Advances in Immunology Volume 74

ADVANCES IN

Immunology VOLUME 74

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ADVANCES IN

Immunology EDITED BY FRANK J. DIXON The Scripps Research Institute La Jolla, California ASSOCIATE EDITORS

Frederick Alt K. Frank Austen Tadamitsu Kishimoto Fritz Melchers Jonathan W. Uhr

VOLUME 74

ACADEMIC PRESS San Diego San Francisco New York Boston London Sydney Tokyo

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Copyright 0 2000 by ACADEMIC PRESS All Rights Resewed. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the fmt page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the US. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2000 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2776/00 $30.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

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Academic Press 24-28 Oval Road, London NWl 7DX, UK http://m.hbuk.co.uk/ap/ International Standard Book Number: 0-12-022474-7 PRINTED IN THE UNITED STATES OF AMERICA 99 0 0 0 1 02 03 04 EB 9 8 7 6 5

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CONTENTS

CONTRIBUTORS

ix

Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers

KIMISHIGEISHIZAKA, YASUYUKIISHII, TATSUMI NAKANO, AND KATSUJISUGIE I. 11. 111. IV. V.

Introduction Historical Background Controversial Issues on Antigen-Specific TsF and Possible Answers Possible Roles of GIF as a Subunit of TsF Discussion and Summary References

1 1 17 33 51 55

The Role of Complement in B Cell Activation and Tolerance

MICHAEL C. CARROLL I. Introduction 11. Complement C3 Alters Fate of Antigen 111. Complement Enhancement of Humord Immunity IV. Negative Selection of Self-Reactive B Cells V. Complement Deficiency and SLE VI. Summary References

61 61 65 72 78 82 83

Receptor Editing in B Cells

DAVIDNEMAZEE

I. Introduction 11. Receptor Selection versus Clonal Selection 111. Antigen Receptor Gene Assembly by V(D)J Recombination IV. Recombination Signals and the 12/23 Rule V. Ig Genes of Mouse and Man VI. Secondary Rearrangements V

89 89 89 90 91 92

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CONTENTS

VII. Recombinational Accessibility VIII. Quasi-ordered Rearrangements IX. Allelic Exclusion X. Mechanisms of Allelic Exclusion XI. Ig-L-Chain Allelic Exclusion XI. Receptor Editing Monitored in Vivo in Transgenic Models of Immune Tolerance XIII. Analysis of Receptor Editing in Gene-Targeted Antibody Gene Mice X N . Central B Cell Tolerance Is Associated with Developmental Block XV. Locus Specificity of B Cell Receptor Editing XVI. Developmental Stage S ecificity of Receptor Editing XVII. A Role for Receptor E ‘ting in Receptor Diversification? XVIII. Editing in Mature, Antigen-Reactive B Cells: Receptor Revision XIX. Locus S ecificity of Receptor Revision XX. Potentia! Functions of Receptor Revision XXI. Problems with the Receptor Selection Concept XXII. Conclusion References

ce

94 95 96 97 99 100 102 104 106 107 108 108 110 111 112 114 114

Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection

PIUSLOETSCHER, BERNHARD MOSER,AND MARCO BAGGIOLINI I. 11. 111. IV. V.

Introduction Expanding Functional Implications of Chemokines Chemokines and Chemokine Receptors in Lymphocytes Chemokine-Mediated Signal Transduction HIV Infection References

127 127 129 144 146 158

Escape of Human Solid Tumors from T-cell Recognition: Molecular Mechanisms and Functional Significance

FRANCESCO M. MARINCOLA, ELIZABETH M. JAFFEE,DANIEL J. HICKLIN, SOLDANO FERRONE

AND

I. 11. 111. N.

Introduction Tumor Cell-Escape Mechanisms Inadequacy of Tumor Cells as Targets Potential Role of HLA Class I Polymorphism in Tumor Cell Escape V. Inhibitory Signals Provided by the Tumor Microenvironment VI. Inadequate Immunogenicity of the Tumor Microenvironment VII. Escapin Esca e Mechanisms: Possible Immuno ogc . a f Alternatives References

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CONTENTS

vii

The Host Response to Leishmania Infection

WERNER SOLRACH AND TAMAS LASKAY I. Introduction 11. The Parasite 111. The Host’s Innate Response IV. The Host’s Adaptive Immune Response V. Persistence VI. Genetic Background of Resistance and Susceptibility VII. Concluding Remarks References

INDEX OF RECENTVOLUMES CONTENTS

275 277 281 285 299 300 302 303 319 327

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CONTRIBUTORS

Numbers in parenthem indicate the pages on which the authors’ contributions begin.

Marco Baggiolini (127), Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland Michael C. Carroll (61), The Center for Blood Research, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 Soldano Ferrone (181),Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263 Daniel J. Hicklin (181),Department of Immunology, ImClone Systems Incorporated, New York, New York 10014 Yasuyuki Ishii (l),La Jolla Institute for Allergy and Immunology, San Diego, California 91121 Kimishige Ishizaka (l),La Jolla Institute for Allergy and Immunology, San Diego, California 91121 Elizabeth M. Jaffee (181),Johns Hopkins Hospital, Baltimore, Maryland 21205 Tam& Laskay (275), Institute for Medical Microbiology and Hygiene, University of Luebeck, 23538 Luebeck, Germany Pius Loetscher (127),Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland Francesco M. Marincola (181), Surgery Branch, Division of Clinical Sciences, National Cancer Institute, The HLA Laboratory, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892 Bernhard Moser (127), Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland Tatsumi Nakano (l),La Jolla Institute for Allergy and Immunology, San Diego, California 91121 David Nemazee (89),The Scripps Research Institute, La Jolla, California 92037 Werner Solbach (275), Institute for Medical Microbiology and Hygiene, University of Luebeck, 23538 Luebeck, Germany Katsuji Sugie (l),La Jolla Institute for Allergy and Immunology, San Diego, California 91121 ix

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ADVANCES IN IMMUNOLOGY, VOL. 74

Biochemical Basis of Antigen-Specific Suppressor T Cell Factors: Controversies and Possible Answers KlMlSHlGE ISHIZAKA, YASWUKI ISHII, TATSUMI NAKANO, AND KATSUJI SUGIE l a Jdla lnsrihrb For Allergy and Immundogy, Son Diego, California

1. Introduction

Generation of antigen-specific suppressor T (Ts) cells following antigen administration was demonstrated by Gershon and Kondo in 1971, and immunological function of these cells was studied extensively by many investigators for the next decade. In these studies, antigen-specific T cellmediated immunosuppression could be transferred from one animal to another syngeneic animals either with intact suppressor cells or with soluble factors derived from the cells. Studies on antigen-specific Ts cells and suppressor T cell factors (TsF) became a major subject of research in cellular immunology from 1971 to 1983. After identification of T cell receptors (TCR) on helper and cytotoxic T cells, however, fundamental questions were raised on T cell-mediated suppression with the failure to identify TCR and TCR gene rearrangement in representative Ts hybridomas. In addition, the ability of Ts cells and antigen-specific TsF to bind specifically to nominal antigen, independently of MHC gene products, created skepticism on the nature of Ts cells and the origin of TsF. Furthermore, in spite of extensive studies and great efforts by many investigators, biochemical characterization of TsF has not been achieved, and genes encoding any of the TsFs have not been cloned. If TsF does not exist, however, why have many immunologists reproducibly observed antigenspecific suppression of immune responses by T cell factors which could be absorbed by antigenhapten-coupled immunosorbent. The purpose of this article is to review previous observations on TsF and to make some interpretations for controversial issues on this factor. II. Historical Background

A. CONDITIONS FOR THE GENERATION OF ANTIGEN-SPECIFIC SUPPRESSOR T CELLS

After the first description of Ts cells by Gershon and Kondo (1971), similar observations were made by Tada and Takemori (1974). They treated C57B1/6 mice with keyhole limpet hemocyanin (KLH) and demonstrated that transfer of spleen cells or thymus cells of the KLH-treated mice to 1

Copyright 0 2000 by Academic Press. All rights of reproduction in any form reserved. 0065-2776/@3$30.00

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unirradiated syngeneic mice resulted in suppression of anti-hapten antibody response of the recipients to dinitrophenyl (DNP) derivatives of KLH. In this system, treatment of the cells from the KLH-treated donor with anti-Thy-1 and complement abolished the ability of inducing suppression, indicating that antigen-primed T cells were responsible for immunosuppression. In general, suppressor cells could be generated by intravenous injections of a high dose protein antigen, such as KLH, ovalbumin (OVA), and deaggregated human or bovine IgG, which is an immunizing regimen unfavorable for the production of antibodies. Howie and Feldmann (1977) then succeeded in generating antigen-specificTs cells by culture of normal spleen cells with a relatively high concentration of a variey of antigens. A concentration of antigen about 100-fold higher than that optimal for the in vitro priming of helper cells was required for in vitro induction of Ts cells. If macrophages were partially depleted from spleen cells, however, a much lower dose of the antigen was capable of preferentially inducing Ts cells (Ishizaka and Adachi, 1976; Howie and Feldman, 1977). Suppressor T cells could be generated in high responder mouse strains by using modified antigens. For example, ovalbumin-specific Ts cells could be generated in BDFl mice by repeated intravenous injections of ureadenatured OVA, which can prime Th cells but lacked the major B cell epitope in the native antigen (Takatsu and Ishizaka, 1976).Similarly, conjugation of polyethylene glycol (PEG) to protein antigens such as OVA and human IgG (HGG) diminished the immunogenicity of the antigen and facilitated the generation of Ts cells, A single intravenous injection of 1 mg of OVA-PEG into BDFl mice prior to immunization with alumabsorbed DNP-OVA abolished the anti-DNP antibody responses (Lee and Sehon, 1978), and transfer of splenic T cells of the OVA-PEG treated mice into syngeneic mice resulted in suppression of anti-DNP antibody response of the recipients to DNP-OVA (Lee et al., 1981). A series of experiments by Benacerraf and his co-workers using a linear synthetic polymer poly (L-glutamic acid@'-~-alanine~~-~-tyrosine~~) (GAT) as an antigen provided much information on Ts cells (Kapp et al., 1974a, b, 1976). They have shown that an injection of GAT into nonresponder mouse strains to GAT (e.g., H-2s and H-2s mice) made these animals refractory to the subsequent immunization with GAT complexed with methylated-BSA, an immunogenic carrier to which all nonresponder strains could mount a substantial anti-GAT PFC response. This immunosuppression was proved to be due to the development of GAT-specific suppressor cells in nonresponder mice. GAT-specific Ts cells could be generated in vitro even in high responder spleen cells by culture of the cells with a high concentration of GAT (Theze et al., 1977a; Pierres and Germain, 1978). It was also found that partial depletion of macrophages from high

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responder spleen cells led to the preferential induction of Ts cells with normally immunogenic concentration of GAT. Treatment with ultraviolet light, which eliminated the Ia-bearing antigen-presenting cells (APC), resulted in the selective induction of antigen-specific Ts cells (Greene ef al., 1979b). T cells capable of suppressing immune response have been described in a wide range of systems by many investigators (reviewed in Tada and Okumura, 1979; Dorfand Benacerraf, 1984).Suppressor T cells can modulate various immune responses, involving both humoral and cellular immunity, the latter of which includes delayed type hypersensitivity (Bach et al., 1978; Weinberger et al., 1979) and contact sensitivity (Asherson and Zembala, 1974). Particularly, Ts cells in contact sensitivity were extensively studied. Zembala and Asherson (1974) reported that mice injected intravenously with picrylsulfonic acid failed to develop contact sensitivity upon subsequent immnization with picrylchloride by skin painting, and that passive transfer of lymph node cells of the picrylsulfonic acid-treated mice to normal mice prevented the development of contact sensitivity of the recipients to picrylchloride. Claman and his colleagues (Miller and Claman, 1976; Moorehead, 1976) have shown that pretreatment of mice with dinitrobenzene sulfonic acid (DNBSOJ or with dintroflurobenzene (DNFB)treated syngeneic lymph node cells induced tolerance to contact sensitization with DNFB, and that this tolerance could be transferred to normal recipients by their lymph node cells. The method to induce hapten-specific tolerance by an intravenous injection of hapten-coupled syngeneic spleen cells was applied by Bach et al. (1978) to induce tolerance specific for p-azobenzenearsonate (ABA) in A/J strain mice. They found that subcutaneous injection of ABA-coupled syngeneic spleen cells resulted in sensitization of the animals for delayed type hypersensitivity (DTH) to the hapten or hapten-coupled spleen cells. However, spleen cells and thymus cells from the tolerant mice, which had been established by an intravenous injection of the same hapen-coupled spleen cells, were capable of suppressing the development of DTH in syngeneic recipients that were subcutaneously sensitized with the ABA-coupled spleen cells. An intravenous injection of hapten-coupled syngeneic spleen cells was employed to induce Ts cells specific for 4-bydroxy-3-nitrophenylacetyl (NP) group as well (Weinberger et al., 1979). Spleen cells of mice injected 7 days previously with NPcoupled syngeneic spleen cells contained NP-specific T cell populations capable of suppressing DTH-mediated footpad swelling responses to NP. B. PROPERTIES OF Ts CELLS Antigen-specific Ts cells found in many experimental systems were CD8' cells (Feldman et al., 1975; Cantor et al., 1976; Tadaet al., 1977). However,

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it became clear that very complex cell interactions are involved in the generation of effector Ts cells, and each of the interacting cells had a different phenotype. In general, the Ts cell system consists of at least two and in some cases three distinct Ts subpopulations (Germain and Benacerraf, 1981; Sunday et al., 1981; Green et al., 1983). The first acting Ts cells are CD4' inducer cells (Tsi or Ts 1) (Weinberger et al., 1979; Benacerraf and Germain, 1979; Claman et al., 1980), and these cells are involved in the generation of effector Ts cells which express CD8 (Feldman et al., 1975, Sy et al., 1979; Baxevanis et al., 1982). A cell surface marker that appeared to be unique for Ts cells was described in 1976 (Murphy et al., 1976; Tada et al., 1976).An antiserum raised by immunization of the congenic strain BlO.A(3R) with cells from BlO.A(5R) (or vice versa) seemed to react exclusivelywith Ts cells. Genetic analysis indicated that the gene encoding the antigenic determinant was located in the I region of the class I1 MHC, called 1-3. Monoclonal antibodies to I-J haplotypes were generated as well (Kanno et al., 1981; Waltenbaugh, 1981; Uracz et al., 1985a). Using monoclonal anti-I-J antibody, however, Tada et al. (1978)demonstrated that some helper T cells express I-J determinant. Nevertheless, Ts cells in various antigen systems could be depleted by treatment of splenic lymphocytes with anti-I-J antibody and complement (Kanno et al., 1981; Trial et al., 1983). A unique feature of Ts cells is their ability to bind nominal antigen. In many systems, antigen-specific Ts cells could be obtained by setthng B cell-depleted spleen cells in the antigen-coated polystyrene surfaces (Okumura et al., 1977; Taniguchi and Miller, 1977).Enriched population of Ts cells could be recovered from the antigen-coated plates following temperature shift. The yield of cells recovered from the plates was less than 0.5% of original splenicT cells, but the majorityof the antigen-specific suppressor activity was recovered. Indeed, the human IgG(HGG)-specificTs hybridomas (Taniguchiand Miller, 1978),KLH-specificTs hybridomas (Taniguchi et al., 1979) and NP-specific Ts hybridomas (Kuchroo et al., 1990) were established using the Ts cell populations enriched by using antigen-coated plates. The KLH-specific Ts hybridomas derived from C57B1/6 (H-2b) mice bound lZI-labeled KLH and expressed I-Jb determinant (Taniguchi et al., 1982). SUPPRESSOR T CELLFACTOR (TsF) ACTMTY C. ANTIGEN-SPECIFIC Another development in Ts cells was the finding that extract of Ts cells contained soluble factor(s)which replace the function of Ts cells. Takemori and Tada (1975) primed mice with a relatively high dose of KLH for a generation of Ts cells, and lysed the thymus cells and spleen cells by sonication. An injection of the sonicated cell-free material to syngeneic

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mice at the time of primary immunization with DNP-KLH resulted in suppression of IgG anti-DNP antibody response of the recipients. However, the same extract was ineffective in suppressing the anti-hapten antibody response of the same strain mice to DNP coupled to unrelated carrier, such as DNP-BGG and DNP-OVA. The splenic and thymic extract of the KLH-treated mice was also effective in suppressing the in vitro secondary anti-hapten antibody response of the DNP-KLH-primed spleen cells to the homologous antigen (Taniguchi et al., 1976). The effect of the extract was not only antigen (carrier)-specific but also MHC-restricted. If the factor was raised in H-2”’dF1 mice, it would suppress antibody responses of each parental haplotype (i.e., H-2kand H-2d)but not the strains of other MHC haplotypes. As expected, Ts hybridomas contained TsF. Lysate of the KLH-specific Ts hybridoma cells obtained by freeze-thaw treatment or ascitic fluid from hybridoma-bearing mice suppressed the in vitro secondary antibody response to DNP-KLH of syngeneic or semisyngeneic but not allogeneic spleen cells (Taniguchi et al., 1979). Thus, the hybridoma-derived suppressor factor suppressed the antibody response in the antigen-specific fashion and did not appear to act across the H-2 barrier (Taniguchi et al., 1982). Our studies on isotype-specific regulation of IgE antibody response lead to identification of a suppressor T cell factor that had affinity for homologous antigen. In the course of the studies, we described T cell factors that had affinity for IgE, and either enhanced or suppressed IgE synthesis in an isotype-specific manner ( Ishizaka, 1984). The IgE-potentiating factors and IgE suppressive factors share a common structural gene but are different in their carbohydrate moieties in the molecules (Martens et al., 1987). Under physiological conditions, both the carbohydrate moieties and bioactivities of IgE-binding factors are controlled by two T cell factors that either enhance or inhibit the posttranslational glycosylation process of IgEbinding peptide during their biosynthesis (Ishizaka, 1988).The glycosylation inhibiting factor (GIF) inhibited N-glycosylation of IgE-binding peptide, and rendered the latter factors to selectively suppress the IgE synthesis. As the major cell source of GIF was antigen-specific Ts cells (Jardieu et al., 1984), we constructed OVA-specific Ts hybridomas from spleen cells of BDFl mice which had been treated by intravenous injections of OVA, and selected hybridomas which constitutivelysecreted GIF activity (Jardieu et al., 1985). The GIF activity in culture supernatant of unstimulated hybridomas had a molecular size of about 13-14 kDa, and did not bind to OVA. Upon stimulation with OVA-pulsed syngeneic APC, however, some of the hybridomas produced GIF having afinity for OVA which could be purified using OVA-coupled Sepharose (Jardieu et al., 1987). The molecular size of the OVA-specific GIF was approximately 80 kDa, as

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estimated by gel filtration. The affinity-purified OVA-specific GIF suppressed the in vivo IgE and IgG antibody responses of syngeneic mice to DNP-OVA in a carrier-specific manner. Thus the antigen-specific GIF appears to represent an antigen-specific TsF. Kapp et al. (1976) obtained GAT-specific suppressor cells by immunizing nonresponder H-2s and H-2s mice with GAT and disrupting their thymus cells and spleen cells by sonication. An intravenous injection of extracts of the spleedthymus cells into syngeneic or allogeneic nonresponder mice resulted in suppression of IgG anti-GAT antibody response of the recipients to the immunogenic GAT-MBSA conjugate. If the nonresponder mice were immunized with the immunogenic GAT-MBSA conjugate, however, extracts of their spleen cells failed to show the suppressive activity. The GAT-specific suppressor activity of the extracts of GAT-primed nonresponder lymphoid cells was demonstrated in an in vitro system as well. The primary in vitro IgG antibody response of the spleen cells of unprimed nonresponder mice to GAT-MBSA was suppressed by the addition of the extract to the cultures (Kapp et al., 1977). Hapten-specific TsFs involved in contact sensitivity were described by several investigators. Zembala and Asherson ( 1974) induced tolerance to picryl chloride in mice by treatment with picryl sulfonic acid and sensitized them by skin painting with immunogenic picrylchloride. Several hours after the skin painting, their lymph node cells were obtained and cultured without further addition of antigen. Culture supernatnat of the lymph node cells were then injected into irradiated hosts together with picrylchloridesensitized lymph node cells. They found that the lymph node cells of the tolerant mice released a factor that specifically suppressed the ability of sensitized lymph node cells to transfer contact Sensitivity to irradiated recipients. This factor failed to suppress the sensitivity to trinitro chlorobenzene (TNCB), a sensitizer differing only on a single NOz group on benzene ring, indicating strict antigen specificity of the factor. Similar findings were obtained by Moorhead (1977) in DNFB-specific contact sensitivity system. Lymph node cells from the mice, which had been pretreated with DNBS03 and then skin-painted with DNFB, were cultured to obtain culture supernatant. The ability of DNFB-sensitized lymph node cells to transfer contact sensitivity was greatly diminished by incubating the cells with the culture supernatant. In this system, skin-painting of the tolerant mice was essential for the formation of suppressor factor by their lymph node cells, suggesting that antigenic stimulation was required for the formation of the antigenspecific factor. Indeed, subsequent experiments showed that culture of spleen cells from DNBS03-treated mice with DFNB-coupled syngeneic spleen cells resulted in the formation of the antigen-specific TsF (Moorehead, 1982).An intersting finding on the DNFB factor was that the factor

