galactosidases by monoclonal antibodies produced by somatic cell hybrids. Proc. Natl. Acad. Sci. USA 78:2478–82 178. Jemmerson R, Paterson Y. 1986. Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes. Science 232:1001–4 179. Simitsek PD, Campbell DG, Lanzavecchia A, Fairweather N, Watts C. 1995. Modulation of antigen processing by bound antibodies can boost or suppress class II major histocompatibility complex presentation of different T cell determinants. J. Exp. Med. 1815:1957– 63

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Figure 1 Schematic representation of DM function class II-CLIP complexes (top view; 1) are generated in the endosomal system. DM (2) binds to the class II-CLIP complexes, thus stabilizing a transient, more open, conformational state in the class II molecule (3). This conformational change results in the disruption of the hydrogen bond network that maintains CLIP in the peptide binding site and therefore the release of CLIP. The empty class II molecule is stabilized by DM (4). In the absence of peptides, DM may dissociate from the class II molecule, causing this to collapse and become functionally inactivated (5). This is not likely to be a common scenario in vivo, and instead the open DM-stabilized peptide binding site can bind other peptides generated by proteolysis in the endosomal system (6). Good stably binding peptides have anchor residues that fit in binding pockets on the class II molecule, and when DM is released from the class II molecule (7-8), these peptides become trapped in the binding site (8), while DM is free to catalyze another round of peptide exchange. Poorly stabilizing peptides (other than CLIP) trapped in the binding site of the class II molecule can be released in subsequent rounds of peptide exchange, thus resulting in a peptide editing effect of DM where stably binding peptides are favored.

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Figure 2A A model of H2-O/DO-mediated differences in antigen processing between distinct APCs. Internalized extracellular antigens pass through the endocytic system becoming denatured (by acidic pH) and degraded (by proteolytic enzymes, including cathepsins (Cat)) as their migration proceeds through early endosomes (EE), late endosomes (EE), and lysosomes (Lys), where proteolysis continues. Antigens taken up by fluid phase internalization (blue) are subject to proteolysis generating peptides in early and late endosomes, as well as in lysosomes. Within endosomes, proteolytic enzymes degrade Ii generating class II-CLIP (DR-CLIP) complexes. In dendritic cells and macrophages (Figure 2A) antigens are internalized by fluid phase uptake, or by nonclonal receptors. In these cells, which do not express DO, DM activity is relatively high throughout the endosomal system (indicated by the bar to the left; yellow indicates low peptide exchange activity by DM, red indicates high activity), facilitating efficient CLIP release and loading of class II molecules wherever peptides become available. After peptide acquisition class II-peptide complexes (DR-pep) are transported to the cell surface.

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Figure 2B Internalization of exogenous antigens by B cells occurs by fluid phase and mIg receptor-mediated uptake (brown). As in other cell types, antigens internalized by fluid phase uptake are subject to proteolysis, generating peptides in early and late endosomes, as well as in lysosomes. Peptides generated in endosomes, however, are not favored for binding to class II molecules, since peptide exchange is poor at this pH when DM is complexed with DO (DMDO). mIg receptor-mediated uptake affords protection to antigens in the earlier parts of the endosomal pathway such that Ig-complexed antigens proceed to lysosomal compartments. Dissociation from the Ig molecule through proteolysis is favored in the acidic, highly proteolytic environment found in lysosomes. Here processing proceeds and, since downregulation by DO of DM-mediated peptide exchange is poor at lysosomal pH, the generated peptides can effectively bind class II molecules. Hence, peptides generated in lysosomal compartments may be favored for loading onto class II molecules, after which stable class II-peptide (DR-pep) complexes are transported to the B cell surface for presentation to CD4+ T cells.

Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:143–164 Copyright q 2000 by Annual Reviews. All rights reserved

NEGATIVE REGULATION OF CYTOKINE SIGNALING PATHWAYS Hideo Yasukawa, Atsuo Sasaki, and Akihiko Yoshimura Institute of Life Science, Kurume University, Aikawamachi 2432–3, Kurume 839–0861, Japan, e-mail: [email protected]

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Key Words JAK, STAT, CIS, JAB, SOCS, SSI, SH2 domain, tyrosine kinase, negative regulation, interferon gamma Abstract The Janus family of protein tyrosine kinases (JAKs) and STAT transcription factors regulate cellular processes involved in cell growth, differentiation, and transformation through their association with cytokine receptors. The CIS family of proteins (also referred to as the SOCS or SSI family) has been implicated in the regulation of signal transduction by a variety of cytokines. Most of them appear to be induced after stimulation with several different cytokines, and at least three of them (CIS1, CIS3/SOCS3, and JAB/SOCS1) negatively regulate cytokine signal transduction by various means: CIS1 inhibits STAT5 activation by binding to cytokine receptors that recruit STAT5, whereas JAB/SOCS-1 and CIS3/SOCS-3 directly bind to the kinase domain of JAKs, thereby inhibiting tyrosine-kinase activity. Therefore, these CIS family members seem to function in a classical negative feedback loop of cytokine signaling. Biochemical characterization as well as gene disruption studies indicate that JAB/SOCS1/SSI-1 is an important negative regulator of interferon c signaling. The mechanisms by which these inhibitors of cytokine signal transduction exert their effects have been extensively studied and will provide useful information for regulating tyrosine-kinase activity.

INTRODUCTION Growth, differentiation, and other functions of immune and hematopoietic cells are controlled by multiple cytokines, including interleukins (ILs) and colonystimulating factors (CSFs). Although lacking catalytic domains, members of the cytokine receptor superfamily mediate ligand-dependent activation of protein tyrosine phosphorylation through their association with and activation of members of the Janus kinase (JAK) family of protein tyrosine kinases. Cytokines induce homo- or heterodimerization and activation of their cognate receptors, resulting in the activation of associated JAK kinases (JAK1, JAK2, JAK3, and Tyk2). The activated JAKs phosphorylate the receptor cytoplasmic domains, thereby creating docking sites for SH2-containing signaling proteins. Among the substrates of tyrosine phosphorylation are members of the signal transducers and 0732–0582/00/0410–0143$14.00

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activators of the transcription family of proteins (STATs) (1). Although this pathway was initially found to be activated by interferons(s) (IFNs), it is now known that a large number of cytokines, growth factors, and hormonal factors activate JAK and/or STAT proteins. Various cytokines induce the tyrosine phosphorylation and activation of one or more of the seven STAT family members. Cytokines also activate Ras-MAP kinase pathways, phosphatidyl-inositol-3 (PI3) kinase, and phospholipase C-c (PLCc). The targeted disruption of genes for a number of Jaks and STATs in mice has revealed specific biological functions for many of them (for a review, see 2–4). For example, mice lacking JAK1 failed to manifest biologic responses to cytokines that bind to three distinct families of cytokine receptors: all class-II cytokine receptors, cytokine receptors that utilize the c(c) subunit for signaling, and the family of cytokine receptors that depend on the gp130 subunit for signaling. JAK2 gene disruption causes embryonic lethality due to the absence of definitive erythropoiesis. JAK3 knockout causes severe immunodeficiency. Mice in which the genes for STAT1, STAT4, and STAT6 are disrupted are viable but lack functions mediated by IFNs, interleukin 12 (IL-12), or IL-4, respectively, suggesting that these STATs perform very specific functions in immune responses. In contrast, the two highly related STAT5a and STAT5b are activated by various cytokines, including growth hormone (GH), prolactin (PRL), erythropoietin (EPO), interleukin-2 (IL-2), IL-3, granulocyte macrophage-colony stimulating factor (GMCSF), and thrombopoietin (TPO). The phenotypes of both STAT5a and STAT5b single-gene knockout mice and double knockout mice revealed an important role of STAT5s in PRL and GH signaling as well as in IL-2-dependent T cell functions (5–9). Over the years, the majority of investigations on cytokines have focused on the mechanisms by which they exert their actions. However, it is clear that the actions of cytokines are limited in both magnitude and duration, making it important to understand the mechanisms by which their actions are negatively controlled. Moreover, the downmodulation of one cytokine action by several factors, including another cytokine, are often found in important physiological circumstances. For example, Th1 and Th2 cytokines suppress each other (10). cAMP, calcium ionophore, and GM-CSF inhibit JAK1-STAT1 or STAT3 pathways (11), although the molecular basis of such modulation has not been well characterized. The deregulation of the negative feedback of the JAK-STAT pathway has been implicated in hematopoietic disorders, autoimmune and inflammatory diseases, interferon (IFN)-resistance, and cancer. For example, the constitutive activation of JAK and/or STATs has been implicated in transformation by v-src (12, 13). A constitutively active TEL-JAK fusion gene is implicated in T cell leukemia (14, 15). Mutations in the erythropoietin (EPO) receptor gene causing the deletion of the C-terminal region have been implicated in familial erythrocytosis, which is inherited as an autosomal dominant trait (16). In this disease, the disruption of the SHP-1 binding sites of the erythropoietin (EPO) receptor at the C-terminal

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region has been shown to cause prolonged activation (phosphorylation) of JAK2 (17). In Table 1, the major regulatory mechanisms of JAK-STAT pathways are listed. The degradation of STATs and receptors should be involved in the regulation of signaling (18, 19). Naturally occurring dominant negative variants of STAT5 have also been reported (20, 21). Specific STAT1 and STAT3 inhibitors PIAS1 and PIAS3 have also been reported; however, their physiological meaning has not been clarified because these two genes are highly expressed without cytokine stimulation (22, 23). Tyrosine phosphatases containing the SH2 domain (SHP-1 and SHP-2) also regulate cytokine signaling, both positively and negatively. SHIP and Cbl have been implicated in the negative regulation of tyrosinekinase signaling and could also negatively regulate cytokine signaling (see below). However, recently, a new family of cytokine-inducible SH2 proteins (CISs) was identified and shown to be involved in the negative regulation of cytokine signals, especially JAKs and STATs.

SHP The activation of JAK tyrosine-kinase activity is positively regulated by the transphosphorylation of a critical tyrosine within the activation loop of the kinase domain (24–28). Considerable evidence suggests that one mechanism that terminates JAK activation involves the recruitment of a tyrosine phosphatase containing an SH2 domain (SHP-1) to receptor complexes resulting in the dephosphorylation of JAKs (28–30). The direct binding of JAK1 or JAK2 with SHP-1 has been demonstrated (31, 32). The potential importance of this mechanism is strongly suggested by the phenotype of motheaten (me/me) mice lacking SHP-1 that die from a disease with components of autoimmunity and inflammation (33). SHP-1 is a negative regulator of JAK kinases implicated in familial erythrocytosis (familial polycythemia) inherited as an autosomal dominant trait (16). In this disease, the disruption of the SHP-1 binding sites of the EPO receptor, as noted above, resulted in prolonged activation (phosphorylation) of JAK2 (17). SHP-1 is also implicated in familial hemophagocytic lymphohistiocytosis TABLE 1 Regulatory mechanism of the JAK-STAT pathway 1. Receptor-mediated endocytosis and degradation in lysosome 2. Dephosphorylation by tyrosine phosphatases, SHP and SHIP 3. Degradation by ubiquitin-proteasome systems, Cbl? SOCS box? 4. PIAS? 5. Dominant negative STATs 6. Expression of signaling molecules 7. CIS/SOCS/SSI family

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(FHLH) (34). However, SHP-1 regulates a number of receptor and nonreceptor tyrosine kinases negatively, including c-kit and ZAP-70 (35, 36). Thus, it remains to be determined which kinases are specifically responsible for the phenotype. SHP-2, which is relatively ubiquitously expressed, has been implicated in positive regulation in cell growth and development. However, Kim et al found that SHP-2 negatively regulates gp130-mediated induction of acute-phase plasma protein genes in hepatic cells (37). You et al reported that SHP-2 also functions as a negative effector in IFN-induced growth-inhibitory and apoptotic pathways (38). They showed that treatment of mouse fibroblast cells lacking a functional SHP2 with IFN-a or IFN-c resulted in augmented suppression of cell viability compared to that of wild-type cells, probably by sustained activation of JAKs and STATs. Therefore, SHP-2 could function as a negative regulator of cytokine signaling in certain circumstances.

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SHIP SH2-containing 5’-inositol phosphatase (SHIP) has been implicated in inhibitory signals not only from the immunotyrosine-based inhibition motif (ITIM) but also from some cytokine receptors. SHIP, which is widely expressed in hematopoietic cells, was first identified as a tyrosine phosphoprotein associated with Shc in response to numerous cytokines. Using SHIP–/– mast cells, Huber et al (1998) demonstrated that SHIP restricts the IgE-induced extracellular calcium entry by hydrolyzing phosphatidylinositol 3, 4, 5-trisphosphate [PI(3, 4, 5)P3] (39). SHIP–/– splenic B cells displayed prolonged Ca2` influx, increased proliferation in vitro, and enhanced mitogen-activated protein-kinase (MAPK) activation in response to B cell receptor–Fcc receptor IIB coligation (40). In addition, increased numbers of granulocyte-macrophage progenitors were observed in both the bone marrow and spleen of SHIP–/– mice, perhaps as a consequence of hyperresponsiveness to stimulation by the macrophage colony-stimulating factor, the granulocyte-macrophage colony-stimulating factor, interleukin-3, or the Steel factor as observed in vitro (41). Liu et al showed that engagement of the IL-3 and the GM-CSF receptors in SHIP –/– cells leads to increased and prolonged PI3 kinase-dependent PI(3, 4, 5)P3 accumulation and protein-kinase B (PKB) activation (42). These data indicate that SHIP negatively regulates the growth-factor and cytokine-mediated PKB activation as well as the cell survival of myeloid cells.

Cbl The adaptor molecule c-Cbl has also been demonstrated to be tyrosine phosphorylated in response to many stimuli, including cytokines (43, 44). However, genetic studies showed it to be a negative regulator of several tyrosine kinases. In C. elegans, the c-Cbl homologue Sli-1 acts as a negative regulator of the Ras homologue Let60 (45, 46). Moreover, several recent studies have demonstrated that mammalian c-Cbl has a negative regulatory function in intracellular signal

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transduction, such as Syk tyrosine-kinase activation in mast cells (47), T cell receptor (TCR)-induced ERK2, and AP-1 activation (48). Cbl-b, a member of the Sli-1/c-Cbl protein family, has also been demonstrated to inhibit Vav-mediated cJun kinase activation (49). Indeed, c-Cbl gene knockout mice exhibited enhanced TCR signaling via Syk and ZAP-70 (50). The acceleration of ubiquitin/ proteasome-dependent degradation of receptor tyrosine kinases could be a mechanism of downregulation by c-Cbl (51, 52). Although the negative regulation of cytokine signals by c-Cbl has not been demonstrated, Ueno et al have reported that suppression of c-Cbl expression accelerated the EGF-dependent JAK/STAT pathway (54). Therefore, c-Cbl could also be a regulator of cytokine signaling. Previously, we cloned and characterized a novel adaptor molecule containing a Pleckstrin-homology (PH) domain and an Src homology-2 (SH2) domain, APS (55). APS was cloned using the yeast twohybrid system with the oncogenic c-kit kinase domain as bait. This protein forms a new subfamily of SH2 proteins with Lnk and SH2-B. APS is tyrosine phosphorylated in response to c-kit or B cell receptor stimulation; a single major tyrosine phosphorylation site, which is highly conserved in this family, was found at its C-terminus. The phosphorylation of the C-terminus creates a binding site for c-Cbl. Recently, an isoform of SH2-B (SH2-Bb) was shown to be a good substrate for JAK2; it was phosphorylated in response to IFNc and growth hormone (GH) (56). We also found that APS was highly phosphorylated in response to cytokine stimulation and suppressed erythropoietin (EPO)-induced STAT5 activation (57). Using the 293 cell-reconstitution systems, we showed that APS and c-Cbl synergistically inhibit cytokine-dependent STAT5 activation. Therefore, APS was suggested to function as a negative regulator of cytokine signaling in collaboration with c-Cbl.

CIS: Prototype of Cytokine-Induced SH2 Proteins The first member of this family was denoted as CIS (now called CIS1), for cytokine-inducible SH2 containing protein (58, 59) (Figure 1). CIS1 was cloned originally as an immediate early gene that was induced by IL-2, IL-3, and EPO. CIS1 was found to associate with the tyrosine phosphorylated EPO receptor and the IL-3 receptor b chain following stimulation with EPO or IL-3, respectively (58). Forced expression of CIS1 could partially suppress IL-3- or EPO-induced proliferation as well as STAT5 activation (58, 60). CIS1 binds specifically to the phosphorylated tyrosine residue of the EPO receptor, Y401 (61). This residue is one of the major STAT5 binding sites. Therefore, one possibility is that CIS1 might function by directly blocking phosphotyrosine motifs on receptors, preventing their coupling to stimulatory signaling pathways (Figure 2). Alternatively, given that CIS1 was observed to have a relatively short half-life, CIS1 might be a scavenger of tyrosine phosphorylated proteins, targeting them for degradation as well. It has been demonstrated that CIS1 itself is ubiquitinated (61). Thus, it is possible that CIS1 is involved in the ubiquitin/proteasome-dependent degradation

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Figure 1 Schematic structure of the CIS/SOCS/SSI family and other CH domain/SOCS box-containing proteins. The numbers indicate amino acid length.

of the activated cytokine receptors by functioning as a ubiquitin-conjugating enzyme, an E3-like molecule. This is also supported by the recent finding that the C-terminal region of the CIS (CH domain or SOCS box) interacts with the Elongin B, C complex (62, 63) and probably with Rbx1, which activates cdc34like E2 molecules (64) (see below).

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Figure 2 Signaling pathways activated in response to cytokines and action of three CIS family members. A schematic structure of the cytokine receptor dimer after ligand binding is shown. The shaded boxes in the cytoplasmic domain of the receptor represent boxes 1 and 2, which share homology among cytokine receptors. Two major signaling pathways, Ras-MAP kinase and JAK-STAT, are activated through cytokine receptors. CIS1 binds to the receptor and CIS3 and JAB bind to JAKs, thereby inhibiting signaling.

The CIS promoter contains two pairs of tandem TTCNNNGAA motifs that are capable of binding STAT5, and a CIS1 promoter reporter construct was induced by EPO in cells transfected with EPOR and STAT5 (60). The essential role of STAT5 in CIS expression was confirmed by the observation that CIS1 expression was not observed in the ovaries or thymus of STAT5a and b double knockout (KO) mice (8, 9). Therefore, CIS1 acts as a kind of negative feedback regulator of the JAK-STAT5 pathway. The negative effects of CIS1 on STAT5 activity were confirmed by the phenotypes observed in CIS1-transgenic mice (65). We created CIS1-transgenic mice under the control of b-actin promoter. These transgenic mice developed normally; however, their body size was smaller and their weight lower than those of the wild-type mice, suggesting a defect of growthhormone signaling. Female transgenic mice failed to lactate after parturition because of a failure of the terminal differentiation of the mammary gland, suggesting a defect of prolactin signaling. IL-2-dependent upregulation of the IL-2receptor a chain and proliferation were partially suppressed in splenocytes. These phenotypes remarkably resemble those found in STAT5a and/or STAT5b knockout mice (5–9). Indeed, STAT5 tyrosine phosphorylation was suppressed in the mammary gland and liver in CIS1 transgenic mice. Furthermore, IL-2-induced activation of STAT5 was markedly inhibited in T cells in transgenic mice, while leukemia inhibitory factor-induced STAT3 phosphorylation was not affected. We

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also found that the number of cdT cells as well as that of natural killer (NK)-T cells was dramatically decreased in transgenic mice. Moreover, Th1/Th2 differentiation was also altered in CIS1-transgenic mice, although the mechanism is not clear at present. These data suggest that CIS1 functions as a specific negative regulator of STAT5 in vivo and plays an important regulatory role in T cell differentiation. Although there are no detailed data presented, CIS1-deficient mice have been shown to be phenotypically normal in all regards (mentioned in references 9 and 98). This strongly suggests a functional redundancy of CIS by related gene(s). Indeed, CIS turned out to be a member of a large family that is probably involved in regulation of cytokine signaling.

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Identification of JAB/SOCS-1/SSI-1 Three groups, including ours, independently cloned the second CIS family member by entirely different experimental approaches. Starr et al cloned suppresser of cytokine signaling-1 (SOCS-1) as an inhibitor of IL-6-induced differentiation and growth arrest of the murine monocytic leukemia cell line M1 (66); Endo et al cloned the same protein as a JAK-binding protein (JAB) that could interact with the catalytic (JH1) domain of JAK2 (67); and Naka et al identified it by using an antibody that recognizes a common sequence of the SH2 domain of STATs and that was not a STAT protein but, rather, a STAT-induced STAT inhibitor-1 (SSI-1) (68) (Figure 2). When overexpressed, JAB/SOCS-1/SSI-1 can inhibit any signaling utilizing JAKs, such as STAT5 activation by EPO, STAT3 activation by leukemia inhibitory factor (LIF) or IL-6, and c-fos induction by IL2. However, since JAB did not inhibit either fibroblast growth-factor (FGF) induced c-fos activation or c-kit phosphorylation, although it binds to the FGF receptor and c-kit, JAB/SOCS-1/SSI-1 seems to be specific to JAK tyrosine kinases (69). Ohya et al also cloned the same gene (also called TIP-3) by twohybrid screening using a Tec kinase domain as bait (70). JAB/SOCS-1/SSI-1 was found to suppress Tec kinase activity. However, this inhibitory effect was marginal compared with that on JAKs. Recently, JAB/SOCS-1/SSI-1 has been shown to bind to the c-kit receptor tyrosine kinase via its SH2 domain (67, 71). Although JAB did not inhibit the catalytic activity of the c-kit tyrosine kinase, JAB inhibited c-kit mediated proliferation signals, probably by binding to the signaling proteins Grb-2 and the Rho-family guanine nucleotide-exchange factor Vav, via its SH3 domain (71). However, such effects seem to be observed only when JAB/SOCS-1 is extremely overexpressed at nonphysiological levels, and study of knockout mice suggests no connection between c-kit and JAB/SOCS-1 (see below). Losman et al reported that JAB/SOCS-1 potentially inhibits the activation of JAK1 kinase and STAT6 in response to IL-4 (72), suggesting that JAB/SOCS-1 is a negative regulator of IL-4 signaling. However, the most important physiological function of JAB/ SOCS-1 is the regulation of IFNc signaling (see below). Thus, as suggested by

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Figure 3 Structure and model of kinase inhibition by JAB/SOCS-1 and CIS3/SOCS-3. (A) Schematic model of the functions of JAB and CIS3 domains. Essential amino acids in the kinase inhibitory region (KIR) and the extended SH2 subdomain are highlighted with bold circles. (B) The model of JH1 activation and inhibition by JAB/SOCS-1. Binding of JAB to the activation loop prevents the access of substrates and/or ATP to the catalytic pocket.

Venkataraman et al (73), JAB/SOCS-1, which is induced by IFNc, could play a key role in modulating IL-4 signaling, therefore in Th1/Th2 differentiation.

Involvement of JAB in Interferon-c Signaling IFNc is the most potent inducer of JAB/SOCS-1/SSI-1 in a wide variety of cell lines (74, 75). IFNb has no inducible effect. Previously, JAB/SOCS-1/SSI-1 was reported to be induced by IL-6 in mouse liver and M1 cells, suggesting that JAB/

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SOCS-1 is a negative feedback regulator of IL-6/STAT3 (68). However, our experiments and those of others have suggested that IL-6 or LIF induces the expression of JAB much less efficiently than IFNc. JAB/SOCS-1 could be a negative feedback regulator of IFNc/STAT1 rather than IL-6/STAT3. Our recent study suggests that the dysregulated overexpression of JAB can confer IFN-resistance to cells (75). NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFNb and IFNc. M1 leukemic cells stably expressing JAB were also resistant to IFNc and IFNb-induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFNc did not induce tyrosine phosphorylation or DNA-binding activity of STAT1. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant LoVo and Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, the overexpression of JAB could be, at least in part, a mechanism of IFNresistance with a dominant phenotype. It is clinically important to address the question of whether or not JAB is involved in the interferon resistance frequently found in patients with chronic myelogenous leukemia or those carrying the hepatitis type-C virus. Using M1 cells, we proposed that JAB/SOCS-1 induced by IFNc could be a mechanism of the antagonistic effect of IFNc against IL-6 or LIF (74). Pretreatment of M1 cells with IFNc increased the level of JAB/SOCS1 and prevented LIF- or IL-6-induced differentiation of M1 cells. Similarly, Venkataraman et al reported that IFNc stimulation results in a loss of IL-4-induced STAT6 tyrosine phosphorylation, nuclear translocation, and DNA binding (73). Using the fibrosarcoma cell-line U3A, which lacks Stat1, they demonstrated that the transcription-activation function of Stat1 is required for the IFNc-mediated repression. Treatment with IFNc, but not IL-4, specifically upregulates the expression of JAB/SOCS-1, and overexpression of JAB/SOCS-1 effectively blocks IL4-induced STAT6 phosphorylation and transcription. This suggests that the IFNc-mediated repression of several cytokine actions is at least in part mediated by JAB/SOCS-1.

Physiological Function of JAB/SOCS-1 Elucidated from Gene Disruption The most striking finding came from the phenotype of JAB/SOCS-1/SSI-1 KO mice (76–78). JAB/SOCS-1/SSI-1 -/- mice displayed growth retardation and died within three weeks after birth before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. Lymphocytes in the thymus and spleen of the KO mice exhibited accelerated apoptosis with aging, and their number was 2–25% of that in wild-type mice at 10 days of age. Among various proand anti-apoptotic molecules examined, upregulation of Bax was found in the lymphocytes of the spleen and thymus of KO mice. In addition, a progressive loss occurred of maturing B lymphocytes in the bone marrow, spleen, and periph-

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eral blood. Thus, JAB/SOCS-1/SSI-1 is required for the in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival. Some of these phenotypes resemble those found in IFNc-transgenic mice (79, 80), and constitutive activation of STAT1 was found in JAB/SOCS-1/SSI-1–/– mice liver (J.C. Marine et al, unpublished data). Hematopoietic progenitor cells from JAB/SOCS-1/SSI-1–/– mice were hyperresponsive to inhibition by IFNc, the degree of inhibition varying markedly with the stimulating factor used (78). Importantly, much of the pathology of JAB/SOCS-1 –/– mice can be eliminated by injecting mice with antibodies to IFNc or by crossing the mice with IFNc knockout mice (81, 81a). Therefore, JAB/SOCS-1 is indeed a negative regulator of IFNc/STAT1 and probably plays an important role in the prevention of apoptosis induced by STAT1. Physiologically, it is also very interesting to examine the involvement of JAB/SOCS-1 in chronic inflammation.

Molecular Mechanism of Kinase Inhibition by JAB/SOCS-1 The activation of JAK tyrosine-kinase activity is positively regulated by the transphosphorylation of a critical tyrosine within the activation loop of the kinase domain. In the case of JAK2, that is the tyrosine residue Y1007. Recently, we demonstrated that JAB/SOCS-1 specifically binds to phosphorylated Y1007 (pY1007) in the activation loop of JAK2 (82). Extensive mutational analysis revealed the existence of three functional domains. Binding to the phosphorylated activation loop requires the JAB/SOCS-1 SH2 domain and an additional Nterminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB/SOCS-1 related proteins. An additional N-terminal 12 amino-acid region (kinase inhibitory region) of JAB/SOCS1 also contributes to high-affinity binding to the JAK2 tyrosine-kinase domain and is required for the inhibition of JAK2 signaling and kinase activity (Figure 3). The importance of the N-terminal region of JAB/SOCS-1 was also pointed out by Nicolson et al (83). These studies define a novel type of regulation of tyrosine kinases and may provide a basis for the design of specific tyrosine-kinase inhibitors (Figure 3). Without the kinase inhibitory region, JAB/SOCS-1 binds to the JAK2 kinase domain (JH1) with similar affinity to pY1007 phosphopeptide, suggesting that this region is a second binding site for JH1. We noticed that the kinase inhibitory region somewhat resembles the activation loop of JAKs. Therefore, we hypothesized that the kinase inhibitory region may mimic the activation loop by functioning as a pseudosubstrate. A similar structure and function relationship was found in CIS3/SOCS3, another JAK inhibitor (84). However, CIS3/SOCS3 did not inhibit JH1 kinase activity in vitro very efficiently (82, 83). Therefore, the true inhibitory mechanism by CIS3/SOCS-3 is still an open question. This hypothesis must be verified by kinetic analysis or more directly by co-crystal structure analysis using purified JAB/SOCS-1 and JH1.

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Recently, other SH2 proteins, Grb10 and Grb14, have been shown to contain the second novel domain that interacts with the insulin receptor and insulin-like growth factor receptor in an activation loop-dependent manner (85, 86). These studies suggest that Grb10 and Grb14 may interact within or near the activation loop of the insulin receptor kinase domain, thereby physically blocking tyrosinekinase activity. Although there is no sequence similarity between the kinase inhibitory region of JAB and any domains of Grb10 or Grb14, it would be interesting to determine whether other tyrosine kinases possess their own specific inhibitory SH2 protein that interacts with the activation loop. It may also be possible to design a specific JAB-like tyrosine-kinase inhibitor acting against a given tyrosine kinase by modifying the SH2 domain to bind to the kinase activation loop as well as the kinase inhibitory region to the catalytic pocket of the kinase.

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The CIS Family By database search, Masuhara et al reported five additional CIS family members (CIS2–6) (69) (Figure 1). Hilton’s group also identified one additional member (SOCS-7/CIS7; partial cDNA) (87). A partial cDNA of CIS5 was also cloned by Takenawa’s group as a binding protein for the SH3 domains of Nck, Ash (Grb2), PLCc (88). This gene was expressed mainly in brain. Others are expressed in hematopoietic and lymphoid tissues such as bone marrow, spleen, and thymus, and their expression is induced by a subset of cytokines in some hematopoietic cells (69). Thus, it is reasonable to call this family ‘‘the CIS family.’’ Figure 1 shows the structure of CIS family members (CISs). While the Nterminal regions share little sequence similarity, all seven contain an SH2 domain and a conserved C-terminal region that we designate as the CH (CIS-homology) domain, or others referred to as SOCS-box or SC-motif. The consensus motif in the CH domain is LXXPXXRX5SLQHLCRXXIX6–9IXXLPLPXXLXDYLXXYXY/F (X: any amino acid), although the entire length of the C-terminal region was varied. The SH2 domain exhibited 23–58% identity among CISs. Relatively high identity was seen between CIS1 and CIS2 (about 50%), CIS4 and CIS5 (about 58%), and JAB and CIS3 (about 40%). The -5 and -1 positions of the SH2 domain are fixed as L and G, respectively. Interestingly, the SH2 domains of STATs also contain the same amino acids at these positions (89). The Nterminal region is only 40–80 amino acids in length except for CIS4–7 which has a quite long N-terminal region (more than 350 amino acids). Among CIS family members, only CIS3/SOCS-3 and JAB/SOCS-1 can bind to JAK2-JH1 (69, 74, 84). Binding was confirmed by using two-hybrid analysis as well as co-precipitation assays in vitro and in vivo. The interaction of JAK2JH1 with JAB was stronger than that with CIS3/SOCS-3. By using a simple reporter-gene assay system, we found that JAB/SOCS-1 and CIS3/SOCS-3 almost completely blocked cytokine-dependent STAT3 and STAT5 reporter-gene activation (69). CIS2 and CIS4 had no effect or rather enhanced the reporter-gene activation. However, CIS3/SOCS-3 and JAB/SOCS-1 did not affect FGF- or

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PDGF-induced c-fos promoter activation, suggesting that the effect of CIS3/ SOCS-3 and JAB is quite specific to JAK tyrosine kinases. CIS3/SOCS-3 is physiologically interesting, because it is expressed in a wide variety of tissues, including brain. Bjorbaek et al found that peripheral leptin administration to ob/ ob mice, but not db/db mice, rapidly induced CIS3/SOCS-3 mRNA in the hypothalamus, but had no effect on CIS1, JAB, or CIS2. In mammalian cell lines, CIS3/SOCS-3, but not CIS1 or CIS2, blocked leptin-induced signal transduction (90). The expression of CIS3/SOCS-3 mRNA in the arcuate and dorsomedial hypothalamic nuclei is increased in Ay/a mice, a model of leptin-resistant murine obesity. CIS3/SOCS-3 may be a leptin-inducible inhibitor of leptin signaling, and a potential mediator of leptin resistance in obesity. Auernhammer et al also found that injection of LIF or IL-1b increased CIS3/SOCS-3 mRNA in the pituitary, and, in AtT-20 cells, stable overexpression of CIS3/SOCS-3 inhibited basal and LIF-stimulated ACTH secretion (91). We also found that CIS3/SOCS-3 inhibited prolactin-induced STAT5 activation (92). CIS3/SOCS-3 was also implicated in the negative regulation of EPO signaling (84), IL-10 signaling (93, 94), and IL2 signaling (95). Recently, the promoter region of CIS3/SOCS-3 was characterized, and STAT3 binding sites were found in it (96). However, we recently reeported that CIS3/SOCS-3 is highly expressed in erythroid lineage cells during embryonic development (96a). And CIS3/SOCS3 deletion results in an embryonic lethality at 12–16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of erythroid progenitors is greatly increased in response to EPO. These results demonstrate that CIS3SOCS3 is critical in negatively regulating fetal liver hematopoiesis, probably by negatively regulating EPO receptor/JAK2 signaling. Functions of CIS3/SOCS-3 in adult tissues remain to be clarified. CIS2/SOCS-2 binds to the insulin-like growth factor I (IGF-I)-receptor cytoplasmic kinase domain (97), but the functional meaning of this interaction is not clear.