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derived from CBA(H-2k)lymphocytes could suppress the contact sensitivity of syngeneic mice but not allogneic H-2d or H-2b mice. Further studies

by Moorehead (1979) to solve the genetic restriction of his antigen-specific factor using congenic mouse strains indicated that the identity of K or D region of H-2 complex between the donor of the factor and responding T cells was required for the suppression of contact sensitivity. The results suggested that this factor recognized the haptenic determinant associated with the products of either the H-2K or H-2D locus. Greene et al. (1979a) demonstrated ABA-specific suppressor activity in the extract of ABA-tolerant spleen and thymus cells. The activity was also obtained by culturing spleen cells of mice which received an intravenous injection of ABA-coupled syngeneic spleen cells followed by challenge with ABA diazonium salt 24 hr before sacrifice. The factor was capable of abolishing the DTH response if injected for 5 consecutive days after sensitization with ABA-coupled spleen cells. However, the factor failed to affect the DTH responses to TNP-coupled spleen cells and showed the antigen specificity. OF ANTIGEN-SPECIFIC TsF D. PROPERTIES A unique property of TsFs is that the factors have affinity for the homologous antigen. The KLH-specific TsF activity in the extract of spleen cells of KLH-treated mice bound to KLH-coupled immunosorbent, and could be recovered by elution of the column at acid pH. When the extract was applied to a similar column coupled with the other antigens, such as OVA or BSA, the activity failed to retain in the column and was recovered in the flow through fraction (Takemori and Tada, 1975). The same principle applied to OVA-specific GIF/TsF (Jardieu et al., 1987), HGG-specific TsF (Chaouat, 1978), and hapten-specific TsF activities. The specific binding of the GAT-specific TsF from nonresponder mice to homologous antigen was established by absorption with GAT-Sepharose and elution of the activity from the column with 0.6 M KC1, which was employed for elution of anti-GAT antibodies bound to the same column (Theze et al., 1977a). Similarly, picryl chloride-specific TsF activity bound to picryl-albumincoupled Sepharose (Zembala et al., 1975), DNFB-specific factor was absorbed with and eluted from the TNP-column (Moorehead, 1977), and the ABA-specific suppressor activity was absorbed with and eluted from ABA-coupled Sepharose column (Greene et al., 1979a). A common property of TsF activity in the extracts and culture supernatants of 1-J+ Ts hybridomas is that the activity could be absorbed with anti-I-J antibodies. Thus, C57BUG-derived KLH-specific TsF could be absorbed on columns of alloantisera anti-H-2b or anti-I-Jb (BlOA(5R) antiBlOA(3R))but not with anti-I-Jk(BlOA(3R)anti-BlOA(5R))nor with rabbit

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antibodies against mouse immunoglobulins or those specific for the Fab portion of mouse immunoglobulins (Taniguchiet al., 1976, 1982).Association of an I-J determinant with TsF was confirmed in other studies on various antigen-specific TsF (Taniguchi and Miller, 1978; Theze et al., 1977a; Greene et al., 1977; Waltenbaugh et al., 1977; Aoki et al., 1983). BDF1-derived OVA-specific GIF also bound to anti-I-Jbbut not to antiI-Jk immunosorbent (Jardieu et al., 1987). Furthermore, the activity of TsF from H-2bbut not from H-2kmice was abrogated by the addition of a minute quantity of anti-I-Jb antibody in the in vitro PFC responses (Taniguchi et al., 1984). Reciprocally, the activity of H-2'-derived, GTspecific TsF was abrogated by monoclonal anti-1-Jkantibody (Waltenbaugh et al., 1981). E. SUPPRESSOR T CELLCASCADE Evidence has been presented that GAT-specific suppression is mediated by a cascade of T cell interactions. It was found that the generation of Ts cells by primary immunization of nonresponder mice with GAT or GT was prevented, if the animals were treated with cyclophosphamide 2-3 days prior to immunization. By this treatment, the animals lost nonresponsiveness to GAT, or GT and became a responder to the antigen. However, if the cyclophosphamide-treated animals were injected with cell-free extract of spleen cells from another GAT- or GT-immunized nonresponder mice, which should contain TsF, the recipients developed Ts cells. These results suggested the presence of two distinct populations of Ts cells. Further analysis of this phenomenon revealed that an injection of GATspecific suppressor factor from the inducer or first-order Ts cells (Tsi or Tsl), together with a minute dose of antigen, elicited the generation of effector or second population of GAT-specific Ts cells (Tse or Ts2) (Germain et al., 1978). Ts2 hybridomas have been established by fusion of splenic T cells of mice that had been treated with GAT-specific TsFl and GAT. TsF2 obtained from the hybridoma is antigen-specific in suppressive activity and in its antigen-binding capacity (Kapp et al., 1980). Extensive studies on the Ts cell cascade were carried out by Dorf and his associates using NP system. The cascade proposed by their work is schematically shown in Fig. 1. Similar to DNFB and ABA systems, spleen cells of mice injected 7 days previously with NP-coupled syngeneic spleen cells contained NP-specific T cells capable of specifically suppressing NPspecific contact sensitivity (Weinberger et al., 1979).The suppressor cells, called Tsl, displayed suppressive activity if they were transferred into syngeneic mice during priming of the recipient (inductive phase) for contact sensitivity response but failed to suppress the response if the cells were transferred into antigen-primed mice one day before antigen chal-

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y ldiotype mAnt i -Idi otype

FIG.1. A summary of the NP suppressor cascade. Intravenous administration of NP on I-At I-J' APC induces CD4+ Tsl cells. Tsl release TsFl that is presented on I-J-bearing factor presenting cells (FPC) to induce CD8+ Ts2, which in turn release TsF2. The TsF2 activates previously primed Ts3 cells in an I-J and Igh-restricted fashion. Once Ts3 cells are activated, they release TsF3, which after binding antigen on an I-J' FPC, mediates nonspecific suppression of CS responses either directly or through T acceptor cells (Tacc). (From Dorf and Benacerraf, 1984, from the Annual Review of Immunology, Vol. 2, 0 1984, by Annual Reviews, www.annualreviews.org, with permission.)

lenge. Sherr et al. (1980) gave an iv injection of either antigen-coupled adherent cells or nonadherent cells to mice and found that antigen-coupled adherent cells were 1000-fold more efficient at inducing Tsl activity than antigen-coupled nonadherent cells. These findings suggested that presentation of antigen by the adherent cells resulted in the generation of Ts cells. In the NP system, the adherent cell population responsible for Tsl induction carried Ia determinant and lacked T cell markers (Usui et al., 1984). Further characterization of the adherent cells indicated that the antigen-presenting cells for the induction of Tsl had the properties of macrophages. It was also found that the induction of Tsl cells required presentation of antigen in the context of H-2 coded determinants. In the NP and ABA systems, Tsl cells have been hybridized to generate Tsl hybridoma cell lines (Oknda et al., 1981a). These hybridomas were CD4+,constitutively secreted TsFl which functionally substituted for Tsl cells. Namely, TsFl suppressed contact sensitivity, if adminsitered on the day and the day after antigen priming (inductive phase). This factor specifically bound to anti-1-J alloantibodies, heterologous anti-idiotypic antibodies and an appropriate hapten-conjugated protein. If injected into

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normal mice, the TsFl could induce second population of Ts cells (Ts2) (Whitaker et al., 1981; Okuda et al., 1981b). The induction of Ts2 cells required 4-7 days which accounts for the fact that TsFl must be administered early to show suppressive activity. Ts2 cells were capable of suppressing responses when added in the effector phase of the DTH, contact sensitivity, or PFC responses. The Ts2 cells could be generated in vitro as well by incubating normal syngeneic spleen cells with NP-specific Tsl hybridoma supernatant (Sherr et al., 1983). An important property of TsFl is that the factor is able to induce Ts2 cells in any strain of mouse without genetic restriction (Okuda et al., 1981b; Kapp et al., 1983). It was also found that the induction of Ts2 cells required presentation of TsFl by factor-presenting cells which appeared to be I-J' macrophages (Aoki et al., 1983). The Ts2 cells are anti-idiotypic CD8+ cells, do not bind antigen, but bound to NPb-related idiotype-coated plates (Weinberger et al., 1980). Hybridomas with the properties of Ts2 cells have been established. These cells constitutivelyproduced soluble factors (TsF2) with functional properties similar to the Ts2 cells (Minami et al., 1981).Thus, TsF2 could suppress contact sensitivity when it was administered one day before and the day of antigen challenge (i.e., effector phase of the response). TsF2 did not have affinity for NP but reacted with allele-specific idiotypic antibodies and anti-I-J antibodies. At this point, Ts2 cells were thought to be the final effectors of suppression. However, an injection of Ts2 cells or TsF2 did not suppress the immune response, if the recipient mice had been treated with low dose of cyclophosphamide shortly after immunization, suggesting that the target of TsF2 is highly sensitive to cyclophosphamide. Evidence was obtained that the target cells were induced as a consequence of conventional immunization and that they were highly sensitive to cyclophosphamide. These cells are precursors of Ts3 cells and appear to be inactive until appropriately triggered by Ts2 or TsF2 (Sunday et al., 1981; Minami et al., 1982; Sherr and Dorf, 1982). Such precursor cells of Ts3 contained cytoplasmic TsF, but did not constitutively secrete the factor. The spleen cells responsible for the presentation of TsF2 for induction of Ts3 cells were Thyl- and adherent FcR' cells. They were absent in nonadherent cell and dendritic cell populations, and I-at I-Jt cells (Nakamura et al., 1982; Lowry et al., 1983). The Ts3 cells in the NP and ABA systems were antigen-specific, bound to antigen-coated plates (Sherr and Dorf, 1982), and their effects were H-2 restricted (Minamiet al., 1982).They expressed CD8 and reacted with anti-I-J alloantisera (c.f. Fig. 1).The Ts3 cells produced an antigenspecific TsF3, which mediated suppression in the effector phase of the immune response. The suppressive activity of Ts3 cells and TsF3 could be demonstrated in cyclophosphamide-treated recipients and was re-

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stricted by I-J and Igh genes (Minami et al., 1982). Ts3 hybridomas which constitutively secrete TsF3 have been obtained in the NP system (Okuda et al., 1981~). Some TsFs described by the other investigators probably belong to the effector type TsF. All of the DNP-specific TsF described by Moorehead (1979), picryl chloride-specific TsF described by Asherson and Zembala (1974),KLH-specific TsF described by Taniguchi et al. (1982) and effector type GT-specific TsF (designated as TsF2) described by Kapp and Araneo (1982) have affinity for respective antigen and may correspond to TsF3 in the NP system.

F. BIOCHEMICAL CHARACTERISTICS OF TsF Since most of the antigen-specific TsFs had affinity for nominal antigen and possessed an antigenic determinant recognized by anti-I-J antibodies, these two properties were employed to purify TsF. Taniguchi et al. (1980, 1981) fractionated either the extract of KLH-specific Ts hybridoma or ascitic fluid from the hybridoma-bearing mice on KLH-coupled Sepharose or anti-I-Jb coupled Sepharose and found that the TsF consisted of two distinct polypeptide chains. One of them had the capacity to specifically bound to KLH, and another chain contained I-Jbdeterminant. It was also found that the association of the two chains is required for the expression of TsF activity. For biochemical identification of the polypeptide chains, Saito and Taniguchi (1984)biosyntheticallylabeled the hybridoma products with =S-methionine and fractionated the extract of the hybridoma on KLHcoupled immunosorbent or anti-1-Jb coupled immunosorbent. Analysis of the acid eluates from the columns by SDS-PAGE indicated that the TsF consisted of the 45 kDa antigen-binding peptide and 27 kDa polypeptide having the I-J determinant. Attempts to identify the second-order GAT-specificTsF by Kapp and her associates gave somewhat similar results. They found that Ts2 hybridomaderived TsF was composed of two disulfide-linked polypeptide chainsone of which was antigen-binding and I-J- and the other chain which lacked affinity for GAT but bore 1-J determinant-and that both chains were required for TsF activity (Kapp et al., 1983). They tried to isolate GAT-specific TsF2 from a large quantity of culture supernatant of a Ts2 hybridoma by combination of ammonium sulfate precipitation, gel filtration through a Sephadex GlOO column, followed by isoelectric focusing and reverse phase column chromatography (Turck et al., 1985). The protein isolated by these procedures had a molecular weight of approximately 66,000 and isoelectric point of 6.8 to 6.9. Association of TsF activity with the protein was confirmed by analysis of the final preparation by SDSPAGE under nonreducing conditions. The TsF bioactivity was recovered

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by elution of the gel slice containing the 66-kDa band. The purified TsF bound to GAT-coupled Sepharose and anti-I-Jq antibodies. To obtain more information on the biochemical properties of TsF, the TsF2 from the same hybrdoma was biosynthetically labeled with =S-methionine, and analyzed by 2D nonreducingheducing SDS-PAGE. The results of the experiments revealed that the TsF was a disulfide-linked heterodimer, composed of a basic and an acidic polypeptide chains, both having molecular weight of 30,000. The basic polypeptide reacted with anti-I-J antisera, whereas the acidic chain had antigen-binding capacity (Turck et al., 1986). The NP-specific TsF3 may have similar biochemical characteristics to the KLH-specific TsF and GAT-specific effector type TsF. Reduction with 5 mM DTT did not affect the suppressive activity of NP-specific TsF3. After fractionation of the reduced material on NP-BSA-coupled Sepharose or anti-1-J immunosorbent, neither eluate nor flow-through fraction had suppressive activity. However, the activity was restored by combining the eluate and flow-through fractions (Furusawa et al., 1984). Thus the TsF3 appears to be composed of disulfide-linked antigen-binding polypeptide chain and I-J' chain, and such basic structure appears to be common for the effector type TsF in the three antigen systems. The inducer type TsFs appear to be structually different from the effector type TsFs. TsFl in both GAT and NP systems bound to antigen-coupled immunosorbent and anti-I-J immunosorbents. Molecular size of the affinity purified TsF was in the range of 45,000to 60,000, as estimated by gel filtration (Theze et al., 197%; Furusawa et al., 1984). After reduction, however, the two determinants could not be separated. Thus, the TsFls were believed to be a single polypeptide chain, which has affinity for respective antigen and bear 1-J determinant. Based on such information, Krupen et al. (1982) tried to isolate GAT-specific TsFl in culture supernatant of a Ts hybridoma. The TsF was affinitypurified using GAT-Sepharose, and eluates from the immunosorbent column with 2.0 M KCl was fractionated by reverse phase, followed by ionexchange HPLC. The purified factor was a single polypeptide chain of Mr 24,000,and had the ability to suppress antibody and T cell proliferative responses to GAT. The preparation gave a single 24-kDa band in SDS-PAGE under both reducing and nonreducing conditions, and extract of the gel slice showed bioactivity. In their experiment, the concentration of purified TsF required for 50% suppression of the GAT-stimulated PFC was only 0.013 pg. G. MONOCLONAL ANTI-TsF Attempts were made to prepare monoclonal antibodies specific for TsF. One of them is the rat IgM monoclonal antibody 14-12, which is believed to be specific for effector type TsF. The hybridoma was established from

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spleen cells of rats immunized with partially purified murine SRBC-specific TsF. The SRBC-specific TsF in the serum of mice hyperimmunized with SRBC could be affinity purified using the mAb coupled to Sepharose (Ferguson et al., 1985). Analysis of the eluates from the column, after radiolabeling, gave a single band of 68,000 Da in SDS-PAGE under reducing conditions, followed by autoradiography. The mAb 14-12coupled Sepharose bound TsF of different specificities such as OVA-specific GIF from the Ts hybridoma 231F1 (Iwata et al., 1989a), NP-specific TsF3 (Steele et al., 1989) and bee venom PLA2-specificTsF (Mori et al., 1993). Ferguson and Ivenson (1986) obtained another rat IgM mAb 14-30, which was specific for TsiF. In addition to SRBC-specific TsiF, which has been used for immunization of rats and for the selection of hybridoma, the monoclonal antibody coupled to Sepharose absorbed the NP-specific TsFl (Okuda et al., 1981a),ABA-specific TsFl (Whitaker et al., 1981),and D10G4- derived conalbumin-specificTsiF activities (Green et al., 1987). On the other hand, DNP-specific TsF activity did not bind to either the 14-12- or 14-30coupled Sepharose (Fairchild et al., 1988). Another monoclonal antibody, B16G, was established from spleen cells of BALB/c mice immunized with affinity-purified TsF specific for the murine mastocytoma P815. This monoclonal antibody bound polyclonal TsF obtained from the spleen of tumor-bearing animals and the TsF released from a P815-specific Ts cell hybridoma. Furthermore, the monoclonal Ab B16G coupled to Sepharose could absorb NP-specific TsF1, but neither TsF2 nor TsF3 (Steele et al., 1987).Thus, the tumor-specific TsFl appears to share serological determinant with hapten-specific TsF1. Devens et al. (1991) established two rat monoclonal antibodies 984 and 2441, using partially purified GAT-specific TsFl and chemically homogeneous TsF2, respectively, as immunogens. The monoclonal antibody 984 coupled to Sepharose removed GAT-specific TsFl from both crude and purified cell extracts or culture supernatant, whereas monoclonal antibody 2441 was specific for TsF2. By allogeneic mixed lymphocyte cultures, they generated alloantigen-specific Ts cells, which suppressed the generation of allospecific CTL responses in vitro and found that the Ts cells in this system were depleted by treatment either with the mAb 984 plus complement or with mAb 2441 plus complement. However, recombination of the antibody 984 plus complement-treated cultures with the antibody 2441 plus complement-treated cultures lead to restortion of suppressive activity, suggesting that two populations of Ts cells are involved in the suppressive pathway. The results also suggest that TsFl and TsF2 are associated with the plasma membrane of Tsl and Ts2, respectively.Another important finding was that the epitope recognized with the mAb 984 was detected on Ts hybridomas, Ts clones, and some T cell leukemias, but

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none of the Th clones, Th lines, CTL lines, B cells, or macrophage lines display this antigen. One of the reasons for skepticism regarding Ts cells is that there was no phenotypic marker to distinguish Ts cells from other T cell subsets. However, the molecules recognized by the mAb 984 may well be a unique cell surface marker for Ts cells. H. I-J PARADOX Initially, “I-J” was described in two independent systems. Murphy et al. ( 1976)demonstrated that Ts cells expressed unique antigenic determinants which were detected by alloantisera prepared in H-2 congenic mice. Using conventional genetic mapping techniques, they localized the subregion encoding for the determinant in the I-region and designated the subregion as I-J. Independently, Tada et al. (1976) carried out genetic analysis of the antigenic determinant associated with the KLH-specific TsF. They demonstrated that the I-J region controlled the expression of the determinant, and that alloantisera prepared in BlOA(SR)-BlOA(5R)combination specifically absorbed the KLH-specific TsF. A large number of alloantisera produced by the combination of various intra-H-2 recombinant mice having different background genes were utilized to map I-J between I-A and I-E subregions. Tada and Okumura (1979) have shown that the anti-I-J antisera did not contain antibodies against the Ia molecules on B cells. They also demonstrated that expression of TsF activity requires H-2 homology between the source of the TsF and the target cells and localized the homology region to the I-J segment. The presence of the I-J determinant on some helper T cells was reported by Tada et al. (1978). It was also suggested that some macrophage-like cells express I-J determinants which may be involved in the interaction between the antigen-presenting cells and a subset of T cells (Niederhuber and Allen, 1980). Murphy et al. (1981) demonstrated that the I-J determinant ( J l ) expressed on Ts cells were distinct from the 1-J determinants (J2) present on the accessory cells. These findings suggest that multiple I-J determinants exist. Dorf and Benacerraf (1984) reported that sharing of the homologous I-J gene between APC and T cells was required for the generation of Ts cells and suggested that I-J molecules on macrophages function as antigen-presenting entities for Ts cells in the way class I1 MHC molecules function in helper T cells. According to their model, T cells would express I-J complementary structure, but not I-J on APC. Furthermore, different I-J determinants have been determined by Green et al. (1983) on T cells in the regulatory circuit, such as Ts inducer and Ts effector. As already described, TseF activity and TsiF activity from Ts hybridomas appear to be associated with distinct molecules. Thus, I-J is not a single molecule but the antigenic determinants existing in a series

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of heterologous molecules expressed on functionally different T cells (Kurata et al., 1984). A big blow to the findings and theories described earlier was the results of recombinant DNA approaches which suggested that there is not enough DNA in the putative I-Jregion to account for the presence of one structural gene (Steinmetz et al., 1982). Furthermore, attempts to identify mRNA in Ts hybridomas with cosmid clones from the putative segment of I-J DNA failed to uncover evidence of any mRNA encoded by this portion of the H-2 complex in a large number of hybridoma cell lines secreting I-J' TsF (Kronenberg et al., 1983). It is therefore clear that the I-J gene does not exist within the region between I-A and I-E. This led some investigators to conclude that I-J may map outside of H-2 region (Hayes et al., 1984) or I-J may not exist (Klein et al., 1983). Interesting findings in this respect are that the I-Jkdeterminanuepitope could be adaptively acquired by T cells of different genotype differentiated under the influence of the H-2k environment. Thus, Asano et al. (1987) established radiation bone marrow chimera where stem cells of different genotype differentiated into T cells in the different H-2 environment. They primed chimeric mice with KLH and determined the effect of monoclonal anti-I-Jk antibodies on the helper function of their T cells for in vitro antibody response. In this experiment, they obtained evidence that Th cells with H-2b genotype (B6 cells), but differentiated in the environment of B6C3F1, could be removed by some anti-I-Jkantibodies. More strikingly, they found that helper T cells of H-2' (B6) genotype differentiated under the H-2k(C3H)environment could be removed by anti-I-Jk,whereas helper T cells of the C3H + B6 chimera were not affected by the antibody. Adaptive aquisition of I-J epitopes on T cells from chimeric animal was confirmed in mixed lymphocyte reactions (MLR). Uracz et al. (1985a) showed that monoclonal anti-1-Jk antibody blocked the syngeneic and allogeneic MLR of H-2kand H-2" haplotypes but not that of their H-2 congenic partners. Thus, they extended this finding to T cells of bone marrow chimera (Uracz et al., 1985b). They observed that some monoclonal antiI-Jk antibodies inhibited MLR of C3H T cells, but not B6 T cells, to SJM9 cells which could stimulate both C3H and B6 T cells. The same anti-I-Jkantibodies inhibited MLR of T cells from B6C3F1+ C3H chimera but not the T cells from B6C3F1+ B6 chimera, even though the responding cells carried H-2k determinants as detected by cytotoxic testing with appropriate anti-MHC class I antibodies. From these experiments, they concluded that I-J epitopes are adaptively expressed on helper T cells under the influence of environmental H-2 and suggested that I-J epitopes may be associated with the MLR recognition sites of T cells.

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Possibility of adaptive acquisition of I-Jkdeterminant in TsF was investigated by Sumida et al. (1985) using chimeric mice. They immunized chimera with KLH, obtained KLH-specific TsF from their thumus, and determined the suppressive effect of the TsF on the in vitro secondary antibody response of DNP-KLH-primed spleen cells of C57B1/6 or C3H mice. Distinct from the observations by Uracz on MLR, Sumida et al. found that KLH-specific TsF from the C3H + C57B1/6 chimera suppressed the C3H but not the C57B1/6 response in KLH-specific manner, while the TsF from the C57B1/6 C3H chimera suppressed the B6 response but not the C3H response. Thus, the host-specific restriction specificity of TsF was not aquired in chimera. However, if the chimeric mice were given APC of the host type at the time of immunization with KLH for the generation of Ts cells, KLH-specific TsF from either the C3H + B6 or B6 + C3H chimera specifically suppressed the responses of both B6 and C3H, but not the third party BALB/c mice. It was apparent that the restriction specificity of TsF generated in chimera depends on the H-2 type of the APC available at the time of antigen stimulation. Thus, Sumida et al. suggested the presence of class II-like molecules on APC, which are essential for expression of I-J phenotype on Ts/TsF. In any event, their results also indicated that I-J determinant on Ts could be adaptively acquired to recognize self MHC or related structures. Biochemical characterization of I-J molecules on T cells was achieved by Nakayama et al. (1989) using CD4+ T cell clones, which expressed I-Jkdeterminant on their cell surface. The cells were surface labeled with ‘%I,and 1-Jk+molecules in NP40 lysates of the cells were immunoprecipitated with monoclonalanti-I-Jkantibody. Analysis of the precipitates by twodimensional gel electrophoresis gave a radiolabeled 86-kDa homodimder of approximately 44 kDa glycopeptide, whose isoelectric point was pH 5.3 to 6.4. This 1-Jk+protein was immunoprecipitated from the lysate of T cell clones with I-Ak and I-Ek restriction specificity but not in the lysates of antigen-specific T cell clones from H-2b mice. BlO.A(5R)-derived T cell clones having either I-Abor I-Ekrestriction specificities bore the I-Jkmolecule, but similar molecules in the lysate of an BlOA(3R)-derived I-Ab restricted T cell clone were not immunoprecipitated by the anti-I-Jk. It was also found that KLH-specific T cell clone from B6 + B6C3F1 chimera bore the 86-kDa 1-Jkt molecule, whereas KLH-specific T cell clone from B6C3F1 + B6 chimera did not. Thus the results of the experiments confirmed that expression of the 1-Jk+molecules on T cells was not determined by the genotype of the T cells but was dictated through an adaptive process in radiation bone marrow chimeras. It was expected that “I-J puzzle” may be resolved by the biochemical identification of the 1-Jk+molecules on T cell clones. It should be noted,

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however, that either the 86kDa 1-Jktdimer protein or the 44 kDa monomer glycopeptide on Ts cells does not appear to correspond to the I-J' chain of TsF formed by the cells. These findings indicate that the same I-J determinant is associated with multiple proteins and suggest that isolation of cDNA clone encoding the I-J' molecule may not solve the I-J paradox. 111. Controversial Issues on Antigen-Specific TsF and Possible Answers

A. ARE SUPPRESSOR CELLST CELLS?

After identification of T cell receptors (TCR) on helper and cytotoxic T cells, many controversial issues were raised on Ts cells and TsF. One of the questions was whether the Ts cells actually belong to T cells. This question was raised because the major population of representative Ts hybridomas did not express a detectable level of CD3, and Ts cell clones had not been established for a long time. However, Weiner et al. (1988) subcloned CD3+ cells in ABA-specific Ts hybridoma line, F12.23, and demonstrated that conditioned media of CD3+ clones, but not the CD3clones, contained the TsF activity. Similarly, Kuchroo et al. (1988) established CD3' clones from three NP-specific Ts3 hybridoma clones for surface expression of CD3 using either anti-CD3 or NP-BSA-coated plates and found that TsF activity in the culture supernatant of the CD3' hybridoma clone was at least tenfold higher than the activity in the culture supernatant of parent Ts hybridoma line. These results indicated that failure of detecting CD3 on the original hybridoma cells was due to loss of TCWCD3 on the majority of the cells after many passages without selection, and that CD3'/TCRt cells were actually the source of TsF. The KLH-specific hybridomas (Taniguchi et al., 1982) and OVA-specific GIFproducing TS hybridomas (Iwata et al., 1989a) also express CD3 and TCRaP. Furthermore, several investigators established Ts cell clones bearing CD3 and TCRaP. Although the suppressive effect of some of the Ts clones was neither MHC restricted nor antigen-specific (Hisatsune et al., 1990; Hu et al., 1992), both the HGG-specific Ts clones (Takata et al., 1990) and OVA-specific Ts clone (Chen et al., 1992) suppressed in vitro antibody responses in an antigen-specific and MHC-restricted manner and expressed TCRaP, CD3, and CD8 on their cell surface. It was also found that TCRaP heterodimers were expressed on leprosy-specific human Ts clones (Modlin et al., 1987) and virally transformed hen egg lysozymespecific murine Ts clone (De Santis et al., 1985; Ballinari et al., 1985). Lack of TCR on the majority of previously established Ts hybridoma cell line cells may partly explain the findings of Hedrick et al. (1985) that TCRP chain gene was deleted or not rearranged in most of the representative Ts hybridomas. After subcloning of the hybridomas for

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CD3+ cells, rearrangement of TCRa gene was demonstrated in the KLHspecific Ts hybridoma (Imai et al., 1986), OVA-specific GIF-producing hybridoma (Ishii et al., 1996a),NP-specific Ts3 hybridomas (Collinset al., 1990),and DNP-specific Ts hybridoma (Barbo et al., 1995).None of these TCRa genes was derived from fusion partner. It was also found that the OVA-specific GIF-producing hybridoma (231F1 cells) and DNP-specific Ts hybridoma utilized Vp8.3 and Vp8.2 gene segment, respectively. Evidence was presented that the TCR on the NP-specific Ts3 hybridoma was composed of a unique a chain and BW5147 (fusion partner)-derived p chain; however, the hybridoma utilized such TCRaP to recognize antigen (Collins et al., 1990). There is no doubt that most of the effector type Ts hybridomas employed by several investigators in recent years have characteristics of T cells. TCR and CD3 are expressed on Tsl or Tsi cells as well. In the series of experiments by Green and his colleagues, Zheng et al. (1988)found that the synthetic peptide (P18)-specifichybridoma, Al. 1,which constitutively secreted TsiF, bore CD3 and TCRaP, and recognized the peptide in the context of I-Ad product. They also indicated that a representative Th2 clone, D10G4, released TsiF when they were stimulated by homologous antigen (conalbumin)processed by W-treated macrophages, and that the TsiF released from the Th2 clone, together with “accessoryfactor,” induced the generation of CD8+ effector type Ts cells (Green et al., 1987). Subsequently, Kuchroo et al. (1990)constructed several NP-specific Tsl hybridomas using the TCRap- BW thymoma cells as a fusion partner and demonstrated that the hybridomas expressed CD3-associated TCRap and CD4. These hybridomas recognize antigen in the context of I-Ek. TCR genes utilized by two of the three hybridomas were Va4JaTA19, Vp8.3DP2Jp2.4 for CKBTsl-81 and Va8JaLB2, Vp6DP2Jp2.6 for CKB Tsl-38, respectively. These hybridomas produced 11-2 or IL-2 and IL-4 upon antigenic stimulation in the presence of appropriate APC. These properties are the same as Th cells. Only the difference between the Tsl cells and Th cells was that all of the Tsl hybridomas constitutively secreted NP-specific TsFl which suppressed DTH when given in induction phase. Thus the Tsl cells are indistinguishable from Th cells in terms of cell surface markers, suggesting that Tslnsi cells are related to Th cells.

B. How CANTs CELLSAND TsF BINDNOMINALANTIGEN? It is well established that TCR on helper and cytotoxic T cells do not recognize nominal antigen but bind processed antigen associated with the product of MHC (Marrack and Kappler, 1986). If this principle applies for TCR on Ts cells, why representative Ts cells, such as KLH-specific Ts and HGG-specific Ts cells bound to antigen-coated plates, and TsF derived

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from these cells had affinity for nominal antigen coupled to immunosorbents? At some points, several investigators thought about the possibility that Ts cells might have unique receptors for antigen, or that TCR on Ts cells may be distinct from those on Th cells (Weiner et al., 1988; Kuchroo et al., 1988). However, subsequent experiments indicated that TCR on Ts cells are the same as those on Th cells. We speculated that the capacity of producing antigen-binding factor may be restricted to T cells having a certain epitope specificity, and determined the epitope specificity of TCR on OVA-specific GIF(TsF )-producing hybridomas (Iwata et al., 1989a). Among 14 GIF-producing hybridoma clones derived from spleen cells of OVA-primed BDFl mice, 8 hybridoma clones produced GIF having affinity for OVA upon stimulation with OVApulsed APC. Determination of the epitope recognized by the T cell hybridomas showed that all of the 8 hybridoma clones responded to a common synthetic peptide representing amino acid residues 307-317 of OVA molecules in the context of either I-Ad or I-Ab product. In contrast, none of the remaining 6 hybridomas responded to the peptide in the presence of APC. Some clones in the latter group were specific for the major immunogenic determinant, representing amino acid residues 323-339 in the OVA molecules (Shimonkevitz et al., 1984), but GIF produced by these cells upon antigenic stimulation failed to bind to OVA. Thus, only the hybridomas specific for the peptide 307-317 in the context of I-Ad or LAb product produced GIF having affinity for OVA. It was found that binding of the OVA-specific GIF from a representative Ts hybridoma, 231F1 cells, to OVA-coupled Sepharose could be inhibited by the peptide 307-317. Indeed, the OVA-specific GIF bound to the peptide coupled to Sepharose and could be recovered by acid elution (Iwata et al., 1989b). Specific binding of the factor not only to OVA but also to p307-317 suggested that the stretch of amino acid sequence represented by the peptide is exposed to the surface of the OVA molecules. This idea is supported by X-ray crystal structure of OVA, which indicated that the p307-317 sequence represents a portion of a loop in OVA molecules (Stein et al., 1991). Thus, we speculated that the epitope for the TCR on the cell source of antigenspecific TsF represents an external structure of the antigen molecules. Molecular cloning of TCRa and p chains on the 231F1 cells support the concept that the TCR recognizes the epitope (Ishii et al., 1996a). Among the cDNA clones encoding the TCRa chain on the 231F1 cells, two types of the cDNA clones were identical to either of the two TCRa genes from the fusion partner, BW5147, but the third type of the cDNA clones consisted of Vall.3, a unique Ja and the complete C a segment. The Va11.3 gene segment was originally identified in Th clone A10 cells, which specifically responded to OVA presented by I-Ak-bearingAPC

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(Malissen et al., 1988). Subsequently, we have cloned the cDNA of the TCRa chain from an OVA-specific Th hybridoma 12H5 cells and a Ts hybridoma 71B9 cells, both of which recognize the same epitope as that recognized by the 231F1 cells (Iwata et al., 1989a) and found that TCRa on all of the three hybridomas utilize Va11.3 gene segment. Only three amino acid differences were found among the deduced amino acid sequences of the V a region of TCR on the three hybridomas specific for p307-317. More recently, we cloned TCRP chain gene of all of the three hybridomas and found that Vp8.3 segment is utilized in all of the three hybridomas. These findings may be similar to previous observations that the murine TCR Va3 segment is associated with reactivity of T cells to p azobenzene arsonate, irrespective of MHC restriction. Based on these findings, Tan et al. (1988) suggested that Va3 may encode a protein sequence with a binding site for the arsonate hapten. One may speculate that the Va11.3 and/or Vp8.3 region play essential roles in binding of the TCR on the 231F1 cells to the epitope p307-317 in OVA molecules. We extended the studies on the epitope specificity of TCR on Ts cells to the Ts hybridomas specific for bee venom phospholipaseA2 (PLA,), whose X-ray crystal structure has been reported (Scott et al., 1990). All hybridomas that produced PLA2-specificGIF upon antigenic stimulation were specific for the peptide representing amino acid 19-34 in the PLAz molecules in the context of I-Ad,and the antigen-specific GIF formed by the hybridomas had affinity for the peptide (Moriet al., 1993).X-ray crystal structure of bee venom PLA2 indicated that the sequence of amino acid 19-34 consists of a loop and a a helix (Fig. 2). We found that the a helix portion (p25-34) of the sequence was required for binding of the peptide to Ia molecule, whereas the loop portion (p19-24) contained amino acid residues involved in interaction with TCR. Evidence was obtained that the same loop sequence is involved in binding of the peptide to antigenspecific GIF. Furthermore, the molecular model, shown in Fig. 2, indicates that this loop is exposed to the surface of the PLA2 molecule. Thus, the TCR expressed on the cell source of PLAz-specificGIF is actually specific for the sequence representing an external structure in the antigen molecule, and the antigen-specific GIF shares the epitope specificity with the TCR of its cell source. In both the OVA and PLAz systems described above, treatment of antigen-pulsedAPC with anti-IAdantibody prevented antigenic stimulation of Ts cells. The response of the hybridoma cells to the synthetic peptide in the presence of glutaraldehyde-treated APC was also prevented by pretreatment of APC with the anti-IAdantibody, indicating that participation of class I1 MHC product on APC is required for recognition of the epitope by the TCR. However, antigen-specific GIF from the cells bound

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FIG.2. Molecular model of the amino-terminal one-third of bee venom PLAzas compared with bovine pancreatic PLAz (dotted line). (From Mori et a,!., 1993, with permission.)

to the peptide coupled to Sepharose. Similar findings were obtained by Zheng et al. (1988) on the Ts cell hybridoma A 1.1 which specifically responded to a synthetic peptide of a defined sequence (P18)in the context of I-Ad for the formation of IL-2. This hybridoma constitutively released antigen-specific TsFl having affinity for P18 itself. Correlation between the fine epitope specificity of TCR on Ts cells and the epitope recognized by the antigen-specific TsF formed by the cells in the three antigen systems described earlier suggested a possible relationship between TCR and the antigen-specific factors. It has been believed that TCR does not bind “processed antigen” without participation of a MHC product. However, Silliciano et al. (1986) reported that multivalent hapten conjugated with synthetic polymers bound to TCRaP on MHC-restricted FITC-specific T cell clones, and that the binding could be inhibited by a high concentration of monovalent hapten. In many experimental works on Ts cells, hapten coupled to syngeneic cells or multivalent synthetic peptides were employed as an immunoged tolerogen to generate Ts cells. In these systems, the epitope recognized by TCR would be an external structure of the immunogen. Indeed, Kuchroo et a2. (1988) demonstrated that CD3’ NP-specific Ts3 hybridoma cells formed rosettes with NP-coupled sheep erythrocytes, and the rosette formation was inhibited by NP-BSA conjugate. It was also found

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that rosette formation of the Ts3 hybridoma depended on the expression of TCRaP on the cells, indicating that TCRs are involved in the rosette formation with NP-coupled SRBC (Collins et al., 1990). These findings suggest that the hapten itself, without participation of MHC product, can bind to TCR of the hapten-specific Ts cells, although the participation of a MHC product is required for activation of the cells. This principle may not be unique for Ts cells. Some helper T cell clones specific for ABA have been reported to directly bind the antigen in the absence of MHC product (Rao et al., 1984). It was suggested that an immunodominant T cell epitope is an amphipathic CY helix or has the ability to fold as an amphipathic structure (Delisi and Berzotsky, 1985). Indeed, the immunodominant epitopes in sperm whale myoglobin and that in egg white lysozyme (Allen et al., 1987) represent CY helix in the native antigen molecule. In these immunodominant epitopes, Ia-contact residues are intermingled with residues that are involved in the engagement of the TCR. It was also found that the immunodominant OVA peptide 323-339 is in a P sheet in the OVA molecules and is not exposed to the surface of the molecules (Buus et al., 1987). Failure of nominal antigen to bind to the TCR specific for the immunodominant epitope may partly be due to the hidden nature of the epitopes. As will be discussed in a later section, antigen-specific TsF appears to be a derivative of TCR. We speculate that the real ligand for TsF is an antigen fragment associated with a MHC product, which is recognized by the TCR of its cell source, but the factor shows some affinity for nominal antigen because the T cell epitope for TCR is an external structure of the antigen molecule. Although the binding to nominal antigen is a common property of most of the antigen-specific TsF, some of the antigen-specific TsF do not have affinity for the antigen. Such a TsF was obtained from OVA-specific Kd-restricted CD8 Ts clone which was established from spleen cells of BDFl mice treated with OVA-PEG conjugate (Chen et al., 1992). This factor suppressed PFC response of DNP-OVA primed spleen cells in carrier-specific manner, and the effect of the factor was Kd-restricted. Nevertheless, the factor failed to bind to OVA-coupled immunosorbent (Chen et al., 1994). Since the tolerogenic OVA-PEG conjugate, which was employed for the generation of Ts cells, does not react to antibodies against OVA, the conjugate appears to lack the major antigenic determinant in native OVA molecules. It is reasonable that the T cell epitope for the Ts cells generated by the treatment with OVA-PEG is not an external structure in native OVA molecules. Presence of antigen-specific TsF having no affinity for nominal antigen supports the concept that both TsF and TCR recognize processed antigen associated with a MHC product.