Function of the CH-Domain/SOCS Box Database search also revealed that a similar CH domain is present in several proteins which contain ankyrin-like repeats, in the Ras-like GTPases and in proteins containing a WD domain (69). Hilton’s group called this domain ‘‘SOCS box’’ (66). They have identified 16 other proteins that contain this motif by using database search (87). These proteins fall into five classes based on the protein motifs found in the N-terminal of the CH domain. In addition to four new CIS proteins (SOCS-4 to SOCS-7) containing an SH2 domain, they describe four new families of proteins that contain either WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3), or ankyrin repeats (ASB-1 to -3) as well as G proteins. The CH domain may be involved in signal transduction because these domains in the N-terminal region are frequently found in signaling molecules, although

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TABLE 2 List of CIS family members Other names CIS1 JAB CIS2 CIS3 CIS4 CIS5 CIS6 CIS7

CIS SOCS-1, SSI-1 SOCS-2, SSI-3 SOCS-3, SSI-3 SOCS-6 SOCS-7, NAP4 SOCS-5 SOCS-4

Amino Acids 257 211 197 225 535 .485 536 .521

Inductiona

Targetb

yes; STAT5 yes; STAT1 yes yes; STAT1,3 yes ? yes ?

cytokine receptors JAKs IGF-I receptor JAKs ? Grb2, PLCc ? ?

a

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Induction of mRNA by various cytonkines in some hematopoietic cells. STATs possibly involved in induction are also indicated. b Apparent tyrosine phosphorylated proteins that bind to CISs. ? means not done or not identified.

this region is apparently not necessary for kinase inhibition by JAB or CIS3 (63, 82, 83, 84). Recently, Kamura et al reported that the Elongin BC complex interacts with the CH domain/SOCS box and prevents proteasome-dependent degradation of JAB/SOCS-1 (63). Elongin B is a ubiquitin-like protein, and Elongin C is a Skp1like protein that binds to a BC-box motif that is present in the Elongin A and CH domain/SOCS box. On the contrary, Zhang et al suggested that the SOCS box accelerates degradation of target proteins (including JAKs), from the analogous to the family of F-box-containing proteins, which mediate ubiquitin-proteasome degradation (62). The SOCS-box sequence has similarity to the a-domain of von Hippel-Lindau (VHL) tumor-suppressor gene product as well as the Skp2 F-box protein (98). The VHL protein is part of a complex that includes Elongin B/C, Cullin-2, and Rbx1, an evolutionarily conserved protein that contains a RING-H2 finger-like motif and interacts with Cullins (99). The yeast homologue of Rbx1 is a subunit and potent activator of the Cdc53-containing SCF-Cdc4 ubiquitin ligase (E2) required for the ubiquitination of the cyclin-dependent kinase inhibitor Sic1. Therefore, it is probable that JAB/SOCS-1 recruits the ubiquitin-ligation system into JAK2 by forming a complex with Elongin B/C, Rbx1, and Cullin-2. The CH domain/SOCS box-mediated degradation of JAK and cytokine receptors should be studied more intensively in the near future.

NOMENCLATURE Since CIS was the first member of this family to be identified and most of the family members are cytokine-inducible genes, we have referred to these proteins as the CIS family proteins. However, since SOCS is the most commonly used

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one, we have also referred to SOCS by number in this review. Although SOCS, JAB, and SSI are all nomenclatures that have merit, the nomenclature should be agreed upon as soon as possible. In Table 2, the different names of each CIS member are listed. Three of the CIS family members, CIS1, JAB/SOCS-1, and CIS3/SOCS-3, have been extensively characterized. CIS1 is induced via STAT5 and then binds to the activated receptor, thereby inhibiting STAT5 activity by masking the STAT5 binding sites and/or promoting the degradation of the activated receptor. JAB/SOCS-1, and probably CIS3/SOCS-3, are induced by several cytokines (either STAT1 or STAT3 is probably involved in this induction) and then bind to JAK kinases directly, thereby inhibiting kinase activity. Although little is known about other CIS family members, a study of their physiological functions has been started. Understanding the mechanisms by which JAKs and STATs are inhibited by CISs/SOCSs is very important for developing specific pharmacological inhibitors of cytokine signaling as well as tyrosine kinases. ACKNOWLEDGMENTS We would like to express our appreciation to Ms. Miho Chikushi for preparing the manuscript. Visit the Annual Reviews home page at www.AnnualReviews.org.

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Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:165–184 Copyright q by Annual Reviews. All rights reserved

T CELL ACTIVATION AND THE CYTOSKELETON Oreste Acuto1 and Doreen Cantrell2 1 Molecular Immunology Unit, Department of Immunology, Pasteur Institute, 75724, Paris, cedex 15, France; e-mail: [email protected] 2 Lymphocyte Activation Laboratory, Imperial Cancer Research Funds, Lincolns Inn Field, London WC2A 3PX, United Kingdom; e-mail: [email protected]

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Key Words T cell antigen receptor, CD28, protein tyrosine kinases, protein kinase C, Rho GTPases Abstract Ligation of the T cell antigen receptor (TCR) stimulates protein tyrosine kinases (PTKs), which regulate intracellular calcium and control the activity of protein kinase C (PKC) isozymes. PTKs activated by antigen receptors and costimulatory molecules also couple to phosphatidylinositol-3 kinase (PI3K) and control the activity of Ras- and Rho-family GTPases. T cell signal transduction is triggered physiologically by antigen in the context of antigen presenting cells (APC). The formation of stable and prolonged contacts between T cells and APCs is not neccessary to initiate T cell signaling but is required for effective T cell proliferation and differentiation. The stabilization of the T cell/ APC conjugate is regulated by intracellular signals induced by antigen receptors and costimulators. These coordinate the regulation of the actin and microtubule cytoskeleton and organize a specialized signaling zone that allows sustained TCR signaling.

INTRODUCTION The coordinated activation of T cells in response to foreign antigen ensures antigen-specific T cell clonal expansion and differentiation. Lymphocyte activation is controlled by signaling pathways initiated by antigen receptors and costimulatory molecules. The physiological ligand for T cell activation is a foreign peptide bound to major histocompatibility complex (MHC)-encoded molecules presented on the surface of professional APC such as dendritic cells (DC). The TCR comprises a/b subunits that recognise peptide/MHC and the signal transducing subunits e, c, d, and f (CD3-f complex). Biochemical signals initiated by this receptor determine the specificity of T cell activation, but events initiated by other membrane proteins such as the MHC receptors CD4/CD8, costimulators like CD2, CD28, and integrins modulate the intracellular signaling thresholds required to initiate a T cell immune reaction (1, 2). It is generally recognized that the integrity of the actin and microtubule cytoskeleton provides an essential structural support for T cell activation (3–6). The first part of this review describes 0732–0582/00/0410–0165$14.00

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the T cell signaling pathways identified biochemically in experiments with agonistic anti-TCR/CD3 antibodies. The second part will document the morphological changes that occur during T cell activation and cover recent progress towards understanding how symbiotic interactions between TCR signal transduction pathways and the cytoskeleton drive T cell activation.

THE INDUCTION OF TCR SIGNALING

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The Regulation of Protein Tyrosine Kinases: the Importance of ZAP70/Lck Interaction Conventional biochemistry has established that the TCR initiates signaling by recruiting and activating protein tyrosine kinases (PTKs) of the Src, Syk, and Tec families. Moreover, genetic studies in mice and humans reveal that these PTKs are indispensible signaling molecules for T cell activation. An exhaustive description of the molecular events of this phase of the activation process has been reported (7–9). In this review we focus on the essential facts pertinent for understanding how TCR signaling initiates. Immunoreceptor tyrosine-based activation motifs (ITAMs) of the signal transducing subunits CD3 c, d, e and f chain are phosphorylated by Src PTKs, essentially Lck, thus making the ITAMs competent to associate with ZAP70 via tandem Src-homology 2 (SH2) domains characteristic of Syk family PTKs (7). ITAMbound ZAP70 is tyrosine phosphorylated and Lck binds to it via its own Srchomology 2 (SH2) domain (10, 11); ZAP70 catalytic activity is considerably increased as a consequence of Lck phosphorylation of the ZAP70 activation loop (12, 13). This initial process leads to the phosphorylation of ZAP70 substrates (e.g. SLP76, LAT) (14, 15) and may induce and/or amplify the activation of other PTKs (e.g., Src and Tec) downstream of ZAP70 (16). The LckSH2 domain binds Tyr319 in the linker region of ZAP70 (17) and predictibly, this tyrosine is essential for ZAP70 activation (18). Moreover, mutation of ZAP70 Tyr319 into a motif with three to fourfold increase in affinity for Lck (17) (and microcalorimetry data, F. Schaeffer, V. Di Bartolo, O. Acuto, unpublished data) causes a twenty to thirty fold increase in ZAP70 functional activity (17). Hence the SH2-mediated interaction of Lck with ZAP70 is of central importance; it promotes continued ITAMs phosphorylation with additional recuitment of ZAP70 and other lck substrates such as the Tec PTK Itk (19).

ITAM Phosphorylation Pre-Exists before T Cell Activation The stoichiometry of ITAM and ZAP-70 tyrosine phosphorylation is regulated by antigen and will be low if the TCR interaction with peptide/MHC is weak (20, 21). Phosphorylation is only led to completion, by strong TCR-ligand interactions or by the help of co-receptors CD4/CD8 (22). In quiescent T cells phosphorylation

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of ITAMs by a pool of active Src PTKs is an ongoing reaction masked by the overwhelming repressive capacity of one or more tyrosine phosphatases (14, 15, 17, 23). CD45 is a good candidate (Figure 1). TCR ligation by antigen shifts this equilibrium with the driving force for amplification of the response almost certain to be exclusion of phosphatase molecules away from the TCR-ligand complex. This model was proposed by Davis & van der Merwe (24), who noted that the size of the TCR/MHC complex will ensure membrane approximation within 150 nm during T cell activation by antigen on the surface of an APC. Consequently, membrane molecules with large extracellular domains will be segregated out of the TCR/MHC contact whereas small-sized costimulatory receptors such as CD2 and CD28 would not (Figure 2). The tyrosine phosphatase CD45, which acts as a powerful negative regulator of TCR signaling (27), has a large extracellular domain that would be excluded on a size basis from proximity of a TCR/MHC complex. A sudden change in the local distribution of CD45 will promote a sudden increase in phosphorylation of ITAMs by proximal Src kinases. Note that CD45 also has a positive role in T cell signaling because this phosphatase is responsible for maintaining a pool of active Lck in quiescent cells (25, 26). T cell signal transduction should thus be viewed as a perturbation of an existing

Figure 1 A model of T cell activation based on a dynamic phosphorylation/dephosphorylation equilibrium. (Top) Steady-state level of ITAMs phosphorylation by Lck is of very low stoichiometry in an unstimulated T cell as it is counteracted by protein tyrosine phosphatases (PTPs). (Bottom) When the Ag/MHC: TCR interaction has a sufficient halflife, the balance shifts towards accumulation of phosphorylated ITAMs against a negative effect by PTPs. This effect may be reached by sudden local inactivation/physical exclusion of PTPs. Once ITAMs are sufficiently stably phosphorylated, ZAP70 binds, protects the ITAMs from dephosphorylation and all the downstream steps can proceed to completion (e.g. activation of ZAP-70 by Lck and substrates phosphorylation).

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Figure 2 A model explaining how CD28 can facilitate TCR engagement and signaling. Once ligated by B7, CD28 provides the T cell with an initial adhesion capable of approximating the T cell and APC membranes. Concomitantly, Fyn/Lck PTKs phosphorylate Vav and activate its GEF activity. Rac, the main Vav target then induces marked cortical actin changes that may favor an initial membrane re-organization leading to the formation of SMACs (e.g., receptor segretation and focalization of signaling protein complexes associated to coalescing membrane rafts). This process induced by CD28 may enhance membrane proximal signaling from the TCR.

phosphorylation/dephosphorylation equilibrium that will lead to amplification of ITAMs phosphorylation with subsequent propagation of the signal, provided that ZAP70 is activated by Lck (Figure 1).

How Are Coreceptors Involved in TCR Signal Transduction? A Model for Coreceptor Function Based on Lck/ZAP70 Association It was originally thought that the co-receptors CD8 and CD4, which are constitutively associated with Lck, are responsible for chaperoning this PTK to the ITAMs thus provoking their phosphorylation and ZAP70 binding. Two mechanisms have been proposed, one in which a simultaneous binding of TCR and coreceptor to the same peptide/MHC complex takes place (28) and a more recent sequential model in which first the TCR binds to peptide/MHC followed by the co-receptor (22, 29). However, these models do not explain how ITAMs can be phosphorylated in cells lacking CD4/CD8 co-receptors (e.g., most cd TCR- and some ab TCR-bearing T cells, early pre-T cells). Moreover, any model for coreceptor function has to explain how CD4 and CD8 recognize the very rare TCR/ peptide/MHC complexes and ignore all the other free TCRs and MHC molecules.

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Three-dimensional structural information finds no evidence for a conformational differences between isolated MHC and TCR molecules or their complexes which would favor coreceptor interaction with triggered TCRs nor does the binding geometry of CD8 to class I MHC provide a basis for such a conformational change mechanism (30, 31). An alternative model (32) for coreceptor recruitment into activated TCR complexes is based on the evidence that the SH2 domain of Lck is important for co-receptor function (33) and on the SH2-mediated association of Lck with ZAP70 after TCR triggering (10, 11). In this model, Lck binding to ITAM-bound tyrosine phosphorylated ZAP70 is the chaperone that forces coreceptors to recognize the engaged TCR and the MHC molecule bound to it. The latter interaction will take place even with both strong and weak agonists (34). When the TCR binds to a weak agonist and the co-receptor is absent, phosphorylation of ITAMs and ZAP70 as well as Lck/ZAP70 association will be of very low stoichiometry. The presence of the co-receptor (whose affinity for MHC is in the three-digit lM range (1) will shift a short half-life association of Lck with ZAP70 into a more stable one, with consequent increase of phosphorylation reactions by a reciprocal positive feed-back loop between intracellular and extracellular interactions.

SIGNALING AFTER TYROSINE KINASES IN T CELLS Tyrosine kinases initiate a cascade of signaling pathways during T cell activation; the key events are the activation of Ras- and Rho-family GTPases signaling networks and regulation of inositol phospholipid metabolism. The latter response controls intracellular calcium and the activity of diverse serine/threonine kinases including members of the PKC family and phosphatidyl inositol-3 kinase (PI3K)controlled serine kinases.

The Regulation of Phospholipase C, Calcium and Protein Kinase C TCR coupling to phospholipase C gamma 1 (PLCc1) allows the TCR to regulate hydrolysis of phosphatidylinositol (4,5) biphosphate (PI(4,5)P2) and hence control the production of inositol 1,4,5-triphosphate and diacylglycerol. Initial models for TCR regulation of PLCc1 were simple and proposed that tyrosine phosphorylation of the enzyme resulted in its activation. Now, it is clear that antigen receptor activation of PLCc1 normally requires at least three classes of PTKs; Lck, ZAP70, and Tec kinases such as Rlk and Itk in T cells (Btk in B cells) (35–37). There is also a requirement for two adapter molecules that are both substrates for ZAP70: LAT (Linker for activation of T cells) and SLP76 (SH2-domain containing leukocyte protein of 76 kD) (38–41). LAT is an integral membrane protein with a short extracellular region and a long cytosolic tail with nine tyrosine residues conserved between mouse and human (42). LAT is selectively tyrosine phos-

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phorylated in response to triggering of antigen receptors and interacts with the PLCc1 SH-2 domain thereby recruiting this enzyme to the plasma membrane. The importance of SLP76 for PLCc1 activation is unequivocal but quite why is not yet established. One hypothesis is that PLCc1 tyrosine phosphorylation is mediated by a TEC family kinase and that interactions between these kinases and SLP76 are required to coordinate the TEC/PLCc1 interaction. LAT almost certainly acts as the scaffold for assembly of the PLCc1 signaling complex with SLP76 binding to LAT via the newly described Grb2-like adaptors Gads, GrpL, or Grf40 (43–45). The importance of PLCc1 regulation for lymphocyte activation stems from the fact that this pathway controls the production of inositol polyphosphates and diacylglycerols, which regulate increases in intracellular calcium and activate serine/threonine kinases of the protein kinase C family, respectively. Sustained elevation of intracellular calcium concentration ([Ca2`]i) is absolutely critical during the initial phases of T cell activation both for induction of cytokine gene expression and for controlling T cell cytolytic function (46, 47, 48). It must be emphasised that cytokine gene induction requires co-ordination of calcium signals with signals mediated by the Ras/Rho GTPases and by serine/threonine kinases of the PKC family (49). The potency of calcium and PKC signaling pathways for lymphocyte activation is underlined by the ability of pharmacological agents that elevate intracellular calcium levels and activate PKC (calcium ionophores and phorbol esters respectively) to mimic many aspects of antigen receptor triggering. It should also be emphasized that T cells express multiple calcium and diacylgycerol regulated molecules. For example, T cells express multiple functionally distinct PKC isoforms that can be classified into distinct groups: the classical PKCs (a, bI, bII and c), which are regulated by calcium, DAG and phospholipids; novel PKCs (d, e, g and h), which are regulated by DAG and phospholipids; the atypical PKCs (n and k), which lack calcium or diacylglycerol binding domains (50–52). Stunning confocal microscope images reveal the specific recruitment of PKCh to the plasma membrane at the contact zone formed between T cells and APCs (53). This spatial-temporal regulation of serine kinases during antigen receptor engagement has refocused attention on the relevance of PKCs for lymphocyte activation. The functional diversity of PKCs highlights the potential for PKCs to have a pleotropic function in lymphocytes.

p21ras Regulation in T Cells The guanine nucleotide binding protein p21ras rapidly accumulates in its active, GTP-bound form in antigen receptor activated T cells (49, 55). Ras function is essential for cytokine gene induction and has a critical role in thymocyte development (56, 57). The guanine nucleotide binding cycle of Ras is controlled by guanine nucleotide exchange proteins (GEFs), which promote formation of active GTP-bound Ras complexes, and GTPase activating proteins (GAPs), which stimulate the intrinsic GTPase activity of Ras resulting in hydrolysis of bound GTP

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to GDP. The nucleotide exchange reaction switches Ras on whereas the hydrolysis of GTP turns it off (58). There has been much descriptive biochemistry about the regulation of Ras in lymphocytes (for review see 8, 59). The Ras GEF Sos, the mammalian homologue of the Drosophila ‘Son of Sevenless’ protein associates constitutively with the SH3 domains of an adapter molecule Grb2. In TCR-activated cells the Grb2 SH2 domain interacts with tyrosine-phosphorylated residues in the cytoplasmic tail of the adapter LAT thereby forming protein complexes that regulate the membrane localization and catalytic activity of Sos. One complication to this simple model is that another adapter molecule, SLP76, is also needed for TCR regulation of Ras although exactly why is not clear (41, 60). Once Ras is activated it couples to multiple biochemical effector signaling pathways including the Raf-1/MEK/ ERk1,2 kinases and signaling pathways controlled by the Rac/Rho GTPases (61, 62). In peripheral T cells Ras is important for pathways that activate transcription factors involved in cytokine gene induction. Surprisingly, despite the known importance of Ras, there is little solid information about the important targets for Ras action in the thymus.

Phosphatidylinositol-3 Kinase in T Cells TCR regulation of inositol phospholipids by the action of PLCc1 was one the earliest defined signaling pathway in lymphocytes. However, TCR triggering also stimulates inositol lipid turnover by controlling the activity of phosphatidyl inositol 3-kinase (PI3K) which phosphorylates PI(4,5)P2 on the D-3 position of the inositol ring to produce PI(3,4,5)P3 (63). There are multiple isoforms of PI3K and antigen receptors are thought to stimulate the activity of a PI3K complex that comprises a regulatory p85 and a catalytic p110 subunit. T cells express p110a and p110d and both these catalytic subunits appear to participate in lymphocyte signaling responses (63). Models for PI3K activation invoke p85 binding to adapters, which recruit the enzyme to the plasma membrane; constitutive membrane targeting of p110 catalytic subunits of PI3K creates a constitutively active enzyme that generates PI(3,4,5)P3 and PI(3,4)P2 when expressed in cells (64). The candidate adapters for recruiting PI3K to the membrane in TCR-activated cells are TRIM (65) or LAT (42). Genetic evidence for the importance of PI3K for B lymphocytes has been illustrated by the phenotype of mice lacking expression of the p85a regulatory adapter protein for PI3K that show profound defects in B cell function (66, 67). T lymphocytes lacking p85a appear normal but this is because they still contain functional PI3K. These data suggest that the regulation of PI3K in T cells is not mediated by p85a; whether p85b is important or whether there is a completely different adapter for PI3K regulation in T cells is unknown. The products of PI3K, PI(3,4,5)P3 and PI(3,4P)2, bind to the plextrin homology (PH) domains of proteins and either allosterically modify their activity or induce relocalization of the protein to defined areas of the plasma membrane where activation can occur (68). Recent studies have identified both tyrosine

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kinase and serine kinase regulation by PI3K in lymphocytes: the TEC family tyrosine kinases BTK and ITK (35, 69) and a serine/threonine kinase protein kinase B or Akt/PKB (70). These have PH domains that bind D-3 phosphoinositides and recruit these enzymes to the plasma membrane (71–73). TEC kinases function at the membrane to regulate PLCc1 activity. The other PI3K target, Akt/ PKB has several possible functions in T cells: It has been shown to play an important role in maintenance of cell survival in fibroblasts and epithelial cells (for review see 71–73). In T cells, PKB can stimulate the activity of E2F transcription factors, which are important components of the mechanisms that control the mammalian cell cycle (74). PKB also phosphorylates and inactivates GSK3 (71–73), an enzyme initially identified as a regulator of glycogen metabolism but which also has broader functions: GSK3 can also control the nuclear export of Nuclear Factor of Activated T cells (NFAT) transcription factors involved in cytokine gene induction (75).

PI3K is a Link to Rho Family GTPases. A third class of PH domain containing proteins regulated by the products of the PI3K cascade are GEFs for Rho GTPases such as Rac, Rho and Cdc42, (for review see 76). The best characterized Rho family GEF in lymphocytes is Vav1: The binding of PI(3,4,5)P3 to the PH domain of Vav-1 recruits it to the plasma membrane where tyrosine kinase mediated activation of the molecule occurs. Rho GTPases regulate lymphocyte survival, proliferation, and differentiation (55). They interact with multiple effectors and can initiate diverse signals. However, the first biological role ascribed to these GTPases was the dynamic organization of the actin cytoskeleton and the assembly of associated integrin structures. Experiments with constitutively active PI3K mutants have shown that the D-3 phosphoinositide products of PI3K are sufficient to induce Rac- and Rho-mediated cytokeletal responses (64). Cdc42, Rac1, and RhoA are responsible for distinct patterns of actin reorganization: RhoA regulates the formation of actin stress fibers and focal adhesions; Rac1 controls lamellipodia formation and focal complex assembly and subsequently can activate Rho-mediated cytoskeletal changes. Cdc42 first triggers induction of microspikes and filopodia, but consecutively can induce Rac- and Rho-mediated responses. T cells lacking the Rac GEF Vav-1 show defects in actin polymerization suggesting that the link between Rac and the maintenance of the actin cytoskeleton is maintained in lymphocytes (78, 79). An example of the ability of Vav-1 to control the actin cytoskeleton in T cells is shown in Figure 3 (see color insert), which illustrates how a T cell line overexpressing Vav-1 has abundant lamellipodia compared to control cells. There is also genetic and biochemical evidence for Cdc42 regulation of the actin cytoskeleton in lymphocytes. The genetic defect in Wiscott-Aldrich immunodeficiency syndrome (WAS) patients maps to an effector protein for Cdc42, termed WASP (80). The defects in WAS patients include cytoskeletal and cell activation abnormalities in lymphocytes. WASP-deficient

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cells have a paucity of microvilli on the cell surface. WASP has been implicated in the control of in actin depolymerization (81), and recent data also indicate that Cdc42/WASP family complexes are strong stimulators of the actin nucleating activity of the Arp2/3 complex (82, 83). Moreover, Cdc42 can control T cell polarization toward APCs (84), a process that is critical for efficient T cell-APC contact and the directed release of cytokines.

SPATIO-TEMPORAL RESOLUTION OF GENERAL CYTOSKELETAL CHANGES PRECEDING AND ACCOMPANYING T CELL ACTIVATION

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Morphological Modifications of T cell during the Encounter with APC The T cell signaling pathways described above were identified biochemically in experiments with agonistic anti-TCR/CD3 antibodies. Genetic studies have shown them to be important for T cell activation, but it should always be remembered that the physiological trigger for T cell activation is antigen presented to the T cell by an APC. Stimulation by agonistic anti-TCR/CD3 antibodies thus bypasses an important regulatory step in T cell activation; one that relies on the capacity of the T cell to establish an interaction with APCs, a process controlled by changes in cell cytoskeletal architecture. T cell activation is accompanied by a dynamic re-organization of cortical actin, exemplified by the increase in actin polymerization (e.g. increase in filamentous actin, F-actin) (85–87). Analysis at the single-cell level by time-lapse videomicroscopy has revealed the highly dynamic cytoskeletal and morphological modifications taking place in a T cell interacting with an APC (88–92). Trautmann and co-workers initially defined three main stages in the T cell/APC interaction: The first stage is the establishment of T cell/APC physical continuity, the second stage is characterized by T cell rounding, and it is followed by a third stage of T cell spreading (88) resulting in the formation of what has been termed by Dustin and colleagues as an immunological synapse (2). The initial area of contact between the T cell and the APC is formed within a few seconds to 1 minute (89, 91). Once established, the area of contact remains stable while moderate T cell membrane ruffling restricted to the contact zone can be observed (90–92). In the absence of antigen or at low Ag concentration, the T cell maintains its motility and continues to crawl on and around the APC and may leave in search of another partner (88, 89, 91). Initial contact and crawling is likely to depend on loose adhesion forces (1) generated by receptor couples such as CD28/B7, CD2/ CD58(CD48), or LFA1/ICAMs (93). Receptors on the APC (B7.1 and B7.2 may be the best candidates) may convey a ‘‘danger signal’’ (94) so that the T cell will pay particular attention and explore a professional APC (e.g., activated DC) in search of antigen, perhaps by receiving some intracellular signaling already at

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this stage (see below). These loose interactions favor fast serial scanning of different APCs by the T cell.

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Signals That Control the Formation of the Contact Zone Pre-treatment of T cell with cytochalasin D, a drug that prevents actin polymerization, prevents the formation of T cell/APC contacts (91). Moreover, the integrity of the cytoskeleton needs to be maintained throughout signaling for T cell commitment toward differentiation and proliferation (95). Actin cytoskeletal changes are detectable in T cells making a contact with an APC during the short time before (or in absence of) antigen recognition (90, 91). The changes in the actin cytoskeleton that accompany formation of the first T cell/APC contacts cannot therefore be regulated by the TCR but must be induced by coreceptors, e.g CD28, CD2, or LFA1/ICAMs. Molecules like CD28 are good candidates for promoting the initial modifications because the relative abundance of the CD28 ligands CD80/CD86 on the APC (.104) is much higher than the levels of MHC/ peptide complexes that the TCR would likely encounter (100–200). Moreover, CD28 has a real kinetic advantage over the TCR when considering the affinity of the interaction of these two receptors with their respective ligand(s) on the APC (i.e CD80/CD86 versus peptide/MHC). CD28 association rate constant (Kon) for CD80 is at least two orders of magnitude above the Kon so far determined for most TCR/peptide/MHC interactions (1, 96). Although CD28/CD80 Koff is faster than those measured for TCR/peptide/MHC complexes, perhaps to leave sufficient flexibility at the cell cell interphase during the formation of the contact zone (1), the equilibrium dissociation constant (KD) of the CD28-CD80/CD86 complex is similar, if not lower. Similar data have been obtained with CD86 (S. Davis and A. van der Merwe, personal communication). These kinetic and mass data make it likely that CD28 will engage its ligand faster and at a higher stoichiometry than the TCR (Figure 2). CD28 ligation certainly induces an increase in F-actin, which concentrates at the contact site (87). The molecular basis for CD28 regulation of the actin cytoskeleton is probably explained by the connection between CD28 and PI3K. CD28 interaction with its ligands CD80 or CD86 results in tyrosine phosphorylation of the CD28 cytoplasmic domain and the recuitment of PI3K to the plasma membrane with a corresponding activation of PI3K activity that generates D3phosphoinositides (63). Membrane targeting of PI3K is sufficient to induce Racand Rho-mediated dynamic reorganization of the actin cytoskeleton (64). It is also striking that Vav-1 is rapidly tyrosine phosphorylated by the initial contact between CD28 and its physiological ligands CD80 or CD86 (97, 98). Tyrosine phosphorylation of Vav-1, which activates the catalytic function of this Rac GEF, is TCR autonomous in that it does not require TCR expression (98) but appears to depend on Src PTKs and in particular on Fyn (99). In this context, it should be emphasized that Vav-1 was originally identified as a substrate for antigen receptor activated tyrosine kinases but this induced phosphorylation was transient.

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In contrast, CD28 induces a rapid and sustained tyrosine phosphorylation of Vav-1 in the absence of simultaneous ligation of the TCR (97). CD28 regulation of PI3K and Vav-1 would inevitably induce Rac/Rho-mediated actin cytoskeleton rearrangements of the type seen during the first stage of the T cell/APC contact. Recent studies have found that CD28 can influence proximal TCR-mediated signaling events such as the phosphorylation of the TCR f chain and ZAP70 (100). CD28/B7 ligation lowers the TCR activation threshold and potentiates T cell tyrosine phosphorylation pathways (101, 102). Ongoing work indicates that the promotion of TCR signaling during the initial stage of the T cell-APC encounter depends not only on CD28-mediated adhesion but also on CD28 signaling through Vav-1 (103), (F. Michel, L. Tuosto, G. Mangino and O. Acuto, unpublished data). These data force revision of previous models of T cell costimulation. It is unequivocal that signals generated by CD28 are essential for immune function, and it was proposed initially that CD28 initiates signaling to the nucleus in pathways parallel to the TCR. Now it seems that antigen-independent signals mediated by costimulatory molecules such as CD28 may promote signaling through the TCR complex by allowing the changes in the actin cytoskeleton that tighten and stabilize the T cell/APC conjugate.

Signals that Form a Stable T Cell/APC Contact Zone The second stage of the formation of the T cell/APC contact is antigen dependent and associated with a sudden widening of the contact zone characterized by a massive membrane protrusion from the T cell (in a pulsatile fashion) toward the APC (88, 89, 91). The signals that drive the second phase of the T cell/APC interaction are not known, but it occurs under conditions of poor or no [Ca2`]i elevation and requires the integrity of the actin cytoskeleton (89, 91). The only clue about how signal integration from the TCR and costimulators might control the actin cytoskeleton comes from reports that a unique protein complex consisting of tyrosine phosphorylated Vav-1 and tyrosine phosphorylated SLP76 assembles in TCR-triggered and CD28-costimulated T cells (98, 99, 104). SLP76 binds simultaneously to Vav-1 and p21-activated kinase (Pak) via the adaptor Nck (105), and the SLP76/Vav-1/Pak complex may facilitate physical proximity between activated Rho family GTPases and Pak. There is an enormous body of data from studies in fibroblasts that Pak co-ordinates the re-organization of the actin cytoskeleton. In particular, studies showing Pak regulation of myosin light chain kinase (106) indicate that the Vav-1/SLP76/Pak complex has the potential to dynamically control the type of changes in the actin cytoskeleton (107) essential for prolonging the T cell/APC contact. Cdc42 and/or Rac regulation of Pak activation in T cells has been described (60).

The Structure of the Stabilized T Cell/APC Contact Zone The final stabilization of the T cell/APC contact requires durable TCR engagement and is thought to be regulated by Ca2` (89), i.e. elevation of intracellular [Ca2`]i seems to initiate a signal that tells the T cell to stop and continue receiving

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signals from the APC (88, 108). As the T cell stops, it stabilizes contact with the APC. The stable contact zone between a T cell and the APC is not just a random distribution of receptors-counterreceptors. Rather, receptors and signaling molecules are organized within this area termed SMAC (for supramolecular activation cluster) or mature immunological synapse (6, 109). There is a specific pattern of receptor segregation containing in a concentric fashion two subregions, one internal where TCR, CD4, (together with Src PTKs Lck and Fyn and PKCh) are confined and the external one containing the integrin molecule LFA-1 with talin, which connects through vinculin to actin filaments (6, 109). The external structure may be analogous to focal complexes or adhesions (110). It has also been noted by Alcover and co-workers that the external zone of the SMAC contains ezrin (A. Roumier and A. Alcover personal communication), a cytoskeletal protein member of the ERM (ezrin, radixin, moesin) family, whose function is to connect membrane proteins to actin filaments (111). There will inevitably be intense interest in how the SMAC forms in such a precise and organized way. One clue comes from observations that the T cell/ APC contact zone is apparently enriched in plasma membrane glycosphingolipidenriched microdomains (GEMs, also referred to as lipid rafts) (102, 112, 113, 114). A number of molecules involved in antigen receptor signaling have been found concentrated within the GEMs including Src family PTKs (112, 115) and adaptor molecules such as LAT (116). Importantly, antigen receptors are transiently targeted to these lipid microdomains during an activation response, and it is proposed that this is where interactions with tyrosine kinases and adapter molecules occur (112, 117, 118). It has also been suggested that GEMs are the site of inositol lipid metabolism since both PLCc and PIP2 have been found concentrated within GEMs (114, 119). The signaling mechanisms that cordinate the reoganization of GEMs are not known but it has been described that GEMs interface with the actin cytoskeleton (120, 121), and in this regard it is noteworthy that CD28 signaling has been shown to induce GEM polarization toward the site of CD28 ligation (102). CD28 is uniquely coupled to the regulatory networks that control the activity of Rho family GTPases. The type of changes in the actin cytoskeleton controlled by Rho GTPases could control the polarization of GEMs into the SMAC. The GEMs would then spatially organize the key signaling molecules in the SMAC.

CONCLUDING REMARKS TCR Signal Transduction and the Actin Cytoskeleton? The complicated TCR-induced biochemical responses discussed in the first part of the review can be detected within seconds of T cell contact with APCs yet it takes 10–15 min to form a stable T cell/APC contact zone or SMAC (6, 109). Hence, the known TCR proximal signaling events are not dependent on stable T

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cell/APC contacts or on the formation of a SMAC or immunological synapse. Rather, the signaling pathways initated by costimulators and antigen receptors organize the SMAC; the rapid changes in cell morphology, adhesion, and motility that form the SMAC are suggestive of an effective adaptive program to ensure that once the T cell identifies the appropriate ligand for its TCR clonotype on an APC a sustained lasting signal is established. Is the initiation of TCR signaling regulated by the actin cytoskelton? Yes in the sense that without dynamic changes in actin structures it is unlikely that the T cells will make sufficient effective contacts with APCs to enable TCR ligation with peptide MHC complexes. It should also be remembered that T cells do not randomly contact APCs but probably respond to chemokine gradients. The coordinated regulation of the actin cytoskeleton will be important for the motility of T lymphocytes and will be critical to allow T cells to respond to the chemokine gradients that will ensure the initial T cell contact with the APC (5). T cell activation and coordinated changes in the actin cytoskeleton are symbiotic processes. The first signals to control the actin cytoskeleton when a T cell contacts an APC are antigen independent and mediated by costimulators such as CD28; there are also likely contributions from other molecules such as CD2 or LFA-1 (122, 123, 124). Once the TCR signaling cascade is established changes in calcium, PI3K, and GTPases triggered by antigen receptors develop the actin dynamics that stabilize the T cell/APC conjugate and organize a signaling zone within the contact zone. This allows sustained T cell signaling to promote proliferation and differentiation.

ACKNOWLEDGMENTS We would like to thank past and present members of the Lymphocyte Activation Laboratory and of the Molecular Immunology Unit for their contributions that helped to forge our knowledge of T cell activation mechanisms. A special thanks to F. Michel and V. Di Bartolo for helping with confocal microscopy and the artwork pictures shown in this review; to A. Trautmann and A. van der Merwe for helpful discussions and suggestions and to Kim Rowan and Wendy Houssin for assistance in the preparation of this manuscript. We would also like to thank A. Alcover, A. Roumier, A. Shaw, M. Bachmann, A. van der Merwe and S. Davis for sharing results before publication. Work from our laboratories was made possible by support to D.C. from the Imperial Cancer Research Fund, the EEC and the Human Frontier Science Programme and to O.A. from the Institut Pasteur, Association pour la Recherche sur le Cancer, Centre National pour la Recherche Scientifique, and Human Frontier Science Programme. Visit the Annual Reviews home page at www.AnnualReviews.org.

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Figure 3 Overexpression of Vav-1 in Jurkat cells induces formation of lamellipodia. Jurkat cells were transfected with a vector expressing myc-tagged Vav-1. © (C) Differential interference contrast (DIC) microscopy image shows the formation of large lamellipodia only in the cell at the far right. (A) Anti-myc staining to reveal Vav-1 (green). (B) F-actin staining by phalloidin (red). It is clear that only the cell overexpressing Vav-1 has altered morphology. The white arrow indicates a cell not expressing transfected Vav-1. Note the strong increase in F-actin in the Vav-1 overexpressing cell and the coincident staining of Vav-1 and F-actin.

Annual Review of Immunology Volume 18, 2000

CONTENTS

Annu. Rev. Immunol. 2000.18:165-184. Downloaded from arjournals.annualreviews.org by HINARI on 09/01/07. For personal use only.

Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:185–216 Copyright q by Annual Reviews. All rights reserved

THE SPECIFIC REGULATION OF IMMUNE RESPONSES BY CD8~ T CELLS RESTRICTED BY THE MHC CLASS IB MOLECULE, QA-1 Hong Jiang and Leonard Chess Department of Medicine and Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032

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Key Words TCR, Qa-1, CD8, CD4, immunoregulation Abstract Over the last three decades considerable evidence has accumulated that CD8` T cells regulate peripheral immune responses, in part, by specifically controlling the outgrowth of antigen-triggered CD4` T cells. This regulatory function of CD8` T cells has been shown, in vivo, to control the emergence of autoreactive CD4` T cells as well as CD4` T cells reactive to conventional antigens, including alloantigens. In this review, we summarize the evidence that this immune suppression mediated by CD8` T cells is dependent, in part, on specific cognate interactions between MHC class I–restricted regulatory CD8` cells and antigen-activated CD4` T cells. Moreover, we review the evidence that regulatory CD8` T cells recognize antigen-activated CD4` T cells in a TCR specific manner restricted by the MHC class Ib molecule, Qa-1. The Qa-1 molecule may be uniquely qualified to serve this MHC restrictive function because, unlike conventional MHC molecules, it is preferentially and transiently expressed on activated and not resting CD4` T cells. This may assure that only recently antigen-activated CD4` T cells expressing Qa-1/TCR peptide complexes will induce regulatory CD8` T cells and subsequently become susceptible to regulation. Because Qa-1 also binds to self Qdm peptides that trigger NK (CD94/ NKG2) receptors on CD8` T cells, the machinery for homeostatic regulation of regulatory CD8` T cells can be envisioned. Finally, we propose a model by which these TCR specific, Qa-1-restricted regulatory CD8` T cells selectively downregulate antigen-activated T cells expressing TCRs of certain affinities. Ultimately these regulatory CD8` T cells control the peripheral TCR repertoire during the course of immune responses to both self and foreign antigens.

INTRODUCTION Immune responses are initiated when resting precursor CD4` T cells are triggered by MHC/peptide complexes in concert with costimulatory molecules on the surface of antigen presenting cells (APCs) (1–3). As a consequence of this triggering the CD4` T cells proliferate, begin to secrete cytokines (IL-2, IFN-c, IL-4, etc.) 0732–0582/00/0410–0185$14.00

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and express important cell surface molecules including the IL-2 receptor (CD25), CTLA-4, and CD40 ligand. In order to regulate the immune response and to dampen the potential for autoimmunity, the immune system has also evolved several mechanisms to downregulate and control the outgrowth, differentiation, and function of peripheral antigen-activated CD4` T cells. One level of control resides at the initial clonal activation of the T cell receptor itself by MHC/peptide complexes. T cell signaling via this tri-molecular interaction is thought to be dependent on the affinity and duration of binding of the TCRs with MHC/peptide complexes (4, 5) and will subsequently influence many events during the evolution of both primary and secondary immune responses. In this regard, TCR triggering induces not only activation and differentiation events but, in addition, induces apoptotic pathways. These apoptotic signals are regulated, in part, by cell surface molecules, like FasL, expressed during antigen triggering (6). During the initial T cell/ MHC/peptide interaction, other receptor ligand interactions become pivotal in ultimately dictating the functional fate of T cells. For example, one of the earliest antigen activation–induced cell surface molecules expressed by T cells is CD40L (7). A critical consequence of the interaction of CD40L with CD40 expressed on APCs is the upregulation of other key costimulatory molecules, including CD80 and CD86 (8, 9). These molecules interact with T cell CD28 or CTLA-4 molecules to determine whether the outcome of antigen triggering will be either functional T cell activation or, alternatively, anergy and tolerance induction (3, 9–11). Thus, antigen triggering in the absence of CD80 or CD86 triggering is known to induce anergic T cells (3, 12, 13). Similarly, blockade of the CD4L/CD40 pathway can lead to tolerance induction (14). Another consequence of MHC/peptide triggering of CD4` T cells is the further differentiation of the CD4` T cells into the functional distinct TH1 and TH2 subsets (15–17). The elaboration of distinct cytokines by the TH subsets provides a second level of control intrinsic to the outgrowth and function of CD4` T cells (18). For example, IFN-c secreted by TH1 cells is known to downregulate the differentiation and function of TH2 cells, and conversely, IL-4 and IL-10 inhibit TH1 cell differentiation (17–19). In addition to these regulatory pathways, which are intrinsic to CD4 T cells, recent data strongly suggests that CD8` T cells also interact with CD4` T cells to regulate immune responses in a profound manner. For example, CD8` T cells have been shown to control the physiological outgrowth and function of autoreactive CD4` T cells in vivo (20–27). These regulatory interactions between CD4` and CD8` T cells are complex and involve both antigen specific and nonspecific mechanisms. In principle, one can envision three distinct but not mutually exclusive models by which CD8` T cells specifically regulate antigendriven CD4` T cells. First, CD8` T cells could be triggered by antigens to secrete lymphokines, which, in turn, control the CD4` T cells activated by the same antigens. Indeed, cytokines including IL-4 and IFN-c secreted by CD8` T cells have been shown in some systems to regulate CD4` T cell function (28). Because of the potential proximity of CD4` and CD8` T cells at the site of initial antigen

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activation and because antigen-activated T cells may preferentially express the receptors for these cytokines, the effect of even these antigen nonspecific cytokines will be relatively antigen specific. Second, antigen triggering induces CD4` T cells to transiently express nonpolymorphic membrane activation molecules unrelated either to antigen or TCR that are recognized by regulatory CD8` T cells. The recognition of these activation molecules (sometimes referred to as ergotypic structures) may induce CD8` T cell differentiation into effector cells that have the potential to delete or inactivate antigen-activated CD4` T cells (29, 30). Third, T cell receptor related structures, including TCR-derived MHC/ peptide complexes expressed on antigen-activated CD4` cells may induce regulatory CD8` T cells (24, 25, 31–33). These CD8` T cells could then differentiate and recognize the TCR-derived peptide/MHC class I complexes expressed on the activated CD4` inducer cells. The effector phase of regulation mediated by these putative TCR peptide-recognizing CD8` T cells may involve either conventional cell-mediated cytotoxic (CTL) functions and/or the release of cytokines. This latter view of immune regulation involving recognition of TCR structures was initially suggested, in principle, by Jerne in his idiotype-driven network hypothesis (34). In addition, many of the suppressor cell interaction models proposed during the latter part of the 1970s and early 1980s involved complex interactions between T cell subsets (which now include the CD4` and CD8` subsets) as well as the recognition of TCR related structures by CD8` T cells (28, 35– 37). In this chapter we review recent evidence that CD8` T cells are generated during immune responses, in vivo and in vitro, which downregulate CD4` T cells in a TCR Vb–specific manner. We appreciate that this work resurrects two immunological hypotheses (i.e. TCR specific network regulation and control of immunity by suppressor T cells) that were more prevalent and/or popular two to three decades ago, at a time when the scientific revolution that has occurred in the molecular characterization of the immune system was in its infancy. We briefly review the history and experimental basis of these ideas. Moreover, we focus on and review some recent experiments that permit us to synthesize some of the old and new experiments and construct a general model of immune regulation by CD8` T cells.

HISTORICAL CONSIDERATIONS: THE INITIAL RISE AND FALL OF THE CD8` T CELL SUPPRESSOR PARADIGM The idea that immune responses must be tightly regulated was implicit in the early ideas of clonal selection as proposed by Ehrlich in 1900. He envisioned that during the ontogeny and outgrowth of the immunocompetent clones responsive to foreign antigens there had to be mechanisms to control the outgrowth of clones

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reactive with self (38). In Ehrlich’s conception the failure to control the outgrowth of autoreactive cells would lead to a state of ‘‘horror autoxicus’’ or autoimmunity. Ehrlich’s ideas were amplified and refined in the 1950s by MacFarlane Burnet and Neils Jerne, with the elaboration of the Clonal Selection Hypothesis (34, 39, 40). This hypothesis states that (a) multiple clones of immunocompetent cells displaying unique antigen specific receptors exist prior to the introduction of foreign antigens; (b) following exposure to a particular foreign antigen, the antigen interacts with its unique receptor on the cell surface of an immunocompetent cell, and this specific receptor-antigen interaction triggers cells to divide and express their immunological programs; and (c) the majority of cells bearing receptors for self-antigens must be eliminated during differentiation or carefully regulated. These tenets of clonal selection have proven, over the years, to be essentially correct. A corollary of these notions is that autoimmune diseases arise from either the failure to eliminate or inactivate immunocompetent cells during their ontogeny and/or the failure of the immune system to control the outgrowth or function of self-reactive clones in the periphery. Thus, immunocompetent selfreactive cells circulating in the periphery either had to be incapable of responding to self-antigens (tolerant) or alternatively had to be specifically suppressed. The idea of suppressor T cells arose in the late 1960s and early 1970s at a time when it was already clear that immune responses were effected by lymphoid cells (41) and that an interesting subdivision of labor existed among populations of lymphocytes resident in distinct lymphoid organs (41–43). For example, it was discovered that thymic-derived T cells effected cell-mediated immune responses, whereas bone marrow–derived B cells produced antibodies (44–46). Moreover, B cell antibody production was found to be dependent on help from thymicderived T cells (47–50). In this setting, classic experiments at Yale University, in the Gershon laboratory, showed that adoptive transfer of T cells from animals made tolerant to antigen X could specifically suppress anti-X antibody production in recipient animals (51, 52). These experiments were confirmed in a variety of settings during the 1970s (53–55). It was also found that this suppressor activity of T cells could also downregulate delayed type hypersensitivity responses (DTH) and tumor immunity (56, 57). An important conceptual development in immunology in the 1970s that gave further credence to the notion of suppressor T cells was the idea that distinct T cell functions were mediated by phenotypically stable subsets of T cells expressing distinct cell surface molecules. Thus, the seminal experiments of Cantor and Boyse showed that the genetically well-defined Lyt allo-antisera could be used to define functionally distinct subsets of cells expressing either helper (inducer) function (Lyt1) or alternately cytotoxic functions (Lyt2) (58, 59). Moreover, the suppressor functions were found to be largely contained in the Lyt2 subset (60, 61). These findings were later extended to other species including man (62, 63), and with the advent of monoclonal antibodies the CD4 and CD8 surface molecules were identified and used to study T cell differentiation and immunoregulation in more precise detail (64, 65). The inducer Ly1 population was exclusively found in the CD4` population and the cytotoxic and suppressor

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populations were largely contained within the CD8` population (66, 67). In addition to these functional distinctions, CD4` and CD8` T cell subsets also differed in a cognitive sense. Thus, CD4` T cells were found to be MHC class II restricted, whereas CD8` T cells were found to be MHC class I restricted (65, 68, 69). In both mice and man suppression was shown in vitro to require interactions between CD`8 cells and CD4` cells (69–72). These experiments led to the findings that a subset of activated CD4` T cells were required to induce CD8` suppressor cells. Moreover, the CD8` effector cells then could mediate suppression by suppressing the activity of the CD4` T cell inducing population. From these experiments emerged models in which extraordinarily complex cellular circuits were envisioned (37, 73–76). For example, a CD4` suppressor inducer T cell subset was characterized by the expression of two additional cell surface molecules identified by alloantisera, one termed I-J (77–80) and the other termed Qa-1 (72, 81). The precise genetic or molecular description of I-J has remained mysterious, in part because the cell surface structure recognized by I-J alloantisera was never identified and the original evidence that I-J was encoded in the MHC class II region was not correct (82, 83). In contrast, the Qa-1 alloantisera mapped to the 38 end of MHC class I region on chromosome 17 in a region known to encode other genes, like TL (84, 85). It is of interest that eventually Qa-1 was cloned and found to be a MHC class Ib molecule capable of presenting endogenous as well as exogenous peptides (86–88). However, the potential significance of Qa-1 expression on suppressor inducer cells was not delineated until the mid 1990’s (25, 33) (see sections below). In this regard, it is important to re-emphasize that the original suppressor cell circuits were initially conceived and/or deduced at a time when molecular immunology was in its infancy. For example, the nature of the TCR receptor was unknown as was the precise structure and function of MHC molecules in restricting T cell activity. In addition, the great majority of the cytokines that are now known to regulate immune functions and T cell differentiation were largely unknown. Clearly, understanding the precise role of these lymphokines in suppression would have significantly influenced the interpretation of data suggesting that an array of antigen specific and nonspecific suppressor factors were uniquely secreted by suppressor inducer and effector cells. To this day the precise biochemical definition of these putative antigen specific suppressive factors has not been resolved. An alternative hypothesis that will be discussed in depth below is the idea that the specificity of immune suppression is mediated not by the putative specific soluble factors but instead by cognate interactions between CD8` regulator cells and antigen-activated CD4` T cells. In this view, conventional ab TCRs expressed on the surface of suppressor CD8` T cells recognize activated CD4` T cells in an antigen-specific manner and are triggered to inactivate or delete the CD4` T cell. We in fact review below the evidence that the ‘‘uniqueness’’ of suppressor cells and their TCRs may simply be that they are endowed with the capacity to recognize self TCR peptides presented by MHC class I molecules expressed by antigen activated inducer clones. The consequences of TCR

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triggering of ‘‘suppressor’’ CD8` T precursors could then be quite conventional and involve differentiation into specific cytotoxic lymphocytes (CTL), which lyse targets (inducer CD4` T cells) and/or secrete lymphokines that downregulate these inducer cells. Indeed, the cautionary notes concerning suppressor T cells and the doubts concerning the very existence of suppressor T cells that emanated in the middle to late 1980s were based on the lack of evidence that suppressor cells were a unique subset of T cells distinguished from helper and cytotoxic T cells (28, 89– 91). These cautionary notes emphasized that there was no evidence that suppressor T cells were endowed with unique sets of antigen receptors (distinct from the ab and cd TCRs), unique antigen specific soluble factors or unique I-J encoded suppressor structures. Indeed these arguments remain essentially valid. These concerns led to general skepticism for the role of T-T interactions in the downregulation of immune responses. On the other hand, evidence has continued to emerge during the last two decades that has substantiated many of the central ideas of immune suppression and particularly the idea that the immune system, not unlike all other complex physiologic and biochemical systems, is homeostatically regulated and often involves feedback suppressor mechanisms. In the next sections we will first briefly review the substantial in vivo data that unequivocally show that during immune responses regulatory suppressor CD8` T cells are induced that profoundly effect the outcome of autoimmunity and allograft responses by downregulating immunoreactive CD4` T cells.

THE RESURRECTION OF THE CD8` T SUPPRESSOR PARADIGM IN VIVO T Cell Vaccination Induces Regulator Cells, Including CD8` T Cells, Which Prevent Autoimmunity and Abrogate Immune Responses to Alloantigens One prediction of the model that suppressor T cells recognize TCRs expressed on antigen-activated CD4` T cells is the notion that CD4` T cell clones specific for antigen X may be used to induce regulatory CD8` T cell suppressors in vivo, which would specifically downregulate the immune responses to CD4` T cells expressing a TCR specific for antigen X. Early suggestions that this prediction is correct came from experiments that show that specific unresponsiveness to transplantation antigens could be induced by auto-immunization with syngeneic, antigen-specific T lymphoblasts (92, 93). Subsequent experiments provided evidence that these allospecific lymphoblasts serving as an immunogen can induce antiidiotypic immunity and tolerance induction. For example, immunization of F1 rats with alloreactive T-cell populations of parental strain origin, induces a hostmediated T-cell response that is specific for anti-MHC receptors on parental T cells. This protective immunity is rapid in onset and once induced, was shown to

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provide highly effective, specific resistance to lethal GVH disease. Moreover, this resistance could be adaptively transferred to syngeneic recipients (94–96). Furthermore, Irun Cohen in the early 1980’s initiated a series of experiments that demonstrated that autoimmune T cell lines and clones can be used to vaccinate animals to prevent autoimmunity (97, 98). For example, the experimental autoimmune rodent model of multiple sclerosis, experimental allergic encephalomyelitis (EAE), can be induced either by injection of myelin antigens including myelin basic protein (MBP) or alternatively, by inoculation with ‘‘encephalogenic’’ MBP reactive T cell lines. However, if these encephalogenic T cells are attenuated by irradiation prior to injection, they do not cause disease but instead induce protection from subsequent induction of disease. As a consequence of these experiments, Irun Cohen, coined the term ‘‘T cell vaccination’’ (TCV) to denote the use of attenuated autoimmune T cells as vaccines to prevent autoimmune disease. Unlike conventional immunotherapy, T cell vaccination is selectively immunosuppressive and unlike other forms of immunotherapy TCV does not necessarily require precise knowledge of the autoantigen, which is often clinically difficult to define. Moreover, TCV has been widely used to ameliorate autoimmune diseases in a variety of animal models (99–102) and studies in some human autoimmune diseases have already been initiated (103–105). Although the precise mechanisms by which T cell vaccination abrogates autoimmunity are unknown, it has generally been thought that T cell vaccination augments the normal mechanisms employed by regulatory T cells to control the pathogenic potential and/or outgrowth of disease-causing T cells (106). In this regard, several lines of evidence have suggested that regulatory T cells mediate the protective effect of T cell vaccination by downregulating autoimmune T cells. For example, adoptive transfer of lymph node cells obtained from T cell vaccinated rats to naive rats, led to acquisition of EAE resistance by the recipient rats (20, 31). In vitro studies of these regulatory cells provided evidence for CD8` T cells that recognize and downregulate the vaccine T cells. Moreover, in the studies of multiple sclerosis in humans (104) the majority of the regulatory T cells isolated from CD4` T cell–vaccinated patients were CD8` T cells. These CD8` T cells were shown to inhibit the proliferation of vaccine T cell clones upon antigen stimulation and also specifically lysed the vaccine CD4` T cell clones in vitro (31, 104). Recently, experiments from our laboratory have shown that CD8` T cell depletion completely abolished TCV-induced protection of EAE induced by encephalogenic clones, providing direct evidence that CD8` T cells are required for TCV-induced protection from EAE in mice (107).

Direct Evidence that CD8` T Cells Downregulate Immune Response to Both Autoantigens and Foreign Antigens In Vivo Three sets of experiments in the early 1990’s provided further direct evidence that CD8` T cells regulate immune responses in vivo (22, 23, 108). The first experiment employed the EAE model in B10PL animals in which mice immu-

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nized with an encephalitogenic peptide of MBP develop acute EAE and recover from the first episode of disease and do not develop relapsing EAE. Importantly, the first episode of EAE renders the animals resistant to the re-initiation of EAE by secondary immunization. In contrast, animals depleted of CD8` T cells in vivo, using anti-CD8 monoclonal antibody (Mab)–mediated clearance are no longer resistant to a second induction of disease with MBP (22). These data demonstrate that CD8` T cells play a major role in the resistance to a second induction of EAE. Furthermore, as noted above, TCV-induced protection of EAE is abrogated in mice depleted of CD8` T cells (107). In a second set of experiments designed to investigate the role of CD8` T cells in EAE, CD8-/- ‘‘knockout’’ mice were generated and bred with the EAEsusceptible PL/J H-2u strain. The onset of disease and susceptibility were similar in both the CD8-/- mice and wild-type mice. However, the mutant mice had more chronic EAE, reflected by a higher frequency of relapse (23). These experiments provide direct evidence that CD8` T cells play a role in both inducing resistance to autoimmune disease and in abrogating recurrent relapsing episodes of autoimmunity. These experiments are of clear clinical interest because it is known that the great majority of human autoimmune diseases manifest acute attacks of autoimmunity variably interspersed with periods of remission. In the third set of experiments, it was shown that CD8` T cells control the immune response of H-2d mice to the conventional antigen, hen egg lysozyme (HEL) (108). Thus, Vba haplotype mice that lack 10 TCR Vb gene segments respond efficiently to the HEL peptide 74–96. In contrast, normal Vbb H-2d mice do not respond to the HEL peptide 74–96. Importantly, the normal Vbb H-2d mice depleted of CD8` T cells in vivo (by anti-CD8 Mab treatment) respond vigorously to this antigen. These studies demonstrate CD8` T cells control and normally suppress the immune response to certain peptides derived from conventional antigens like HEL. These studies also suggest that the TCR Vb region of the genome encodes proteins involved in this CD8` T cell regulation (see below). These studies taken together with the experiments reviewed above suggest that CD8` T cells govern immune responses to both conventional and autoantigens.

EVIDENCE THAT CD8` T CELLS, IN VIVO, CONTROL THE OUTGROWTH OF CD4` T CELLS IN A Vb SPECIFIC MANNER Studies of TCR Vb Peptide Immunization A variety of studies have provided indirect evidence that the TCR or peptides derived from the TCR of autoimmune T cells are part of the target structure recognized by regulatory CD8` T cells. For example, regulatory CD8` T cells isolated from T cell–vaccinated animals preferentially recognize the vaccine T

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cells activated by the specific antigen but not other T cells activated by different antigens in vitro (31, 104). Moreover, other studies have shown that pathogenic populations of immune cells are restricted in terms of TCR gene usage and the self peptide epitope recognized (109, 110). In addition, the classical TCV studies reviewed above have been extended in a significant manner to show that vaccination with TCR Vb peptides also efficiently prevents the development of EAE in rats and mice. Although TCR peptide immunization may function via more distinct mechanisms than ‘‘whole’’ T cell vaccination, it provides additional evidence that the TCR is involved in immune responses that control autoimmunity (111–113). The potential mechanism of this protection was investigated in experiments of Gaur et al who showed that vaccinating mice with a TCR Vb8.2 CDR2 peptide induces a state of TCR Vb specific unresponsiveness. Thus, the in vitro proliferative response to immunogens that usually induce an immune response mediated by Vb8.2 T cells are inhibited by prior TCR peptide vaccination. Moreover, the activation of T cells by anti-Vb8.2 crosslinking was also inhibited in a Vb specific manner in vaccinated mice. Importantly, depletion of CD8` cells before TCR peptide vaccination blocked such inhibition (24). Thus, the inhibition was dependent on CD8` T cells providing support for the idea that protection from EAE by TCR Vb peptide immunization is CD8` T cell dependent. Taken together, these TCR peptide immunization experiments provide substantial in vivo support for the prediction of some of the early notions of immune suppression. For example, one consequence of immunity is the induction of CD8` regulatory cells which are capable of specifically downregulating CD4` T cells through recognition of TCR related structures and suppressing immune responses to autoantigens and to alloantigens, in vivo. While the detailed mechanisms of this protection induced by vaccination with TCR Vb peptides is currently being actively investigated, preliminary clinical trials using relevant TCR Vb peptides have been initiated in patients with autoimmune disease, including multiple sclerosis (114–117).