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23

C. POSSIBLE RELATIONSHIP BETWEENTsF AND TCR 1. Requirement of TCR Gene for the Formation of TsF Expression of TCRaP on Ts hybridomas and Ts clones, as well as common epitope specificity shared by the antigen-specific TsF/GIF and TCR of their cell sources, strongly suggest the relationship between TCR and TsF. Indeed, OVA-specific TsF (GIF) activity from an OVA-specific I-Ad restricted Ts hybridoma bound to the monoclonal anti-TCRa chain (H28.710) (Becker et al., 1989) coupled to Sepharose and could be recovered by elution at acid pH (Iwata et al., 198913). Similarly, all of the HGGspecific TsF activity in the cell lysate of the MHC class I-restricted, HGGspecific Ts clone (Takata et al., 1990), DNP-specific TsF activity in the culture supernatant of MHC Class I-restricted DNP-specific Ts hybridoma (Fairchild et al., 1990), NP-specific TsF3 activity released from NP-specific Ts3 hybridoma (Collins et al., 1990), NP-specific TsFl activity from I-Ek restricted NP-specific Tsl hybridomas (Kuchroo et aE., 1991), the peptide Poly 18-specific TsFl activity from I-Ad-restricted Tsl hybridoma, Al.l (Green et al., 1991) and bee venom PLA2-specificGIF/TsF (Mori et al., 1993) bound to the monoclonal anti-TCRa antibody coupled to Sepharose and could be recovered by elution of the immunosorbent either at acid pH or with 2 M KCl. Chen et al. (1994) also observed that the OVAspecific TsF from OVA-specific H-2Kd-restricted Ts clone bound to the same anti-TCRa antibody. Thus, association of the TCRa chain determinant with TsF appears to be a common property of TsF. Among the TsFs described earlier, the OVA-specific GIF, DNP-specific TsF and OVA-specific TsF (Chen et al., 1994), and NP-specific TsFl (Kuchroo et al., 1990) activities could be absorbed not only with the antiTCRa but also with the monoclonal anti-TCRP chain antibody (H-57-597) coupled to Sepharose, suggesting that these TsFs consisted of both TCRa and p chains. However, the other TsFs such as NP-specific TsF3 and Poly 18-specific TsFl failed to be retained in the anti-TCRP immunosorbent. It is known that the monoclonal antibody H-57-597 binds to the TCRP chain associated with TCRa chain but not to the isolated or denatured TCRP chain (Kubo et al., 1989; Gascoigne, 1990). Thus, failure of the monoclonal anti-TCRP chain to bind some TsF does not necessarily exclude the presence of TCRP chain in the preprations. In order to obtain more evidence for the hypothesis that the TCRa and/ or chain genes may also encode for the antigen-specific TsF, Zheng et al. ( 1989) studied the effect of antisense oligonucleotides corresponding to genes encoding the V region of the TCRa or P chain on the formation of antigen-specific TsF by the Poly 18 peptide-specific Ts hybridoma Al.l. As expected, prevention of either TCRa or TCRP chain gene expression

24

ISHIZAKA d aJ.

by an appropriate antisense oligonucleotide inhibited the expression of TCRaP and CD3 on the cell surface. They found that antisense oligonucleotides corresponding to the Al.1 Va gene blocked the constitutive production of antigen-specificTsF activity by the cells, whereas antisense oligonucleotide corresponding to A l . 1VP gene had no effect. Their results strongly suggested that transcription of TCRa gene, but not the TCRP gene, is required for the formation of the antigen-specific TsF1. The same conclusion was reached by Kuchroo et al. (1991) who had established a series of TCRa- or TCRP- expression variants of NP-specific Tsl hybridomas. They demonstrated that loss of TCRa mRNA, but not the loss of TCRP mRNA, was accompanied by the concomitant loss of suppressor bioactivity of conditioned medium. Furthermore, homologous transfection of TCRa cDNA into a TCRa-P+ clone reconstituted both CD3-TCR expression and TsFl bioactivity. Suppressor activity from the TCRP- variants retained in NP-KLH-coupled Sepharose and anti-TCRa antibody-column. However, the TsFl activity was not absorbed either with the anti-CD3 or with anti-TCRP chain affinity column, suggesting that TCRP chain is not required for the formation of NP-specific TsF. A similar principle may apply for the relationship between the NPspecific TsF3 and TCR of its cell source. The DNA sequence of a TCRa chain cDNA clone from the NP-specific Ts3 hybridoma CKB 3-3.11was found to encode a Va2 segment rearranged in frame to a JaTA28 segment (Collins et al., 1990). Only the functionally rearranged P chain gene found in the cDNA library of the hybridoma was that derived from the fusion partner. The antigen-binding molecules on the hybridoma, as determined by rosette formation with NP-coupled SRBC, were the TCRaP dimer composed of the Va2 a chain and the BW5147-derived P chain, and the level of surface expression of the antigen-binding molecules correlated with the levels of mRNA of the TCRP chain. In contrast, TsF activity in the conditioned medium of the subclones did not correlate with the expression of TCRaP. No difference in the suppressor factor activity was found among the subclones in which TCRP mRNA level differ by 250-fold. Furthermore, TsF activity in the conditioned medium of both the TCRP' and TCRP- subclones failed to be retained in anti-TCRP chain (H57-597) column but bound to anti-TCRa (H-28-710) column. The results indicated that a product of TCRP chain gene is not required for the formation of NP-specific TsF3. In contrast to the findings on NP-specific TsFs, a product of TCRP chain gene appears to be essential for the formation of DNP-specific, Kdrestricted TsF which can suppress the effector phase of contact sensitivity to DNFB. Fairchild et al. (1993) generated a panel of TCR variants of DNP-specific Ts hybridoma Mts 79.1 by y irradiation. In 7 of the 12

BIOCHEMICAL BASIS OF ANTIGEN-SPECIFIC SUPPRESSOR T CELL FACTORS

25

variants, the parental VP 8 chain transcript was absent. Deletion of the TCRP chain gene in these subclones was confirmed by Southern blot. None of the 7 TCRP chain gene deletion mutants and 5 TCRa chain gene deletion mutants produced DNP-specific suppressive activity. However, expression of the parental TCRP chain gene in one of the /3 chain gene deletion mutants reconstituted the ability to produce the DNP-specific TsF activity. Similarly, transfection of the TCRa chain gene in the TCRa gene deletion mutants reconstituted both the surface TCR expression and production of DNP-specific TsF (Barbo et al., 1995). They found that the TsF activity in the transfected cells, as well as the original hybridoma Mts 79.1 bound not only to the monoclonal anti-TCRa (H-28-710) but also to anti-TCRP (H-57-597) and anti-VP8 antibodies. In order to confirm the difference between their DNP-specific TsF and NP-specific TsFs by Kuchroo et al. (1991), Barbo et al. (1995) transfected the cy chain gene construct into an a-/P- chain gene deletion mutant. As expected from their findings, the transfection did not restore the production of TsF. Thus they concluded that the production of DNP-specific/class I MHC-restricted suppressor molecule is linked to the expression of both a and p chain genes and speculated that the immunosuppression is mediated by a soluble form of TCR. Evidence for the relationship between TCR and TsF was obtained at the mRNA level as well, using mouse Ts clone obtained by virus-induced transformation of hen egg-white lysozyme-specific Ts cells (De Santis et al., 1985).The Ts clone expressed TCRaP and constitutively released TsF which suppressed the antibody response to the lysozyme (Adorini et al., 1982). To test the hypothesis that mRNA of the a and P chains of the TCR could also direct the synthesis of TsF molecules, De Santis et al. (1987) enriched mRNA of the TCR a and /3 chains using the cDNA encoding constant regions of a and P chains and translated the mRNA in Xenopus oocytes. Injections of translation products of a mixture of cy and P chain-homologous mRNA into mice at the time of antigen-priming suppressed PFC responses of their lymph node cells in antigen-specific manner. Translation products of either a chain mRNA or P chain mRNA alone failed to exert suppressive activity. They also found that mixing of separately translated a and P gene products exhibited only marginal suppressive activity on the anti-HEL antibody response, suggesting that appropriate posttranslational modification, such as the formation of TCRaP heterodimer, is involved in the formation of TsF. As described in a previous section, molecular cloning of TCR genes from many Ts hybridomas proved gene rearrangement in Ts cells, and provided evidence that the gene segments are actually utilized for the expression of TCR recognizing T cell epitopes. It is suspicious, however,

26

ISHIZAKA

d

al.

whether the TCRa gene cloned from KLH-specific Ts hybridomas (Imai et al., 1986) and DNP-specific Ts hybridoma (Barbo et al., 1995) actually encode the TCR recognizing respective antigen. In all of 9 KLH-specific Ts hybridoma clones studied, the entire Va and J a sequences of the TCRa gene were identical and encoded by Val4 and Ja281 gene segments with one nucleotide N region (Koseki et al., 1989). Subsequent experiments have shown that Val4' T cells represent a unique subset expressing the invariant TCRa chain encoded by these gene segments, and that the frequency of the a chain expression was >1.5% of the total a chain found in laboratory strains (Koseki et al., 1990). Another important development in this line of the work was that NKT cells express this invariant TCRa chain Val4 Ja281 and either Vp8.2, Vp7 or VP2 TCRP chain (Lantz and Bendelac, 1994; Makino et al., 1995). Using the Val4 NKT mice expressing the invariant Val4 and Vp8.2 transgenes, the ligand for the TCR on NKT cells was identified as a-galactosylceramide associated with CD1 (Kawano et al., 1997). Barbo et al. (1995) reported that DNP-specific Kd-restricted Ts hybridoma, MTs79.1 expressed the Val4 Ja281 invariant a chain and Vp8.2 chain, both of which were identical in their sequence to those expressed on NKT cells of the transgenic mice. Although Fairchild et al. (1993) and Barbo et al. (1995) indicated that expression of both the TCRa and TCRP genes are required for the formation of DNP-specific TsF by the.Mts 79.1, there is no evidence that the TCR on the Ts hybridoma is involved in recognition of DNP group associated with class I MHC molecule. It is hard to believe that the TCR consisting of the invariant Val4 Ja281 chain and Vp 8.2 chain recognize both the a-galactosylceramide associated with CD1 and DNP group associated with class I MHC molecule. It is not known why the invariant Val4 Ja281 gene segments were obtained in the molecular cloning of TCR on KLH-specific Ts hybridomas and the DNP-specific Ts hybridoma. In view of unique functions of NKT cells, which produce relatively large quantities of IL-4 and IFNy (Bendelac et al., 1997), possibility may be considered that Ts cells may actually be NKT cells. However, this possibility is unlikely because the invariant Val4 gene segment was not obtained in the molecular cloning of TCR genes in the other Ts hybridomas or Ts clones. 2. Biochemical Identijication of TsF Accumulated evidence described earlier suggests that TsF is a product of TCR gene(s) or a derivative of TCR. However, biochemical identification of TsF had only limited success. It is evident that TCRa chain gene of NP-specific Tsl is essential for the formation of NP-specific TsF1, but a product of TCRa chain gene was not detected in conditioned medium of Tsl hybridomas. In an attempt to identify TsF1, Gallina et al. (1990)

BIOCHEMICAL BASIS OF ANTIGEN-SPECIFIC SUPPRESSOR T CELL FACTORS

27

adapted NP-specific Tsl hybridoma CKB Ts-17 to serum-free medium and purified the TsFl in the conditioned medium using NP-BSA-coupled Sepharose. Acid eluate of the column contained a protein which reacted with the monoclonal anti-TsF1, B16.G, in ELISA. SDS-PAGE and Westem blot analysis of the acid eluate from NP-BSA Sepharose column gave a 43-kDa band which bound monoclonal antibody B16.G. Similarly, fractionation of culture supernatant on B16.G-coupled Sepharose and analysis of acid eluate from the column by SDS-PAGE and immunoblotting gave a 43-kDa band which reacted with alkaline phosphatase-coupled NP-BSA. Although the authors failed to use the monoclonal anti-TCRa chain antibody for immunoblotting, the 43-kDa protein probably represents TCRa chain or its derivative. Subsequently, Bissonette et al. (1991) provided clear evidence for the relationship between Poly 18-specific TsFl and TCRa chain. They have shown that TsFl activity in the culture supernatant of A l . 1 cells bound to anti-TCRa (H28-571) coupled to Sepharose and was recovered by acid elution. Thus, they metabolically labeled the products of Al.l cells by culture of the cells in the presence of =S-methionine, and proteins in culture supernatant were subjected to affinity chromatography on antiTCRa (H28-710) Sepharose. SDS-PAGE analysis of the acid eluate from the column, followed by autoradiography, gave a single radioactive band of 46 kDa. Association of immunosuppressive effect with a 46-kDa protein was confirmed by testing the extract of gel slices for immunosuppressive effect. The results suggested that the Al.1-derived TsF is a soluble form of TCRa chain. This idea is supported by subsequent findings by Green et al. (1991) that expression of the A l . l TCRa cDNA in other T cell hybridomas conferred the recipient cells the ability to produce the soluble antigen-specific factor. However their observations are in conflict to the fact that a soluble form TCRa chain is not formed by Th cells, and to the findings by Kuchroo et al. (1995) on transfection of TCRa cDNA in Ts and Th hybridomas. They expressed the TCRa cDNA from a NP-specific Ts hybridoma into a TCRa- mutant of the ABA-specific Th cell hybridoma and observed that culture supernatant of the stable transfectant did not suppress DTH responses to NP. In contrast, transfection of the TCRa chain cDNA from the ABA-specific Th clone into a TCRa- mutant of the NP-specific Ts hybridoma resulted in the formation of TsF activity. The specificity of TsF formed by the transfectant was decided by the cDNA transfected but not by the host cells. The results suggest that the cellular environment of the Ts cells is essential either for the formation or release of suppressor molecules. Another important finding was identification of an OVA-specific TsF from the OVA-specific, Kd-restricted Ts clone (Chen et al., 1994). These

28

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cells contained TsF, which could be obtained by freezing and thawing treatment, and released the factor in culture supernatant upon activation of the cells with anti-CD3. Fractionation of the cell extract on anti-TCRa column, followed by analysis of acid eluates from the column by SDSPAGE and Western blotting, revealed 84- and 42-kDa proteins which bound the monoclonal anti-TCRa antibody (H28-710). Analysis of the same eluate by SDS-PAGE under reducing conditions gave only the 42-kDa band. Fractionation of the same cell extract on anti-TCRP (H57597) column followed by Western blot analysis with the anti-TCRa antibody showed that eluates from the column contained the 84-kDa protein but not the 42-kDa protein. Fractionation of the cell extract on the monoclonal anti-VP8 antibody-coupled Affigel also showed that the 84-kDa component bound to the anti-VP8 antibody as well. SDS-PAGE analysis of culture supernatant of anti-CD3-treated cells under nonreducing conditions and immunoblotting with the mAb H28-710 demonstrated the 84-kDa protein, but not the 42-kDa component. In contrast, culture supernatant of resting, unstimulated Ts cells did not contain either the TsF activity or any component reacting with the monoclonal anti-TCRa antibody. From these findings, they concluded that TsF is the 84-kDa protein, consisting of disulfide linked two subunits of 42 kDa. As the 84-kDa protein bound to both the anti-TCRa chain and anti-TCRP chain antibodies, they suggested that the TsF represents a soluble form of the a/3 heterodimer of TCR. Our studies on biochemical characterization of antigen-specific GIF (TsF) gave somewhat different results. The experiments were carried out based on previous findings that the OVA-specific Ts hybridomas and PLA2specific Ts hybridomas constitutively secrete 13-kDa nonspecific GIF and formed antigen-specific GIF upon stimulation with antigen-pulsed APC or with anti-CD3 ( Jardieu et al., 1987; Mori et al., 1993).The OVA-specific GIF and PLA2-specific GIF suppressed in vivo IgE and IgG antibody responses to DNP-OVA and DNP-PLA2, respectively, in canier-specific manner. Both factors bound to the mAb 14-12, but not to mAb 14-30, indicating that the antigen-specific factors are effector type TsF. Thus, we cultured the OVA-specific Ts hybridoma, 231F1 cells, or PLA2-specific Ts hybridoma, 3B3 cells, with antigen-pulsed A20.3 cells, and culture supernatants were fractionated on respective antigen coupled to Sepharose. The majority of GIF activity in the culture supernatants was retained in the antigen-coupled immunosorbent and was recovered by acid elution. We expected that the 13-kDa GIF would be detected in the acid eluate fraction by SDS-PAGE analysis under reducing conditions, followed by Western blotting with anti-GIF. However, analysis of the fraction gave a

BIOCHEMICAL BASIS OF ANTIGEN-SPECIFIC SUPPRESSOR T CELL FACTORS

29

55-kDa protein rather than a 13-kDa protein (Fig. 3). It was also found that the 55-kDa protein bound not only anti-GIF but also the anti-TCRa chain antibody (H28-710) in immunoblotting (Nakano et al., 1996). Treatment of the Ts hybridomas with anti-CD3, followed by culture of the cells in Protein A-coated flasks also induced the formation and secretion of the 55-kDa protein. However, the 55-kDa protein was not detectable in their cytosol. Only 18kDa GIF peptide was detected in cytosol. It was also found that culture supernatants of unstimulated cells contained the 13kDa GIF, but no 55-kDa GIF. Thus it appears that the 55-kDa GIF is formed by the Ts hybridomas only after stimulation of the cells with antigen-pulsed APC or by cross-linking of CD3. If the 55-kDa protein was synthesized as a single peptide, one might expect the presence of mRNA encoding the peptide. Thus we stimulated the 231F1 cells with anti-CD3 and determined the size of mRNA which could hybridize with GIF cDNA(Nakano et al., 1996). Fractionation of culture supernatants confirmed that essentially all GIF bioactivity in the 24-h culture supernatant bound to OVA-Sepharose. However, in both antiCD3-stimulated cells and unstimulated cells, only the mRNA hybridizing with GIF cDNA was 0.6 kb mRNA which encodes the 13-kDa GIF (Mikayama et al., 1993). Absence of a mRNA encoding the 55-kDa GIF suggested A rGF

B

OVA -__

kDa

- 68-•

-

- 31 _I

46

-20.1

-

-14.4

-

-*

FIG.3. Biochemical identification of 55-kDa GIF in antigen-specific molecules from Ts hybridomas. (A) PLA2-specificGIF preparation (PLA,) was obtained from culture supernatant of 3B3 cells by affinity purification using PLA2-coupled Sepharose. E. coli-derived 13-kDa human rGIF was applied to lane 1 as a control. (B) OVA-specific GIF (OVA) was obtained from culture supernatant of 231F1 cells by af€inity purification, followed by DEAE-Sepharose column chromatography. They were analyzed by SDS-PAGE under reducing conditions and probed with anti-GIF. (From Nakano 69 al., 1996, J. Immunol. 156, 1730, Copyright 1996, The American Association of Immunologists, with permission.)