Studies of the TCR Vb–Specific CD8` T Cell Downregulation of Antigen Activated CD4` T Cells In Vivo To develop direct evidence that CD8` T cells normally function to control the outgrowth of specific sets of CD4` T cells in vivo attention was turned to the study of superantigen-induced outgrowth of CD4` T cells (25). Superantigens bind to particular TCR Vb molecules and as a consequence activate a relatively large set of T cells expressing the particular TCR Vb to proliferate, differentiate and secrete cytokines. Administration of superantigen in vivo is followed by rapid changes in the populations of circulating T lymphocytes expressing the particular TCR Vbs. For example, after injection of the superantigen staphylococcus enterotoxin B (SEB), there is an initial deletion (12–24 h) followed by a proliferative expansion and a second phase of deletion (after day 4) of CD4` and CD8` T cells that express TCR Vb8 chains (118–120). The increase in Vb8` T

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cells reaches a maximum on day 4 and by day 10 returns to background. However, the CD4`Vb8` but not the CD8`Vb8` T cell population becomes reduced to about 30%–40% below baseline and remains at this reduced level for at least 21 days. The mechanisms responsible for the initial depletion of CD4` Vb8` cells are thought to be predominately a consequence of apoptosis because it is known that triggering of the TCR alone by either superantigen or anti-TCR antibodies induces programmed cell death (121–123). On the other hand, the mechanism of the prolonged deletion of CD4` Vb8` T cells was of interest and we entertained the possibility that this reflected CD8` T cell regulation. To test this idea the regulation of CD4` Vb8` T cells was studied in SEB primed CD8` T cell–deficient mice. These experiments specifically showed that the downregulation of CD4`Vb8` T cells below baseline is not observed in mice depleted of CD8` cells or in CD8` T cell–deficient b2 M-/-mice. Further, the effects of CD8` T cell depletion were TCR Vb specific because CD8` T cell depletion had no effect on populations of CD4` T cells unaffected by SEB. Taken together, these experiments provided evidence that CD8` T cells, in vivo, control the outgrowth of CD4` T cells in a TCR Vb– specific manner (25). In experiments designed to determine possible mechanisms of the CD8` T cell regulation of CD4` Vb8` T cells, CD8` T cells derived from SEB primed mice, were found to be cytotoxic to CD4` T cell targets based on their TCR Vb expression. For example, Vb8` but not Vb81 targets were killed. Furthermore, this TCR Vb–specific cytolysis was b2-microglobulin (b2l)– dependent because target CD4`Vb8` T cells from b2 M-/-mice were not lysed by these CD8` CTL. Taken together, these data provided evidence that the Vb8– specific regulatory functions of CD8` T cells in vivo and in vitro is dependent on b2l-associated molecules. Moreover, in other experiments it was shown that Vb8 specific CD8` T cells are also induced by T cell vaccination with SEB– activated CD4` Vb8` T cells (33). Thus, the fact that CD8` Vb specific CTL can be induced in vivo by activated CD4` T cells strongly suggests that these CD8` Vb–specific CTL may contribute significantly to the protective effects of T cell vaccination. Further evidence for the in vivo protective role of CD8` Vb–specific T cells in T cell vaccination has come from preliminary experiments in an EAE model system in which a MBP-specific encephalogenic CD4`Vb8` T H1 clone, termed 1AE10, was used to initiate EAE (107). EAE is induced in virtually all mice within 10 days and TCV employing 1AE10 gives complete protection. This model permitted the direct analysis of the Vb specificity of TCV-induced protection as well as the effects of CD8` T cell depletion on this protection. For example, CD8` T cell–depleted animals were compared to control groups that either did not receive TCV or were not CD8` T cell depleted. As noted above, CD8` T cell depletion completely abolished TCV-induced protection in EAE animals showing that CD8` T cells are required for efficient protection from EAE in this model. Moreover, the TCR Vb specificity of this CD8` T cell–mediated protec-

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tion was studied. Thus, MBP specific, TCR Vb8` and TCR Vb6` clones, were compared with respect to their capacity to serve as vaccine T cells and induce protection from EAE. These studies showed that Vb6` clones did not protect animals, whereas the Vb8` 1AE10–inducing clone and other MBP-specific TCR Vb8` clones protected animals from EAE. Therefore, TCV-induced in vivo protection from EAE is CD8` T cell dependent and Vb specific (107).

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EVIDENCE THAT TCR Vb8–SPECIFIC REGULATORY CD8` T CELLS ARE RESTRICTED BY THE MHC CLASS IB MOLECULE, QA-1 Because the Vb8-specific regulatory functions of CD8` T cells were dependent on b2l-associated molecules it was expected that anti-sera to conventional H2 molecules would inhibit function. However, quite surprisingly, the M1/42.39 antibody, which is a rat anti mouse H-2 monoclonal antibody known to be specific for all H-2 haplotypes including H-2Kd and H-2Dd and H-2Ld MHC class I antigens (124), did not block the function of the Vb-specific CD8` CTL in BALB/ c mice despite the fact that the antibody efficiently blocked control of allospecific CTL. These experiments led us to the hypothesis that the Vb specific CTL were, perhaps, restricted by non-classical MHC class 1b molecules. Moreover, because of the experiments reviewed above showing that the Qa-1 molecule was prominently expressed on cells involved in T cell suppression, and because it was known that Qa-1 was preferentially expressed on activated lymphoblasts, we entertained the idea that Vb specific CD8` regulatory cells were Qa-1 restricted. Three distinct types of experiments subsequently showed that Qa-1 could function as restriction elements for CD8` TCR Vb–specific regulatory T cells. First, in a series of blocking studies using well characterized sera that initially defined the Qa-1a and Qa-1b alleles it was shown that the Qa-1b but not the Qa-1a determinant was expressed on the antigen-activated CD4` target cells derived from BALB/c mice. Moreover, the anti-Qa-1b but not the anti-Qa-1a antiserum efficiently blocked Vb8-specific CD8` CTL generated from the spleens of SEBprimed mice. The same anti-Qa-1b did not block the killing of target cells by CD8` anti-MHC class Ia allogeneic CTL. These conventional allospecific CTL, as expected, were blocked by the rat anti-mouse H-2 monoclonal antibody (25). In the second set of experiments a human B cell line was transfected with either Qa-1 alone or Qa-1 together with TCR Vb8 or Vb6 cDNAs. These transfectants were then used as targets for CD8` Vb-specific CTL induced by T cell vaccination with activated CD4` Vb8` T cells (33). These CTL efficiently killed only those target cells expressing both murine Qa-1b and Vb8. Transfectants expressing Vb8 or Qa-1b alone were not lysed and transfectants expressing Qa1b and Vb6 were also not killed. These results demonstrate that co-expression of murine Qa-1b and Vb8 cDNAs is necessary and sufficient for human B cells to

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serve as targets for the murine CD8` anti-Vb8 CTL. It is important to emphasize that the target transfectants are B cells that do not express cell surface TCRs. Thus, Vb8 sequences do not need to be expressed on the cell surface as part of a mature TCRb polypeptide in order to be recognized by the Vb specific CD8` CTL. In this regard, it is well known that MHC class I molecules can efficiently present processed intracellular protein antigens. Moreover, it is also known that Qa-1 molecules can present foreign antigens (125) as well as self MHC class Ia peptides (126). In light of these considerations, these results are most consistent with the idea that CD8` anti-Vb8 CTL recognize TCR Vb peptide/Qa-1 complexes expressed on target T cells (33). The third line of evidence that Qa-1 molecules can serve as targets for CD8` TCR Vb specific regulatory cells stems from experiments employing CD8` T cell hybridoma clones derived from B10.PL mice that were vaccinated with an irradiated syngeneic MBP-specific, encephalogenic CD4` Vb8` T cell clone (33). For example, a CD8`Vb-reactive, Qa-1-restricted hybridoma clone (21– 5A9) was isolated that preferentially reacts with syngeneic target T cells expressing TCR Vb8, but not Vb6, in a Qa-1-restricted manner. In the course of these studies it was observed that antigen-activated but not resting CD4`Vb8` Qa-1 targets could trigger the hybridoma. Kinetic studies showed that activated CD4` T cells were most stimulatory on days 4–8 following activation and were nonstimulatory by day 12. One interpretation of this kinetic data is that it may reflect the kinetics of the activation-dependent expression of Qa-1/TCR peptide by CD4` T cells. In support of this, the level of Qa-1-expression increases with T cell activation and peaks around day 4 to 7 and is absent by day 10. Moreover, the responses induced by the activated CD4` T cell targets were inhibited in an allele specific manner by Qa-1 antisera. Taken together, these studies show at a clonal level that Qa-1-restricted, Vb-specific CD8` T cells are generated by T cell vaccination (33, 127). In summary, these experiments support the view that Qa-1 can function as a restriction element for regulatory CD8` TCR Vb–specific cells. These studies do not exclude the possibility that other MHC class Ib molecules, or even some MHC class Ia molecules, may also subserve this function. However, the inhibition of TCR Vb–specific regulatory CD8` cells by anti-Qa-1 but not MHC class Ia antibodies, suggest that Qa-1 is the predominant restriction element. On the other hand, the studies above also do not rule out the possibility that other types of regulatory cells including CD4` T cells may downregulate immune responses in a Vb-specific fashion and use entirely different MHC restriction elements (see below). Nevertheless, we will point out in subsequent sections, that there are a number of biologic features of the Qa-1 molecule that make it particularly suitable to function as a restricting element to present self-TCR peptides to regulatory CD8` T cells. For example, the preferential expression of Qa-1 on antigenactivated T cells would preclude resting T cells from being susceptible to downregulation.

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EVIDENCE THAT TCR Vb–SPECIFIC REGULATORY CD8` T CELLS INDUCED BY TCV PREFERENTIALLY RECOGNIZE TH1 BUT NOT TH2 CD4` CELLS Further analysis of the Qa-1-restricted Vb-specific T hybridoma clone, 21–5A9, revealed that it preferentially recognized CD4`Vb8` TH1 clones but was not reactive with CD4`Vb8` TH2 clones (127). This was the case even for pairs of TH1 and TH2 clones expressing identical TCR Va and Vb chains. The mechanism that accounts for this differential recognition is not clear, but preliminary analysis suggests that it is not secondary to differential cytokine secretion. However, preliminary TCV studies suggest that the differential recognition of TH1 and TH2 cells by CD8` T cell hybridomas in vitro may represent a mechanism by which CD8` T cells regulate immune responses in vivo (107). Thus, mice were vaccinated with either the CD4`Vb8` T cell clones, 1AE10, (TH1) or the TCR identical CD4`Vb8` TH2 clone, 3AD2. The TH1 clone 1AE10 completely protected mice from subsequent induction of EAE, whereas the TH2 clone 3AD2 did not protect. Moreover, CD8` T cell depletion significantly abrogated this protection. This data is consistent with the model that Vb8` TH1 cells but not Vb8` TH2 cells are able to induce TCR Vb8-specific, Qa-1-restricted regulatory CD8` T cells that specifically downregulate TH1 T cell–induced disease in vivo. In this regard, it is of interest that TCV employing cDNA encoding Vb8 has been shown to protect animals from EAE. Although it is unknown if this form of TCV is also CD8` T cell–dependent, the protection observed is accompanied by a shift in the MBP-specific TH functional phenotype from TH1 to TH2 (128).

EVIDENCE THAT TCR Vb–SPECIFIC REGULATORY CD8` T CELLS EXIST IN HUMAN PERIPHERAL BLOOD A variety of studies in the human immune system provided evidence that recognition of the TCR is integral to the specificity of immune regulation between T cells subsets. For example, a series of experiments have shown that it is possible to generate human CD4` T cell clones that proliferate specifically to autologous CD4` clones that either show a particular antigen specificity or specific Vb expression (129–131). Moreover, human CD8` T cell clones raised against autologous allo-reactive CD4` T cell lines inhibit fresh autologous CD4` T cells from proliferating to the same alloantigen (132). These results are consistent with the idea that T-T cell interactions may involve all or part of the TCR as a target of recognition. Evidence that Vb-specific CD8` T cells exist in human peripheral blood came from studies showing that CD8` T cells raised to CD4` Vb2` T cell clones, in vitro, differentiated into CTL that specifically lyse independently isolated

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autologous CD4` T cell targets expressing Vb2` but not Vb21 TCRs. Moreover, in reciprocal cloning experiments, CD8` T cells raised against autologous CD4` T cell clones with different TCR Vb usage were cytotoxic only to CD4`T cell targets expressing TCR Vbs expressed by the inducing clone. Taken together, these experiments indicate that CD8` T cells can kill autologous CD4` T cells based, at least in part, on the recognition of the TCR Vbs expressed. Interestingly, this TCR Vb–specific cytotoxicity was not blocked by the monoclonal antibody W6/32, which reacts with nonpolymorphic determinants present on HLA A/B/C Class Ia molecules. This data suggests that like in the mouse, MHC class Ib structures might serve as the restricting element (133). In contrast, Zhang et al showed that human CD8` T cells, isolated from T cell–vaccinated patients, which specifically recognize and kill potentially encephalogenic autologous MBPreactive CD4` T cell clones, could be blocked by the conventional HLA class Ia antibody, W6/32 (104). Taken together, these data suggest the possibility that under certain conditions MHC class Ia as well as MHC class Ib molecules may present TCR Vb peptides, at least in the human system.

REGULATORY CD4` T CELLS In this section we briefly review immunoregulatory functions effected by CD4` T cells. First, in a series of experiments, Kumar and Sercarz identified a CD4` T cell population in EAE-recovered mice, which specifically respond to a peptide derived from TCR Vb 8.2. These CD4` T cells downregulate 1–9Nac MBP proliferative responses, in vitro, and in vivo protect mice from EAE. For example, they isolated a CD4` T cell clone, specific for a peptide derived from framework region 3 of TCR Vb8.2 chain (B5). When adaptively transferred, this clone protects animals from subsequent induction of EAE. Moreover, immunizing animals with the B5 peptide protected animals from EAE (134–137). These CD4` T cells are analogous to the TCR Vb–specific CD8` T cells described above; however, as expected the CD4` regulatory T cells are restricted by MHC class II molecules instead of MHC class Ib molecules. Because activated murine T cells do not express MHC class II, they obviously cannot process and present MHC class II/ Vb peptide to regulatory CD4` T cells. Thus, the precise means by which the MHC class II restricted regulatory CD4` T cells are induced and function is unclear. Two distinct mechanisms were considered (138). First, it is possible that the TCR Vb peptides derived from antigen-activated CD4` T cells might be processed and presented by conventional antigen presenting dendritic cells or macrophages. This could occur, for example, if apoptotic CD4` T cells or if membrane fragments containing TCR Vb, are ingested by dendritic cells. These dendritic cells could then present TCR peptides complexed to either MHC class II or MHC class I and subsequently induce both Vb-specific CD4` or CD8` regulatory T cells. The CD4` regulatory T cells would not be able to directly downregulate antigen-activated CD4` T cells which do not express MHC class

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II molecules. However, these cells could locally secrete modulatory cytokines, such as TGF-b or IL-10 at the site of CNS inflammation and as a consequence downregulate MBP-specific CD4` T cells. Alternatively, it is possible that dendritic cells that can present Vb peptides on either class I or class II MHC molecules might locally focus both CD4` regulatory cells with TCR Vb–specific CD8` regulatory T cells on the same cell surface in a CD40L-dependent manner analogous to mechanisms employed by CD4` T cells to help conventional cytotoxic CD8` CTL (139–141). This possibility is supported by experiments that show that the function of TCR Vb–specific regulatory CD4` T cells, in vivo, is dependent on CD8` T cells (134). The second set of studies demonstrate that under certain experimental conditions CD4` T regulatory cells can downregulate antigen specific CD4` T cells independent of other T cells. For example, it was shown that TCR transgenic mice (T/R`) specific for MBP rarely develop spontaneous EAE. However, when these T/R` mice are crossed with RAG-1-deficient mice to obtain mice (T/R1) that have T cells expressing the transgenic TCR almost all mice develop spontaneous EAE (142). Because both T/R` and T/R1 mice have large numbers of the potentially encephalitogenic CD4` anti-MBP T cells these results suggest that a very small number of nontransgenic lymphocytes, that are present in T/R` but absent in T/R1 mice can potently suppress the in situ activation of CD4` anti-MBP T cells mediating EAE. To identify the cellular regulatory mechanisms important in this suppression, T/R` mice were crossed into mice deficient in either B cells, CD8` T cells, NK1.1 CD4` (NKT) cells, c/d cells or a/b cells (143, 144). Only mice that were deficient in CD4` a/b T cells developed EAE. Moreover, T/R1 mice were protected from EAE by the early (prior to the onset of EAE) adoptive transfer of purified CD4` T cells from normal donors. These results support the view that under certain experimental conditions CD4` cells alone can suppress the initiation of autoimmunity. The specificity or mechanisms of this suppression is currently unknown. It is conceivable that the CD4` T regulatory cells are non-specifically activated to secrete cytokines known to downregulate EAE activity. In this regard, several groups have demonstrated that CD4` T cells secreting TGF-b IL-4 or IL-10 can down regulate a variety of autoimmune diseases including EAE, murine inflammatory bowel colitis models and NOD diabetes models (143, 145–149). It is of interest that both CD4` and CD8` regulatory T cell clones, which specifically recognize potentially encephalogenic autologous MBP-reactive CD4` T cell clones, have been observed in patients with multiple sclerosis treated by TCV with attenuated MPB-reactive clones (104, 150). In these experiments, although the majority of specific regulatory T cells were CD8`, approximately 10% were CD4`. Because activated human CD4` T cells can express MHC class II, it is conceivable that these regulatory CD4` T cells downregulate target CD4` T cells through recognition of TCR peptides presented by MHC class II molecules. If this were the case these regulatory CD4` T cells would be analogous to the Qa-1 restricted, TCR Vb–specific regulatory CD8` T cells described

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above. Speculating further, it is of interest that some MHC class II molecules, including HLA-DQ are less polymorphic then the more prevalent HLA-DR molecules and could potentially serve to present a restricted set of TCR peptides. In this regard suppressor cells restricted by HLA-DQ class II molecules have been observed in patients with lepromatous leprosy who are selectively unable to mount cellular immunity to Mycobacterium leprae (151). Although it is unclear what factors govern whether CD4` or CD8` regulatory cells are induced, as noted above it is possible that dendritic cells that can present Vb peptides on either class I or class II MHC molecules may serve to induce either CD4` or CD8` TCR-specific regulatory cells. In this regard, it is known that dendritic cells can express both MHC class I and II molecules, including the less polymorphic HLA-DQ molecules (152, 153).

THE POTENTIAL FUNCTIONAL SIGNIFICANCE OF RESTRICTING T REGULATORY CELLS BY THE MHC CLASS IB MOLECULE, QA-1 There are a number of biologic features of the Qa-1 molecule that may make it particularly interesting with respect to its potential function as a restricting element to present self TCR peptides to CD8` T cells. First, although Qa-1 is expressed on a variety of hematopoietic tissues, it is only minimally or not expressed at all on resting T cells and thymocytes and its expression is significantly increased following peripheral T cell activation by antigen (154). For example, analysis of Qa-1 isolated from detergent lysates of surface labeled cells indicates that Qa-1 displays little charge heterogeneity on resting lymphocytes, but the level of expression and degree of charge heterogeneity are both increased on activated lymphocytes (155). Moreover, because the peripheral Qa-1 expression is short lived and persists for only a few days on activated CD4` T cells, this may exclude resting T cells from downregulation by Qa-1-restricted CD8` regulatory cells. In this way the relatively short-lived upregulation of Qa-1 on antigen-activated T cells is analogous to the transient expression of CD40L on antigen-activated T cells (7). The transient expression of both of these functional molecules, one for help (CD40L) and the other for suppression (Qa-1) on the surface of antigen-activated T cells would serve to confine the regulatory functions of these molecules to a time frame of specific antigen activation and may consequently prohibit promiscuous help or suppression. In addition, the rather limited expression of Qa-1-in the thymus may account for the fact that Qa-1restricted CD8` T thymocytes are not negatively selected in the thymus. The mechanism for the potential positive selection of Qa-1-restricted T cells in the thymus is currently unknown. It is conceivable that other MHC class Ia or class Ib molecules may serve to positively select those Qa-1-restricted T cells in the thymus.

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Second, Qa-1 is known to be a MHC class Ib molecule of limited polymorphism with the potential to present self and foreign peptides to CD8` T cells (87, 88). The predominant self peptide presented by Qa-1 is termed Qdm (for Qa-1– determinant modifier), derived from the signal leader sequence of certain murine class Ia molecules (156–159). This peptide (AMAPRTLLL) binds with high affinity and accounts for the majority of the peptides associated with this molecule (159, 160). However, Qa-1 can also bind other self peptides including those derived from heat shock proteins (161) and preproinsulin leader sequences (162). In addition, data reviewed here suggests that CD8` T cells recognize TCR Vb motifs restricted by Qa-1. In addition, Qa-1 can bind exogenous peptides including bacterial derived peptides (160, 163–165). Many features of the Qa-1 binding cleft are also conserved in the rat RTBM.1 and in human HLA-E molecules, suggesting that these molecules may associate with structurally similar peptides (87). Thus, it is perhaps not surprising that HLA-E, like Qa-1, also binds to Qdm as well as Qdm-like leader sequence peptides derived from HLA class Ia molecules. The functional significance of the Qa-1/Qdm complex as well as similar HLAE/leader peptide complexes has recently begun to be illuminated by the finding that both Qa-1/Qdm complexes and HLA-E/leader peptide complexes interact with the CD94/NKG2 heterodimeric receptors expressed on NK cells (166). This set of receptors is comprised of an invariant C type lectin chain, CD94, that is disulfide-bonded with the NKG2 glycoproteins (167–169). The CD94/NKG2 receptor complexes are expressed on both human and murine NK cells as well as a subset of T cells (166, 170). The human CD94/NKG2 complex recognizes HLA-E/MHC class Ia leader sequence peptides (171), whereas the murine CD94/ NKG2 complex binds the homologous Qa-1/Qdm complex (170). As a consequence, tetramers of HLA-E or Qa-1 bind to a large fraction of human and murine NK cells, respectively (170–173). The CD94/NKG2 receptors is of interest in that they appear to gauge the level of MHC class I expression in targets, indirectly, by recognizing leader sequences of MHC molecules. Functionally these receptors are thought generally to limit NK killing of normal targets. The regulatory functions of CD94/NKG2 receptors are largely dependent on signaling through the NKG2 molecules. For example, the NKG2A/B molecules possess two immunoreceptor tyrosine-based inhibition motif (ITIM) sequences in their cytoplasmic domain, responsible for the inhibitory function of these receptors, whereas other NKG2 proteins lack ITIMs and transmit positive or activating signals (168, 174). For example, the human CD94/NKG2C binds to HLA-E, and noncovalently associates with DAP12, a membrane receptor containing an immunoreceptor tyrosine-based activating motif (ITAM). Efficient expression of CD94/ NKG2C on the cell surface requires the presence of DAP12, and charged residues in the transmembrane domains of DAP12 and NKG2C are necessary for this interaction (175). Phosphorylated DAP12 peptides bind ZAP-70 and Syk protein tyrosine kinases, suggesting that this activation pathway is similar to that of the T and B cell antigen receptors (176). Taken together, these various MHC class I

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receptors expressed by NK cells are endowed with molecular mechanisms (ITIMs and DAP12-related ITAMS) not only to function as killer inhibitory receptors but perhaps more importantly as receptors maintaining homeostasis. (174, 177).

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CD94/NKG2 RECEPTORS REGULATE THE FUNCTIONAL SIGNALING OF a/b AND c/d TCRS In this regard, the CD94/NKG2 receptors are also expressed on subsets of T cells expressing either a/b or c/d TCRs and therefore potentially, in concert with these TCRs, may regulate not only NK function but also antigen specific functions of more conventional T cells. For example, human c9/d2 T cells known to recognize phosphoantigens expressed by certain bacteria, mycobacterium and tumor cells have recently been shown to co-express CD94/NKG2 receptors, and signaling through the CD94/NKG2 receptor regulates the synthesis of cytokines (IFN-c and TNF-a) as well as antigen-induced cytotoxic functions (178). CD94/NKG2 receptors may also be co-expressed and regulate conventional MHC class I restricted a/b CD8` CTL specific for tumor or viral peptide/MHC complexes (179–182). Of interest, the co-expression of CD94/NKG2 receptors on a/b CD8` T cells is upregulated by cytokines including TGF-b and IL-10 (180, 181). The potential functional significance of the co-expression of a/b TCR with CD94/NKG2 receptors on CD8` CTL was suggested in experiments showing that ligation of CD94 on these cells can either activate or inhibit killing mediated by the a/b TCR (179, 183). These data support the idea that the co-expression of a/b TCR and CD94/ NKG2 receptors on conventional CD8` CTL homeostatically regulate CTL functions. It is of interest to speculate on how CD94/NKG2 receptors may functionally interact with the a/b TCRs expressed by Vb-specific, Qa-1-restricted regulatory CD8` T cells. First of all, recent data assaying Qa-1/Qdm teramers binding to T cell subsets suggest that a significant number of murine CD8` T cells bind to these tetramers and, thus, presumably express CD94/NKG2 receptors (170, 172). This suggests that clones of the regulatory CD8` T cells, in fact, co-express two distinct types of Qa-1 receptors. One type is an a/b TCR that recognizes Qa-1/ TCR peptide complexes and the other is the CD94/NKG2 receptor that recognizes Qa-1/Qdm. These two receptors, as noted above, could be differentially regulated on the cell surface of CD8` T cells and functionally regulate one another. Moreover, depending on whether CD94NKG2 receptors have ITIMs or alternatively bind DAP12 they may either enhance or downregulate CD8` T cell function. In addition, during CD4` T cell activation both Qa-1/Qdm and Qa-1/TCR Vb ligands may be differentially upregulated. For example, resting CD4` T cells may preferentially express Qa-1/Qdm, and with activation, other peptides (presumably TCR Vb peptides) may bind to Qa-1. This hypothesis is consistent with biochemical data that show that the Qa-1 molecule displays little charge

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heterogeneity on resting lymphocytes but that the level of expression and degree of charge heterogeneity are both increased on activated lymphocytes (155). This increase in charge heterogeneity may reflect the presence of newly formed Qa-1/ TCR Vb complexes expressed during activation. The functional consequence of this model is that resting T cells will preferentially express Qa-1/Qdm, which following ligation of CD94/NKG2 may for the most part trigger inhibitory signals. At a point in time during T cell activation the prevalence of Qa-/TCR Vb expressed on target CD4` T cells may reach a threshold sufficient to trigger positive signals through the a/b TCR expressed on CD8` T cells. In this way the two types of Qa-1 receptors expressed on CD8` T cells may ultimately provide homeostatic control of the regulatory T cells.

GENERAL MODEL OF T CELL REGULATION BY CD8` T CELLS IN THE CONTEXT OF PERIPHERAL TCR REPERTOIRE SELECTION In this review we have summarized some of the pertinent evidence that CD8` T cells regulate ongoing peripheral immune responses, at least in part, by cognate interactions with antigen-activated CD4` T cells in a TCR-specific manner restricted by the MHC class Ib molecule, Qa-1 (see Figure 1, color insert). We have also reviewed the data that suggest that the Qa-1 molecule is uniquely qualified to serve this MHC restrictive function because unlike conventional MHC molecules it is preferentially expressed on activated and not resting CD4` T cells. In addition, Qa-1 expression in the thymus is either low or non-existent. Thus, precursor a/b TCR–expressing CD8` T cells restricted by Qa-1 are not negatively selected in the thymus. The conceptual origins of the current studies emerged from the experimental data and hypothesis that arose during the last three decades suggesting that suppressor CD8` T cells regulate peripheral normal immune responses and play a critical role in the regulation of autoimmune responses. In particular, key experiments defining the role of CD8` T cells in controlling autoimmunity stemmed from studies showing that mice that have recovered from the first episode of MBP peptide–induced EAE are resistant to re-induction of disease and that this resistance is CD8` T cell–dependent. More recent experiments have shown that this protection or resistance can be induced by TCV with activated MBP-reactive, CD4` T cells that induce Qa-1restricted TCR Vb–specific regulatory CD8` T cells. These data are compatible with the hypothesis that the regulatory CD8` T cells scan the surface of CD4` T cells and selectively delete or inactivate those T cells expressing certain TCR Vb peptides bound to Qa-1. This hypothesis would predict that the peripheral T cell repertoire of CD4` T cells reactive to MBP should be different in naı¨ve (EAE-susceptible) compared to EAE-recovered (EAE-resistant) animals. In this regard, elegant studies by Lehmann and Sercarz have shown that determinants of

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MBP that are cryptic after primary immunization can become immunogenic in secondary responses and during the course of EAE (184–186). This diversification of the autoreactive T-cell repertoire has led to the concept of ‘‘determinant spreading’’ during the course of T-cell–driven autoimmune disease. Moreover, recent experiments in our laboratory have also investigated the peripheral TCR Vb repertoire of CD4` T cells to the 1–9 Nac MBP peptide in naı¨ve compared to EAE-recovered mice. These studies showed a significant reduction of certain Vb8.2-expressing MBP-reactive CD4` T cells in EAE recovered animals, accompanied by the emergence of other CD4` MBP-reactive T cells expressing other TCR Vbs, as well as the persistence of some MBP-reactive Vb8.2 clones. This change in the TCR repertoire was not observed in CD8` T cell–depleted animals. It is of interest that although EAE-recovered mice are resistant to disease, there is a persistence of some MBP-reactive CD4` T cells, including some TCR Vb8.2 clones. The persistent MBP-reactive CD4` clones may express distinct MBPspecific TCRs that escape TCR Vb-specific CD8` T cell recognition. For example, the TCRs on these persistent clones may be of too low affinity for MBP to trigger the mechanisms that normally permit downregulation. These observations lead us to propose the following hypothesis. Because regulatory CD8` T cells downregulate CD4` T cells based predominately on the recognition of Qa-1/TCR peptide complexes expressed on the surface of CD4` T cells, the cognitive distinction by which certain CD4` T cells are downregulated by CD8` T cells and others are not, is dictated by the ability of the CD4` T cells to process and present the relevant Qa-1/TCR peptide complexes on the cell surface. It is likely that the major determinant of whether or not a CD4` T cell will process and present Qa-1/TCR peptide will be the initial cognitive encounter between the particular TCR expressed by CD4` T cells and the MHC/ peptide complex presented on antigen-presenting cells. We envision that T cells expressing TCRs with either low affinity or high affinity interaction with MHC/ peptide will not signal Qa-1/TCR peptide presentation. Only T cells with ‘‘intermediate’’ affinity, above and under certain thresholds, will trigger Qa-1/TCR peptide presentation. The precise threshold for these CD4` T cells to process and present self TCR peptide coupled to Qa-1 molecules will not only be a function of the quality, intensity and duration of the initial tri-molecular complex interaction between antigen-specific CD4` T cells and antigen-presenting cells, but may also be influenced by signaling via co-stimulatory molecules including CD80, CD86 and CD40. Moreover, these same factors will determine whether the surface Qa-1 molecules are predominately comprised of Qa-1/Qdm or both Qa-1/Qdm and Qa-1/TCR peptide. The expression of Qa-1/TCR peptide will induce specific functional responses from regulatory CD8` T cells which, as discussed above, may be further regulated by Qa-1/Qdm expression. These considerations can be viewed as an ‘‘affinity model’’ of peripheral T cell regulation. If we consider this model with respect to self-reactive CD4` T cell clones, it is likely that high affinity clones will have been deleted in the thymus, so that the TCR affinity of clones for self determinants in the periphery

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will either be low or intermediate. The T cells expressing TCRs with intermediate affinity to self are potentially pathogenic and capable of inducing auto-immune disease. These T cells will have been positively selected on self antigens in the thymus but escape negative thymic selection because they possess TCRs with intermediate affinity to self, sufficiently low enough not to induce apoptosis and thymic death. This type of T cells in the periphery may be triggered by self antigen to process and present Qa-1/TCR peptides and be subject to CD8` T cell– mediated downregulation. One biological consequence of this downregulation of self reactive T cells is to provide an additional mechanism to ensure peripheral self tolerance. In contrast, the T cells with very low affinity to self will have escaped negative selection in the thymus and also will not be subject to CD8` T cell–mediated downregulation in the periphery. We envision that this type of T cells may have high affinity to foreign antigens and thus be preserved as part of the peripheral T cell repertoire to foreign antigens. The MBP-reactive clones that persist in EAE-recovered mice or newly emerge in these mice may fall into this category of cells. These same principles can be readily extended to the CD8` T cell–mediated downregulation of the vast majority of T cells specific to foreign antigens. Thus, we propose that only T cells with intermediate affinity TCRs to MHC/foreign peptides will induce Qa-1/TCR peptide surface expression and be subject to CD8` T cell–mediated downregulation in periphery. The low affinity T cells may not receive sufficient TCR triggering to proliferate and/or differentiate and be diluted out by higher affinity T cell clones during the course of immune responses. We envision that T cells with high affinity TCRs to foreign antigens will also escape CD8` T cell–mediated downregulation. Perhaps the net effect of homeostatic mechanisms involving both Qa-1/Qdm and Qa-1/TCR peptide is to inhibit the downregulation of these high affinity T cells in periphery. Taken together, these considerations would predict that during immune responses T cells with intermediate affinity TCRs for either self peptides or foreign peptides would be downregulated by the Qa-1-restricted CD8` T cells. T cell clones with high affinity TCRs for foreign peptides would be most likely to survive this type of down-regulation in periphery. In this regard, it is known that during the evolution of an immune response and following repeated antigenic exposure there is a change in both the antigenspecific B cell immunoglobulin and the T cell receptor primary repertoire leading to a higher affinity repertoire. In the case of B cells, the special mechanisms of somatic hypermutation permit the creation of higher affinity receptors and the preferential selection of higher affinity receptors in secondary responses is a direct consequence of antigen triggering, although T-B contact–dependent help involving CD40 may also dictate which B cell clones will survive in germinal centers. In contrast to B cells, T cells do not undergo somatic hypermutation, and therefore antigen triggered peripheral T cells retain germline-encoded VDJ segments. Nevertheless, recent studies have shown that although the TCR repertoires between naı¨ve and primarily activated T cells are not significantly different there is a

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significant change in the TCR Vb repertoire following secondary immunization (4, 187, 188). Molecular correlation of this change in TCR repertoire between primary and secondary responses has recently been evaluated in studies assaying MHC/peptide tetrameric complex binding to specific TCRs. These studies have shown that during the secondary response there is a selection against TCRs with the highest dissociation rates for MHC/peptide binding and for TCRs that bind MHC/peptide for a long duration. These parameters generally correlate with selection for higher affinity binding (4, 187, 188). We propose that superimposed on this direct antigen (MHC/peptide)–driven selection of the TCR repertoire, fine tuning of the TCR repertoire is made by regulatory Qa-1/TCR peptide–specific CD8` T cells. As emphasized above, these regulatory CD8` T cells are induced by and regulate only those activated CD4` T cells with intermediate affinity TCRs to foreign antigens that express surface Qa-1/TCR peptide–complexes. Thus, the peripheral TCR repertoire to foreign antigens changes during the course of immune responses, in the absence of somatic hypermutation mechanisms, by direct antigen (MHC/peptide)–driven TCR selection and additionally by regulatory CD8` T cells that selectively downregulate T cells expressing intermediate affinity TCRs. ACKNOWLEDGMENTS The authors wish to thank Drs. Ned Braunstein, Harvey Cantor, Lorraine Flaherty, Jim Forman, Mark Soloski, Alan Stall, and Robert Winchester for helpful discussions, support, and reagents. The research was siupported by NIH grants AI39630, AI30675 and National Multiple Sclerosis Society grant RG2938A to HJ, and NIH grants AI 39675, Ai40226A to LC. Visit the Annual Reviews home page at www.AnnualReviews.org.

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182. Speiser DE, Valmori D, Rimoldi D, Pittet MJ, Lienard D, Cerundolo V, MacDonald HR, Cerottini JC, Romero P. 1999. CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients. Eur. J. Immunol. 29:1990 183. Bellon T, Heredia AB, Llano M, Minguela A, Rodriguez A, Lopez-Botet M, Aparicio P. 1999. Triggering of effector functions on a CD8` T cell clone upon the aggregation of an activatory CD94/ kp39 heterodimer. J. Immunol. 162:3996 184. Sercarz EE, Lehmann PV, Ametani A, Benichou G, Miller A, Moudgil K. 1993. Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 11:729 185. Lehmann PV, Forsthuber T, Miller A, Sercarz EE. 1992. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358:155 186. Lehmann PV, Sercarz EE, Forsthuber T, Dayan CM, Gammon G. 1993. Determinant spreading and the dynamics of the autoimmune T-cell repertoire. Immunol. Today 14:203 187. Busch DH, Pamer EG. 1999. T cell affinity maturation by selective expansion during infection. J. Exp. Med. 189:701 188. Busch DH, Pilip I, Pamer EG. 1998. Evolution of a complex T cell receptor repertoire during primary and recall bacterial infection. J. Exp. Med. 188:61

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Figure 1 Model of cognate interactions in the induction and function of regulatory CD8+ T cells. CD8+ T cells regulate peripheral immune responses by cognate interactions with antigen activated CD4+ T cells in a TCR specific, Qa-1 restricted manner. Antigen activated CD4+ T cells begin to express surface Qa-1 molecules associated with TCR Vβ peptides. These Qa-1/Vβ complexes expressed on the surface of CD4+ T cells trigger the differentiation of regulatory CD8+ T cells, which subsequently downregulate activated CD4+ T cells in a Qa-1 restricted, Vβ specific manner.

Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:217–242 Copyright q by Annual Reviews. All rights reserved

THE BIOLOGY OF CHEMOKINES AND THEIR RECEPTORS Devora Rossi1 and Albert Zlotnik2 1

Pharmingen Inc., 10975 Torreyana Road, San Diego, California 92121–1111; e-mail: [email protected] 2 DNAX Research Institute, 901 California Avenue, Palo Alto, California, 94304; e-mail: [email protected]

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Key Words chemokines, inflammation, trafficking, metastasis, angiogenesis Abstract During the last five years, the development of bioinformatics and EST databases has been primarily responsible for the identification of many new chemokines and chemokine receptors. The chemokine field has also received considerable attention since chemokine receptors were found to act as co-receptors for HIV infection (1). In addition, chemokines, along with adhesion molecules, are crucial during inflammatory responses for a timely recruitment of specific leukocyte subpopulations to sites of tissue damage. However, chemokines and their receptors are also important in dendritic cell maturation (2), B (3), and T (4) cell development, Th1 and Th2 responses, infections, angiogenesis, and tumor growth as well as metastasis (5). Furthermore, an increase in the number of chemokine/receptor transgenic and knock-out mice has helped to define the functions of chemokines in vivo. In this review we discuss some of the chemokines’ biological effects in vivo and in vitro, described in the last few years, and the implications of these findings when considering chemokine receptors as therapeutic targets.

INTRODUCTION In the last few years, we have witnessed the development of EST (expressed sequence tag) databases and the widespread application of bioinformatics for new gene discovery. As a result, the pace of novel gene discovery has accelerated dramatically. One of the best examples of the impact of these technologies has been in the number of members of the chemokine superfamily identified in this time. The chemokines are ideal molecules to be discovered through bioinformatics because they are small secreted molecules that exhibit very specific cysteine motifs in their amino acid sequence. Most chemokines have four characteristic cysteines, and depending on the motif displayed by the first two cysteines, they have been classified into CXC or alpha, CC or beta, C or gamma, and CX3C or delta chemokine classes. The only exception to the four Cys rule is lymphotactin (6), which has only two Cys residues. Two disulfide bonds are formed between 0732–0582/00/0410–0217$14.00

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the first and third Cys and between the second and fourth. Thus, lymphotactin manages to retain a functional structure with only one disulfide bond. In addition, the CXC or alpha subfamily has been divided into two groups depending on the presence of the ELR motif preceding the first cysteine: the ELR-CXC chemokines and the non-ELR-CXC chemokines. Chemokine receptors are G-protein coupled, seven-transmembrane receptors. Based on the chemokine class they bind, the receptors have been named CXCR1, 2, 3, 4, and 5 (bind CXC chemokines); CCR1 through CCR9 (bind CC chemokines); XCR1 (binds the C chemokine, Lptn); and CX3CR1 (binds the CX3C chemokine, fractalkine or neurotactin) (Table 1). Along with the accelerated rate of discovery of chemokines has come the realization that these molecules not only control hemopoietic cell migration, but also are involved in a number of other physiological and pathological processes. Figure 1 summarizes our current understanding of the involvement of chemokine biology in other areas. Also, the characterization of new chemokines has uncovered new roles of these molecules, for example, in lymphoid cell development. The mouse and human genome/EST projects have generated large sequence databases, which resulted in the identification of most of the members of the chemokine superfamily. When viewed in this light, we can discern certain trends.

TABLE 1 Summary of the known chemokine receptors and some of their known human ligands Chemokine receptors

Human chemokine ligands

CXCR1 CXCR2 CXCR3 CXCR4 CXCR5

IL-8, GCP-2 IL-8, GCP-2, Gro a, Gro b, Gro c, ENA-78, PBP MIG, IP-10, I-TAC SDF-1/PBSF BLC/BCA-1

CCR1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9

MIP-1a, MIP-1b, RANTES, HCC-1, 2, 3, and 4 MCP-1, MCP-2, MCP-3, MCP-4 eotaxin-1, eotaxin-2, MCP-3 TARC, MDC, MIP-1a, RANTES MIP-1a, MIP-1b, RANTES MIP-3a/LARC MIP-3b/ELC, 6Ckine/LC I-309 TECK

XCR1 CX3CR1

Lymphotactin Fractalkine/neurotactin

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Figure 1 Biological functions of chemokines and chemokine receptors.

For example, among the first ones discovered were those chemokines produced by many cellular sources. Conversely, the last ones found exhibit restricted tissue and cellular specificity. This specificity suggests organ-specific functions, as in the case of TECK described below. This suggests that other chemokines that remain to be discovered will likely exhibit limited tissue distribution and will not be identified until their cellular source is analyzed by EST production. This chapter does not aim to be a comprehensive review on chemokines; rather, we highlight those areas of chemokine biology with new developments or therapeutic potential.

ROLE OF CHEMOKINES IN ANGIOGENESIS/ ANGIOSTASIS AND METASTASIS Angiogenesis is a biological process through which blood vessels are generated. Although it is a strictly controlled and transient event during wound healing, angiogenesis is also associated with several chronic inflammatory diseases, such as psoriasis, rheumatoid arthritis (pannus formation), and idiopathic pulmonary fibrosis (7) as well as with tumor growth and metastasis (8), where neovascularization appears to be aberrantly upregulated. Solid tumor growth requires the presence of neovascularization to guarantee an adequate supply of oxygen and nutrients (9). Strieter (10) and others have proposed that an imbalance between angiogenic and angiostatic factors may be responsible for tumor growth and the development or progress of some chronic inflammatory diseases. It is well established that ELR-CXC chemokines are potent angiogenic factors, able to stimulate endothelial cell chemotaxis, while most non-ELR-CXC che-

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mokines are strong angiostatic factors, which inhibit the endothelial cell chemotaxis induced by ELR-CXC chemokines (10). In fact, CXC chemokines act as angiogenic or angiostatic factors depending solely on the presence of the ELR motif (10). ELR-CXC chemokines bind to CXCR2 and few to CXCR1, while non-ELR-CXC chemokines bind to CXCR3, CXCR4, and CXCR5 (Table 1). Several examples of diseases in which the balance between angiogenic and angiostatic chemokines is altered appear in the literature. For example, Keane et al (7) demonstrated that lung tissues from IPF (idiopathic pulmonary fibrosis) patients constitutively express more IL-8 and less IP-10 than those from healthy individuals. This suggests that a net angiogenic balance of produced factors may be established in IPF patients, which causes fibroplasia and deposition of extracellular matrix, leading to progressive fibrosis and loss of lung function. Similar observations (high ELR-CXC chemokines vs. low non-ELR-CXC chemokines) have been described for other inflammatory diseases such as chronic pancreatitis, inflammatory bowel disease (11), and psoriasis (12). In the latter case, high levels of IL-8 and low levels of thrombospondin-1 (an angiogenesis inhibitor) produced by psoriatic keratinocytes directly correlated with their angiogenic capacity, and moreover, correlated with the overexpression of its receptor, CXCR2, but not CXCR1 (13). This suggests a tight relationship between ELRCXC chemokine expression and its receptor, CXCR2, in angiogenesis. Interestingly, CXCR2 shares a high degree of homology with a G-proteincoupled receptor (ORF74) encoded by the Kaposi’s sarcoma–associated herpesvirus-8 (KSHV/HHV8) (14). Bais et al (14) have shown that conditioned media from cells stably transfected with KSHV-GPCR stimulate the growth of human umbilical vein endothelial cells (HUVEC), and they have also suggested that VEGF (vascular endothelial growth factor) might mediate this response. These results are in accordance with earlier studies done by Arvanitakis and collaborators (15), in which they show that this constitutively activated (agonistindependent) KSHV-GPCR signals through the phosphoinositideinositoltrisphosphate-protein kinase C pathway. Thus, PKC (protein kinase C) stimulates the transcription of genes with promoters containing AP-1 sites, such as angiogenic factors (e.g. IL-8 and VEGF) (16, 17). In summary, this KSHVGPCR with high CXCR2 sequence identity is promoting endothelial cell growth and angiogenesis. Therefore, it is possible that CXCR2 mediates angiogenesis in a similar fashion: in a controlled manner during normal wound repair and an abnormal upregulated mode during tumorigenesis.

METASTASIS Tumor development and progression depend heavily on the presence of angiogenic factors. This group of factors includes chemokines as well as growth factors such as VEGF, FGF (fibroblast growth factor), EGF (epidermal growth factor), PDGF (platelet-derived endothelial growth factor), angiogenin, HGF/SF

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(hepatocyte growth factor/scatter factor), and many others not discussed in this review but having equal importance in tumor growth (for a review, see 18). Some of these angiogenic factors, i.e. the chemokines, display a pattern of expression that appears to be imbalanced during tumor development. Luan et al (19) have described an abnormally increased expression of MGSA/GRO (melanoma growth stimulatory activity/growth-related proteins) and decreased levels of IP-10 in melanoma lesions. Similar results were reported by Arenberg et al (8) in non–small cell lung cancer (NSCLC). Interestingly, two subtypes of NSCLC, adenocarcinoma and squamous cell carcinoma, behave very differently, and this behavior correlates with a different chemokine expression pattern. Squamous cell carcinomas show higher levels of IP-10, have lesser metastatic potential, greater patient survival, and less vascularization than adenocarcinomas, which are associated with a worse prognosis. These data support the model established by Strieter et al (10), in which a shift in the balance of ELR vs. non-ELR-CXC chemokine expression determines whether a tumor grows and metastasizes or regresses. Besides the role of chemokines and their receptors in angiogenesis, they also seem to be involved in the process of tumor cell migration, invasion, and metastasis. It is known that certain tumors exhibit certain patterns of metastasis (or invasion) to certain organs; in other words, tumor cells do not migrate randomly. One explanation for this phenomenon is that this specific migration of tumor cells may be determined by the chemokine receptors they express and by the chemokines expressed in the target organs. There is some evidence supporting this hypothesis. Youngs et al (20) have reported that different breast carcinoma cell lines respond differentially to distinct chemokines. Indeed, some of them were unresponsive to the chemokines tested, indicating that tumor cells are not uniform in their ability to migrate in response to chemokines. Kleeff et al (21) have shown that MIP-3a and its receptor CCR6 were expressed by all pancreatic cancer cell lines tested. However, it may be too simplistic to envision that chemokines alone determine the site/s where metastasis develop. Other molecules, such as adhesion molecules (integrin, cadherins), proteases, angiogenic factors, etc, may also be involved in the metastatic fate of tumor cells. Although the involvement of these other molecules in metastasis is beyond the scope of this review (for a review, see 22), an example for this process is given. High levels of the b2 integrin CD11b/CD18 have been detected on an adherent T lymphoma cell line that had high metastatic potential in the kidney. This cell line was very responsive to the chemokines RANTES and MCP-1, produced by a normal kidney cell line. In contrast, the nonadherent parental cell line did not migrate in response to these chemokines, even though both cell lines were able to bind to them. Interestingly, the nonadherent cell line expressed lower levels of CD11b/CD18, which explains its inability to migrate and metastasize in response to RANTES and MCP-1 (23). This suggests that while locally produced chemokines (in this case, by normal kidney cells) may guide these metastatic tumor cells preferentially to the kidney, adhesion molecules expressed by these tumor cells also contribute to their final homing site.

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In conclusion, these results indicate that not all tumor cells respond equally to chemokines, and they also suggest that chemokines and chemokine receptors work in a coordinated fashion with adhesion molecules to determine the ability of tumor cells to invade and colonize other tissues.

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ANGIOSTASIS Angiostasis is another potential antitumor therapeutic effect of chemokines. The expression of most non-ELR-CXC chemokines (IP-10, MIG, I-TAC, PF4) (24– 27) and the receptor they bind (CXCR3) (28) are induced by IFN-c. The angiostatic activity of these molecules seems to be associated with binding to CXCR3. Mouse 6Ckine/SLC (29, 30), a CC chemokine that can bind mouse CXCR3, is also angiostatic (28). However, the mechanism of the angiostatic phenomenon is not known. Luster et al (31) have shown that only IP-10 and PF-4 binding inhibited endothelial cell proliferation in a calcium flux–independent fashion and required the presence of a specific proteoglycan, HSPG, on the surface of the endothelial cells. Whether the HSPG or the CXCR3 is the receptor responsible for chemokine-induced angiostasis is not clear and will require further study.

How Do Cytokines Regulate the Angiostatic/Angiogenic Balance? Several cytokines affect chemokine expression (Figure 2). For example, IL-12 and IL-18, cytokines that induce IFN-c production, synergistically induce tumor regression by inhibiting angiogenesis (32). Moreover, IFN-c is known to be angiostatic, not only because it induces the expression of non-ELR-CXC chemokines, but also because it suppresses the expression of angiogenic CXC chemokines (33, 34). In addition, IFNs are known to inhibit wound healing (35, 36), probably due to their inhibitory effects on endothelial cells (37). Another important cytokine that can affect angiogenesis is IL-10. This cytokine downregulates the expression of MHC II on antigen-presenting cells, which results in the inhibition of proinflammatory cytokine production, such as IFN-c by Th1 cells (38–40). Hence, IL-10 may induce angiogenesis by suppressing IFNc production by T cells and, indirectly, the expression of angiostatic CXC chemokines (41). However, there is evidence that IL-10 may suppress tumor growth by inhibiting angiogenesis. Huang et al (42) have shown that IL-10 blocks macrophage-derived angiogenic factors (e.g. VEGF) and, therefore, tumor growth and metastasis. In summary, an understanding of how the cytokine network regulates this delicate balance of angiostatic and angiogenic chemokines is a promising approach for the control of chronic inflammatory diseases and neoplasias.

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Figure 2 Biological processes and molecules involved in the regulation of angiogenesis.

CHEMOKINES AS THERAPEUTIC AGENTS Chemokines are likely to be primarily responsible for the cell infiltration observed in many disease states. The presence of proinflammatory chemokines may not be beneficial in a chronic inflammatory disease, but it is desirable in diseases where the immune response needs to be promoted, (e.g. cancer). In theory, any chemokine capable of inducing the migration of T, NK cells, dendritic cells, and/or macrophages could promote the regression or even eradication of a tumor mass by boosting the immune response against the tumor. Therefore, several chemokines are now being studied for their potential use as adjuvants in antitumor immune responses. Recent findings have demonstrated that IP-10, MIG, and Lptn have antitumor activity, as well as MCP-1, MCP-3, TCA-3 and others (see below, Antitumor Activity).

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Conversely, a chronic deleterious immune response can be reduced by interfering with the proinflammatory actions of chemokines. Based on this idea, three types of antagonists have received attention: (a) small molecule inhibitors of chemokine receptors, (b) modified chemokines or N-terminal peptides, and (c) neutralizing monoclonal antibodies against chemokines or their receptors (see below, Chemokines in Infectious and Inflammatory Diseases).

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AntiTumor Activity: Chemokines as Agonists In a mouse syngeneic tumor model, Lptn (lymphotactin) has shown antitumor activity only when combined with IL-2 (43). The most likely explanation for this effect is that Lptn induces T and NK cell infiltration (44) to the tumor site, while IL-2 expands the T cell clones upon TCR activation, enhancing a specific immune response. Similar results were obtained by Emtage and collaborators combining Lptn with IL-2 or IL-12 delivered using adenoviral vectors (45). These methods attempt to increase the probability of encounter between T lymphocytes and malignant cells to induce a specific antitumor response. Another approach using chemokines in cancer immunotherapy is DC (dendritic cell)–based vaccines, which favor DC antigen presentation and induce Agspecific CTL responses. For example, Cao et al (46) transduced DC with an adenovirus expressing Lptn and then pulsed the cells with tumor peptides. The modified DC were more effective than uninfected pulsed peptide-DC in reducing metastasis in the 3LL tumor model. These results illustrate the potential use of chemokines as antitumor agents. In addition to Lptn, other chemokines display antitumor activities. IP-10 and MIG are potent chemoattractants for Th1 cells and induce tumor infiltration with CD8` lymphocytes. Several laboratories have demonstrated that IP-10 and MIG are responsible, at least in part, for the antitumor effect of IL-12, mediated through IFN-c (47, 48). Either expression or inoculation of these chemokines or cytokines intratumorally results in tumor regression, associated with extensive vascular damage and necrosis (49–51). Evidently, both the inhibition of angiogenesis and the increased T cell recruitment (52, 31) are a result of the concerted actions of IP-10 and MIG through IL-12, IFN-c, IL-18 (32), and possibly other factors yet to be determined. Other examples of antitumor activity by chemokine gene transfer are MCP-3 (53), MIP-1a (54), MCP-1 (55), and TCA-3 (56). In most cases, augmented leukocyte infiltration occurred in the perivascular areas, peritumoral tissue, or within the tumor that led to tumor necrosis. Various approaches have been used to deliver chemokines in vivo. Some examples, chemokine-transfected tumors (54) or dendritic cells (46), protein injection, and adenoviral vectors, have been mentioned here. Although gene transfer technology is still in its early stages, it has large therapeutic potential, particularly for chemokine delivery. We conclude that inducing chemokine expression in tumors

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and inhibiting it in chronic inflammatory diseases may restore the normal cytokine and chemokine balance and reverse the disease process.

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Chemokines in Infectious and Inflammatory Diseases Chronic inflammatory diseases are characterized by the presence of cell infiltrates, and chemokines are likely to be involved in this phenomenon. The most common way to resolve, or at least ameliorate, this type of disease is to decrease the inflammatory response. Traditionally, this has been accomplished by administering corticosteroids, cyclosporin A, and similar drugs. But due to the success of small molecule inhibitors of GPCRs (G protein–coupled receptors) in the treatment of various diseases, and the fact that chemokine receptors are GPCRs expressed on hematopoietic cells, the pharmaceutical and biotechnology industries have demonstrated particular interest in the production of small-molecule inhibitors of chemokine receptors. In view of the critical role of CCR5 and CXCR4 in HIV infection (1), the search for and development of antagonists against these two chemokine receptors have been the focus of interest of many laboratories. A successful example of antagonists is NSC 651016, a distamycin analog that inhibits HIV-1 replication by downregulating CCR5 and CXCR4 expression (57). Unfortunately, some HIV strains (HIV-2) appear to also use other chemokine receptors as co-receptors for cell entry, such as CCR2b, CCR3, GPR15/BOB (58), US28 (59), and others. Still, the most relevant co-receptor in HIV infection seems to be CCR5, since individuals with defective CCR5 alleles exhibit resistance to HIV-1 infection (60–62). However, the discovery and development of pharmacological antagonists is a long and expensive process. For this reason, other avenues are being explored. For example, modified chemokines and N-terminal peptides can be engineered to allow them to retain binding specificity and affinity to a receptor while blocking it. Several examples have been described in the literature. Plater-Zyberk et al (63) have shown reduced incidence of arthritis in DBA/1 mice treated with the modified chemokine MetRANTES. A similar antagonist, (AOP)-RANTES (amino oxypentane), inhibits HIV-1 infectivity in macrophages and lymphocytes (64). However, the success of modified chemokines or N-terminal peptides as antagonists depends mostly on their capacity to fully occupy the chemokine receptor/ s at nanomolar concentrations, competing with the natural ligand/s binding and thus blocking signaling. One of the advantages of using a modified ligand is that most of the receptors used by that ligand can be blocked or partially blocked by a single antagonist (65). Finally, a third type of antagonist is monoclonal antibodies against chemokines or their receptors. Eosinophils and Th2 lymphocytes are main players in allergic responses. Both cell types express CCR3 (66, 67) and respond through this receptor to several chemokines (RANTES, eotaxin, MCP-4, MCP-3, etc), which makes it a good target for development of antagonists. Heath et al (68) have made a

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monoclonal antibody against CCR3 that blocked chemotaxis and calcium flux induced by all CCR3 ligands. Furthermore, monoclonal antibodies have been proven to work successfully as antagonists in vivo, for example, the anti-TNF-a antibody for the treatment of rheumatoid arthritis (69, 70). This effect may be due, at least in part, to the inhibition of chemokine production brought about by the neutralization of excess TNF-a in the joints of these patients. In sum, all these types of antagonists are promising candidates to block inflammation and/or HIV infection.

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CHEMOKINES IN ORGANOGENESIS AND LYMPHOCYTE TRAFFICKING The proinflammatory activities of chemokines during immune responses have been extensively described in the literature (71). However, in recent years we have learned that chemokines are also involved in noninflammatory functions such as the regulation of lymphocyte trafficking, T and B cell development, and particularly cell compartmentalization within lymphoid tissues (T cell vs. B cell areas). The redundancy of chemokine expression plus the promiscuity of ligandreceptor binding have made it very difficult to understand how the cell migration process works in vivo. Nevertheless, recent findings suggest that chemokine/ receptor expression changes in cells during development and between organs (Figure 3), as do the chemotactic responses.

THYMUS Various chemokines are expressed in the thymus at significant levels: MDC, TARC, 6Ckine/SLC/TCA-4, TECK, SDF-1, and MIP-3b/ELC. Some thymusexpressed chemokines attract mature lymphocytes. For example, MDC (72) attracts activated T cells among other cells (73); TARC attracts peripheral blood T cells (74); TCA-4/6Ckine attracts mature T cells (75). Therefore, this group of chemokines may coordinate the trafficking of mature T cells into the thymus. However, chemokines such as TECK, MIP-3b, and SDF-1 are also able to attract immature T cells and thymocyte subsets with varying efficacy (76). Thus, the specific localization of thymocyte subsets within the thymus (cortex or medulla) may possibly be explained by the differential chemotactic response of these subsets. Mouse TECK is expressed by thymic dendritic cells and induces the migration of thymocytes, but not mature peripheral T cells (4). Recently, GPR9–6/CCR9, a thymus-expressed GPCR, has been reported to be the receptor for TECK (77) and was specifically detected in immature and mature thymocytes. Based on these

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Figure 3 Chemokine expression in lymphoid organs.

data, TECK could attract T cell progenitors from bone marrow to fetal thymus and retain them until T cell development is completed. Probably, once T cells are ready to move to the periphery, they may lose GPR9–6 expression and be able to leave the thymus microenvironment. It is possible that this receptor may be downregulated in peripheral T lymphocytes, since Vicari et al (4) did not detect any chemotactic response with these cells, nor did Wilkinson et al (78). In contrast, there is evidence that TECK may not be responsible for the migration of T cell progenitors to the thymus, since Wilkinson et al (78) have shown that antibodies to TECK did not prevent thymus recolonization by T cell precursors. GPR9–6-deficient mice may help resolve this paradox. SDF-1 and MIP-3b are also chemoattractants for thymocytes. Kim et al (76) have done an extensive characterization of the chemotactic responses of thymocyte subsets to these two chemokines. Thymocytes can be divided into four subpopulations based on their expression of CD4 and CD8 that comprise the immature double negative (DN) CD41 CD81, and double positive (DP) CD4` CD8` subsets and the mature CD4` and CD8` single positive cells (SP). Interestingly, immature DN and DP thymocytes are more responsive to SDF-1 and are located in the thymic cortex, while mature CD4` SP and CD8` SP are more responsive to MIP-3b and are located in the medulla, from where mature T cells migrate to the peripheral blood. These results suggest that chemokines may control the compartmentalization seen within lymphoid organs and coordinate, along with other molecules, T cell development.

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It is very likely that chemokines will be found to regulate intrathymic T cell migration in a very precise manner. As developing T cells achieve specific stages of development (79), they may change their chemokine receptor expression and become responsive to other chemokines that will induce their migration to new intrathymic locations where their subsequent development may proceed. These observations, however, raise the question of whether chemokines merely control intrathymic cell migration or also have direct differentiation effects on the developing thymocytes.

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BONE MARROW SDF-1 is a chemoattractant for T cells, B cells, and megakaryocytes (80) that selectively binds to CXCR4. SDF-1 is a growth factor for progenitor B cells, and chemotactic for human pre- and pro-B cell lines, as well as mature B cells (81, 82). Bone marrow (BM) stromal cells produce SDF-1, which attracts B cell progenitors and places them in contact with the stromal cells. These cells release growth and differentiation factors that are necessary for B cell maturation (81). Also, SDF-1 induces bone marrow colonization by hematopoietic precursor cells (CD34` cells) during embryogenesis (83). These data suggest that SDF-1 and CXCR4 expression are essential for B cell development and maturation in the BM. This has been confirmed by analysis of SDF-1- and CXCR4-deficient mice, which show defects in the hematopoietic, nervous, and vascular systems (84– 86). Ma et al (87) and Kawabata et al (88) have reported the phenotype of the CXCR4-deficient mouse. In these mice, B cell precursors are decreased in fetal liver and BM and abnormally increased in blood. Granulocytes are also decreased in BM and elevated, but immature, in blood. Evidently, B cell and granulocytic precursors are released prematurely into the periphery. Although T cells express CXCR4, their development does not seem to be impaired in the CXCR4-deficient mice. Interestingly, these mice show normal T and B cell localization in secondary lymphoid organs. In summary, CXCR4 may regulate B lymphopoiesis and myelopoiesis by retaining the precursors within the fetal liver and BM microenvironment. MIP-3b is also expressed by bone marrow stromal cells, but only upon LPS (lipopolysaccharide) stimulation. It specifically attracts macrophage progenitors (76) and may increase the number of macrophage progenitors in the BM during inflammatory responses. In this way, more macrophages are generated and exported to the blood during inflammation. Other chemokines expressed in the BM are HCC-1 (89), MCP-2 (90), and MIP-1a (91). However, the B cell–specific chemoattractant BCA-1/BLC is not expressed in BM (92). The role that each chemokine plays in the BM microenvironment is still under investigation. Nevertheless, some chemokines have shown inhibitory effects on the proliferation of hematopoietic progenitors. Some

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examples are MPIF-1, MPIF-2 (93), MIP-1a (94), PF4, and IL-8 (95). Some of these antiproliferative chemokines may be useful therapeutically by preventing cellular damage during anticancer therapies such as chemotherapy or radiation.

SECONDARY LYMPHOID ORGANS

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B Cells The B cell homing chemokine BLC/BCA-1 (96, 92) is expressed in follicles in the spleen, Peyer’s patches, and lymph nodes, specifically in FDC (follicular dendritic cells). Its receptor, BLR-1/CXCR5 (97) is mainly expressed on B cells, Burkitt’s lymphoma cells, and to a lesser extent, in T cells (3) and monocytes (98). Forster et al (97) demonstrated that BLR-1/CXCR5 is required for B cell migration into splenic and Peyer’s patch follicles and for germinal center formation, but not for migration into lymph node follicles. This correlates with a report by Gunn et al (96) who did not detect BLC expression in lymph node follicles. Evidently, other B cell chemoattractants may be responsible for B cell migration into lymph node follicles. One candidate is MIP-3b, which is expressed in lymph nodes and induces B, T, and dendritic cell chemotaxis (76, 99, 100). Interestingly, MIP-3b attracts these cell populations to lymph nodes that are involved in the antigen presentation process, assisting in the initiation of the immune response. Another probable candidate responsible for the B cell migration into lymph node follicles is MIP-3a, which also attracts B cells (101). Another B cell chemoattractant expressed in secondary lymphoid organs is SDF-1. Bleul et al (82) showed that naive and memory B cells are responsive to SDF-1; however, germinal center B cells are not. These cells are responsive to BCA-1, which retains them within the germinal center to undergo somatic hypermutation and affinity maturation. These data correlate very well with chemokine receptor expression, since only naive and resting B cells express CXCR5 (BCA1/BLC binds to CXCR5). Once B cells have been activated, the cells surrounding GC (germinal center/s) express SDF-1 (SDF-1 binds to CXCR4), which probably attracts them out of the GC. Although B cells express CXCR4 at all stages, GC B cells are not responsive to SDF-1a. This could be due to the downregulated surface expression of CXCR4 on GC B cells being much slower than on naive or memory B cells, when incubated with SDF-1. In addition, when GC cells are differentiated in vitro into memory B cells, they increase L-selectin, ICAM-1, LFA-3, and B7.1/CD80 expression as well as responsiveness to SDF-1a (82). This indicates that other molecules, such as adhesion molecules, can also be responsible for changes in chemokine responsiveness of cells.