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ISHIZAKA et al.

that the 55-kDa protein is a posttranslationally formed conjugate of the 13-kDa GIF with a peptide having TCRa determinant (Nakano et al., 1996). In order to obtain evidence that the 55-kDa protein is a derivative of TCRa chain, the TCRa cDNA from the 231F1 cells was cloned and was transfected into the 231F1 cells for overexpression of the TCRa chain (Ishii et al., 1996a). A representative stable transfectant clone 211a was treated with anti-CD3 and cultured in protein A-coated flasks. Analysis of culture supernatants by SDS-PAGE and immunoblotting with the mAb H28-710 indicated that the quantity of the 55-kDa GIF produced by the stable transfectant was more than tenfold of the same protein produced by the untransfected 231F1 cells. As expected, unstimulated transfectant failed to form the 55-kDa GIF. To confirm that the 55-kDa peptide is actually a product of the TCRa cDNA, we attached a nucleotide sequence encoding a peptide of six histidine residues at the 3’ end of the TCRa chain cDNA, and the TCRa-tag gene was transfected into the 231F1 cells. Upon stimulation with anti-CD3, stable transfectants of the mutant cDNA of the TCRa chain with histidine tag produced the 55-kDa peptide having affinity for Ni2+nitrilotriacetic acid (Ni-NTA) agarose. The majority of this peptide in the culture supernatant was retained in the Ni-NTA agarose column and was recovered by elution with 100 mM imidazole. As expected, the 55-kDa protein recovered from the column bound both anti-TCRa chain and anti-GIF in immunoblotting. None of the 55-kDa protein and 13-kDa GIF from untransfected 231F1 cells and the 13-kDa GIF constitutively secreted from the stable transfectants of the mutated TCRa cDNA was retained in Ni-NTA agarose. These findings definitely prove that the 55-kDa GIF formed by the transfectant is a product of the TCRa cDNA transfected. The experiments also provided definitive evidence that GIF bioactivity was associated with the 55-kDa protein. Association of the GIF antigenic determinant and GIF bioactivity with the 55-kDa protein indicated that the protein is not the TCRa chain itself. The size of the protein was significantlylarger than that of the mature TCRa chain, which is in the range of 40-44 kDa (Klausner et al., 1990). The stable transfectant of TCRa cDNA contained both GIF mRNA and TCRa mRNA, but mRNA of the 55-kDa GIF was lacking in the anti-CD3-stimulated cells. These findings collectively indicate that the 55-kDa protein is a posttranslationally formed conjugate of GIF peptide with TCRa chain. Lack of mRNA of the 55-kDa GIF may explain why cDNA encoding TsF has never been obtained in spite of great efforts by several investigators. It was noted that essentially all GIF bioactivity in culture supernatant of anti-CD3-treated 231F1 cells and 211a cells bound to anti-TCRa(H28710)-coupled AffiGel, and that the activity in the culture supernatant of

BIOCHEMICAL BASIS OF ANTIGEN-SPECIFIC SUPPRESSOR T CELL FACTORS

31

anti-CD3-stimulated cells was comparable to or slightly higher than that of unstimulated cells. These findings suggested that the majority of bioactive 13-kDa GIF peptide synthesized in the anti-CD3-stimulated cells selectively associated with the TCRa chain to form the 55-kDa protein. Structural basis for the linkage between the two peptides is not known. However, failure to detect the 13-kDa GIF peptide in SDS-PAGE analysis of the antigen-specific GIF preparation under reducing conditions, even in the presence of 6 M urea, suggested that a covalent linkage other than interchain disulfide bond may be involved in the formation of the TCRa-GIF conjugate (Nakano et al., 1996). Our next question was whether the 55-kDa GIF represents antigenspecific GIF or its subunit. The question was answered by using a stable homologous transfectant of TCRa cDNA in the 231F1 cells (Ishii et al., 19964. The transfectant (21la cells) was stimulated with anti-CD3, and culture supernatant was fractionated on OVA Sepharose. As expected, acid eluate fraction from the immunosorbent contained the 55-kDa GIF, as determined by SDS-PAGE and immunoblotting with anti-TCRa chain (H28-710). Because the antigen-specific GIF activity from the 231F1 cells retained not only in the anti-TCRa chain antibody column but also in the anti-TCRP chain antibody column (Iwata et al., 1989b), we fractionated aliquots of the OVA-eluate fraction on anti-TCRP chain column or antiTCRa chain column and determined the distribution of the 55-kDa GIF between the flow-through and acid eluate fractions by SDS-PAGE and Western blot with the anti-TCRa antibody (c.f. Fig. 4).The results clearly showed that the 55-kDa GIF in the OVA-eluate fraction was retained in both the anti-TCRP chain column and anti-TCRa chain column and was recovered by acid elution. The flow-through fractions from the anti-TCR antibody columns did not contain a detectable amount of the 55-kDa GIF. The results indicated that the OVA-specific GIF consisted of the 55-kDa GIF peptide and another peptide having the TCRP chain determinant. These two polypeptide chains appear to be linked by disulfide bond, because after reduction of the OVA-elute with 10 mM DTT and alkylation with iodoacetamide, the 55-kDa GIF in the preparation failed to be retained in anti-TCRP chain column but was bound to the anti-TCRa chain column. Upon stimulation with anti-CD3, the 211a cells secreted not only antigen-specific GIF but also the 55-kDa GIF lacking affinity for OVA. When the culture supernatant of anti-CD3-stimulated 211a cells was fractionated on OVA-Sepharose, flow-through fraction contained a substantial quantity of the 55-kDa GIF. However, the 55-kDa GIF in the fraction was not retained in the anti-TCRP column, but was retained in anti-TCRa chain column, and was recovered from the latter column by acid elution

32

ISHIZAKA et al.

A

Culture supernatants of anti-CD3-stimulated21 la cells

1

OVA-Sepharose I

El,',te

H67-Affi

H28-AfE

A

A

J?lC H67-AfE

Eluate NOW Eluate mow Eluate (+) through (+) through

(-1

(-1

F~OW

throlugh

(-I

H28-m

A

Eluate mow (+) through

(-1

FIG.4. OVA-specific GIF contains the 55-kDa GIF peptide and binds to both anti-TCRP (H57-597)-and anti-TCRa (H28-710)-coupledAffiGel. (A) Protocol of the experiment. (+) (-) indicate presence or absence of GIF bioactivity. (B) Immunoblot analysis of each fraction using the mAb H28-710. The 55-kDa peptide in the eluate from OVA-Sepharose (OVA-EL) bound to H-57-597-Affi and was recovered in acid eluate (H57 EL), but absent in the flow-through fraction (H57 FT). The 55-kDa peptide in the flow-through fraction from OVA-Sepharose (OVA-FT)failed to be retained in H-57-597 Affi (H57 EL) but bound to H28-710 Affi (H28 EL). (From Ishii et al., 1996a, J. Immunol. 156, 1740, Copyright 1996, The American Association of Immunologists, with permission.)

BIOCHEMICAL BASIS OF ANTIGEN-SPECIFIC SUPPRESSOR T CELL FACTORS

33

(Fig. 4).When the untransfected 231F1 cells were stimulated with antiCD3 to form antigen-specific GIF, all 55-kDa in the culture supernatant bound to OVA-Sepharose and anti-TCRP chain column. Thus, the formation of the 55-kDa GIF lacking affinity for OVA by the 211a cells appears to be due to overexpression of TCRa chain in the homologous transfectant. Nevertheless, the findings obtained in the experiment shown in Fig. 4 clearly indicated that the 55-kDa GIF itself does not have affinity for OVA, but that the association of TCRP chain is not required for the secretion of the 55-kDa GIF from Ts cells. Previous experiments provided evidence that NP-specific TsF1, which is believed to be a single chain factor, has GIF bioactivity and shares a common antigenic determinant with the 13-kDa GIF (Steele et al., 1989). If TCRa chain plays a dominant role in determination of the epitope specificity of TCR in this system (Collins et al., 1990; Kuchroo et al., 1991), it is possible that the 55-kDa GIF, which is a product of TCRa cDNA, may represent a single chain TsF. Formation of the 55-kDa GIF peptide appears to be unique for Ts cells. Upon stimulation with anti-CD3, a representative Th hybridoma DO11.10 cells did not secrete a detectable amount of any peptide having the TCRa determinant. Failure of Th cells to form the 55-kDa GIF was confirmed by transfection of the 231F1-derived TCRa cDNA into the Th cell line 175.2 (Glaichenhaus et al., 1991), which did not contain TCRa mRNA. The 175.2 cells did not express TCR or CD3, but stable transfectants of the TCRa cDNA in the cells expressed TCR and CD3 on their surface. The TCRa chain in the cells lysate of a representative stable transfectant could be recovered by using H28-AffiGe1, and was identified as a 35-kDa peptide by immunoblotting. After stimulation of the stable transfectant with anti-CD3, however, a peptide having the TCRa chain determinant could not be detected in culture supernatant (Ishii et al., 1996a). This finding was in agreement with the observations by Kuchroo et al. (1995) that NP-specific TsFl activity was not formed by transfection of TCRa chain cDNA of Ts hybridoma into TCRa- mutant of Th cells and showed that Th cells cannot secrete a soluble from TCR even after antigenic stimulation. IV. Possible Roles of GIF as a Subunit of TsF

Many investigators involved in antigen-specific immunoregulation would accept the concept that antigen-specific TsF may be a soluble form TCR or its derivative (Dorfet al., 1992). However, this concept raised several questions as follows. (1)It is well known that TCRa chain rapidly degrades in endoplasmic reticulum (ER) and lysozomes unless the polypeptide chain associates with TCRP chain and CD3 complex peptides (Klausner et al.,

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1990).Thus, an immediate question is how could TCRaP heterodimer or TCRa chain have avoided degradation and be released from Ts cells? (2) Because of binding of TsF to nominal antigen, it has been believed that TsF exerted its antigen-specific immunoregulatory activities through binding to homologous antigen. If TsF is a soluble form of TCR, the structures responsible for antigen-binding must be variable portion of TCR chains. Extensive studies on the affinity between TCR and an antigenic peptide associated with a MHC product indicated that the equilibrium dissociation constant (b)of the binding would be on the order of to 10-'M (Syklev et al., 1994; Matsui et al., 1994; Alam et al., 1996). One may expect that the kd of the binding between nominal antigen and TsF would be even higher than the value. Considering low concentration of both TsF and antigen in lymphoid tissues, TsF may not bind nominal antigen under physiological conditions. If this is the case, antigen-specific effects of TsF are difficult to explain. (3) If TsF is either a soluble form TCR or TCRa chain, why do such molecules have immunosuppressive activities, and how could these molecules have I-J determinant? As described earlier, antigen-specific GIFRsF is a conjugate of TCR with GIF. In view of our previous observations that nonspecific GIF suppressed in vivo antibody responses (Akasaki et al., 1986), we speculated that GIF is a functional subunit of TsF. Extensive studies on GIF described later actually suggest that GIP portion of TsF plays essential roles in the formation and function of TsF. A. STRUCTURAL BASISOF BIOACTIVITY OF GIF Since the major cell source of GIF activity was I-J' Ts cells (Jardieu et al., 1984),we speculated that formation of GIF is unique for Ts cells. Thus, we affinity-purified the 13-kDa GIF molecules in the culture supernatant of the 231F1 cells and isolated a cDNA clone encoding the protein (Mikayama et al., 1993). Nucleotide sequence of the cDNA indicated that the cDNA encodes a 13-kDa peptide of 115 amino acids. Expecting homology between murine GIF and human GIF, the human GIF cDNA clone was isolated using murine cDNA as a probe. Human GIF has 90% homology with mouse GIF at the amino acid level. Polyclonal antibodies against E. coli-derived recombinant murine (rm) GIF bound Ts hybridoma-derived bioactive GIF, indicating that the cDNA actually encodes GIF. However, we obtained unexpected findings as follows. (i) GIF did not have a signal peptide sequence, although a substantial quantity of the 13-kDa peptide was secreted from Ts hybridomas and stable transfectants of the cDNA. (ii) The nucleotide sequence of mouse GIF cDNA was exactly the same as that of a growth factor-induced delayed early response gene (Lanahan et al., 1992), and the nucleotide sequence of human GIF cDNA was

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35

identical to that of macrophage migration inhibitory factor (MIF) cDNA (Weiser et al., 1989), except for one base. (iii) Not only Ts cell hybridomas but also various cell line cells, including Th hybridomas and B cell lines, contained mRNA that hybridized with GIF cDNA, and culture supernatants of these cell line cells contained a 13-kDa peptide which bound anti-GIF in immunoblotting. However, only the 13-kDa peptide from Ts hybridomas had GIF bioactivity (Liu et al., 1994). Recombinant GIF expressed in E. coli and COS 1 cells were also inactive. Nevertheless, neucleotide sequence of the GIF cDNA from Th hybridomas and B cell lines was identical to that of the GIF cDNA from Ts cells, indicating that the Ts-derived bioactive GIF and inactive GIF peptide from nonsuppressor cells have an identical amino acid sequence. These findings suggested that bioactivity of GIF peptide was generated by posttranslational modifications of the peptide. To test the hypothesis, a device was made for the secretion of the GIF peptide through the constitutive secretory pathway. It was found that transfection of cDNA encoding a fusion protein consisting of the Nterminal proregion of calcitonin precursor and human GIF (proCT-GIF) in BMTlO cells resulted in secretion of bioactive GIF, whereas stable transfectants of human GIF cDNA alone in the same cells secreted inactive GIF peptide (Liu et al., 1994). In the former system, evidence was obtained that the fusion protein went through the endoplasmic reticulum (ER) and was cleaved by a furin-like endoproteinase at Golgi for secretion of mature 13-kDa GIF peptide. In the latter transfectant, a large quantity of GIF peptide was present in cytosol, but the peptide was not detectable in the particulate fraction, indicating that the peptide did not go through the constitutive secretory pathway. One might speculate that certain posttranslational modifications of GIF peptide in ER or Golgi were responsible for the generation of bioactivity. However, if one transfects the human GIF cDNA into mouse Ts hybridoma (e.g., 231F1 cells), stable transfectants of the human GIF cDNA secreted bioactive human GIF, indicating that murine Ts cells have a machinery for posttranslational modifications of GIF peptide for generation of bioactivity (Liu et al., 1994). It was also found that the Ts hybridomas as well as those transfected with human GIF cDNA contained a large amount of 13-kDa GIF peptide in cytosol; however, their cytosolic GIF lacked bioactivity. No difference was detected between bioactive GIF and inactive cytosolic GIF in SDS-PAGE analysis, and evidence was obtained that amino acid sequence of the inactive cytosolic GIF was identical to that of bioactive homologue (Nakano et d., 1995). However, we happened to find that inactive cytosolic GIF has affinity for AffiGel 10 itself, whereas bioactive GIF does not. Fractionation of culture supernatant of the Ts hybridoma on AffiGel 10 revealed that

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the culture supernatant contained not only bioactive GIF but also inactive GIF. These findings suggested to us the possibility that the heterogeneity of GIF in bioactivity is due to conformationaltransition of the same peptide. This idea was supported by subsequent studies in which inactive GIF could be converted to bioactive derivatives by chemical modifications. Deduced amino acid sequence of recombinant GIF indicated that the GIF peptide contains three cysteine residues (Cys-57, -60, and -81). X-ray crystal structure of E. coli-derived recombinant human (rh) GIF showed that no intrachain &sulfide bond exists in the molecules (Kato et al., 1996). In view of some evidence that cysteine residue may be involved in particular conformation of some proteins (Turner et al., 1990), experiments were undertaken to determine the effect of sulfhydryl reagents on the bioactivity of GIF. It was found that carboxymethylation of the SH group of Cy-60 in the inactive, E. coli-derived rhGIF with iodoacetate resulted in the generation of bioactivity, although the activity of carboxymethylated GIF was 10- to 20-fold less than that of Ts hybridoma-derived GIF. Furthermore, treatment of E. coli-derived inactive rhGIF with 5,5'-dithiobis(2nitrobenzoic acid) (DTNB) resulted in the generation of a derivativewhose specific bioactivity was almost comparable to that of the Ts-derived bioactive GIF (Table I). Treatment with DTNB converted inactive cytosolic GIF as well to bioactive derivative,while bioactive GIF in culture supernatant of Ts hybridomas was inactivated by reduction with 10 mM DTT. Isolation and chemical analysis of the DTNB-treated GIF derivative revealed that binding of the 5-thio-2-nitrobenzoic acid group with Cys-60 was responsible for the generation of the highly bioactive derivative (Nakano et al., 1997). It should be noted that specific bioactivity of the rGIF derivatives is TABLE I GENERATION OF BIOACTIVITY BY MUTATION AND CHEMICAL MODIFICATIONS OF rGIP Chemical Modification Mutated GIF

None

Iodoacetate*

Wild type GIF C57A C60A C57A/N 106s

>1000

120 10 ND 10

100 200 100

DTNB~ 8 3 ND ND

Numbers in the table are the minimum concentration (ng/ml) of GIF species required for the detection of bioactivity. Under the experimental conditions, iodoacetate and DTNB react exclusively to the SH group of Cys-60. From Sugie d al., 1997, with slight modifications.

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quantitatively different depending on the chemical group bound to the same sulfur atom in Cys-60. Our more recent experiments showed that not only the size but also the presence of charged group(s) in the compound attached to Cys-60 is important for generation of highly bioactive GIF. X-ray crystal structure of GIF protein may explain the preferential reaction of the SH group in Cys-60 with a SH reagent. E. cob-derived rGIF molecules form a barrel-shaped trimer of the 13-kDa GIF protein, and the barrel consists of three /3 sheets (Fig. 5A). Six a helices, two of which belong to each monomer, surround the outside of the barrel (Kato et al., 1996). Ribbon diagram of the monomer is shown in Fig. 5B. In this structure, the SH group of Cys-57, which is in a loop, is directed toward the inside of the barrel, and the SH group of Cys-81, which is in one of the a helices, is directed toward the /3 sheet. In contrast, the SH group in Cys-60, which is in one of the /3 strands, is sticking out between the two a helices. Reaction of a sulfhydryl reagent with this SH group indicates that the reagent could go between the two a helices. X-ray crystal structure of GIF indicated high conformational flexibility in the a helices and adjacent loop regions in the rhGIF molecules. One may speculate that incorporation of a certain substance or a chemical group in the grove between the two a helices may change conformation of the a helices (c.f. Fig. 5B). Separate experiments demonstrated that replacement of Cys-57 or Cys-60 in the inactive rhGIF with Ala by site-directed mutagenesis also resulted in the generation of bioactivity, whereas replacement of Cys-81 with Ala failed to do so (Tomura et al., 1999). Specific bioactivity of the Cys-57 + Ala mutant (C57A) and Cys-60 --f Ala mutant (C60A) were comparable to that of carboxymethylated GIF. However, replacement of Cys-57 with Ala and either carboxymethylation of SH group in Cys-60 or binding of 5-thio-2-nitrobenzoic acid group to Cys-60 synergistically increased the GIF bioactivity (Table I). These findings suggested that replacement of Cys-57 with Ala and the binding of a chemical group to Cys-60 may cause a common change in conformational structure of GIF, and that such changes in conformation are responsible for the generation of bioactivity. Direct evidence for posttranslational modifications of GIF in Ts cells was obtained by reverse phase chromatography and mass spectrometric analysis of affinity-purified GIF from human Ts hybridoma (Tomura et al., 1999). It was found that GIF in culture supernatant consisted of four species of which molecular weight are 12,346, 12,429, 12,467 and 12,551, respectively. In contrast, inactive, cytosolic GIF in the same cells was homogeneous and represented the species of molecular weight 12,346, which is identical to the theoretical value of the molecular weight calculated from their amino acid sequence. It was also found that inactivation of GIF

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41.

FIG.5. Crystal structure of recombinant human GIF. (A) Ribbon diagrams of GIF trimer. N and C indicate N terminal and C terminal, respectively, of each GIF monomer. The trimer is in a barrel shape formed by three p sheets. Each p sheet consists of six /3 strands-four /3 strands in a monomer and the other two stands from the adjacent subunits. Six a helices surround the outside of the barrel. (B) Ribbon diagram of monomeric GIF structure. Numbers indicate the positions of amino acids. The positions of Cys-57, Cys-60 and Asn-106 are indicated.

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39

in culture supernatant by reduction with 10 mM DTT was accompanied by conversion of the 12,467 and 12,551 species to the 12,346 and 12,429 species, respectively. The results indicate that posttranslational modifications in the Ts cells consist of two steps: (1)covalent binding of a chemical group of molecular weight 83, and (2) the binding of a group of molecular weight 121 to a cysteine residue through disulfide bond, (the latter modification is essential for generation of GIF bioactivity). Recent experiments by Watarai et al. (unpublished results) provided evidence that this latter modification is cysteinylation of Cys-60, whereas the former modification is phosphorylation of Ser-91. Cysteinylation of Cys-60 in rhGIF could be achieved in vitro by incubation of rhGIF with cystine. Indeed, the cysteinylated rhGIF had bioactivity, indicating that this modification is an important step for the generation of bioactive GIF. Because only Ts cells can produce bioactive GIF (Liu et al., 1994), we anticipated that the synthesis of GIF peptide is unique for Ts cells. However, a series of experiments described earlier indicated that a unique feature of Ts cells was not the synthesis of the peptide but the capacity for posttranslational modifications of the peptide. Considering that cysteinylation and phosphorylation of the peptide occur in subcellular organelles (Hwanget al., 1992),one may speculate that Ts cells may have a unique transporter protein which facilitates translocation of the GIF peptide to subcellular organelles, where the posttranslational modification of the peptide takes place.