T Cells SLC/6Ckine/Exodus-2 (30, 29, 102) is expressed in HEV (high endothelial venules) and in T cell areas in the spleen, Peyer’s patches, and lymph nodes, and the

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marginal zone of follicles. Indeed, Willimann et al (103) demonstrated that the cells expressing SLC/6Ckine in human lymph nodes were interdigitating dendritic cells. Although SLC/6Ckine is expressed in other nonlymphoid organs, Gunn et al showed that this is due to its expression on lymphatic endothelium (104). Probably, the role of SLC/6Ckine on lymphatic endothelium is to recruit lymphocytes from tissues to draining lymphatics. SLC/6Ckine attracts naive T cells (104), dendritic cells (100), and less efficiently, B cells (104). In addition, it induces firm adhesion of naive T cells via b2 integrin binding to ICAM-1 (104). Moreover, Nakano et al (105) have described a T cell–homing defect in plt mice ( paucity of lymph node T cell) that is related to a gene localized on mouse chromosome 4, where SLC/6Ckine gene maps. Recently, Gunn et al (106) have reported that these mice do not express SLC/6Ckine mRNA, though the sequence of SLC/6Ckine introns and exons is normal. In addition, DCs do not accumulate in the T cell zones of lymph nodes and spleen in the plt mice, which correlates with the DC-chemoattractant ability of SLC/6Ckine described by Kellerman et al (100). These data indicate that SLC/ 6Ckine mediates the homing of naive T lymphocytes and dendritic cells to secondary lymphoid organs. Interestingly, DC-CK1/PARC also attracts naive T lymphocytes. It is expressed by dendritic cells of lymph node–germinal centers and in T cell areas of secondary lymphoid organs (107, 108). One possibility is that SLC/6Ckine directs the migration of naive T cells to lymph nodes through HEV, and then DC-CK1/PARC guides them to the germinal centers. DC-CK-1/ PARC is expressed by a specific subpopulation of human DCs (107) located in close contact with T cells in germinal centers (in human tonsils, spleen, and lymph nodes). However, this human DC subset appears to be involved in maintaining the activation state of GC memory T cells and promoting T-B cell interaction (109) rather than activating naive T cells, since no naive T cells could be detected in the GCs (109). In fact, it is more likely that MDC, TARC, or MIP-3a perform this function. MDC is expressed by monocyte-derived-DC and lymph node-DCs (Langerhans cells) (72, 110) and attracts activated T lymphocytes. TARC is also produced by monocyte-derived DCs and attracts activated T cells (111). In fact, it may be the most DC-specific chemokine. MIP-3a/LARC is expressed in lymph nodes (112, 113) and attracts B cells, activated T cells (114), and CD34`-derived DCs (115). In summary, most of the chemokines previously mentioned attract T, B, and dendritic cells, cells that are involved in the antigen presentation process. This suggests that the seeming redundancy of chemokine expression in lymphoid organs may serve the purpose of attracting different cell subsets into different microenvironments and, consequently, may regulate the antigen presentation process. In addition, this redundancy can also be explained by the fact that chemokines can work sequentially, guiding each cell subpopulation to a specific site (116). Further investigation will be needed to clarify the individual role of each of these chemokines in lymphocyte trafficking and antigen presentation.

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HOMEOSTATIC VERSUS INFLAMMATORY CHEMOKINES When discussing the function of chemokines, it is possible to make a distinction between those whose expression suggests more of a homeostatic function than a regulatory function in inflammation. Many chemokines show an expression pattern that strongly suggests a role in inflammation. They are typically induced in either monocytes or macrophages or in epithelial, endothelial, or fibroblastic cells by proinflammatory cytokines (IL-1, TNF-a, or IFN-c) or stimuli (LPS). This is in fact the most common perceived role for chemokines, a proinflammatory function, frequently associated with a Th1 cytokine expression profile (IFN-c, IL-2, IL-12) and thus with a Th1 cell infiltrate at the inflammation site. However, not all chemokines fit this pattern. For example, other chemokines [e.g. C10 (117), DC-CK1/AMAC-1/PARC (118)] are specifically induced by Th2 cytokines (IL4, IL-10, IL-13) in monocytes or other cells. We recently reported that a new member of the HCC subfamily of chemokines, HCC-4 (119), is induced by IL10 in monocytes (IL-10 is generally considered an anti-inflammatory cytokine). The latter observations raise the possibility that the production of certain chemokines will be associated with Th1 responses (proinflammatory) while others will be associated with Th2 responses. Recently, it has been reported that Th1 cells produce more chemokines than Th2 (120). It is therefore very likely that there will be a segregation of Th1 vs Th2 chemokines depending on the nature of the developing immune responses. This is an area that deserves further investigation. While the identification of chemokines associated with Th1 or Th2 responses is a developing story, the association of chemokine receptor expression with the Th1 or the Th2 phenotypes is well established (reviewed by Lanzavecchia et al in this volume). Several chemokine receptors are associated with the Th1 phenotype (including CXCR3 and CCR5), while others are associated with the Th2 phenotype (CCR3, CCR4, and CCR8) (for a review, see 121). However, some receptors are associated more than others. For example, CCR8 expression is far more abundant at the mRNA level in activated Th2 cells than CCR3. It is likely that CCR3 may actually define a subset within the Th2 population of cells. The expression of chemokine receptors in T helper cells depends on the state of activation of these cells. For example, CXCR3 is present in resting Th1, while CCR8 is only present in activated Th2. Finally, the issue arises of whether the expression of these receptors is only for the purposes of directing the migration of developing Th1 or Th2 cells to the appropriate sites where these responses are occurring, or whether the corresponding ligands may actually have effects on the differentiation of Th1 or Th2 cells. In support of the latter possibility, it has been reported that MIP-1a (a CCR5 ligand) promotes differentiation toward a Th1 phenotype (122). In contrast, there are several chemokines whose expression strongly suggests a homeostatic role. Typically, this is due to the fact that they are expressed in

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normal organs or tissues in the apparent absence of inflammatory stimuli, and/or they may be produced by cells that do not typically participate as active inducers of inflammation. For example, the chemokine HCC-1 is present in large concentrations in serum and is constitutively expressed in several organs (89). Several members of this family (HCC-2 and HCC-4—also called LEC) show strong constitutive expression in various organs, suggesting a homeostatic role. In this category, we could also include many other chemokines that tend to be tissue specific, including some described earlier, such as TECK (abundantly expressed in the thymus), which is likely to have an organ-specific function (for example, involvement in T cell development). Still, other chemokines may represent a ‘‘mixed’’ category, with both homeostatic and inflammatory functions. An example of the latter is fractalkine (123), which is highly expressed in the brain and may have a homeostatic function there but is also induced by TNF-a in endothelial cells and may thus participate in inflammatory reactions.

CONCLUSION The chemokines and their receptors have received increasing attention in the last few years. Besides their role in HIV pathogenesis, it is now clear that chemokines participate intimately in many pathological conditions like inflammation and autoimmunity. They also play a very important role in normal homeostasis, including lymphoid development and migration. Some chemokines have potential therapeutic applications, mainly in cancer through their ability to attract subpopulations of lymphoid cells and also through their angiostatic effects. The nature of their receptors (seven-transmembrane G-protein-coupled receptors) also makes them compelling candidates as therapeutic targets in many areas where chemokines are involved. In addition, chemokines are likely key regulators of immune responses. In particular, the emerging picture is that of a discrete network of cells interacting through their specific production of chemokines or expressing a highly specific pattern of chemokine receptors. The emerging view of this specificity has replaced the older view of a redundant system and in turn suggests that chemokine receptors, in particular, have the potential of evolving into specific markers to identify subsets of cells involved in a given immune response. Little effort has so far been directed toward the functional characterization of subsets of immune cells defined by chemokine receptor expression. This situation will probably change when reagents become available. There is an important need for reagents for flow cytometry as well as a need for neutralizing monoclonal antibodies in order to advance this field. We should expect advances (and surprises) to come from the chemokine field. It is one of the first molecular families on which we have witnessed the impact of bioinformatics and genomics. It is therefore also valuable to learn from these

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TABLE 2 Proposed new nomenclature for human chemokines Systemic name

Human ligand

Mouse ligand (if only the mouse ligand is known)

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CXCL CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL15

Groa/MGSA-a Grob/MGSA-b Groc PF4 ENA-78 GCP-2 NAP-2 IL-8 Mig IP-10 I-TAC SDF-1/PBSF BLC/BCA-1 BRAK/Bolekine Lungkine

XCL XCL1 XCL2

lymphotactin/SCM-1a/ATAC SCM-1b

CX3CL CX3CL1

Fractalkine/neurotactin

CCL CCL1 CCL2 CCL3 CCL4 CCL5 (CCL6) CCL7 CCL8

I-309 MCP-1 MIP-1a MIP-1b RANTES C10/MRP-1 MCP-3 MCP-2

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TABLE 2

(continued) Proposed new nomenclature for human chemokines

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Systemic name (CCL9/10) CCL11 (CCL12) CCL13 CCL14 CCL15 CCL16 CCL17 CCL18 CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27

Human ligand

Mouse ligand (if only the mouse ligand is known) MRP-2/CCF18/MIP-1c

Eotaxin MCP-5 MCP-4 HCC-1/HCC-3 HCC-2/leukotactin HCC-4/LEC TARC DC-CK1/PARC/AMAC-1 MIP-3b/ELC/exodus-3 MIP-3a/LARC/exodus-1 6Ckine/SLC/exodus-2 MDC/STCP-1/ABCD-1 MPIF-1 MPIF-2/Eotaxin-2 TECK SCYA26/Eotaxin-3 (MCC)/ALP/CTACK/ESkine

developments so that in the future we will be able to apply some of the lessons learned from the chemokines to other molecular families. ACKNOWLEDGMENTS We thank Elizabeth Oldham and Lewis Lanier for their comments and suggestions, and Dovie Wyler for reviewing the manuscript. DNAX Research Institute is supported by Schering-Plough Corporation. Note Added in Proof: Proposed Nomenclature At the 1998 Gordon Conference on Chemokines, a Committee headed by Dr. Osamu Yoshie (Kinki University, Japan) was formed to study options for a standardized new chemokine ligand nomenclature. It became clear that with the proliferation of new chemokines, there was a problem with their nomenclature. Often, various groups published the same molecule with different names, leading to confusion in the literature. Dr. Yoshie presented his recommendation at the 1999 Keystone Symposium on Chemokines; it was approved by the audience

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composed of chemokine researchers from most leading laboratories worldwide. Table 2 here shows the recommendation for a new systematic nomenclature for human chemokines. It is based on the fact that the genes for chemokines have already received a standardized designation. For example, the CC gene family is called SCYA (small cytokine-A family) followed by a number. The proposal uses the same numbering system already in use for chemokine genes, but replaces the SCY abbreviation with CXCL for the CXC family, CCL for the CC family, XC for the lymphotactin, and CX3CL for fractalkine. In the case of the CC chemokines, there are some spaces for ligands that have been described in the mouse, but not yet in the human. However, it offers the advantage that the number representing a particular gene will be the same as its ligand, which is represented by the letter ‘‘L’’ after the family (CXC, CC, XC, CX3C). Thus, this nomenclature proposal is analogous to the current one in use for receptors and offers the added advantage that each ligand will be immediately recognizable as belonging to a particular subclass. But the main advantage is that it eliminates nomenclature ambiguities when referring to each chemokine. It is recommended that future articles use this nomenclature. Visit the Annual Reviews home page at www.AnnualReviews.org.

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cytes. Proc. Natl. Acad. Sci. USA 95:258–63 Nakano H, Mori S, Yonekawa H, Nariuchi H, Matsuzawa A, Kakiuchi T. 1998. A novel mutant gene involved in Tlymphocyte-specific homing into peripheral lymphoid organs on mouse chromosome 4. Blood 91:2886–95 Gunn MD, Kyuwa S, Tam C, Kakiuchi T, Matsuzawa A, Williams LT, Nakano H. 1999. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J. Exp. Med. 189:451–60 Adema GJ, Hartgers F, Verstraten R, de Vries E, Marland G, Menon S, Foster J, Xu Y, Nooyen P, McClanahan T, Bacon KB, Figdor CG. 1997. A dendritic-cellderived C-C chemokine that preferentially attracts naive T cells. Nature 387:713–17 Hieshima K, Imai T, Baba M, Shoudai K, Ishizuka K, Nakagawa T, Tsuruta J, Takeya M, Sakaki Y, Takatsuki T, Miura R, Opdenakker G, Van Damme J, Yoshie O, Nomiyama H. 1997. A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-1 alpha/LD78 alpha and chemotactic for T lymphocytes, but not for monocytes. J. Immunol. 159:1140–49 Grouard G, Durand I, Filgueira L, Banchereau J, Liu YJ. 1996. Dendritic cells capable of stimulating T cells in germinal centres. Nature 384:364–67 Tang HL, Cyster JG. 1999. Chemokine Up-regulation and activated T cell attraction by maturing dendritic cells. Science 284:819–22 Imai T, Nagira M, Takagi S, Kakizaki M, Nishimura M, Wang J, Gray PW, Matsushima K, Yoshie O. 1999. Selective recruitment of CCR4-bearing Th2 cells toward antigen- presenting cells by the CC chemokines thymus and activationregulated chemokine and macrophage-

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1994. Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages. J. Immunol. 152:5084–91 Kodelja V, Muller C, Politz O, Hakij N, Orfanos CE, Goerdt S. 1998. Alternative macrophage activation-associated CCchemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern. J. Immunol. 160: 1411–18 Hedrick JA, Helms A, Vicari A, Zlotnik A. 1998. Characterization of a novel CC chemokine, HCC-4, whose expression is increased by interleukin-10. Blood 91:4242–47 Bradley LM, Asensio VC, Schioetz LK, Harbertson J, Krahl T, Patstone G, Woolf N, Campbell IL, Sarvetnick N. 1999. Islet-specific Th1, but not Th2, cells secrete multiple chemokines and promote rapid induction of autoimmune diabetes. J. Immunol. 162:2511–20 O’Garra A, McEvoy LM, Zlotnik A. 1998. T-cell subsets: chemokine receptors guide the way. Curr. Biol. 8:R646– 49 Karpus WJ, Kennedy KJ. 1997. MIP1alpha and MCP-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as Th1/Th2 lymphocyte differentiation. J. Leukocyte Biol. 62:681–67 Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, Greaves DR, Zlotnik A, Schall TJ. 1997. A new class of membrane-bound chemokine with a CX3C motif. Nature 385:640–44

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Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:245–273 Copyright q 2000 by Annual Reviews. All rights reserved

DENDRITIC CELLS IN CANCER IMMUNOTHERAPY Lawrence Fong and Edgar G. Engleman Departments of Pathology and Medicine, Stanford University School of Medicine, Palo Alto, California 94304; e-mail: [email protected]

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Key Words clinical trial, tumor immunology, T cell, vaccine Abstract The potential to harness the potency and specificity of the immune system underlies the growing interest in cancer immunotherapy. One such approach uses bone marrow–derived dendritic cells, phenotypically distinct and extremely potent antigen-presenting cells, to present tumor-associated antigens and thereby generate tumor-specific immunity. Support for this strategy comes from animal studies that have demonstrated that dendritic cells, when loaded ex vivo with tumor antigens and administered to tumor-bearing hosts, can elicit T cell–mediated tumor destruction. These observations have led to clinical trials designed to investigate the immunologic and clinical effects of antigen-loaded dendritic cells administered as a therapeutic vaccine to patients with cancer. In the design and conduct of such trials, important considerations include antigen selection, methods for introducing the antigen into MHC class I and II processing pathways, methods for isolating and activating dendritic cells, and route of administration. Although current dendritic cell–based vaccination methods are cumbersome, promising results from clinical trials in patients with malignant lymphoma, melanoma, and prostate cancer suggest that immunotherapeutic strategies that take advantage of the antigen presenting properties of dendritic cells may ultimately prove both efficacious and widely applicable to human tumors.

INTRODUCTION To date, human tumor immunotherapy has met with only limited success. Among the reasons for this have been the limited availability of tumor-associated antigens and the inability to deliver such antigens in a manner that renders them immunogenic in patients with cancer. However, recent insights into the role of dendritic cells (DC) as the pivotal antigen-presenting cells (APC) that initiate immune responses may provide the basis for generating more effective antitumor immune responses. These rare bone marrow–derived cells are uniquely capable of sensitizing naive T cells to protein antigens. The role of DC in initiating or ‘‘priming’’ immune responses to viral and bacterial antigens in vivo is well established (1– 0732–0582/00/0410–0245$14.00

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3). By harnessing the capacity of DC to present tumor antigens to T cells, DC may serve as the centerpiece of an immunotherapeutic approach to cancer. The ability of the immune system to recognize and attack tumors has been demonstrated unequivocally. While both humoral and cellular effector mechanisms can contribute to tumor lysis, the latter are felt to be responsible for tumor regression in the majority of cases. CD8` cytotoxic T lymphocytes (CTL), in particular, have been demonstrated to recognize and kill cancer cells in various tumor models (4 –6). The ability of DC to prime T cells capable of recognizing and killing tumor cells in an antigen-specific fashion has also been demonstrated in various animal models (7–10). Moreover, DC-based immunization can lead to immunologic memory with protection against subsequent tumor challenges (10).

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CONSIDERATIONS IN DC IMMUNOTHERAPY Defining DC Mature DC have a distinct morphology characterized by the presence of numerous membrane processes that can extend for up to hundreds of micrometers. These processes can take the form of dendrites, pseudopods, or veils (Figure 1). Additional morphologic features of DC include high concentrations of intracellular structures related to antigen processing such as endosomes, lysosomes, and the Birbeck granules of Langerhans cells (LC) of the epidermis. DC are also characterized by the presence on their surface of large amounts of class II MHC antigens and the absence of lineage markers including CD14 (monocyte), CD3 (T cell), CD19,20,24 (B cell), CD56 (natural killer cell), and CD66b (granulocyte) (Table 1) (11). Not surprisingly, in light of their antigenpresenting functions, DC also express various adhesion and costimulatory molecules. Examples of the former include CD11a (LFA-1), CD11c, CD50 (ICAM-2), CD54 (ICAM-1), CD58 (LFA-3), and CD102 (ICAM-3), although all of these markers can be found on monocytes and macrophages (12). Costimulatory molecules such as CD80 (B7.1) and CD86 (B7.2), and molecules regulating costimulation such as CD40 are also expressed on mature myeloid DC (13, 14). DC phenotype varies with different stages of maturation and activation. Human DC precursors circulating in the blood initially can express CD2, 4, 13, 16, 32, and 33, but they gradually lose their expression of these antigens with maturation (15). In contrast, adhesion molecules, costimulatory molecules, and MHC antigens increase with maturation. CD80 and 86 are upregulated with activation, particularly with CD40 ligation. CD86 tends to appear earlier in maturation, while CD80, which is almost unmeasurable in blood precursors, appears later (16). Several antibodies have been described that preferentially but not exclusively stain mature DC. Antibodies reactive against human DC include anti-CD83 and CMRF-44. Antibodies to CD83 stain mature activated DC, but not DC precursors, and also cross-react with activated B cells (17). CMRF-44 can stain circulating

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Figure 1 DC morphology. A. Dendritic cells purified from peripheral blood demonstrate characteristic dendrites after several days of culture in vitro. B. Dendritic cells within the skin are evident on silver stain.

blood DC as well as activated DC, but it also cross-reacts with macrophage and monocytes (18). Antibodies directed at mouse DC include 33D1, N418 (antiCD11c), and DEC-205, although these antibodies also stain monocytes to varying degrees (19–21). DC possess various functional properties that distinguish them from other APC in vitro and in vivo. The ability to present antigens to naive T cells represents one of the most crucial functions of DC. While DC priming can be assessed in vitro, these assays can be difficult to perform routinely (22, 23). As a result, many

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TABLE 1 DC surface phenotype

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DC Langerhans Activated MonocyteActivated precursors cells DC derived DC Monocytes B cells MHC Class I Class II

` `

` ``

` ```

` ``

` `

`` ``

Lymphoid CD2 CD3 CD4 CD19 CD56

`/1 1 ` 1 1

1 1 1 1 1

1 1 1 1 1

1 1 1 1 1

`/1 1 1 1 1

`/1 1 1 ` 1

1 1 1 1 `

` 1 `/1 1 `

` ` ` 1 `

1 ` ` 1 `

`/1 `/1 `/1 ` `/1

1 1 1 `/1 1

Costimulatory molecules CD80 1 CD86 ` CD40 `/1

` ` `

`` ``` `

` ` `

1 ` `

`` `` ``

Antigen receptors Mannose receptor Dec-205 CD16, CD32 CD35 CD36

1 1 ` ` `

` ` ` ` `

` ` 1 `/1

` ` ` ` `

` `/1 ` ` `

1 `/1 `/1 ` 1

Adhesion molecules CD11a (LFA-1) CD58 (LFA-3) CD54 (ICAM-1)

` ` `

` ` `

` ` ``

` ` `

` ` `

` ` ``

DC markers CD83 CMRF-44

1 1

`/1 1

` `

`/1 `

1 `

` `

Myeloid CD1a CD1b CD1c CD14 CD33

groups rely on the ability of DC to induce an allogeneic mixed leukocyte reaction (MLR) (24). In this respect, DC are 10 to 100 times more potent than monocytes or B cells at stimulating allogeneic T cells to proliferate. DC can also stimulate

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autologous T cells, potentially presenting either self or exogenous antigens (including those potentially derived from foreign serum such as BSA) (25, 26). Allogeneic and autologous MLR assays, however, do not assess antigen uptake and processing. While macrophages are more efficient at phagocytosis of larger particles, such as cellular debris and bacteria, freshly isolated DC are active at pinocytosis of soluble antigen. Like macrophage, DC possess nonspecific antigen uptake receptors though at lower levels. Some DC express FccR (CD16, CD32) and complement receptors (CD11b, CD11c, CD35). CD11c may also act as a receptor for LPS as DC lack the classical LPS receptor, CD14, and yet respond to this stimulus. DC also can take up antigen through mannose receptors, potentially through the macrophage mannose receptor or through the receptor recognized by the DEC205 antibody (27). With DC activation and migration from the tissues, antigen uptake activity and the associated antigen receptors are downregulated, resulting in a switch in APC function from antigen uptake to antigen presentation (28). DC are capable of processing antigen via classical pathways: endogenous antigens via the proteasome into the MHC class I compartment, and exogenous antigens via endocytic lysosomes into the MHC class II compartment (29). DC also possess alternative pathways of antigen processing and can route exogenous antigen into the MHC class I pathway through a mechanism known as cross-priming (30). DC may also utilize molecular chaperones such as heat shock proteins (e.g. hsp96) to deliver antigens via the class I pathway (31). Sensitizing the immune system to specific antigens is certainly the most pertinent function for DC, and this has been examined both in vitro and in vivo. Early studies in mice demonstrated that DC exposed to infectious influenza virus or influenza nucleoprotein peptide, in vitro, induce a primary proliferative and antiviral CTL response (3, 32). Similarly, DC induced specific CTL for sendai, herpes simplex, and Moloney leukemia viruses (2). We have demonstrated that human DC, but not monocytes or B cells, can sensitize naive T cells to soluble protein antigens, enabling the generation of antigen-specific CD4` helper and CD8` CTL lines, in vitro (22, 23). In vivo priming of CD4` helper and CD8` cytotoxic T cells with antigenloaded DC has been demonstrated in several animal models. DC can also prime immune responses to antigens in vivo in humans (33, 34). Moreover, administration of DC loaded with tumor-associated peptides or proteins can lead not only to specific proliferative responses and CTL, but also to tumor protection in animal models (7–10). These observations provide a compelling rationale for pursuing DC-based immunotherapy for cancer.

DC Ontogeny and Function Although there is general agreement that DC are derived from hematopoietic stem cells, recent studies indicate that DC can arise from at least two distinct lineages. The precise relationship between these lineages remains controversial, partly

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because no specific markers for DC precursors have yet been identified. However, the heterogeneity of DC lineage and the possibility that the resulting DC subpopulations differ in their functions have significant implications when considering DC-based vaccination strategies. In humans, the more classical ‘‘myeloid’’ DC are derived from a committed DC precursor or granulocyte/monocyte precursor. DC from this lineage appear in the blood as typical lymphoid cells that express high levels of MHC class II antigens and lack most lineage-specific markers. Myeloid DC can also be derived from several cell types previously thought to be terminally committed. For example, monocytes and granulocyte precursors can differentiate into DC when exposed, in vitro, to appropriate combinations of cytokines including GM-CSF, TNF-a with or without IL-4 (35, 36). More recently, Shortman and colleagues described a population of DC derived from lymphoid progenitors in mice (37, 38). This cell type appears to arise from CD4`CD8` lymphoid precursors and can be induced to differentiate, in vitro, without GM-CSF (39, 40). Knockout mouse models have also implicated a lymphoid DC lineage. For example, Rel B knockout mice, in addition to their defects in NK and B cell development, have impaired production of mature DC, although LC develop normally in these mice (41, 42). Ikaros knockout mice lack T, B, and NK cells as well as DC, again implicating a role for a lymphoid lineage in the development of DC (43). While these ‘‘lymphoid’’ DC, characterized by their expression of CD8a homodimers, are felt to play a role in negative selection in the thymus, they clearly can prime naive T cells in the periphery. However, CD8a` DC appear to bias CD4` T cell priming to a Th1 response, while CD8a1 DC appear to bias toward a Th2 response (44, 45). No human CD8` DC have been described. DC derived from CD10` lymphoid precursors have been described in humans, although their role in the immune system has not been resolved (46– 48). More recently, reports of a distinct human DC subpopulation expressing high levels of CD123 (IL-3 receptor) and CD4 and lacking the CD11c myeloid DC marker have appeared (48, 49). Identified in blood and tonsil, these CD123` DC precursors require IL-3 for survival and an activation signal, such as CD40L, for maturation. They appear to bias CD4` T cell priming to a Th2 response, in contrast to myeloid CD11c` DC, which preferentially induce a Th1 response (50). These CD123hi DC also appear to be a major source of type I interferons and may, therefore, possess effector immune function as well (51).

Dendritic Cell Life Cycle In order to effectively exploit DC function, the different stages of DC maturation must be understood. DC precursors migrate from bone marrow and circulate in the blood to specific sites in the body where they mature and act as sentinels for the immune system (Figure 2) (52). This trafficking to the tissues is directed by expression of the chemokine receptors CCR1, CCR5, and CCR6 as well as adhe-

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Figure 2 DC life cycle. DC migrate into peripheral tissues through the vascular endothelium, drawn to such sites by chemokines (for example, MIP1a, MIP-3a, and RANTES). Following activation by one or more stimuli [e.g. LPS, IFN-a or c, RANTES, bacteria, viruses, certain unmethylated immunostimulatory oligonucleotides, also known as CpG, MIP-3b. or SLC (secondary lymphoid tissue chemokine)], DC emigrate from the tissues via the afferent lymphatics to lymph nodes, where they interact with T cells.

sion molecules such as the CD62P-ligand (53–55). Tissue-resident DC, including LC in skin, hepatic DC in the portal triads, mucosal DC, and lung DC, sample the environment, processing and presenting antigens in the context of MHC class I and II molecules. Sources of antigen can include viral proteins, bacterial proteins, or apoptotic bodies (1, 56, 57).

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In addition to encountering antigen, DC are thought to require an antigenindependent danger signal in order to become activated. These signals can include LPS, interferon (IFN) a and c, IL-1b, and potentially direct signals from viruses and bacteria. With activation, DC downregulate their antigen uptake and processing functions and shift to antigen presentation and upregulate their expression of MHC, costimulatory, and adhesion molecules as described earlier. With maturation during in vitro culture, MHC class I and II densities on the cell surface increases 10–100-fold, while MHC synthesis is reduced. Class II peptide-MHC complex turnover falls dramatically, as reflected by the change in surface halflife of these complexes from a few hours on immature DC to 2 days on mature DC, providing a stable source of processed antigen when danger signals are received (58). Once activated, DC leave the tissues and migrate via the afferent lymphatics to the T cell–rich paracortex of the draining lymph nodes, drawn by chemokines MIP-3b and SLC through upregulation of their receptor CCR7 (59, 60). Within the secondary lymphoid organs, activated DC elaborate chemokines such as DCCK1 and MDC that attract naive and memory T cells for priming (61, 62). Activated DC also secrete cytokines including IL-7 and IL-12, which contribute to their unique functions. IL-7 can induce CD4 and CD8 T cell proliferation and B cell differentiation (63, 64). IL-12 biases T helper responses toward a Th1 pattern (65, 66). In contrast to DC, monocytes secrete significant levels of IL-4, -5, and -10 and do not secrete IL-7. This combination of cytokines will typically bias T helper responses to Th2 or antibody generation. This stimulatory milieu produced by activated DC, combined with the presentation of epitopes in MHC class I and class II and the expression of costimulatory molecules, contributes to the generation of potent antigen-specific immune responses (67). Within the secondary lymphoid tissues, activated CD4` T cells can further activate DC through CD40/ 40L interactions, and this also provides a survival signal to the DC (68–70). Furthermore, CD40 ligation allows DC to prime CTL, even in the absence of immediate CD4` T cell help. Preliminary evidence also exists for the ability of DC to present antigen to natural killer cells (NK), perhaps through CD1 (71). This may serve as another mechanism through which DC can elicit an effector immune response.

Sources of DC The earliest studied DC were derived from mouse spleen (72). Their isolation relied upon a transient exposure of the cells to tissue culture plates, which allowed for the depletion of adherent monocytes. Lymphocytes were then depleted by passage over a bovine serum albumin (BSA) gradient to isolate low-density DC (73). Related techniques to obtain DC from murine tissues, including lymph nodes and skin, were subsequently developed, but these techniques yield low numbers of DC, making experiments difficult to perform. More recent approaches for obtaining DC have involved their generation in vitro from precursor cells cultured

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in the presence of different cytokines. One method, in which murine bone marrow cells are depleted of differentiated precursors and grown in medium containing GM-CSF and IL-4, has become widely used because of its greater DC yield (74). Human DC can also be enriched as circulating precursors from the blood by density-based purification techniques (75). After a period of in vitro culture and maturation, DC precursors become larger and less dense. Gradient solutions lacking potentially immunogenic proteins such as BSA have been employed including Percollt (Pharmacia Biotech), Nycodenzt (Nycomed Pharma, Oslo, Norway), and metrizamide. These different fluids are osmotically active to varying degrees and have additional stimulatory properties as well. The use of density-based isolation is, however, limited by the low frequency of DC precursors in blood, representing around 1% of peripheral blood mononuclear cells (PBMC) (22, 75). As a result, a leukapheresis must be performed to generate sufficient numbers (on average 5 x 106) of DC for therapeutic vaccination in humans. As with the animal models, more recent approaches for generating human DC from bone marrow precursors utilize CD34` cells cultured in the presence of exogenous GM-CSF, usually in combination with IL-4 and/or TNF-a (76–79). Stem cell factor (SCF) and/or Flt3-ligand (FL) are often added to increase DC yields by inducing the proliferation of DC progenitors (80, 81). Sources for human CD34` precursors include bone marrow, cord blood, and G-CSF-mobilized peripheral blood. Many groups have generated DC-like cells by culturing CD14` monocyteenriched PBMC in vitro. When cultured for 1–2 weeks with media supplemented with GM-CSF and IL-4, monocytes give rise to large numbers of cells that are morphologically and phenotypically similar to the ‘‘classical’’ density purified DC (35, 77). These cytokine-generated DC require additional maturation in vitro with TNF-a or monocyte-conditioned media in order to fully stimulate in an allogeneic MLR or prime antigen-specific T cell responses in vitro and in vivo. Moreover, without this additional maturation step, the DC phenotype can revert to that of a monocyte. Nonetheless, these ‘‘monocyte-derived DC’’ are capable of inducing strong antitumor responses in vivo, in mice (7, 8). Recently, clinical trials using these cells in patients with several malignancies have demonstrated induction of specific immune responses as well as tumor regression (34, 82). One advantage of this approach includes easy access to CD14` monocytes. 50 mL of whole blood is sufficient to generate over 10 x 106 DC for vaccine production. However, this approach requires at least one week of in vitro culture with exogenous cytokines, which increases the risk of bacterial contamination as well as cost. An alternative approach is to expand DC in vivo. Administration of FL to mice in vivo results in preferential mobilization or release of DC precursors from the bone marrow to the periphery and into lymphoid organs (81). Recently, in a clinical protocol that utilizes FL from Immunex Corp., we have observed that administration of this growth factor can increase the number of circulating blood DC 10–30-fold (Figure 3). These DC can be harvested with a leukapheresis pro-

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B.