B. ASSOCIATIONOF I-J DETERMINANT WITH BIOACTIVE GIF Previous experiments have shown that GIF bioactivity in the culture supernatants of C57B1/6 T cells could be removed by incubation with anti-I-Jb alloantibodies or monoclonal anti-I-Jb antibody, H-6, followed by immunoprecipitation of the antibodies, but the activity could not be removed by anti-I-Jk or anti-I-J” alloantibodies. In contrast, CBA-derived GIF activity bound to anti-I-Jk but not to anti-I-Jb alloantibodies. GIF activity from BIO.A (5R) T cells could be removed by anti-I-Jkbut not by anti-I-Jb alloantibodies, while GIF activity from BIO.A (3R) T cells had IJb determinant (Jardieu et d.,1986). Thus the bioactive GIF from TS cells appears to possess an I-J determinant. Subsequent experiments have shown that affinity-purified bioactive 13-kDa GIF peptide from the 231F1 cells was retained in anti-I-Jb (H6)-coupled AffiGel and was recovered by acid elution (Nakano et al., 1995). The 13-kDa GIF in the acid eluate fraction could be demonstrated by SDS-PAGE and immunoblotting with anti-GIF. Because the amino acid sequence of murine GIF peptide from various murine cell line cells appears to be identical, irrespective of the MHC phenotype of cell sources (Liu et al., 1994), we anticipated that the anti-

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I-Jb monoclonal antibody might have recognized structures generated by posttranslational modifications. This possibility was supported by the finding that not only the 231F1-derived murine GIF but also bioactive human GIF peptide, which was produced by the stable transfactant of human GIF cDNA in the 231F1 cells, were retained in H-6-coupled AffiGel, and were recovered by elution at acid pH (Nakano et al., 1995).As described, bioactive 13-kDa human GIF could be obtained by transfection of the cDNA encoding a fusion protein ProCT-GIF in BMTlO cells. However, the 13-kDa bioactive human GIF peptide obtained from the culture supernatant of the stable transfectant in BMTlO cells failed to retain in H-6AffiGel. The results collectively indicate that the I-J phenotype of GIF is decided by the cell source of GIF, but not by the amino acid sequence of the peptide. Since the cytosolic inactive GIF is identical, irrespective of the cell source, the I-Jbdeterminant recognized by H-6 must have been generated by posttranslational modifications of the peptide in the H-2b/d Ts hybridoma. Tada et al. (1985) indicated that the I-Jkdeterminant recognized by a monoclonal anti-1-Jkwas associated with multiple heterologous molecules expressed on functionally different mature T cells. If the structures recognized by anti-I-J antibodies are those generated by posttranslational modifications, multiple peptides with different primary structures may share a common I-J determinant. This hypothesis agrees with the findings by O’Hara et al. (1995) on TsF obtained by transfection of TCRa chain cDNA that I-J restriction of the resulting TsF is decided by the recipient cells rather than by TCRa chain gene. In any event, our findings that the I-J phenotype is determined by the cells involved in posttranslational modifications of certain peptides may explain several unsolved problems of the I-J puzzle, particularly concerning the properties of the I-J determinant of TsF (Taniguchi et al., 1982) and adative acquisition of I-J specificity in allogeneic chimera (Uracz et al., 1985a; Sumida et al., 1985), and justify previous observations on the association of I-J determinant with TsF. However, the epitope recognized by the monoclonal antiI-Jb antibody is unknown. As described, the chemical nature of the posttranslational modifications of human GIF in a human Ts hybridoma was cysteinylation of Cys-60 and phosphorylation of Ser-91, but such modifications cannot explain I-J phenotype. One might speculate that a cell-derived peptide or lipid may associate with the bioactive GIF through a noncovalent bond, and provide with the I-J epitopes. C. CELLULAR MECHANISMS FOR THE FORMATION OF TCR DERIVATIVES BY TSCELLS Biochemical identification of the antigen-specific GIF (TsF) as a conjugate of TCR with GIF immediately raised questions where these complex

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molecules were formed in Ts cells, and how these TCR derivatives could have avoided degradation in ER. As the 13-kDa GIF does not contain a signal peptide sequence, the peptide would not be translocated through ER. On the other hand, TCRa chain sould be translocated into ER immediately after being synthesized on ribosome. If the 55-kDa GIF is the posttranslationally formed conjugate of the 13-kDa GIF with TCRa chain, where do the two peptides met to form the conjugate? Another fundamental question regarding the TCR-GIF conjugate is whether the TCRa chain in the conjugate came from cell surface TCR or it was newly synthesized after stimulation of the cells with anti-CD3 or antigen-pulsed APC. To answer these questions, we first determined the kinetics of the formation of the 55-kDa GIF by the 211a cells (i.e., homologous stable transfectant of the 231F1-derived TCRa cDNA in the 231F1 cells). The 55-kDa GIF in culture supernatant became barely detectable at 24 h after crosslinking of CD3, and increased to maximum by 48 h. It is known that TCR is internalized within 1 h after cross-linking of CD3, and a portion of the internalized TCR molecues are recycled (Krangel, 1987). The relatively fast kinetics of the internalization and recycling of surface TCR suggested that the 55-kDa GIF may not be derived from TCR expressed on the cell surface. This idea was supported by the experiment in which the GIF peptide was biosynthetically labeled. Treatment of the stable transfectant with anti-CD3 and culture of the cells for 24 h in protein A-coated flasks in the presence of =S-methionine, followed by immunoprecipitation of proteins in the culture supernatant with anti-TCRa antibody (H28-710) revealed radiolabeling of a 55-kDa peptide. When the stable transfectant were precultured for 24 h in the presence of =S-methionine, and the cells were then stimulated with anti-CD3 in the absence of %methionine, the 55-kDa protein in the culture supernatant was not radiolabeled. The results indicated that the 55-kDa GIF was synthesized after cross-linking of CD3, and excluded the possibility that the peptide was derived from TCR expressed on the cell surface (Ishii et al., 1996b). These findings may explain previous observations by other investigatorsthat TCRP- expression variants of hapten-specific Ts hybridomas did not express TCR but could release TsF activity (Kuchroo et al., 1991; Collins et al., 1990). In order to get some idea on the secretory process of the 55-kDa GIF, comparisonswere made between the quantity of the 55-kDa GIF in culture supernatants and cell lysate. The homologous stable transfectant of the 23lF1-derived TCRa cDNA (211a cells) were treated with anti-CD3 and cultured for 24 h on protein A-coated flasks. Determination of the 55-kDa GIF in both culture supernatant and cell lysate by SDS-PAGE and immunoblotting with H28-710 showed that the quantity of the cellassociated 55-kDa GIF was less than 5% of that present in the culture

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supernatant. It has been shown that antigen-specific GIF was associated with the plasma membrane of Ts hybridoma which had been stimulated with antigen-pulsed APC (Iwata et al., 1990). Since the antigen-specific GIF associated with the plasma membrane was recovered by treatment of the cells with EGTA, it is quite likely that the membrane-associated factor is the molecules passively bound to GIF receptors (c.f. next section). In this experiment as well, the cell-associated GIF activity was about 10% of that present in culture supernatant. In contrast, distribution of the 13-kDa GIF peptide between cytosol and culture supernatant was quite different. After 48-h culture of unstimulated 211a cells, the quantity of the 13-kDa GIF peptide in cytosol was approximately 40-fold more than that present in culture supernatant. The results indicated that the 55-kDa protein is secreted with high efficiency and suggested that the route of the secretion is probably constitutive secretory pathway. This speculation was supported by subsequent experiments, in which we proved translocation of a portion of the cytosolic GIF peptide into subcellular organelles after cross-linking of CD3 (Ishii et al., 1996b). As shown in Fig. 6, before stimulation with anti-CD3, the paticulate fraction of 211a cells did not contain a detectable amount of 1SkDa GIF. However, the peptide became barely detectable in the particulate fraction at 18 h

FIG. 6. Anti-CD3-induced translocation of the 13-kDa GIF in homologous transfectant of TCRa cDNA in murine Ts hybridoma. The cells were treated with anti-CD3 and cultured for 0,18,42, or 48 h in protein A-coated wells. Both the cytosol fraction and the particulate fraction were obtained from the cells and analyzed by immunoblotting with anti-GIF. Particulate fraction applied per lane was derived from 5 X lo6 cells. The 13-kDa GIF became detectable in the particulate fraction at 42 h. (From Ishii et al., 1996b, Proc. Natl. Acad. Sci. USA 93, 7207, with permission.)

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after the anti-CD3 treatment and markedly increased by 42-48 h. Thus, the time course of the translocation of the 13-kDa GIF into subcellular organelles after cross-linking of CD3 preceded or paralleled the time course of the release of the 55-kDa GIF from the cells. It was also found that the translocation of the 13-kDa peptide is characteristic for Ts cells. The Th hybridomas, such as DO1l.10 cells, contained a substantial quantity of inactive GIF in cytosol but failed to produce 55-kDa GIF upon stimulation with anti-CD3 (Nakano et al., 1996). Stimulation of the DO1l.10 cells with anti-CD3 failed to induce translocation of GIF into subcellular organelles. Similar experiments with a stable transfectant of the 231F1derived TCRa cDNA in the TCRa- mutant of Th cells (175.2 cells) confirmed that upon stimulation with anti-CD3, the 13-kDa GIF peptide in cytosol did not translocate into subcellular organelles, and that these cells failed to form the 55-kDa GIF (Ishii et al., 199613). If the translocation of the cytosolic 13-kDa GIF into ER is a key process for the formation of the 55-kDa GIF, one may expect that transfection of the chimeric proCT-GIF cDNA in Th cells may facilitate the formation of the 55-kDa GIF by the transfectant. Thus we established a stable double transfectant of both the Pro-CT-GIF cDNA and 231F1-derived TCRa cDNA in the TCRa- mutant of Th cell line, 175.2 cells. Northern blot analysis of mRNA of the double transfectant (i.e., NlCG2 cells) with 32Plabeled GIF cDNA showed two bands (i.e., endogeneous 0.6 kb GIF mRNA and 0.8 kb mRNA); the size of the latter corresponded to the mRNA of the fusion protein consisting of N-terminal region of calcitonin precursor and human GIF. As expected, the NlCG2 cells constitutively secreted bioactive GIF. Immunoblotting of the particulate fraction of the NlCG2 clone with anti-GIF showed that the fraction contained the 13-kDa GIF. Indeed, treatment of the NlCG2 cells with anti-CD3, followed by culture of the cells in Protein A-coated flasks, resulted in the formation of the 55-kDa GIF. The 55-kDa protein from the NlCG2 cells bound both antiGIF and anti-TCRa antibody (H28-710) in Western blotting and possessed the GIF bioactivity, indicating that the peptide is identical to the 55-kDa GIF formed by Ts cells (Ishii et al., 1996b). In the NlCG2 cells, the 13-kDa GIF peptide was detected in the particulate fraction prior to anti-CD3 stimulation, and the treatment with anti-CD3 did not enhance the translocation of the protein. Thus, we wondered if the NlCG2 cells may constitutively form the 55-kDa GIF. Indeed, more than half of the GIF activity in the culture supernatant of unstimulated NlCG2 cells was retained in anti-TCRa chain column and was recovered by acid elution. When the acid eluate fraction was analyzed by SDS-PAGE under reducing conditions and immunoblotting, however, the 55-kDa GIF was barely detectable. Instead, the fraction contained a

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substantial quantity of the 13-kDa GIF. The results suggested that in the culture supernatant of the unstimulated NlCG2 cells, the 13-kDa GIF might be associated with the TCRa chain but the two peptides were not covalently linked, and that cross-linking of CD3 or TCR on the cell surface induced the formation of a covalent bond between TCRa chain and GIF. The observations made in the NlCG2 cells may be related to previous findings that NP-specific Ts hybridomas constitutively secreted TsF activity, which could be absorbed with anti-TCRa antibody, but TCRa chain or its derivative was not biochemically identified in the culture supernatant (Kuchroo et al., 1990). In any event, the experiments on the double transfectant provided strong evidence for our hypothesis that the 55-kDa GIF is a posttranslationallyformed conjugate of GIF peptide with TCRa chain. The anti-CD3-stimulated NlCG2 cells contained GIF mRNA and TCRa mRNA but no mRNA encoding the 55-kDa GIF, even when the 55-kDa GIF was being formed. A fundamental problem remaining to be solved is the mechanism through which cross-linking of CD3 or antigen presentation to Ts cells induces or enhances translocation of GIF into ER. It should be noted that both the posttranslational modifications of GIF in unstimulated cells and the translocation of GIF through ER upon antigen stimulation are unique for Ts cells. One may speculate that these two phenomena are related with each other and may be controlled by transporter protein(s) which are unique for Ts cells. Another question remaining to be answered is why the 55-kDa GIF could be released from Ts cells without ER degradation. In our systems, TCRa chain itself has never been detected in culture supernatants of Ts hybridomas or homologous transfectants of TCRa chain cDNA in the Ts hybridoma. This finding may be expected from the extensive studies on the expression of TCR on the cell surface, which showed that TCRa chain is degraded in ER unless the peptide associates with TCRP and CD3 complex peptides (JSlausneret al., 1990). Bonifacino et al. (1990) reported that the transmembrane portion of TCRa chain contains signals for ER degradation. Shin et al. (1993) indicated that TCRa chain appears to be translocated into the ER lumen, and that recognition for ER degradation takes place in the lumen of the ER. Using chimeric molecules consisting of the extracellular and cytoplasmic domains of CD4 and transmembrane domain of TCRa chain, they have shown that molecules with proper folding of the hydrophobic transmembrane peptide escape from rapid ER degradation. One might speculate that conjugate formation of TCRa chain with GIF peptide may change the folding of the transmembrane portion of TCRa chain, so that the conjugate escapes from ER degradation. We suspect that a portion of the 55-kDa GIF may be subjected to ER degrada-

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tion, and that the peptide secreted from the 211a cells may represent a small fraction of the TCRa chain synthesized. We have recently established a stable transfectant of truncated cDNA encoding the extracellular portion of the TCRa chain. Stimulation of the stable transfectant with anti-CD3 resulted in the formation of a 52-kDa peptide, which reacted with both anti-GIF and anti-TCRa antibody. The quantity of the peptide formed by this transfectant was much more than that of the 55-kDa GIF formed by the 211a cells. These findings collectively suggest that the conjugate formation with GIF protected the TCRa chain from ER degradation, and enabled the Ts cells to release antigen-specific TsF. D. TARGET CELLSOF BIOACTIVEGIF Since the GIF bioactivity has been determined by the ability of the cytokine to switch the helper T cell hybridoma, 12H5 cells from the formation of glycosylated IgE-binding factor to the formation of unglycosylated IgE-binding factor, attempts were made to determine the affinity of bioactive GIF for the 12H5 cells. Bioactive GIF was affinity purified from the culture supernatant of a stable transfectant of the human GIF cDNA in the murine Ts hybridoma, 231F1 cells, and radiolabeled with lZ5I.Scatchard analysis of binding data indicated that the 12H5 cells have two kinds of receptors for the bioactive GIF. Equilibrium dissociation constant (kd) between the bioactive GIF and high affinity receptors was in the range of 10-100 pM, whereas that for low affinity receptors was 0.1 to 1pM (Sugie et d.,1997). Possible relationships between the high affinity receptors and low affinity receptors are not known. Nevertheless, the k d values between bioactive GIF and the low affinity receptors suggest that only the high affinity receptors have biologic significance. Inactive GIF in cytosol of the stable transfectant neither bound to the 12H5 cells nor prevented the binding of '=I-labeled bioactive GIF to the cells, indicating that only bioactive GIF binds to the receptors. In view of previous findings that GIF bioactivity was generated by chemical modifications or mutations of rhGIF, various bioactive derivatives of the rhGIF were examined for binding to the 12H5 cells. As expected, E. coli-derived rhGIF failed to bind to the 12H5 cells, but affinity for the target cells was generated by replacement of Cys-57 with Ala(C57A) or replacement of Asn-106 with Ser(N106S) or carboxymethylation of, or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the rhGIF molecules (cf., Fig. 5B, Table I). Furthermore, such independent chemical changes in the molecules had synergistic effects on increasing the affinity of rGIF molecules for the target cells. The k d values between C57A-DTNB or C57A-Nl06S and the high affinity receptors on the 12H5 cells was 1020 pM, which was comparable to the kd between the bioactive GIF from

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Ts cells and the receptors. These findings collectively indicate that the receptors recognize conformational structures in the bioactive GIF, which are lacking in the inactive homologue. However, the affinity of GIF derivatives for 12H5 cells does not parallel their bioactivity to switch the cells from the formation of glycosylated IgE-binding factor to the formation of unglycosylated IgE-binding factor. Although carboxymethylation of Cys60 in C57A increased both the bioactivity and affinity for the receptors of C57A, substitution of Ser for Asn-106 in C57A markedly increased the affinity of the molecules for target cells without increasing its bioactivity (cf., Table I). Basis for the lack of parallelism between the bioactivity of GIF molecules and the affinity for target cells is not known, but the results suggest that the binding of GIF molecules to target cells may not be sufficient for signal transduction. This finding may not be surprising. It is known that TNFa and lymphotoxin are structurally related and bind to the same receptors with comparable affinity, however, TNFa is 200- to 300-fold more effective than lymphotoxin in inhibiting the growth of tumor cell line cells (Browning and Ribolini, 1989). In any event, high affinity binding of highly bioactive GIF derivatives, such as C57A-DTNB to the target cells, and failure of the inactive wild type rGIF to bind to the same cells strongly suggest that high affinity binding to target cells is an essential step for manifestation of biologic activity. Receptors for bioactive GIF were found not only on the Th hybridomas but also on the pigeon cytochrome C-specific murine Thl clone (ADlO), OVA-specific Th2 clone (AlC6) and the B cell line, A20.3 cells (Sugie et al., 1997). The k d values and the number of high affinity receptors on the Th clones were 30-100 pM and 600-1500 per cell, respectively. However, the receptors for bioactive GIF were not detected on naive resting T cells and B cells isolated from normal mouse spleen. To extend this finding, we determined whether the GIF receptors are expressed on lymphocytes after activation. Naive CD4+ T cells were obtained from AD10 mice, transgenic for the TCRaP that recognizes moth and pigeon cytochrome C (Kaye et al., 1992), and the cells were cultured with pigeon cytochrome C peptide in the presence of irradiated BIO.A spleen cells. After 3 days culture, both the high affinity receptors and low affinity receptors for bioactive GIF were detected on the activated T cells. The k d values and the number of the high affinity reaceptors on the activated Th cells were 10-20 pM and 700-1000 per cell, respectively (Sugie et al., 1999). As expected, lZ5I-labeledinactive wild type rGIF failed to bind to both the activated and naive T cells. It was also found that B cells expressed GIF receptors upon activation. After stimulation of naive, resting B cells for 48 h with the F(ab’)zof anti-p chain antibodies in the presence of IL-4, both the high affinity

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receptors and low affinity receptors were expressed on B cells. Culture of resting B cells in the presence of IL-4 alone, which markedly increased the expression of CD23 and MHC class I1 product, failed to induce GIF receptors. LPS alone was sufficient for activating B cells judging from the enlargement of the cells, but barely induced the GIF receptors. As little as 0.37nglml of IL-4, together with 20 puglml LPS, was sufficient for inducing approximately 400 high affinity receptors per cell. The number of the receptors could be increased about threefold by increasing the concentration of IL-4 to 10 nglml. Thus the concentration of IL-4 may be an important factor in determining the density of the receptors. However, stimulation of resting B cells with LPS and dextran sulfate also resulted in the induction of high affinity receptors (1900 receptors per cell), indicating the presence of IL-4-independent pathway for induction of GIF receptors on B cells. The B cells stimulated for 2 days either with IL-4 and LPS or with LPS plus dextran sulfate expressed SIgM and did not contain SIgG1' cells. Thus, the activated B cells bearing GIF receptors would be SIgM' B cells. The GIF receptors were detected on NK1.1' cells obtained from normal spleen (Sugie et al., 1997). However, further studies showed that NKT cells in the population were responsible for binding of bioactive GIF, and that conventional NK cells did not express GIF receptors (Sugie et al., 1999). The number of high affinity receptors on NKT cells in Val4 NKT mice was on the order of 1000 per cell, which was comparable to that on activated T cells. One may speculate that naive NKT cells in normal spleen may have been primed by natural ligand presented by CD1 expressed on Langerhans cells, dendritic cells, or macrophages (Brossay et al., 1997). This idea may be supported by the fact that NKT cells stand out by their bearing several markers of an activated T cell phenotype including HSA", CD44h' and LECAM-1'" (Lantz and Bendelac, 1994). Receptors for bioactive GIF were not detectable on murine and human macrophage or monocyte cell line cells (Sugie et al., 1997) nor on murine dendritic cells obtained by culturing bone marrow cells in the presence of GM-CSF and TNFa (Sugie et al., 1999). Neither the bioactive derivatives of rhGIF described earlier nor inactive rhGIF bound to macrophage/ monocyte cell line cells or dendritic cells. It has been believed that the peptide with the amino acid sequence identical to GIF is MIF (Weiser et al., 1989; Bernhagen et al., 1994). In our experiments, however, neither the affinity-purified bioactive GIF nor inactive rGIF inhibited migration of human monocytes (Mikayama et al., 1993). Herriott et al. (1993) also reported that rMIF/GIF at up to 5 pglml failed to inhibit migration of macrophages. Calandra et al. (1994) claimed that E. coli-derived rmGIF/ MIF induced the macrophage cell line RAW 264.7 cells for the formation

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of TNFa. In our experiments, however, neither the wild type rhGIF nor bioactive derivatives of rhGIF, at the concentration of 0.1-10 pglml, induced the RAW 264.7 cells for the formation of a detectable amount of TNFa (Sugie et al., 1997). Association of MIF activity with the peptide of the GIF/MIF sequence is controversial. However, lack of GIF receptors on macrophages/monocytes eliminated the possibility that the peptide directly act on the cells. E. POSSIBLE MECHANISMS OF IMMUNORECULATION BY TsF Immunosuppressive effects of antigen-specific TsF/GIF suggest that TsF may regulate the immune response through antigen-specific T cells or B cells or APC. High affinity binding of nonspecific GIF to antigenprimed T cells and B cells described earlier suggests that GIF may well be a functional subunit of TsF. 1. Eflect of GZF on Antigen-Primed T Cells Previous experiments showed that repeated injections of partially purified nonspecific GIF into antigen-primed mice facilitated the generation of antigen-specific Ts cells (Akasaki et al., 1986). Indeed, generation of antigen-specific Ts cells by GIF was reproduced in an in vitro system. In this experiment, BDFl mice were immunized with a minute dose of alumabsorbed OVA for the persistent IgE antibody response, and their spleen cells were stimulated with OVA for activation of antigen-primed T cells. Propagation of the activated T cells by IL-2 in the presence of nonspecific GIF resulted in the generation of OVA-specific T cells which constitutively secrete bioactive 13-kDa GIF, and produced antigen-specific GIF upon incubation with OVA-pulsed APC (Iwata and Ishizaka, 1987). Since the antigen-activated T cells express receptors for bioactive GIF (Sugie et al., 1999),we suspect that binding of bioactive GIF to the receptors induced differentiation of the OVA-specific T cells toward Ts cells. The majority of the GIF-producing T cells obtained by this procedure were CD8+,but some of them were CD4'. Hybridomas constructed from the T cells constitutively secreted nonspecific GIF, and some of them formed antigenspecific GIF upon antigenic stimulation. As expected, affinity-purified OVA-specific GIF from the hybridomas suppressed the in vivo IgE and IgG antibody responses of BDFl mice to DNP-OVA in camer-specific manner (Iwata and Ishizaka, 1988).The results indicated that nonspecific GIF facilitated the generation of antigen-specific Ts cells which could produce antigen-specific TsF upon antigenic stimulation. Subsequent studies have shown that culture of Th2 hybridomas, such as 12H5cells, or Th2 clone such as D10G4.1 cells for 3 days in the presence of bioactive GIF facilitated them to produce their own bioactive GIF.