CD3, CD14, CD19, CD56

A.

1.8%

11.6%

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MHC Class II Figure 3 FL administration in vivo expands circulating blood DC. A. DC in circulating blood as identified by expression of MHC class II and lack of lineage markers represent approximately 1% of the PBMC pre-FL administration. B. Following FL administration in vivo, the fraction of DC is increased approximately 10-fold as a percentage of PBMC.

cedure for ex vivo manipulation and used to prime humans to antigens in vivo (manuscript in preparation). Such an approach would avoid the need for prolonged in vitro culture or repeated leukaphereses. In generating DC for use as a cellular vaccine, regardless of source, the infused cells should possess a stable as well as an activated phenotype. In addition to expressing the requisite MHC and costimulatory molecules to prime T cells, the cells should express appropriate adhesion molecules and chemokine receptors to attract the DC to secondary lymphoid organs for priming. Otherwise, ineffective priming may occur, particularly if the DC are administered systemically rather than locally into the relevant draining lymph nodes. Intralymphatic or intranodal injection of DC may be used to deliver DC directly to secondary lymphoid organs, although these routes of administration have not yet been shown conclusively to produce more effective vaccination (83).

DC and Cancer The ability of DC to generate antitumor immune responses in vivo has been documented in many animal models. Most of these experiments have involved in vitro isolation of DC, followed by loading of the DC with tumor antigen and injection of the antigen-bearing DC into syngeneic animals as a cancer vaccine. DC loaded with tumor lysates, tumor antigen-derived peptides, synthetic MHC class I–restricted peptides, and whole protein have all been demonstrated to generate tumor-specific immune responses and antitumor activity (9, 10, 84). Furthermore, antigen-loaded DC can be used therapeutically to induce regression of

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preexisting tumors (7, 85). These observations have established the rationale for evaluating tumor antigen–bearing DC as therapeutic vaccines in humans.

Candidate Target Antigens DC based immunization requires that the cells present one or more tumor antigens to the host’s T cells. Fortunately, a number of antigens associated with various malignancies have been described (Table 2). Some groups have focused upon

TABLE 2 Potential tumor antigens for immunotherapy

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Antigen Class Tumor specific antigens

Antigen Immunoglobulin idiotype TCR Mutant p21/ras Mutant p53 p210/bcr-abl fusion product

Developmental antigens

Viral antigens

Tissue-specific selfantigens

Overexpressed selfantigens

MART-1/Melan A MAGE-1, MAGE-3 GAGE family Telomerase Human papilloma virus Epstein Barr virus

Tyrosinase gp100 Prostatic acid phosphatase Prostate-specific antigen Prostate-specific membrane antigen Thyroglobulin a-fetoprotein Her-2/neu Carcinoembryonic antigen Muc-1

Malignancy B-Cell non-Hodgkin’s lymphoma, multiple myeloma T cell non-Hodgkin’s lymphoma Pancreatic, colon, lung cancer Colorectal, lung, bladder, head and neck cancer Chronic myelogenous leukemia, acute lymphoblastic leukemia Melanoma Melanoma, colorectal, lung, gastric cancers Melanoma Various Cervical, penile cancer Burkitt’s lymphoma, nasopharyngeal carcinoma, post-transplant lymphoproliferative disorders Melanoma Melanoma Prostate cancer Prostate cancer Prostate cancer Thyroid cancer Liver cancer Breast and lung cancers Colorectal, lung, breast cancer Colorectal, pancreatic, ovarian, lung cancer

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tumor-derived protein extracts or RNA as the source for antigens. When these approaches are used, the vaccine contains multiple antigens, increasing the probability of inducing immunity to more than one tumor-associated antigen. Although the target proteins are initially undefined, with this approach, the immunogen can be identified later (6). On the other hand, there is increased potential for the induction of a destructive autoimmune response to antigens expressed on normal tissues. Truly tumor-specific antigens offer theoretical advantages as immunotherapy targets. The immune response induced by such antigens presumably would be limited to tumor cells bearing epitopes of the antigen, limiting the risk of collateral damage to normal tissues (86, 87). If such proteins arise in the tumor following thymic development, antigen-reactive T cells could exist in the repertoire as they may have avoided thymic deletion. Nevertheless, the immunogenicity of these antigens may be limited by the number of epitopes contained within the protein (e.g., p53 or ras possessing a single amino acid mutation). Moreover, tumorspecific antigens may vary between individuals with the same tumor type. Viral tumor antigens can represent desirable immune targets given the inherent immunogenicity of many viral proteins (88). A number of antigens expressed on normal tissues as well as tumors have also been examined as immunotherapy targets. Despite the relatively small number of such antigens identified to date, many have been shown to stimulate T cell responses in vitro when presented by DC (89, 90). This is surprising since most T cells capable of recognizing self-proteins are presumably deleted from the repertoire during T cell differentiation in the thymus. The extent to which this occurs may vary depending on the extent of protein expression during the period of T cell development. However, preexisting T cells reactive to self-antigens have been demonstrated in vivo in cancer patients. Moreover, tumor-specific immune responses have been induced with various immunization strategies in vivo. Tumor-reactive T cells, therefore, apparently exist in the T cell repertoire of cancer patients but are rendered tolerant or anergic (91). Alternatively, the antigenreactive T cells may recognize and respond to subdominant or cryptic epitopes that are not ordinarily presented at levels sufficient to induce immunity (92, 93). While animal models have been used frequently to examine the immunogenicity of tumor-associated antigens, significant caveats exist in extrapolating animal data to humans. Many of the animal tumor antigen models focus upon proteins that are foreign to the animal (e.g. carcinoembryonic antigen, or Her-2/ neu in mice). Transgenic tumor models (e.g. MMTV/neu) could be used to address this issue although differences in T cell receptor repertoire remain as a confounding factor. Other tumor models (e.g. fibrosarcoma) are based upon tumors that are inherently immunogenic. These model systems contrast with cancer in human patients, where many of the tumor antigens are self-antigens, and tumors other than melanoma and renal cell carcinoma are rarely immunogenic. Another consideration in selecting candidate target antigens is tissue or tumor specificity. Most of the tumor-associated proteins being explored in clinical trials

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(e.g. CEA and Her2/neu) are expressed by some normal tissues (albeit at low density) as well as by neoplastic cells. There exists a theoretical risk that DCbased presentation of these individual antigens, or crude mixtures of tumorderived peptides or nucleic acids, may induce autoimmune disease affecting vital organs. Antigens uniquely expressed in non-essential organs (e.g. prostate) may induce an acceptable form of tissue-specific autoimmunity, especially if these organs have been removed as part of treatment (e.g. prostatectomy). Antigen-loss variants leading to tumor escape have been observed with immunotherapy (94, 95). Ideally target antigens should represent antigens universal to the tumor type and required for their survival and growth. Vaccine formulations may also require mixtures of commonly expressed tumor antigens to target heterogeneous expression of antigens within the tumor. Recently, approaches have been devised to increase the immunogenicity of tumor-associated antigens by modifying their structure or using xenogeneic analogues. For example, immunization with xenogeneic prostate acid phosphatase (PAP), e.g. immunizing rats with human PAP, generated autoimmunity to the prostate gland (96). In contrast, immunizing with the self-protein, rat PAP, did not. Other approaches have focused upon modifying peptides to increase HLA binding or T cell receptor interactions and have shown that these modified peptides can serve as agonist peptides (97, 98). In one recent melanoma trial, patients immunized with a modified melanoma-derived peptide developed clinical responses, while those immunized with the native peptide did not (99). The ability to develop more immunogenic antigens will improve as MHC-peptide–T cell receptor interactions are better understood.

Approaches to Antigen Delivery To date, several approaches have been used to arm DC with target antigen for use in clinical trials. Tumor-associated peptides either in cell lysates or acid eluted fractions from MHC class I molecules on tumor cells have been used to load MHC molecules at the surface of the cell. Specific MHC-restricted peptides derived from tumor-associated antigens such as MART-1, MAGE-1, CEA, and prostate-specific membrane antigen (PSMA) have also been used (34, 100, 101). This approach relies on predictive algorithms to identify peptides with high binding affinity to the HLA molecule, most commonly the HLA*A0201 alleles. Those peptides with demonstrated binding to HLA*A0201 are then studied for immunogenicity based upon their ability to generate CTL, in vitro. Although this approach simplifies production of target antigens to a limited number of peptides, selection of patients is restricted to those with this specific HLA haplotype (30– 45% of the population). Moreover, additional biologically important epitopes may be missed, as binding affinity for a particular HLA allele does not translate directly into immunogenicity. Using MHC class I–restricted peptides also ignores the role of MHC class II–restricted T helper cells in initiating and sustaining an immune response.

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An alternative approach is to utilize full-length recombinant proteins as antigens. By introducing whole protein into DC, subsequent immune responses would potentially be restricted by multiple alleles rather than single alleles as they are with peptides. The antigen-processing machinery of DC would be relied upon to stimulate responses to the important epitopes within the antigen. Under normal circumstances, addition of intact soluble proteins to DC would be expected to result in entry of the proteins into MHC class II processing pathway but not the MHC class I processing pathway. Such processing would allow for presentation of antigenic epitopes to CD4` T cells, which are important sources of proinflammatory cytokines such as IL-2 and IFN-c, but would not allow for efficient presentation of antigenic epitopes to CD8` CTL, which are believed to be critical effectors of antitumor immune responses. Although DC may also ‘‘leak’’ exogenous antigen into the MHC class I pathway, which can lead to stimulation of CD8` CTL, this occurs inefficiently. Nonetheless, since most solid tumors do not express MHC class II determinants, the generation of tumor-reactive CD8` T cells may be required for an effective antitumor response, and this would necessitate that antigens be delivered to DC in a manner that assures their access to the class I MHC processing pathway. A number of approaches are being developed to accomplish this goal, including gene transfer methods that result in antigen processing in the MHC class I pathway of DC and presentation to CD8` T cells. Conjugating certain ‘‘transporter’’ peptides onto full-length proteins allows these proteins to translocate across cell membranes and into the MHC class I pathway. One such peptide derived from HIV tat can increase the efficiency of MHC class I–restricted antigen presentation by more than 100-fold (102). Peptides derived from bacterial toxins may also have similar capabilities (103). DNA transfection of DC, with or without liposomal encapsulation, has been tried with varying success (104). RNA derived from tumors or encoding tumor antigens can also be transfected into DC leading to the generation of class I MHC restricted responses (90). Recombinant viral vectors such as adenovirus, vaccinia, or fowlpox expressing the antigen have been used to deliver transgene products into the class I MHC pathway of DC (105, 106). This approach offers the additional advantage of exposing the DC to immune-activating components of the virus, which leads to a more potent APC. Recombinant retroviruses have also been used, although this approach has so far been effective only in DC precursors because proliferation is required and differentiated DC do not proliferate (107). However, retroviral constructs capable of infecting resting, nondividing cells have recently been described (108, 109).

DC Clinical Trials Human trials using DC loaded with tumor antigens are underway at several institutions. In the first reported DC trial, the effect of autologous DC pulsed ex vivo with tumor-specific antigen was investigated in patients with malignant B cell lymphoma for whom conventional chemotherapy had failed. Like other B lym-

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phocytes, the neoplastic cells in these patients express surface immunoglobulin receptors, and because B cell lymphomas are monoclonal, all the cells of a given tumor express identical surface immunoglobulin. Moreover, this immunoglobulin is potentially immunogenic by virtue of its unique idiotypic determinants, which are formed by the combination of the variable regions of immunoglobulin heavy and light chains (110–112). To prepare idiotype proteins for this clinical study, patients underwent tumor biopsies; the immunoglobulin (idiotype) produced by each tumor was ‘‘rescued’’ by somatic cell fusion techniques and purified from hybridoma supernatants (113). Peripheral blood leukocyte preparations containing DC precursors were obtained from patients by leukapheresis, and monocytedepleted mononuclear cells were incubated for 36 h in the presence of either tumor-specific idiotype protein or a control protein, keyhole limpet hemocyanin (KLH). This incubation technique not only enabled the DC precursors to take up and process the exogenous proteins, but also induced their maturation into potent APC. Following this culture period, the idiotype and KLH-pulsed DC were separated from contaminating lymphocytes on the basis of their differences in buoyant density. The DC were then extensively washed to remove free protein, resuspended in sterile saline, and administered to the patients by intravenous infusion. This procedure was repeated three times at monthly intervals with a booster immunization given four to six months later. Two weeks after each DC infusion, the patients received subcutaneous injections of idiotype, which in the absence of DC or a chemical adjuvant would not be expected to induce an immune response (114). Throughout the trial the patients were followed for the development of an immune response to the idiotype and their tumor burden was monitored. A report of the results obtained in our initial four patients has been published (33). All of these treated patients, as well as six not described in our published report, tolerated their infusions well, and none have experienced clinically significant toxicity at any point during the study. In addition, the majority of the patients developed T cell–mediated anti-idiotype and anti-KLH proliferative responses that were not observed prior to treatment initiation. The anti-idiotype responses were specific for autologous tumor immunoglobulin when compared to irrelevant, isotype-matched immunoglobulins. In addition to these proliferative responses, T cells from one patient were expanded for several weeks in vitro in the presence of idiotype protein and shown to lyse autologous tumor hybridoma cells but not an isotype-matched, unrelated hybridoma. In contrast to earlier idiotype vaccination studies that utilized chemical adjuvants rather than DC (111, 114), a humoral response to idiotype was not detected in our patients, consistent with the interpretation that the idiotype-pulsed DC induced a Th1 rather than Th2 response. Most importantly, two of the patients experienced complete tumor regression, including one who entered the trial with bulky disease and remained in complete remission for more than three years. A third patient experienced a partial response while three have had stable disease states and three have experienced disease progression. Recently, a new cohort of patients has been vacci-

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nated while in remission, and their follow-up is ongoing. In the DC trial for B cell lymphoma, we do not know whether the idiotype pulsed DC used induced CD8` CTL. B cell lymphoma is unusual among malignancies in that the neoplastic cells express both MHC class I and II antigens and would, therefore, be susceptible to recognition and attack by either CD4` or CD8` T cells. A Phase I DC trial in prostate cancer has since been reported by Murphy et al (101). DC were derived from peripheral blood–derived monocytes and cultured in GM-CSF and IL-4. Subsets of the 51 patients were treated with unloaded DC or DC pulsed with HLA*A2–restricted peptides derived from prostate-specific membrane antigen (PSMA). Non-HLA*A2 patients were included in the trial, and no consistent T cell responses were detected. Seven patients were scored as partial responders. Of these patients, 33 were subsequently enrolled in a Phase II study utilizing two PSMA peptides loaded onto autologous DC. Nine patients were scored as partial responders, including four who were partial responders in the original Phase I trial (115). Patients were not, however, controlled for concurrent treatment modifications (including hormone withdrawal), so follow-up studies will be very important. Two vaccine trials for melanoma have also been reported investigating DC pulsed with a panel of melanoma-derived, HLA-restricted peptides. Both trials utilized DC-like cells derived from monocytes by culture in GM-CSF and IL-4. Nestle et al limited their clinical trial to patients expressing the HLA*A1 or A2 alleles and reported that 5 of 15 patients developed clinical responses, including two complete remissions which have been durable (34; personal communication). Induction of delayed type hypersensitivity (with skin testing) to the antigen was seen with this vaccination approach. Lotze et al also reported the results of their clinical trial in melanoma with one complete response in their cohort of six patients (82). Our group has recently initiated several new DC trials. One trial, in multiple myeloma patients who have undergone peripheral blood stem cell rescue following ablative chemotherapy, employs the myeloma-specific immunoglobulin idiotype as the tumor antigen. Each patient’s idiotype is purified from serum. Following high dose therapy and bone marrow recovery, these patients are treated with idiotype pulsed DC using a protocol similar to that used in our lymphoma trials. This trial is currently ongoing, although in our initial cohort of 12 patients idiotype-specific T cell responses were detected only in those patients who achieved a complete response with high dose chemotherapy and had no detectable circulating myeloma protein (116). We have also initiated a trial in prostate cancer employing prostatic acid phosphatase (PAP) as the tumor antigen. Utilizing DC purified from peripheral blood by density gradient centrifugation, this protocol seeks to immunize patients with metastatic prostate cancer with PAP-pulsed DC. Based upon our preclinical data, we are utilizing a xenogeneic PAP, derived from rodents, rather than human PAP as the immunogen. Preliminary data demonstrate the ability of xenogeneic antigen-loaded DC to generate cross-reactive immunity to the self-antigen as well

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as clinical responses in a subset of patients (83). Many other clinical trials have been initiated at various institutions (Table 3).

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Future Directions Although the results to date of the various DC trials are provocative, the procedures used for isolating and ‘‘arming’’ DC are tedious and not yet broadly applicable to clinical practice. Indeed, our observation in the original lymphoma trial that a small number of APC could induce an immune response to a weak antigen in tumor-bearing patients, most of whom were leukopenic as a consequence of prior chemotherapy, was surprising. Not all of the treated patients, however , have experienced tumor regression, suggesting that the DC dose, route of administration, choice of antigen, or method of antigen loading may have been suboptimal. As discussed, many groups are already pursuing techniques for in vitro generation of DC from CD34` precursors or CD14` monocytes. These approaches, while expensive, can generate large numbers of cells for use in clinical trials. Administration of growth factors that increase numbers of circulating DC in patients in vivo offers another possibility. In a limited pilot trial using GM-CSF for DC mobilization, we found that yields of monocytes were significantly increased, making DC purification difficult (E Engleman, unpublished results). In contrast to GM-CSF, FL, which appears to preferentially expand DC precursors in humans in vivo, shows significant promise. It remains to be seen whether FL is useful alone as a therapeutic agent, or whether the DC mobilized with FL must be harvested, armed in vitro with antigen, and infused to induce antitumor immunity. Treatment of mice bearing certain immunogenic tumors with FL has been shown to result in tumor regression (117). These responses presumably occur, at least in part, through DC priming of effector T cells, although NK cells have also been shown to be activated by FL. However; whether such an approach will circumvent the inhibitory milieu within cancer patients will require further investigation. Studies to investigate this possibility are currently underway. In addition to questions regarding the best source of DC for use in clinical trials, the choice of tumor antigen with which to ‘‘arm’’ the DC is almost certainly going to have a profound influence on clinical outcome. At present, the choices are limited because only a few antigens have been identified that are either tumoror tissue-restricted in their expression. Only a subset of these antigens are immunogenic. On the other hand, the number of candidate antigens has increased with time and is expected to increase further as a consequence of intensive gene identification efforts. Ultimately, combinations of antigens should be used to reduce the risk of generating antigen-loss variants that evade the immune response. The increasing use of microarray technology in assessing tumors may someday allow for individualized antigen combinations. Antigen delivery also remains to be optimized. Use of protein, whether tumor derived or with recombinant DNA methods, can be cumbersome and potentially limiting, especially at concentrations that may be necessary for MHC class I

262

TABLE 3 Dendritic cell clinical trials

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Malignancy

Antigen

Antigen construct

DC type

Institution

Investigator

Breast, colorectal, Tumor derived pancreas, lung, melanoma Breast, colorectal, lung Mutant Ras derived

Autologous tumor cell lysate

GM-CSF ` IL-4 cultured monocytes

Uni. of Michigan

Mule et al.(84)

Ras peptides

Vanderbilt

Carbone et al.1

Breast, colorectal, lung Mutant p53

p53 DNA

Vanderbilt

Carbone et al.1

Breast, colorectal

CEA

USC

Weber et al.1

Lung, colorectal

CEA

Stanford

Fong, Engleman et al.

Lung

CEA

Duke

Lyerly, Gilboa et al.1

Lung

CEA

HLA*A2 restricted peptide CAP-1 HLA*A2 restricted peptide CAP-1 (610M)3 HLA*A2 restricted peptide CAP-1 CEA RNA

GM-CSF ` IL-4 cultured monocytes GM-CSF ` IL-4 cultured monocytes CF-CSF ` IL-4 cultured monocytes FL mobilized, density purified DC

Duke

Lyerly, Gilboa et al.1

Melanoma

gp100, Mart-1, & Tyrosinase gp100, Mart-1, & Tyrosinase

HLA*A2 restricted peptides HLA*A2 restricted peptides

Uni. of Zurich

Nestle et al.(34)

Univ. Pittsburgh

Lotze et al.(82)

gp100 (210M)2, MART-1, & Tyrosinase

HLA*A2 restricted peptides

NCI

Marincola et al.1

Melanoma

Melanoma

GM-CSF ` IL-4 cultured monocytes GM-CSF ` IL-4 cultured monocytes GM-CSF ` IL-4 cultured monocytes GM-CSF ` IL-4 cultured monocytes or CD34` cells GM-CSF ` IL-4 cultured monocytes

TABLE 3 Continued Malignancy Melanoma

Non-Hodgkin’s lymphoma Prostate Prostate

gp100, Mart-1, MAGE-3 & Tyrosinase gp100 (210M),2 & Tyrosinase Immunoglobulin idiotype Immunoglobulin idiotype Immunoglobulin idiotype Xenogeneic PAP PSMA

Prostate

PAP

Renal

Tumor derived

Melanoma Multiple myeloma Multiple myeloma

1

263

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Antigen

Antigen construct

DC type

Institution

Investigator

HLA*A2 restricted peptides

GM-CSF ` TNF-a cultured CD34` cells

Baylor

Bancerau et al.1

HLA*A2 restricted peptides Purified protein

GM-CSF ` IL-4 cultured monocytes Dendreon density purified DC Dendreon density purified DC Density purified DC

USC

Weber et al.1

Stanford

Levy, Engleman et al.

Dendreon (multicenter) Stanford

Valone et al.1

Purified protein Recombinant protein Recombinant protein HLA*A2 restricted peptides Recombinant fusion protein Autologous tumor cell lysate

Density purified DC GM-CSF ` IL-4 cultured monocytes 5 adjuvant GM-CSF Dendreon density purified DC GM-CSF ` IL-4 cultured monocytes

Stanford Northwest Biotherapeutics (multicenter) Dendreon (multicenter) UCLA

Personal communication. 2gp100 derived peptide with modification at position 210. 3CEA derived peptide with modification at position 610.

Levy, Engleman et al.(33) Fong, Engleman et al. Murphy et al.(129)

Valone et al.(130) Figlin, Belldegrun, et al.1

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delivery. Use of specific peptide conjugates or fusion constructs (e.g. with HIV tat) may increase the efficiency of presenting epitopes from these soluble proteins. RNA, DNA, and viral vectors are more easily produced and may offer a viable alternative, although issues of transfection efficiency and DC viability remain unresolved. The potential benefits of administering cytokines or other DC activators in combination with DC vaccination remain relatively unexplored. Clearly, DC can elaborate their own cytokines for antigen priming as previously discussed. However, supplementing culture media used for generating DC in vitro with additional cytokines may enhance the APC functions of these DC. TNF-a or CD40 ligand are known to activate DC in vitro and could increase the potency of DC-based vaccines (70, 118, 119). The addition of IL-12 may aid in antigen priming and generating Th1 responses in vitro or in vivo (120). A singular study to date has examined the role of IL-2 in augmenting the effector phase of the immune response to peptide vaccination in melanoma patients. This trial is particularly important in that patients who were immunized and given high doses of IL-2 developed immune responses, while those patients who received the vaccine alone did not (99). Synergy between DC vaccination and IL-2 has already been demonstrated in an animal model (121). No consensus exists on the optimal approach to assessment of immune responses in patients undergoing immunotherapy, let alone DC vaccination. Delayed-type hypersensitivity testing with an antigen challenge injected into the skin has been used to assess gross immune reactivity. Cytokine production by both CD4` and CD8` T cells can be detected by cytokine ELISA, ELISPOT, or intracellular cytokine staining. Other techniques for assessing CD4` T cell responses include measurement of antigen-specific proliferation. CD8 T cell responses have also been assessed with CTL assays and more recently tetramer staining. However, these assays assess the functions of circulating T cells but do not necessarily reflect immune responses in lymphoid organs. Improved immunologic assays that better correlate with clinical outcome will be required before these assays can serve as surrogates for evaluation of tumor status. Before closing, it is worth considering the following question. Why does administration of small numbers of antigen-pulsed DC induce antigen-specific T cell responses and tumor regression in patients in whom both the antigen and DC are already plentiful? The explanation that we favor is that most of the circulating DC and DC residing in or near malignant tissues may be defective in their capacity to take up, process, and present tumor-associated antigens because they fail to become activated and differentiate into mature APC. This view is supported by the observation that DC from tumor-bearing animals can be defective in their capacity to stimulate T cells (86, 122). Moreover, tumors secrete various mediators that have recently been found to inhibit DC differentiation and/or maturation in vitro. Examples include VEGF, IL-6, and IL-10 (91, 123, 124). Tumors may even inhibit migration of DC to tumor-bearing tissues through their secretion of IL-10 or downregulation of vascular adhesion molecules (125, 126). Indeed, the

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presence of mature DC in lung, cervical, colon, gastric, nasopharyngeal, thyroid, and prostate cancers is associated with a favorable prognosis (127, 128). By isolating and arming DC with antigen ex vivo, we may be allowing the cells to mature away from an inhibitory milieu. If defective maturation of DC is a common occurrence in malignancy, then identification of agents that induce their maturation, in vivo, may represent an elegant solution to this problem. In the absence of such agents, administration of DC derived from circulating DC precursors, which have been induced to mature in vitro and armed with appropriate tumor antigens, may prove useful in the treatment of a variety of tumors. Visit the Annual Reviews home page at www.AnnualReviews.org.

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Annual Review of Immunology Volume 18, 2000

CONTENTS

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Discovering the Role of the Major Histocompatibility Complex in the Immune Response, Hugh O. McDevitt Receptor Selection in B and T Lymphocytes, David Nemazee Molecular Basis of Celiac Disease, Ludvig M. Sollid Population Biology of Lymphocytes: The Flight for Survival, Antonio A. Freitas, Benedita Rocha Nonclassical Class II MHC Molecules, Christopher Alfonso, Lars Karlsson Negative Regulation of Cytokine Signaling Pathways, Hideo Yasukawa, Atsuo Sasaki, Akihiko Yoshimura T Cell Activation and the Cytoskeleton, Oreste Acuto, Doreen Cantrell The Specific Regulation of Immune Responses by CD8+ T Cells Restricted by the MHC Class Ib Molecule Qa-1, Hong Jiang, Leonard Chess The Biology of Chemokines and their Receptors, Devora Rossi, Albert Zlotnik Dendritic Cells in Cancer Immunotherapy, Lawrence Fong, Edgar G. Engleman CD8 T Cell Effector Mechanisms in Resistance to Infection, John T. Harty, Amy R. Tvinnereim, Douglas W. White Glucocoricoids in T Cell Development and Function, Jonathan D. Ashwell, Frank W. M. Lu, Melanie S. Vacchio Molecular Genetics of Allergic Diseases, Santa Jeremy Ono Immunology at the Maternal-Fetal Interface: Lessons for T Cell Tolerance and Suppression, A. L. Mellor, D. H. Munn Regulation of B. Lymphocyte Responses to Foreign and Self-Antigens by the CD19/CD21 Complex, Douglas T. Fearon, Michael C. Carroll, Michael C. Carroll Regulatory T Cells in Autoimmunity, Ethan M. Shevach Signal and Transcription in T Helper Development, Kenneth M. Murphy, Wenjun Ouyang, J. David Farrar, Jianfei Yang, Sheila Ranganath, Helene Asnagli, Maryam Afkarian, Theresa L. Murphy The RAG Proteins and V (D) J Recombination: Complexes, Ends, and Transposition, Sebastian D. Fugmann, Alfred Ian Lee, Penny E. Shockett, Isabelle J. Villey, David G. Schatz The Role of the Thymus in Immune Reconstitution in Aging, Bone Marrow Transplantation, and HIV-1 Infection, Barton F. Haynes, M. Louise Markert, Gregory D. Sempowski, Dhavalkumar D. Patel, Laura P. Hale Accessing Complexity: The Dynamics of Virus-Specific T Cell Responses, Peter C. Doherty, Jan P. Christensen The Role of Chemokine Receptors in Primary, Effector, and Memory Immune Responses, Federica Sallusto, Charles R. Mackay, Antonio Lanzavecchia Phosphorylation Meets Ubiquiination: The Control of NF-Kappa-B Activity, Michael Karin, Yinon Ben-Neriah Reservoirs for HIV-1: Mechanisms for Viral Persistence in the Presence of Antiviral Immune Responses and Antiretroviral Therapy, Theodore Pierson, Justin McArthur, Robert F. Siliciano

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Regulation of Antibody Responses via Antibodies, Complement, and Fc Receptors, Birgitta Heyman Multiple Roles for theMajor Histocompatibility Complex Class I--Related Receptor, FcRn, Victor Ghetie, E. Sally Ward Immunobiology of Dendritic Cells, Jacques Banchereau, Francine Briere, Christophe Caux, Jean Davoust, Serge Lebecque, Yong-Jun Liu, Bali Pulendran, Karolina Palucka An Address System in the Vasculature of Normal Tissues and Tumors, E. Ruoslahti, D. Rajotte Genomic Views of the Immune System, Louis M. Staudt, Patrick O. Brown

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Viral Subversion of the Immune System, Domenico Tortorella, Benjamin E. Gewurz, Margo H. Furman, Danny J. Schust, Hidde L. Ploegh

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DNA Vaccines: Immunology, Application, and Optimization, Sanjay Gurunathan, Dennis M. Klinman, Robert A. Seder Gamma Delta Cells: A Right Time and a Right Place for a Conserved Third Way of Protection, Adrian C. Hayday

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Annu. Rev. Immunol. 2000. 18:275–308 Copyright q by Annual Reviews. All rights reserved

CD8~ T CELL EFFECTOR MECHANISMS IN RESISTANCE TO INFECTION John T. Harty, Amy R. Tvinnereim, and Douglas W. White Department of Microbiology and Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, Iowa, e-mail: [email protected], [email protected], [email protected]

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Key Words CD8` T cells, cytolysis, cytokines, pathogens, infection Abstract Based on T cell subset depletion studies and the analysis of gene knockout mice, it is evident that CD8` T cells contribute to resistance against intracellular infections with certain viral, protozoan, and bacterial pathogens. Although they are known primarily for their capacity to kill infected cells, CD8` T cells elaborate a variety of effector mechanisms with the potential to defend against infection. Microbes use multiple strategies to cause infection, and the nature of the pathogenhost interaction may determine which CD8` T cell effector mechanisms are required for immunity. In this review, we summarize our current understanding of the effector functions used by CD8` T cells in resistance to pathogens. Analyses of mice deficient in perforin and/or Fas demonstrate that cytolysis is critical for immunity against some, but not all, infections and also reveal the contribution of cytolysis to the pathogenesis of disease. The role of CD8` T cell–derived cytokines in resistance to infection has been analyzed by systemic treatment with neutralizing antibodies and cytokine gene knockout mice. These studies are complicated by the fact that few, if any, cytokines are uniquely produced by CD8` T cells. Thus, the requirement for CD8` T cell– derived cytokines in resistance against most pathogens remains to be defined. Finally, recent studies of human CD8` T cells reveal the potential for novel effector mechanisms in resistance to infection.