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These cells produced antigen-specific GIF upon stimulation with antigenpulsed APC, indicating that culture with nonspecific bioactive GIF switched the helper T cell hybridomahlone to produce antigen-specific GIF (Ohno et al., 1990). The antigen-specific factors formed by the GIFtreated 12H5 cells and D10G4.1 cells bound to the anti-TsF monoclonal antibody 14-30 but not to 14-12, suggesting that they are TsFl(TsiF).The D10G4.1 is a typical Th2 clone, and 12H5 cells are Th2 hybridoma, which produce IL-4 and 11-5 upon antigenic stimulation. Formation of antigenspecific GIF by GIF-treated Th2 cells indicated that, after preculture with bioactive GIF, some helper cells could function as inducer type Ts cells, suggesting that Tsl cells may simply represent a phenotype of Th cells. This idea is supported by the findings that the 12H5 cells cultured for 3 days in the presence of GIF maintained the capacity to produce bioactive GIF even after 24-h culture in the absence of GIF, but this capacity was lost after 72-h culture in the absence of GIF. Nevertheless, preculture of the Th2 hybridoma or Th2 clone for 3 days with bioactive GIF did not have any effect on the formation of IL-4 by the cells. The quantity of IL4 produced by D 1 0 . a . 1 cells upon stimulation with antigen-pulsed AKR macrophages was not affected by preculture of the Th clone with bioactive GIF. One may speculate the mechanisms of switching helper cells to Tsi cells by GIF. It should be noted that the Th clones and Th hybridomas express high affinity receptors for bioactive GIF (Sugie et al., 1997), and contain a substantial quantity of inactive GIF peptide in cytosol. In Ts cells, bioactive GIF is formed by posttranslational modifications of the inactive GIF peptide, which probably takes place in subcellular organelles. It is also known that translocation of the fusion protein consisting of N-terminal proregion of calcitonin precursor and GIF (ProCT-GIF) through ER resulted in the formation of bioactive GIF even in BMTlO cells. We speculate that the binding of bioactive GIF to high affinity receptors on Th cells induces translocation of inactive cytosolic GIF into subceullar organelles, where the peptide is modified and gains bioactivity. We suspect that generation of antigen-specific GIF-producing T cells by bioactive GIF may be related to so-called Ts cells circuit described by German et al. (1978), in which TsFl and antigen induced the generation of antigen-specific Ts2, that in turn produced antigen-specific TsF2. Since the NKT cells express high affinity receptors for bioactive GIF, the possibility may be considered that the NKT cells may be involved in Ts cascade. Dorf and Benacerraf (1984) suggested that TsF3 can act indirectly through another subpopulation of T cells called T acceptor cells (Tacc), which subsequently liberate a nonspecific factor that suppresses immune response (cf. Fig. 1).Considering that NKT cells can produce a

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large quantity of cytokines, such as IL-4, IL-10, and IFNy, posibility may be considered that GIF/TsF may affect cytokine production of NKT cells and regulate the immune response. As described, KLH-specific Ts hybridomas and a DNP-specific Ts hybridoma express invariant TCRa and /3 chains identical to those on NKT cells (Koseki et al., 1989; Barbo et al., 1995). One might speculate on the possibility that these cells were Tacc rather than the effector type Ts cells. 2. Efiect of GIF on Antigen-Primed B Cells Presence of high affinity GIF receptors on activated B cells suggested that bioactive GIF might affect the differentiation of activated B cells into immunoglobulin-forming cells. To test this possibility, normal resting B cells were stimulated with 10 pg/ml LPS in the presence of varying concentrations of IL-4 for 6 days for the production of immunoglobulins, and the effect of a bioactive rGIF derivative, C57A-DTNB was tested in the system. As reported by Snapper and Paul (1987) and Snapper et al. (1988), IgM levels produced by LPS-stimulated B cells progressively declined with increasing IL-4 concentration, whereas the dose response of IgGl synthesis to IL-4 concentration was bimodal; IgGl levels peaked at 0.37 ng/ml of * IL-4, declined to a minimum at 3.3ng/ml IL-4, and then rose with further increase of IL-4. IgE became detectable at 3.3 ng/ml IL-4 and increased progressively at a higher concentration of IL-4. If C57A-DTNB was added at 24 or 48 h after initiation of the culture, both the IgGl and IgE synthesis, but not the IgM synthesis, was suppressed (Sugie et al., 1999). Repeated experiments with different concentrations of C57A-DTNB showed that 100-500 ng/ml of the GIF derivative suppressed both IgGl and IgE synthesis by about 50%. As expected, 500 ng/ml of inactive, wild type rhGIF failed to affect the immunoglobulin synthesis. Direct suppressive effect of the bioactive GIF derivative on B cells for IgGl and IgE synthesis might explain, at least in part, the suppressive effect of Ts-derived nonspecific GIF on in vivo IgE and IgG antibody responses of BDFl mice to alum-absorbed OVA (Akasaki et al., 1986). If the major target of bioactive GIF is antigen-primed B cells, the possibility may be considered that bioactive GIF may suppress antigenspecific T-B interaction. Indeed, recent experiments by Sugie, in which activated B cell population from B cell antigen receptor transgenic mice were employed for antigen presentation to unprimed T cells from TCR transgenic mice, supported the hypothesis. The results indicated that bioactive rGIF derivative, such as C57A-DTNB, prevented either endocytosis or processing of specific antigen by the primed B cells, and markedly suppressed the antigen-induced differentiation of precursor Th cells to Thl/Th2 cells.

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These findings may provide a clue to resolve controversial issues on the mechanisms for immunosuppression by antigen-specific GIF or TsF. As described, antigen-specific GIF is a posttranslationally formed conjugate of TCRaB with bioactive GIF, of which TCR portion may recognize a processed antigen in the context of a MHC product. However, the kd value of the interaction between TCR and MHC-peptide complex is in the range of M. We wonder that such low affinity binding may not have to biological significance under physiological conditions. Because the kd value of the binding between bioactive GIF and GIF receptors is on the order of lo-'' to lo-'' M , we suspect that the GIF portion of the conjugate may play the major role in the binding of TsF to its target cells. If this is the case, possible target cells for TsF would be antigen-primed T cells, activated or primed B cells, and NKT cells, which express high affinity receptors for GIF, rather than macrophages or dendritic cells. Among the three possible candidates, antigen-activated B cells express antigenic peptide associated with class I1 MHC product. One might speculate that binding of TsF to the GIF receptors on antigen-primed B cells will facilitate the interaction between the MHC-peptide complex on B cells and the TCR portion of TsF. This interaction may contribute to the preferential effect of the factor on the B cells specific for homologous antigen and render the immunosuppressive effect of TsF specific for the antigen. Since the TCR portion of antigen-specific TsF may recognize peptide-MHC complex, the hypothesis may also explain MHC restriction of the immunosuppressive effects of the antigen-specific TsF. V. Discussion and Summary

It is now obvious that Ts cells express TCR and release suppressor factors which have identical epitope specificity and a common antigenic determinant with TCR. The antigen-specific T cell factors released from the Ts cells are most likely to be a derivative of TCR or a conjugate of TCR with some cytokine such as GIF. Binding of such TsF to nominal antigen can be explained by the fact that the T cell epitope for TCR on the cell source of TsF represents an external structure of the antigen. Considering that the TsF is a derivative of TCR, we suspect that TsF is specific for a processed antigenic peptide associated with a MHC product, but the peptide itself has some affinity for TsF and TCR. Major problems in the process of biochemical identification of TsF were that the concentration of TsF in culture supernatants of Ts cells is extremely low, and the factors are hydrophobic. It should be noted that either serumfree or protein-free culture medium was employed in all of the experiments in which TCR-like molecules in culture supernatants were biochemically

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identified. Another problem was that culture supernatants of Ts hybridomas contained several species of proteins which nonspecifically bound to Sepharose and/or AffiGel, and these proteins were recovered in acid eluates of immunosorbents. In all of our experiments, therefore, culture supernatants were preabsorbed by recycling the concentrated supernatants through a large column of rabbit-IgG(RGG)-coupled AffiGel overnight prior to affinity purification. The RGG-AffiGel removed several cell-derived proteins including inactive 13-kDa GIF peptide, and this procedure was essential for identification of the 55-kDa GIF, which bound both anti-GIF antibodies and anti-TCRa chain in immunoblotting. Accumulated evidence for the relationship between TsF and TCR suggested that TsF may be formed either by an alternative splicing of the TCR genes or by posttranslational modifications of TCR chain(s). In the case of the 55-kDa GIF, it is clear that alternative splicing of TCRa gene is not involved. The 55-kDa protein formed by a stable transfectant of a mutated cDNA encoding TCRa chain with histidine tag at C terminus bound to Ni-NTA agarose, indicating that the 55-kDa GIF contained the C terminus of TCRa chain. We believe that the 55-kDa GIF is a posttranslationally formed conjugate of TCRa chain with GIF. Evidence was presented that upon antigenic stimulation of Ts cells, GIF is translocated through ER and forms a conjugate with TCRa chain. The conjugate formation would protect TCRa chain from ER degradation, probably prevent association of CD3 complex with TCRaP, and facilitate the secretion of the TCR-GIF conjugate through constitutive secretory pathway. Translocation of the GIF peptide through ER, followed by posttranslational modification of the peptide in EWGolgi would transform the inactive GIF to bioactive GIF. Thus the Ts cells release antigen-specific GIF having bioactivity upon antigenic stimulation. The conjugate formation explains the reason why mRNA of the 55-kDa GIF does not exist even when the 55-kDa GIF is being formed by Ts cells. Lack of the mRNA of the 55-kDa GIF in Ts cells will explain why cDNA clone encoding TsF could not be obtained in spite of great efforts by several investigators. We believe that GIF plays important roles not only in the formation of a soluble form derivative of TCR but also in the function of TsF. We suspect that the TCR-GIF conjugate would bind to antigen-primed T cells and B cells through the high affinity receptors for bioactive GIF and exert immunosuppressive effects. The abilities of the 13-kDa bioactive GIF to suppress antigen-induced differentiation of primed B cells for IgGl/IgE synthesis and to prevent endocytosis or processing of the antigen by the antigen-specific B cells strongly suggest that TsF would suppress the immune responses through similar mechanisms. One might speculate that association of TsF with antigen-primed B cells through GIF receptors will

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facilitate the interaction of the TCR portion of TsF to antigenic peptideMHC complex on the B cells, and that the dual interaction will make the immunosuppressive effect of GIF selective for the homologous antigen system. Formation of TCRa-GIF conjugate is unique for Ts cells. Although inactive GIF peptide is being synthesized in Th cells, cross-linking of CD3 on Th cells induced neither the translocation of GIF peptide through ER nor the formation of TCRa-GIF conjugate. However, Th cells could produce TCRa-GIF conjugate if one transfects the Pro-CT-GIF cDNA into Th cells for translocation of the fusion protein into ER and facilitates the recipient cells to form bioactive GIF. In this connection, it should be noted that culture of Th cells in the presence of bioactive GIF, which facilitate the Th cells to form their own bioactive GIF, confer the Th cells the capacity to form antigen-specific GIF upon antigenic stimulation (Ohno et al., 1990). Since the antigen-primed Th cells express high affinity receptors for bioactive GIF, we speculate that binding bioactive GIF to the receptors on Th cells triggers translocation of cytosolic GIF into subcellular organelles for posttranslational modifications and lets them produce bioactive GIF. Although the antigen-specific GIF formed by the GIF-treated Th cells has not been biochemically identified, we suspect that TCRaGIF conjugate is being formed by the cells upon antigenic stimulation. If this is the case, Ts cells are simply a phenotype of Th cells. The idea is in agreement with the findings by Kuchroo et al. (1990) that no difference between Tsl and Th cells was found in terms of cell surface markers and interleukin production, and with the observations of Green et al. (1987) that representative Th clone D10G4.1 could produce TsFl upon stimulation with antigen-pulsed UV-irradiated macrophages. Our experiments provided evidence that OVA-specific GIF, which is an effector type TsF, consists of the 55-kDa TCRa chain-GIF conjugate and the TCRP chain. The 55-kDa GIF itself did not retain in the OVA-coupled Sepharose. Similarly, OVA-specific TsF characterized by Chen et al. (1994) consists of TCRa and TCRP chains. In contrast, NP-specific TsFl could be formed by the TCRP expression variants of Tsl cells, indicating that TCRP chain is not required for the formation of the antigen-specific TsF1. It is rather surprising that the TCRa chain or its derivative has affinity for NP-hapten. However, the possibility remains that the TsFl is a homodimer of TCRa chain derivative. Evidence was presented that NP-specific TsFl has GIF bioactivity and shares a common antigenic determinant with the 13-kDa GIF (Steele et al., 1989). These findings suggest that the TsFl is a 55-kDa TCRa chain-GIF conjugate, or its homodimer. It is obvious, however, that our hypothesis on the nature of TsF may not be applied to the KLH-specific TsF and GAT-specific TsF, whose relationship to TCR

is unknown. It should be noted that these TsFs were obtained from cell extract and could not be released from the Ts cells. In contrast, antigenspecific GIF appears to be released from Ts cells through constitutive secretory pathway and does not exist in the cytosol. It has been believed that the role of TsFl in the Ts cascade is to generate Ts2 cells. Evidence was presented by Aoki et al. (1993) that the “factor presenting cells” in the cascade are macrophage-like adherent cells bearing I-J determinant (I-J interaction molecules) (cf., Fig. 1).If TsFl is a TCRaGIF conjugate, absence of GIF receptors on macrophage/monocyte cell line cells or dendritic cells raised the question: how can TsFl associate with the “factor-presenting cells”? The possibility exists that the TsFs involved in DTH are conjugates of TCR chain(s) with an unknown cytokine other than GIF, and the cytokine has affinity for macrophages. However, another possibility is that GIF receptors may be expressed on a subset of accessory cells. Kawasaki et al. (1986) indicated that usual macrophage cell line cells, such as P388D1, cannot be the target cells of NP-specific TsF. Using cloned macrophage hybridoma cells, Kuchroo et al. (1989) indicated that 2-h incubation of one of the hybridoma clones or splenic adherent cells with TsF was sufficient for pulsing the factor-presenting cells and that the TsF-pulsed macrophages activated precursors of Ts3. This experimental procedure indicates that TsF2 actually bound to the factor-presenting cells and conferred the cells with the capacity to activate Ts cascade. These findings suggested the possibility that the factorpresenting cells or a subset of macrophages might bear high affinity receptors for TsF/GIF. It is well known that macrophages synthesize inactive GIF. One may speculate that bioactive GIFRsF may trigger the factorpresenting cells to form their own bioactive GIF in a similar manner as that observed in Th cells. Biochemical mechanisms involved in the Ts cascade is unknown. We speculate, however, that GIF-GIF receptor interaction, which may induce translocation of inactive cytosolic GIF peptide into subcellular organelles for the production of bioactive GIF, is involved in the cellular interactions in the suppressor pathway. Many problems on Ts and TsF remain to be solved. Further studies are required for understanding Ts cascade and the basis of I-J restriction. The most fundamental question to be answered is that whether the TsF plays essential roles in immuneregulation. Nevertheless, we believe that series of research carried out by many investigators, including ourselves, provided convincing evidence for the presence of T cell-derived antigen-specific factors. ACKNOWLEDGMENTS A part of our work described in this review was supported by Research Grants AI-11202 and A1 14784 from the US. Health and Human Services. This paper is publication #301 from the La Jolla Institute for Allergy and Immunology.

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ADVANCES IN IMMUNOLOGY, VOL 74

The Role of Complement in B Cell Activation and Tolerance MICHAEL C. CARROLL The Center for Blood Research and Depa-t of Pediatrics, Harwrd h4edical School, Boston, Massachuseffs

1. Introduction

Immunologists have been aware of the innate immune system and its role in host protection against microbial infections for over a century. However the actual mechanisms involved in innate protection are only now becoming clear. A major motive for the recent attention to innate immunity comes from the growing awareness that it also serves to alert or “turn on” the adaptive immune process. The importance of microbial products as adjuvants in boosting the immune response to noninfectious antigens is well accepted. A connection between adjuvants and the innate immune response was made only within the last decade. Janeway proposed that the adjuvant effect resulted from its activation of innate immunity (Janeway, 1989). This general idea was expanded to suggest that innate immunity actually selected those antigens to which T cells should respond (Matzinger, 1994). Similarly, it has been proposed that B cells are also directed to non-self antigens by the innate response (Fearon and Locksley, 1996). In this review, I will discuss recent studies from my laboratory and others that provide results from in vivo experiments supporting the role for innate immunity in directing or “instructing” the humoral response. I will also discuss recent studies that support a role for natural immunity in negative selection of self-reactive B cells. Even though, on the surface, these new findings might seem at odds with the instructive role, I will argue that they provide an explanation for the known presence of selfreactive natural antibodies in the serum of normal individuals. II. Complement C3 Alters Fate of Antigen

A. C3d Is A NATURAL ADJUVANT Strong support for the instructive hypothesis was reported recently by Fearon and colleagues. They found that coupling of multiple copies of complement C3d to hen lysozyme lowered the amount of antigen necessary for an optimal secondary response by as much as 10,000-fold (Dempsey et al., 1996). Remarkably, attachment of three copies of C3d was more effective than mixing hen egg lysozyme (HEL) antigen with complete 61

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Freunds adjuvant. The mechanism responsible for the dramatic enhancement is probably due to multiple effects, and they will be discussed in more detail later. First, it is worth considering how complement is activated and covalently attaches to antigens in uivo. The complement system was “discovered’ early in this century by Jules Bordet who was awarded the Nobel prize in 1919 for his clarification of a heat-sensitive factor of serum (complement) that in conjunction with antibody induced bacteriolysis (Bordet and Gengou, 1901).We now know that complement actually consists of over 20 distinct serum proteins and cell surface receptors (Muller-Eberhard, 1988).Acting in a cascade fashion, the terminal step is formation of a polymer of C9 in the membrane of the target bacteria or cell. One of the most striking events in the cascade is the covalent attachment of activated components C4 (C4b) (Harrison et al., 1981) and C3 (C3b) (Law et al., 1980; Tack et al., 1980) to the antigen. Proteolytic cleavage of C4 or C3 exposes an internal thioester leading to transacylation of OH or amide groups on the acceptor surface. For example, C3b binds covalently to carbohydrate moieties or side chains of amino acids such as Ser, Thr, Tyr, and Lys (Fig. 1).Human C4b, on the other hand, binds to either OH or amino groups depending on the isotype. C4 is highly polymorphic and in humans there are two isotypes, C4A and C4B. The tandem genes encoding these proteins are located in the class 111region of the major histocompatibility complex (MHC),and each locus

FIG.1. Covalent attachment of activated C3 to antigen alters fate of antigen. Activation of classical pathway of complement by antibody-antigen complexes results in proteolytic cleavage of C4 and C3 exposing an internal thioester that reacts with OH and NH2 groups on surface of antigen-antibody complex. Although not shown, C4b also binds covalently to the antigen, and binding is critical to stabilize the C3 convertase (C4bC2a). Complement activation is probably mediated by pre-existing natural antibody followingprimary immunization with neo-antigens.