STIMULATION OF PATHOGEN-SPECIFIC CD8` T CELL RESPONSES CD8` T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan, and bacterial pathogens. During the initial encounter with a microbe, CD8` T cells bearing TCR specific for pathogen-derived antigens are selected to undergo clonal expansion. The specificity of this selection is driven by the interaction of the TCR with short (8–10 amino acid) pathogen-derived peptides presented by MHC class I molecules on the surface of professional 0732–0582/00/0410–0275$12.00

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antigen-presenting cells (APC) (1). APC such as dendritic cells (DC), by virtue of their expression of specific costimulatory molecules, are capable of stimulating naive T cells to proliferate and differentiate in response to antigen (2). Priming of CD8` T cells occurs in response to antigens that gain access to the cytosol of APC for processing by the endogenous MHC class I presentation pathway (3). Thus, CD8` T cells are activated in response to cytosolic infections with viruses, intracytoplasmic bacteria, and protozoa. CD8` T cells also respond to antigens from noncytosolic sources such as phagosomal-bacterial and -protozoan pathogens; these antigens are processed by exogenous MHC class I presentation pathways that may be limited to professional APC (4). Recent evidence demonstrates that DC are also able to ‘‘cross present’’ MHC class I–restricted antigens obtained from apoptotic, infected cells (5). New technologies for detection of antigen-specific CD8` T cells, including staining with tetrameric MHC class I–peptide complexes, intracellular cytokine staining, and ELISPOT analysis, have revised our understanding of the magnitude of CD8` T cell responses to infection (6). As a result of clonal expansion, pathogen-specific CD8` T cells rapidly increase from virtually undetectable in the naive host to levels that are readily detectable (1–2% of splenic CD8` T cells in primary responses to certain bacterial infections) (7) or even dominate the repertoire, reaching 50% or more of splenic CD8` T cells in the primary response to certain viral infections (8, 9). These expanded populations of ‘‘effector’’ CD8` T cells contribute to clearance of the pathogen and then decline in numbers to a memory level that may be maintained at 5–10% of the initial clonal burst size (8). Memory CD8` T cells may be present for the life of the host and are able to mount rapid, heightened responses to reinfection with the specific pathogen (10). Experiments to understand the signals that maintain the memory pool are ongoing, with evidence for and against such mediators as persistent antigen (11, 12), specific cytokines (13), or MHC molecules (14).

CD8` T CELL EFFECTOR MECHANISMS Concurrent with the initiation of proliferation is the instigation of a program of gene expression that arms the CD8` T cell with a veritable arsenal of effector mechanisms to combat the infection. The capacity to mobilize these effector mechanisms, which include cytolysis of infected cells and the production of cytokines, chemokines, or microbicidal molecules, develops over time after the initial stimulation of naive CD8` T cells and manifests upon subsequent encounter of the effector CD8` T cells with infected cells. Since MHC class I molecules are expressed on essentially all nucleated cells, effector CD8` T cells can respond against intracellular pathogens in most tissues and facilitate clearance of the infection. In the case of memory CD8` T cells, mobilization of effector mechanisms is very rapid (15, 16), occurs in response to reduced antigen levels, perhaps due to the increased expression of adhesion molecules on memory T cells (10), and

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is less dependent on the costimulatory signals required for priming antigenspecific naive T cells (17). Activated CD8` T cells are able to induce cytolysis of infected cells by two distinct molecular pathways (18): the granule exocytosis pathway, dependent on the pore-forming molecule perforin, or by the upregulation of FasL (CD95L), which can initiate programmed cell death by aggregation of Fas (CD95) on target cells. Both of these pathways, activated in response to signals from the TCR, stimulate the caspase cascade in the target cell, leading to apoptotic death (19). Efficient lysis by the granule exocytosis pathway requires the coordinated delivery of perforin and granule enzymes, such as granzymes A and B, into the target cell (20, 21). CD8` T cells also elaborate cytokines, including IFN-c and TNF, as well as chemokines that function to recruit and/or activate the microbicidal activities of effector cells such as macrophages and neutrophils (22). Cytokines may also directly interfere with pathogen attachment or pathogen gene expression, or they may restrict intracellular replication. As with cytolytic effector mechanisms, expression of cytokine molecules by CD8` T cells is tightly regulated through TCR-dependent signals.

UNDERSTANDING THE ROLE OF SPECIFIC CD8` T CELL EFFECTOR MECHANISMS It is important to note that many of the effector mechanisms employed by CD8` T cells are also employed by other cells of the immune system. Natural killer (NK) cells and at least some MHC class II–restricted CD4` T cells express perforin and/or FasL and are capable of cytolysis. TH1-type CD4` T cells and NK cells produce IFN-c as do cd T cells. TNF is produced by many cell types, including CD8` T cells, and has a variety of effects on cells expressing one or both of the TNF receptors, ranging from activation to death (23). In addition to their potential as CD8` T cell effector mechanisms most, if not all, of these molecules play regulatory roles ranging from T cell homeostasis to enhancement of antigen presentation. Given the complexity of the CD8` T cell arsenal, the non-exclusive expression of these molecules and their pleiotrophic effects on the immune system, understanding which mechanisms are important in CD8` T cell– mediated resistance to infection is a substantial challenge. In the last several years, gene knockout mice, lacking specific CD8` T cell effector molecules, have become important tools in addressing this significant biological question. In this review, we summarize the results from selected studies of experimental infections of mice with viral, protozoan, and bacterial pathogens as well as from recent experiments in human systems describing potentially novel CD8` T cell effector mechanisms in resistance to infection.

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CD8` T CELLS IN RESISTANCE TO VIRAL INFECTIONS Viruses are obligate intracellular pathogens that depend on host cell molecules to complete their replication cycles. They range in complexity from less than ten to several hundred potential open reading frames, and they infect an enormous range of eukaryotic cells and a variety of tissues. The results of infection include destruction of the host cell (lytic), replication in the absence of host cell damage (nonlytic), or an interaction where viral gene expression is limited or absent (latent) but reactivation can occur. Some viruses exhibit exquisite host specificity, while other viruses are capable of infecting any mammalian cell. Thus, viruses exhibit both shared and unique interactions with their host and, as a group, present a complex challenge to the immune response. By virtue of their dependence on host protein synthesis machinery, all viral proteins are potentially accessible to the endogenous MHC class I–presentation pathway and to the attention of CD8` T cells. Despite the obvious potential for CD8` T cells to provide immunity against viruses, these cells are not always the dominant effectors of antiviral immunity. However, a role for CD8` T cells in resistance to at least some viruses is clearly documented in animal models and is further supported by the finding that several viruses express molecules that specifically interfere with MHC class I antigen presentation (24) or CD8` T cell– induced cell death (25). Given the limited genome size of even the largest viruses, it would be remarkable if such molecules were maintained in the absence of an important biological function.

ROLE OF CYTOLYSIS IN RESISTANCE TO LYTIC AND NONLYTIC VIRAL INFECTION A major attribute of CD8` T cells is their ability to kill target cells expressing specific MHC class I–peptide complexes. Cytolysis was recognized as potentially significant in resistance to viruses because lysis of the infected cell should eliminate the factory for viral production and contribute to resolution of the infection. Experimental support for this notion was convincingly presented upon analysis of perforin gene knockout (PKO) mice, which lack the granule exocytosis pathway of cytolysis (26–28). This early work, which was elegantly summarized in a prior volume of the Annual Review of Immunology (29), revealed that perforin, but not Fas-dependent cytolysis, was critical in clearance of lymphocytic choriomeningitis virus (LCMV), a nonlytic virus that is eliminated by a CD8` T cell–dependent mechanism (26, 27). Interestingly, another study demonstrated that neither perforin nor Fas-mediated cytolysis was required for resistance to vaccinia virus, vesicular stomatitis virus, or Semliki Forest virus–lytic viral infections where antibodies, CD4` T cells, and cytokines also contribute significantly

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to viral clearance (30). These results led to the hypothesis that CD8` T cell– mediated cytolysis may be required for immunity to nonlytic viruses but is not a necessary effector mechanism in resistance to those viruses that lyse their host cell (29). The requirement for perforin-dependent cytolysis in viral clearance has subsequently been analyzed in several murine models. Control of primary influenza virus infection, a lytic infection of lung epithelial cells, involves CD8` T cells (31). Immunity to reinfection, in both mice and humans, requires the participation of antibodies. Using CD4` T cell depletion to prevent a vigorous antibody response, and a series of bone marrow and splenocyte chimeric mice to control perforin expression in CD8` T cells or Fas expression on lung epithelia, Topham et al demonstrated that perforin-deficient CD8` T cells clear influenza virus from Fas-positive epithelia but fail to clear virus from Fas-minus epithelia (32). In addition, wild-type CD8` T cells were able to clear influenza infection from Fasminus epithelia. Thus, either perforin- or Fas-mediated cytolysis was required and sufficient to eliminate primary influenza infection from the lung, indicating that CD8` T cell control of lytic infection with influenza virus is dependent on cytolysis. CD8` T cells are critical in recovery of mice from infection with ectromelia virus, a murine-specific orthopoxvirus that causes lytic infection. Wild-type B6 mice survive footpad infection with doses up to 106 PFU of ectromelia virus. In contrast, B6-PKO mice and B6-PKO/granzyme A double deficient mice succumb to infection with as little as 10 PFU (33). Mortality was attributed to the failure of the B6-PKO mice to control infection, since levels of virus were substantially elevated at 8 days post infection compared to wild-type mice. Consistent with previous studies (30), the absence of perforin had little impact on resistance to cowpox virus, which exhibits less virulence for mice than does ectromelia virus. These results demonstrate that perforin-dependent cytolysis is critical in resistance to lytic infection with ectromelia virus, a highly virulent natural mouse pathogen. One of the proposed functions of perforin is to facilitate the delivery of granzymes into the target cell (34). Granzyme B (GrmB) activates the caspase cascade in target cells and induces apoptosis associated with rapid DNA fragmentation (20). GrmB-deficient CD8` T cells are unable to induce rapid DNA fragmentation and apoptosis of target cells. However, GrmB-deficient CD8` T cells will eventually induce DNA fragmentation. Recently, data have been presented that Granzyme A (GrmA) also induces target cell DNA fragmentation in a caspaseindependent fashion, but with delayed kinetics compared to GrmB (35, 36). Consistent with this information are studies indicating a severe block in cytolysis in GrmA x B double-deficient mice, similar to that observed with perforin deficiency (35). These results indicate that perforin, delivered by CD8` T cells, is not sufficient for cell lysis and that perforin-induced membrane alterations may be repaired in the absence of granzymes. GrmA-deficient mice, in contrast to perforin-deficient mice, do not exhibit defects in clearance of LCMV (21). However, they do exhibit a defect in clearance of ectromelia virus (37). This result may

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involve the expression of protease inhibitors (serpins) by the ectromelia virus, which could inhibit GrmB-mediated activation of the caspase cascade (25). Thus, GrmA may be required for destruction of cells infected with ectromelia virus or other pathogens that are able to block the GrmB-induced caspase cascade (35, 36). Taken together, these results do not support the hypothesis that CD8` T cell– mediated cytolysis is an unnecessary effector mechanism for resistance to lytic viruses. This hypothesis was originally based on analysis of three lytic viruses, vaccinia virus, vesicular stomatitus virus, and Semliki Forest virus, which are not highly virulent, natural pathogens of mice. Consistent with these results, perforin was not required for resistance to cowpox virus infection of mice. In contrast, perforin-dependent cytolysis was clearly required for resistance to lytic infection with the highly virulent mouse poxvirus, ectromelia virus. In addition, either perforin or Fas-mediated cytolysis was required for clearance of lytic infection of lung epithelia with influenza virus, in the absence of CD4` T cells. Thus, either perforin- or Fas-dependent cytolysis may be required to resist highly virulent infection with host-adapted viruses or in situations where CD8` T cells are the sole line of defense. The converse hypothesis, that CD8` T cell–mediated cytolysis is required for clearance of nonlytic viruses, requires further analysis. However, recent data in the LCMV system, described below, suggest that noncytolytic mechanisms may also be effective against this nonlytic virus (38). Finally, it should be noted that the designation of viruses as lytic or nonlytic is generally based on in vitro analysis in cell culture. For instance, the fact that LCMV is lytic for some cell types is the basis for the plaque assay to quantify the virus (39). Thus, lytic or nonlytic designations from in vitro analyses may not reflect the true nature of viral infection in vivo.

PERFORIN-DEPENDENT CYTOLYSIS: PROTECTION, IMMUNOPATHOLOGY, AND HOMEOSTASIS Intracranial infection of adult B6 mice with LCMV results in lethal CNS disease mediated by CD8` T cells (40). In contrast, B6-PKO mice do not develop severe CNS disease after intracranial infection (26). Thus, perforin-dependent cytolysis plays an important role in the immunopathogenesis of LCMV-induced CNS disease. Interestingly, a fraction of perforin-deficient mice succumb to peripheral infection with LCMV (26, 27), indicating that the failure to clear this normally nonlethal infection can be fatal. Infection of resistant B6 mice with Theiler’s virus (TMEV), a murine picorna virus, results in an acute encephalitis that is cleared by a CD8` T cell-dependent mechanism in approximately 10 days post infection (41). TMEV is lytic for murine fibroblasts, but viral replication is restricted in other cell types such as

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macrophages and may be restricted in the CNS as well (42). Intracranial infection of B6-PKO mice with 2 x 104 PFU of TMEV resulted in death within 20 days (43). Mortality was associated with a substantial viral load in the CNS of B6PKO mice at 11 days post infection, a time when virus is cleared from infected B6 mice. Thus, perforin appears to be essential for control of acute CNS infection by TMEV. Studies by another group revealed that intracranial infection of B6PKO mice with 2 x 106 PFU of TMEV resulted in a persistent infection under conditions where the infection was eliminated in B6 mice (44). Interestingly, although these B6-PKO animals exhibit some demyelination, they did not exhibit overt clinical symptoms. The extent of demyelination in these animals and the precise relationship between demylination and overt disease remain to be determined. These data, in conjunction with the finding that b2M-deficient mice develop persistent CNS infection and demyelination without overt CNS disease (45), suggest that perforin-dependent, CD8` T cell-mediated cytolysis contributes to both the clearance of TMEV and the clinical symptoms of CNS disease. The reason for the differences in mortality after infections with the same strain of virus is unknown. Interestingly, survival correlated with the highest challenge dose of virus. This result may be due to small differences in the virus strains, CD8` T cell exhaustion (46), or high-dose intracranial TMEV infection, which may induce a peripheral infection that inhibits migration of CD8` T cell into the CNS, as observed with LCMV (47). Intracranial infection of B6 mice with the neurotropic JHM virus, a member of the mouse hepatitis virus group of coronaviruses, results in demyelination that is at least partially dependent on virus-specific CD8` T cells (48). JHMV is rapidly cytolytic in vitro, but evidence for in vivo cytopathology is lacking. Perforin-deficient mice, infected intracranially with doses of JHMV that are lethal for wild-type mice, exhibit a moderate reduction in morbidity and extended survival compared to wild-type mice, although all perforin-deficient mice eventually succumb to infection (49). CNS viral loads were elevated, and clearance of virus from the CNS was delayed in the perforin-deficient mice. These results suggest that perforin-dependent cytolysis contributes to the efficiency of virus clearance but also contributes to clinical disease. CD8` T cells play a role in clearance of cardiopathogenic strains of the picornavirus, coxsackievirus B3, and also in the pathology of viral myocarditis (50). In contrast to wild-type B6 mice, which die from myocarditis within 10 days post infection, all perforin-deficient mice survive the infection (51). Inflammatory heart lesions were present in infected perforin-deficient mice, but at a reduced level compared to wild-type mice. Perforin-deficiency had no impact on viral replication in heart tissue or clearance of the virus from the host. Thus, perforindependent cytolysis contributes significantly to the immunopathology of myocarditis but not to the clearance of the inducing virus. CD8` T cell-derived perforin may also play a role in T cell homeostasis. B6 mice infected with LCMV clone 13 develop chronic infections associated with deletion of virus-specific CD8` T cells (52). Perforin-deficient mice develop

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chronic infections with LCMV clone 13 that are indistinguishable, based on the titers of virus in tissue, from those seen in B6 mice (53). However, deletion of LCMV clone 13–specific CD8` T cells in perforin-deficient mice is less efficient than in B6 mice, and a substantial fraction of the B6-PKO mice die within 2–4 weeks after LCMV clone 13 infection. This mortality is reversed by depletion of CD8` T cells. Similar results were observed in a model of high dose LCMV infection where deletion of CD8` T cells was less efficient in B6-PKO mice (46). Together, the results suggest that perforin plays a regulatory role in CD8` T cell homeostasis that is apparently independent of antigen levels, at least in LCMV clone 13 infections. Absence of this regulatory mechanism leads to death, perhaps related to the chronic activation of CD8` T cells. While the mechanism of action of perforin in this situation is undefined, the authors speculate that CD8` T cells may develop susceptibility to perforin-mediated damage after a certain upper limit of cell division (53). However, it is equally possible that other constituents of the cytotoxic granules are important in T cell homeostasis during chronic viral infection and that perforin is required for their function. Resolution of this interesting issue will require further investigation. These studies reveal the capacity of perforin-dependent cytolytic mechanisms to exacerbate disease, including cardiac and CNS pathology, after viral infection. Thus, perforin-dependent cytolysis is a two-edged sword, involved in viral clearance but also capable of mediating pathology. In addition, intriguing evidence suggests that the same granule exocytosis pathway may participate in CD8` T cell homeostasis during chronic viral infection.

RESISTANCE TO VIRUSES IN THE ABSENCE OF PERFORIN As mentioned above, perforin-dependent cytolysis was not required for resistance to lytic infections with vaccinia virus, Semliki Forest virus, vesicular stomatitis virus, or cowpox virus (30, 33). CD8` T cells are also required for clearance of acute lung (lytic) infection with murine gamma herpesvirus 68 (MHV-68) and to control latently infected B cells in this natural mouse infection model, with similarities to Epstein-Barr virus infections (54). Intranasal infection of B6 and B6PKO mice with MHV-68 resulted in similar kinetics of viral clearance in the lung, indicating that perforin-mediated cytolysis is not required to clear the acute lytic infection (55). In addition, levels of virus in the spleen up to 21 days post infection were not significantly different between B6 and B6-PKO mice, indicating that establishment of the latent infection was perforin independent. These results, together with the results with coxsackievirus B3 (51), indicate that perforindependent cytolysis is not required for resistance to all murine-adapted viruses. Similarly, IFN-c-deficient mice cleared lung infections and developed latent infection with MHV-68 in a manner that was only slightly, if at all, deficient compared to wild-type mice (56). In contrast, while IFN-c receptor–deficient mice

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clear lung infections with MHV-68, establishment of the chronic infection is delayed and the mice develop an unusual spleen pathology that is reversed upon depletion of CD8` T cells (57). This discrepancy in the nature of infection in IFN-c and IFN-c receptor–deficient mice is potentially interesting and may suggest the existence of alternate ligands for the IFN-c receptor. Consistent with this notion, herpes simplex virus infection is more virulent in IFN-c receptor–deficient mice than in IFN-c-deficient mice (58). The long-term control of latency or the sufficiency of Fas-mediated cytolysis for control of lytic or latent infections with MHV-68 was not addressed. This issue is of interest because Fas-mediated cytolysis clears influenza virus from lung tissue (32). CD8` T cells contribute to clearance of primary rotavirus infection in B cell– deficient mice as demonstrated by development of chronic infections after CD8` T cell depletion (59, 60). In CD8` T cell–depleted mice, or in b2M-deficient mice, clearance of rotavirus is slightly delayed compared to wild-type mice (60). Perforin- and Fas-deficient mice clear rotavirus infection with the same kinetics as wild-type mice, and infection of these mice is similarly exacerbated by CD8` T cell depletion (61). Perforin-deficient mice also clear rotavirus infection when treated with neutralizing anti-mouse IFN-c, and perforin-deficient CD8` T cells clear virus from chronically infected Rag-2 deficient mice with kinetics similar to those of wild-type CD8` T cells. While these results clearly demonstrate a contribution of CD8` T cells in resistance to viral infection in the absence of perforin, Fas, or IFN-c, they are also potentially consistent with the relevance of either perforin- or Fas-dependent lysis as described in the influenza system (32). However, it is also clear that CD8` T cells are not the major mediators of rotavirus immunity in wild-type mice, and thus, it may not be surprising that no single CD8` T cell effector mechanism is required for clearance. Despite the evidence that CD8` T cells participate in control of these viruses, perforin-dependent cytolysis is not required and does not contribute in a detectable fashion to control of a murine-adapted virus, MHV-68, as well as coxsackievirus, rotavirus, vaccinia virus, Semliki Forest virus, vesicular stomatitis virus, and cowpox virus. In the majority of these studies, the role of Fas-dependent cytolysis in the absence of perforin remains to be examined. Each of these viruses is capable of lytic infection, but they represent a broad spectrum in the virulence they bring to mouse infection, and evidence exits that CD8` T cell–independent mechanisms also participate in clearance of infection. Further studies are required to determine which, if any, of the known CD8` T cell effector mechanisms are involved in clearance of these pathogens.

CD8` T CELL CYTOKINES IN RESISTANCE TO VIRAL INFECTION In addition to their capacity to induce cytolysis of infected cells, effector and memory CD8` T cells produce cytokines such as IFN-c and TNF after stimulation through their antigen receptors. A role for CD8` T cell–derived IFN-c in

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resistance to LCMV infection was suggested by the observation that viral clearance was delayed by several days in IFN-c receptor deficient mice compared to controls (62). The IFN-c receptor–deficient mice mounted an LCMV-specific CD8` T cell response that was similar in magnitude to that observed in wildtype mice but which reached peak levels with delayed kinetics, associated with the delayed clearance of the infection. However, in contrast to perforin-deficient mice that fail to clear LCMV and exhibit altered homeostasis of the CD8` T cell compartment (26, 27, 53), IFN-c receptor–deficient mice eventually clear the infection, and homeostasis of the CD8` T cell compartment appears normal. Similarly, IFN-c-deficient mice eliminate acute LCMV infection within 12 days and develop significant CD8` T cell responses (63). Although these studies do not formally demonstrate that CD8` T cell derived IFN-c participates in resolution of LCMV infection they clearly indicate that IFN-c is not a required CD8` T cell effector mechanism for clearance of this virus. Infection of newborn mice with LCMV causes a persistent, lifelong infection (40). Transfer of LCMV-specific memory CD8` T cells, in the form of immune splenocytes, into persistently infected wild-type mice results in clearance of the infection from most tissues by 21 days post transfer. Although clearance is absolutely dependent on CD8` T cells, effective immunotherapy requires both CD8` and CD4` T cells (52). Interestingly, LCMV-immune splenocytes from IFN-cdeficient mice fail to clear the persistent infection from wild-type mice (63). This result suggests that IFN-c from CD4` or CD8` T cells is critical for clearance of the persistent infection, perhaps through its immune regulatory functions. However, recent work demonstrated that CD8` T cells, but not CD4` T cells, from IFN-c-deficient mice survive after transfer into Listeria monocytogenes–infected SCID mice (64). Since clearance of chronic LCMV infection requires both CD4` and CD8` T cells, a role for IFN-c in survival of CD4` T cells could also explain the failure to clear chronic infection. This issue could be resolved by mixing experiments with wild-type and IFN-c-deficient T cell populations. As described above, clearance of JHMV from the CNS is at least partially dependent on CD8` T cell–derived perforin (49). IFN-c-deficient mice also exhibit delayed clearance of JHMV, more severe paralysis, and increased mortality in comparison to wild-type mice (65). IFN-c-deficient mice develop virusspecific CD8` T cells responses at levels similar to those of wild-type mice. Interestingly, although the IFN-c-deficient mice clear virus from most CNS cell types such as neurons, microglia, and astrocytes, they fail to clear virus from oligodendrocytes. This finding may reflect the fact that oligodendrocytes apparently lack MHC class I molecules but do express the IFN-c receptor (66). Viral hepatitis, caused by hepatitis B virus (HBV), is thought to occur through the destruction of infected hepatocytes by MHC class I–restricted CD8` T cells (67). Interestingly, essentially all hepatocytes are infected, yet viral clearance and clinical recovery occur without destruction of the entire liver. Using a transgenic model, in which murine hepatocytes express the entire HBV genome, evidence was provided for a nonlytic mechanism by which CD8` T cells are able to

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extinguish viral gene expression (68). Transfer of MHC class I–restricted antiHBV CD8` T cell clones into HBV transgenic mice eliminated expression of the viral genome with minimal hepatocyte death. HBV-specific CD8` T cell clones from perforin-deficient mice, which caused no discernible hepatocyte death, also mediated the effect. The ability of wild-type CD8` T cells to inhibit viral gene expression was abrogated by neutralization of both IFN-c and TNF and was partially blocked when either cytokine was neutralized. Although these results demonstrate the participation of CD8` T cells, IFN-c, and TNF in the shutdown of viral gene expression, they do not conclusively demonstrate that CD8` T cells are the source of the relevant cytokines, both of which are made by multiple cell types. This issue is relevant to understanding the specificity and mechanism of viral clearance because infection of the same HBV transgenic mice with LCMV, adenovirus, and cytomegalovirus also results in elimination of HBV gene expression (69, 70). Thus, HBV clearance can be independent of CD8` T cell recognition of HBV-infected cells. This model has now been extended to primates infected with HBV, which clear the virus without substantial liver damage and prior to extensive T cell infiltration (71) and persistent LCMV infection, initiated by infection of newborn mice (38). The latter mice harbor LCMV in hepatocytes and Kupffer cells of the liver. As described above, transfer of LCMV-immune CD8` T cells into persistently infected mice causes clearance of infection unless the transferred cells do not express IFN-c (63). CD8` T cell–mediated clearance of LCMV from persistently infected mice occurs in the absence of extensive liver damage. Interestingly, viral RNA and antigen were rapidly cleared from hepatocytes but only slowly cleared from nonparenchymal Kupffer cells. Administration of IL-12 or infection with adenovirus also resulted in clearance of LCMV infection of hepatocytes but not nonparenchymal cells. These data suggest that cytolysis is essential for complete clearance of virus, specifically from those cells that derive from the regenerating macrophage pool. In contrast, viral clearance from hepatocytes appeared to result from a nonlytic mechanism. The authors speculate that such nonlytic mechanisms may be operative to clear infections with extensive involvement of essential tissues such as the liver (38). CD8` T cells are thought to be important in control of HIV infection of humans, although the effector mechanisms involved in viral clearance remain largely undefined (72). However, it was recognized in the 1980s that CD8` T cells from HIV-infected individuals secreted a factor or factors that inhibit HIV replication in vitro, in the absence of cytolysis, and it was suggested that these factors may contribute to control of infection in vivo (73). Shortly before the demonstration by multiple groups that b-chemokine receptors are coreceptors for HIV (74), a study showed that the b-chemokines RANTES, MIP-1a, and MIP1b could be purified from the supernatants of transformed CD8` T cells and inhibit HIV replication in a T cell line in vitro (75). In this study, neutralization of these b-chemokines with an antibody cocktail completely abrogated the ability of CD8` T cell supernatants, from transformed cells and 3 of 4 HIV-infected donors, to inhibit HIV replication. From these data and other similar reports, it

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was concluded that the nonlytic inhibition of HIV replication in vitro was a consequence of interference with HIV binding to the b-chemokine coreceptors. However, addition of antibodies that neutralize these same b-chemokines did not abrogate the ability of CD8` T cells to inhibit HIV replication in macrophages (76) or CD4` T cell/dendritic cell cocultures (77). These results suggest that novel CD8` T cell–derived anti-HIV factors remain to be identified. The discrepancy in the results of these studies may relate to the virus strain and the target cell types analyzed; further studies will be needed to resolve this issue. Together, these studies reveal the potential of CD8` T cell–derived soluble factors (cytokines and chemokines) to influence viral infection in the absence of cytolysis. Signaling through cytokine receptors for IFN-c and TNF results in altered gene expression in the responding cell (78, 79). Since viruses utilize host molecules to transcribe and translate their gene products, it is possible that cytokines can directly inhibit viral infection by influencing host factors required for viral gene expression. Such nonlytic mechanisms to eliminate viruses could potentially lessen the degree of immunopathology in response to viral infection of essential tissues such as the liver or CNS. Despite the strong evidence that bchemokines, and perhaps other unidentified CD8` T cell factors, limit HIV infection of cells in vitro, the challenge lies in determining the role of this mechanism in resistance to HIV infection in vivo and the reason for the ultimate failure of the host to control this infection. Finally, it should be pointed out that none of the studies reviewed here conclusively demonstrated that CD8` T cells are the relevant in vivo source of these cytokines, all of which are produced by other cell types during infection.

CD8` T CELLS IN RESISTANCE TO INTRACELLULAR PROTOZOA Mouse models of infection with intracellular protozoan parasites have been used to analyze the pathogenesis of these organisms and the immune response to infection. These pathogens undergo intricate developmental processes, often involving multiple host species, and the immune response to parasite infection is complex and multifactorial. Experiments with T cell subset depletion and gene-deficient mice reveal that CD8` T cells contribute to immunity against a number of protozoan parasites including Toxoplasma gondii (80), Plasmodium berghei (81–83), Trypanosoma cruzi (84, 85), and Encephalitozoon cuniculi (86). Information on CD8` T cell effector mechanisms is currently limited to these pathogens.

INFECTIONS WITH INTRACELLULAR PROTOZOAN PATHOGENS Each of these parasites has a distinct life cycle that needs to be considered in the context of CD8` T cell effector functions. T. gondii, the causative agent of toxo-

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plasmosis, is an apicomplexan protozoan found in nearly all animals and most birds. Transmission of oocysts, which develop in the cat gut, occurs by consumption of contaminated undercooked meat, in utero by hematogenous spread, or via tissue transplant. The oocysts contain sporozoites that invade host cells and differentiate into tachyzoites, which reproduce rapidly in the parasitophorus vacuole, eventually rupturing the infected cell to release infectious tachyzoites. A normal immune response leads to encystment within brain and muscle tissue; the parasites within such cysts reproduce very slowly, if at all, and persist for years. Loss of immune function, as seen in immunocompromised patients, allows reactivation and/or spread of the disease. Sporozoites of Plasmodium species, the causative agents of malaria in humans and animal models, are transmitted to the host bloodstream via mosquito bite. The sporozoites enter hepatocytes where they mature within a parasitophorus vesicle. Within approximately 2 weeks, the sporozoites mature into tissue schizonts that rupture the infected cell, producing 10,000 to 40,000 merozoites. Merozoites initiate the red blood cell stage of infection and are the source for mosquito transmission. Since hepatocytes express MHC class I molecules, it is assumed that they are the targets of Plasmodium-specific CD8` T cells. T. cruzi, the causative agent of Chagas’ disease, is a sarcomastigophoran protozoan capable of infecting most mammalian species. The parasite, which is naturally transmitted by the bite of an insect, can infect cells such as macrophages and cardiac myocytes. Parasites enter cells in a parasitophorus vacuole but then escape to the cytoplasm where they differentiate into amastigotes and proliferate. The infected cells then burst, liberating trypomastigotes that invade other cells. Major sites of infection include the CNS, reticuloendothelial system, and heart. Disease occurs as an acute febrile phase