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encodes more than 10 alleles (Carroll et al., 1984).Variation in C4 structure alters its function as C4A and C4B preferentially form amide or ester linkages, respectively. This difference in efficiency in binding can have a dramatic overall effect as bound C4b forms part of the catalytic complex of C3 convertase (i.e., C4bC2a). For example, reconstitution of C4-deficient guinea pigs with human C4A protein restores the humoral response to bacteriophage c$X 174. However, pretreatment with a similar concentration of C4B fails to restore the B cell response to this nonglycosylated antigen (Finco et al., 1992). Thus, it was proposed that C4A binds to the protein surface of phage efficiently and forms a stable C3 convertase, whereas C4B is not effective because of inefficient binding to nonglycosylated proteins. An analogy can be made with MHC class I and I1 binding of peptides where polymorphic differences in the binding groove can alter the repertoire of peptides presented. The importance of C3 attachment is obvious from the number of specific receptors (i.e., CR1, CR2, CR3, CR4) that recognize different breakdown products expressed on lymphoid and myeloid cells (Fearon and Wong, 1983). Moreover, regulators of complement activation (RCA; e.g., membrane cofactor and decay activating factor) are expressed on most cells. They bind activated C3 and prevent bystander lysis of self. Many of the receptors that recognize C3b also recognize C4b (Kinoshita et al., 1985; Kalli and Fearon, 1994). Binding of C4b is critical for stabilizing C3 convertase (Reid and Porter, 1981) and, as will be discussed later, appears to be essential in localization of self-antigens (Prodeus et al., 1998).

B. INNATE RECOGNITION AND ACTIVATION OF COMPLEMENT The innate immune system includes multiple families of recognition proteins that activate complement following binding to microbes. Most are lectins which bear carbohydrate recognition domains (CRD). For example, collectins such as mannan-binding lectin ( MBL) recognize carbohydrate structures not commonly found on mammalian cells and activate the lectin pathway of complement (Epstein et al., 1996). Like the classical pathway, the lectin pathway leads to proteolpc activation of complement C2 and C4 and formation of C3 convertase. Although not considered part of the innate immune system, natural antibody is a major recognition protein of microbial and viral antigens leading to activation of complement (Fig. 1). Natural antibody represents rearranged heavy and light chains encoded in the germline which have presumably evolved to recognize pathogenic structures and certain self-antigens. A major source of IgM natural antibody are B-1 cells, which are distinguished from conventional B-2 cells by their phenotype (i.e., CD5+, CDllb/CD18+, CD21/CD35+, IgM'", IgD-, CD23-) (Kantor and Herzenberg, 1993). B-1 cells are not thought to

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undergo somatic hypermutation, and they localize primarily to the peritoneal cavity. The recent finding that mice deficient in secreted IgM have an impaired humoral response to suboptimal doses of protein antigen supports the importance of IgM in humoral responses (Boes et al., 1998), although these experiments could not distinguish between IgM secreted by B-1 or conventional B-2 cells. The enhancing effect of specific IgM was shown to be dependent on its ability to activate the first component of complement (Clq) (Heyman et al., 1988). Thus, it is proposed that when antigen is introduced in uiuo, it is bound by a pre-existing natural antibody which activates the classical pathway (via Clq) resulting in covalent attachment of C3b (which is subsequently degraded to C3d). This model is supported primarily by indirect evidence. Ross and colleagues reported the presence of specific natural IgM and IgG in human serum that binds keyhole limpet hemocyanin (KLH) and enhances its uptake by B cells via CD21 and CD35 (Thornton et al., 1994). Presumably, natural antibody recognizes the KLH and activates complement resulting in coupling of C3 ligand to the antigen. An alternative role for IgM and complement is that IgM secreted by early antibody forming cells (AFC) following primary immunization binds antigen within the lymphoid compartment and activates complement. These two possibilities are not mutually exclusive as it might be that both sources of antibody are essential. In summary, low affinity natural IgM is important in the initial activation and covalent attachment of activated C3 to the antigen. Subsequently, as AFC produce specific IgM and switch to IgG, additional complement is activated enhancing clonal expansion and germinal center formation. C. SOURCEOF COMPLEMENT IN HUMORAL IMMUNITY With the exception of Clq, the early components of complement (i.e., C2, C4, and C3) are synthesized constitutively by hepatocytes. The presence of relatively high levels of complement within the blood provide an immediate source of complement for protection against microbial infections. However, many of the early components of complement, including Clq, are also synthesized by macrophages. Therefore, the question arises as to the importance of extrahepatic or “local” complement synthesis at the site of infection and within the draining lymphoid compartment in adaptive immunity. This is an important question given the significant enhancing effect of C3. Studies with guinea pigs naturally deficient in complement C3 or C4 reveal impaired antibody responses (discussed in more detail later). The impaired responses are restored apparently by reconstitution with the missing component (Bitter-Suermann and Burger, 1989).For example, the impaired primary and secondary humoral response to bacteriophage by C4-deficient guinea pigs (g.p.) was restored following replacement with active g.p. or human C4 protein prior to immunization

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(Finco et al., 1992). Thus, the presence of serum C4 protein at the time of antigen injection is sufficient to enhance a memory response following antigen challenge 3 weeks later even though C4 activity is lost within days of treatment. These experiments suggest that the presence of serum complement within the early stages of the primary response is critical. However, these experiments did not look at long-term memory nor the affinity of the specific IgG antibody. Support for the importance of a local source of C3 comes from recent studies in C3 knockout mice (C3-/-). Reconstitution of lethally irradiated C3-/- mice with wild type (WT) bone marrow (BM) restores local synthesis by donor macrophages within the lymphoid compartment although serum levels of C3 remain undetectable. C3 synthesis by donor macrophages appears to be regulated as C3 synthesis within the white pulp is significantly increased following immunization of the chimeric mice (Fischer et al., 1998b). Thus, even though the chimeric mice have low to undetectable levels of circulating C3, sufficient amounts of C3 are secreted locally within the lymphoid compartment to restore the enhancing effects of complement. It will be important in future studies to clarify the regulation of local C3 production in uiuo. 111. Complement Enhancement of Humoral Immunity

A. ROLEOF CLASSICAL PATHWAY The involvement of complement in the antibody response has been known for several decades, but the actual mechanisms are only now becoming clear. Nussenzweig and colleagues first reported expression of C3 receptors on lymphocytes and proposed that they might be involved in humoral immunity (Nussenzweig et al., 1971). Identification of a role for C3 in the antibody response was first reported by Pepys who showed that treatment of mice with cobra venom factor (which activates and consumes serum C3) led to an impaired response to thymus-dependent (TD) and some thymus-independent (TI) antigens (Pepys, 1972). The mechanism for complement involvement was proposed for localization of antigen based on observations that depletion of serum C3 also altered retention of antibody aggregates on follicular dendritic cells (FDC) within the splenic lymphoid follicles (Papamichail et al., 1975; Klaus et al., 1980). Thus, it was proposed that complement was required for efficient trapping and retention of T D antigens by FDC within the lymphoid compartment. With the identification of guinea pigs with genetic deficiencies in C3 and C4 came both the confirmation that complement was important in humoral immunity to T D antigens and that the classical pathway was more important than the alternative (Ochs et al., 1986; Bitter-Suermann and Burger, 1989). Finding that classical pathway was involved suggested that antibody (or

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MICHAEL C. CARROLL

other activators of classical pathway such as MBL) was involved in recognition of T-dependent antigens and responsible for complement activation. The development of knockout mice deficient in complement Clq, C3, or C4 allowed for a more detailed dissection of the enhancing effect of complement. Like complement deficient g.p., deficient mice have an impaired humoral response to TD antigens (Fischer et aZ., 1996; Cutler et aZ., 1998) (Figs. 2A, B). The defect localizes to the B cells as CD4+ T cells are primed normally (Fischer et a l , 1996). Further insight into the nature of the defect was gained by immunohistochemical analysis of splenic cryosections. The C3- and C4-deficient mice fail to form germinal centers (GC) of normal size and number of following challenge intravenously (i.v.) with soluble antigen (Fischer et aZ., 1996). These findings suggest that complement is important in the survival of activated B cells and that it might play a role in affinity maturation. As suggested by previous studies in cobra venom factor (CVF)-treated mice, retention of antigen on FDC is impaired in the deficient mice (Fischer et aZ., 1996).Therefore, one possible explanation for the defective response is the absence of antigen retention on splenic FDC. Mice deficient in the first component C l q have impaired

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FIG.2. Humoral immune response is impaired in mice deficient in complement C3 or C4 or complement receptors CD21/CD35. Mice (WT or complement-deficient) were

immunized at days 0 and 21 with a T-dependent antigen (2 X logPFU bacteriophage) i.v. and total Ig (A & C) or IgG (B & D) titers determined by plaque assay.

THE ROLE OF COMPLEMENT I N B CELL ACTIVATION AND TOLERANCE

67

C3 activation via the classical pathway; and as would be expected they also have an impaired secondary response to T-dependent antigens. Characterization of the specific antibody isotypes made in response to TD antigen reveals a defect in IgG2a that correlates with a significant reduction in gamma interferon synthesis by antigen-specific T cells (Cutler et al., 1998). THE ENHANCING EFFECTOF COMPLEMENT B. CD21ED35 MEDIATE Two major receptors for activated fragments of complement C3 (and C4) are CR1 (CD35; molecular weight 190 kDa) and CR2 (CD21; molecular weight 150 kDa) (Ahearn and Fearon, 1989). In humans, they are encoded by separate but linked genes on chromosome 1 (Carroll et al., 1988).They are expressed differentially with CD35 on myeloid (red blood cells, monocytes, granulocytes), lymphoid (B cells), and FDC, whereas CD21 is limited to primarily lymphoid (B and T cells) and FDC. On B cells, CD21 forms a signaling complex with CD19, Tapa-1, and Leu-13 (Tedder et al., 1994; Fearon and Carter, 1995) (Fig. 3).Biochemical studies

FDC

FIG.3. Complement receptors CD21 and CD35 are co-expressed primarily on B cells and follicular dendritic cells in mice. On B cells CD21 and CD35 form a coreceptor with CD19 and Tapa-1. Binding of antigens coupled to C3d coligates coreceptor and B cell antigen receptor resulting in lowering of threshold of B cell activation. Signal enhancement is mediated by CD19 as it becomes rapidly phosphorylated providing docking sites for PI3 kinase and vav. CD21/CD35 expression on FDC provide a major antigen trapping function of antigen within the lymphoid follicles.

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MICHAEL C. CARROLL

have demonstrated that co-cross-linking of the CD21/CD19 complex with the B cell receptor (BCR) lowers the threshold for B cell activation as much as several orders of magnitude (Carter and Fearon, 1992). Thus, it is proposed that complement enhances humoral immunity by lowering the threshold of antigen activation of naive B cells (Fearon and Carter, 1995). This hypothesis was tested in vivo in transgenic mice, and the results are discussed later. A potential mechanism explaining the reduction in threshold is based on the finding that cross-linkingthe coreceptor results in rapid phosphorylation of CD19 by src-like kinases and provides a docking site for vav (Sat0 et al., 1997; O’Rourke et al., 1998). Although, cross-linking of the BCR independently of the coreceptor also results in phosphorylation of CD19, biochemical results suggest that this is not sufficient to ensure activation of vav (O’Rourke et al., 1998). This is an important point as it will be argued below that survival of GC B cells requires cross-linking of the coreceptor via C3d-antigen complexes and that survival is independent of antigen affinity (Fischer et al., 1998a). Further support for the importance of coreceptor signaling comes from the observation that mice totally deficient in CD19 have an impaired antibody response to TD antigens and do not form GC to noninfectious antigens (Engel et al., 1995; Rickert et al., 1995). Unlike human, mouse CD21 and CD35 are encoded at a single locus (Cr2) (Kurtz et al., 1990). CD35 which is assembled from 21 short consensus repeats (SCRs) encodes all of CD21 (composed of 15 SCRs) and an additional 6 N-terminal units (Molina et al., 1990, 1994). The additional coding sequence of CD35 provides the binding sites for of C3b and C4b as well as cofactor activity (i.e., conversion of C3b to iC3b by factor I requires cofactor activity). Since CD35 includes all of CD21 including the C3d binding site, it would be expected to form a coreceptor with CD19 on B cells. A definitive role for CD21/CD35 in the humoral response to TD antigens was identified by characterization of knockout mice bearing a disruption of complement receptors CD21 and CD35 (Cr2-/-) (Ahearn et al., 1996; Molina et al., 1996) (Figs. 2C, D). Like C3- and Cpdeficient mice, Cr2-/- mice have an impaired secondary antibody response and a reduced number and size of GC following immunization i.v. with TD antigens (Ahearn et al., 1996). However, the defect in GC formation appears to vary according to the nature of the antigen. Molina and colleagues reported an impaired secondary response to sheep red cells in their Cr2-/- mice but a normal number of GC. It will be important to further dissect the GC response and to answer such questions as the repertoire of V gene usage and affinity maturation of antibody response. In summary, two possible explanations for the impaired response in the deficient mice are reduced coreceptor signaling or antigen retention.

THE ROLE OF COMPLEMENT IN B CELL ACTIVATION AND TOLERANCE

69

C. B CELLCORECEPTOR SIGNALING AND ANTIGENRETENTION ON FDC Evidence in vivo in support of the importance of co-receptor signaling in humoral immunity comes from several recent reports. Croix et al. used the blastocyst complementation approach to obtain chimeric animals in which their FDC were CD21/CD35+ but their B lymphocytes were derived from Cr2-/- embryonic stem cells (Croix et al., 1996). In this approach, lymphocyte-deficient mice (RAG-2-/-) are reconstituted with Cr2-/- embryonic stem (es) cells such that the lymphoid compartment (B cells) is derived entirely from Cr2-/- es cells but nonlymphoid tissues (such as FDC) are chimeric (i.e., mixed Cr2+/+ and Cr2-/-). The chimeric mice failed to make a normal secondary response to the TD antigen NP-KLH, suggesting the importance of coreceptor expression on B cells. In a complementary set of experiments, Ahearn et al. reconstituted Cr2-/- mice with WT BM to generate chimeric mice in which the B cells were CD21/CD35 + but FDC were CD2UCD35-deficient (Ahearn et al., 1996). Despite the absence of expression of complement receptors on FDC, the chimeric animals made a secondary antibody response to TD antigen (bacteriophage) characterized by a normal number and size of GC (Ahearn et al., 1996). Interestingly, it was noted that the specific IgG response declined rapidly following secondary challenge and this suggested that the secondary response was not completely normal. To further identify the importance of coreceptor signaling in the B cell response in vivo, Fischer et al. crossed the Cr2-/- mice with the wellcharacterized hen lysozyme (HEL)-immunoglobulin ( Ig) transgenic (tg) mice (Fischer et al., 1998a). An advantage of the HEL-Ig tg model is that the relative affinities of the hybridoma (Hy-HEL-10) used to construct the tg mice is known (Lavoie et al., 1992). Thus, by varying the form of avian lysozyme used as antigen, one could compare the importance of coreceptor expression with either very high or moderate affinity antigens. For example, turkey (TEL) and duck (DEL) lysozyme bind the Hy-HEL10 hybridoma with approximately 2000-fold difference in relative affinities (i.e,, 2 X 10'' M versus 1 X lo7 M, respectively). An adoptive transfer approach was used such that WT recipients were immunized 7 days prior to transfer with the respective antigen. The advantage of this approach is that the Ig tg B cells are introduced into an ongoing immune response where T cell help and antigen retention on FDC are provided by the recipient (Shokat and Goodnow, 1995). In this manner, the requirement for coreceptor expression could be evaluated independent of antigen retention and priming of T cells. Not unexpectedly, it was found that Cr2-/HEL-Ig tg B cells failed to survive within follicles of mice immunized with

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MICHAEL C. CARROLL

the moderate affinity antigen (i.e., DEL) but survive normally in mice immunized with TEL (Fischer et al., 1998a). These observations support the threshold hypothesis (i.e., the B cell coreceptor is required to lower the threshold for antigen activation in the case of lower affinity antigens) ( Fearon and Carter, 1995). Interestingly, the loss of Cr2-/- HEL-Ig tg B cells from the follicles over 5 days following transfer is not a passive event because when they are transferred into nonimmune recipients a similar number of Cr2+/+ and Cr2-/- tg cells are retained in the follicles. One explanation for the dramatic reduction is that the Cr2-/- B cells are eliminated by antigen-specificCD4+ T cells in a Fas-dependent mechanism (Garrone et al., 1995; Rathmell et al., 1995; Rothstein et al., 1995). According to this model, cross-linking of CD40 on the B cell surface by CD40 ligand on cognate CD4+ T cells upregulates assembly of a trimeric form of Fas (CD95) on the B cell. Engagement of Fas by FasL (expressed by the T cell) results in apoptosis of the B cell unless a survival signal is induced via the BCR. Thus, in the absence of coreceptor expression, subthreshold signaling by the tg B cell might not be not sufficient to block a Fas-mediated death signal. By contrast, binding of the very high affinity antigen TEL apparently induces a sufficiently strong signal via BCR to circumvent the requirement for the coreceptor (Fischer et al., 1998a) (Fig. 4A). Preliminary studies using Fas-deficient (lprllpr) mice crossed with the Cr2+/+ or Cr2-/- HEL-Ig tg as immune recipients reveal that, in the absence of functional Fas, the Cr2-/- Ig tg B cells are not eliminated from follicles of DEL immune recipients (M. Zhang, R. Barrington, and M.C. Carroll, unpublished results). A prediction of the threshold hypothesis predicts that at very high affinity antigens do not require coreceptor signaling to transduce signals effectively. This hypothesis was tested using the adoptive transfer model described earlier except that the WT recipients were immunized with high affinity antigen (TEL). As expected, Cr2+/+ HEL-Ig tg B cells entered existing GC within the splenic follicles and competed for antigen and T cell help. In contrast, Cr2-/- tg B cells failed to survive within the GC, although they did survive within the surrounding follicles ( Fischer et al., 1998a). These results suggest a novel role for the coreceptor in enhancing GC B cell survival irrespective of the affinity of the antigen. A model that would account for these results is that GC B cells require not only T cell help and antigen (MacLennan, 1994; Kelsoe, 1995) but CD21 ligand (i.e., C3d) (Fig. 4B). GC survival of Cr2+/+ B cells is impaired by treatment of mice with a soluble form of the CD21 receptor (CR2)2-IgG.Thus, blocking of C3d-CD21 interaction results in elimination of GCs within 48 hours of treatment (Fischer et al., 1998a). A similar effect is observed on treatment of immunized WT mice with a large concentration of soluble antigen at

THE ROLE OF COMPLEMENT IN B CELL ACTIVATION AND TOLERANCE

Centrocytes

71

contact

0

y

Antigen receptor Antigen Mutated antigen receptor CDPlICDlSTTapa-1 co-receptor

FIG. 4. B cell expression of CD2l/CDlS/Tapa-l coreceptor is important for B cell survival within the lymphoid compartment and clonal selection within the germinal centers. Complement enhances B cell memory by (A) lowering threshold of antigen activation and follicular survival of B cells and (B) providing suMval signal for GC centrocytes.

peak GC period (Han et al., 1995; Pulendran et al., 1995; Shokat and Goodnow, 1995). One interpretation of these results is that soluble antigen blocks the interaction between the GC B cell and FDC; therefore the B cells fail to receive a necessary survival factor (Shokat and Goodnow, 1995). Based on the study of Fischer et al., it is likely that the survival factor is C3d (Fisher et al., 1998a). Combined, these findings demonstrate that clonal selection of B cells within the GC requires contact between the FDC and B cell and that CD21/CD35 and its ligand C3d are critical to survival. A protective role of coreceptor signaling on B cells has also been reported in an in vitro model by Qin et al. (1998). The authors examined the mechanism involved in FDC-mediated enhancement of B cell production of specific antibody in vitro. In this model, the addition of antigencoated FDC promotes long-term antibody production by antigen-specific B cells in vitro in the presence of T cell help (Wu et al., 1996). Blocking

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MICHAEL C. CARROLL

CD21/CD35 on the B cell either by use of sCR2, use of Cr2-/- B cells, or absence of C3d on the FDC results in loss of antibody synthesis (Qin et al., 1998). Presumably, the activated B cells die in culture in the absence of signaling via CD21KD35 and C3d. Although there is strong evidence that coreceptor signaling is important in B cell survival, both in follicles and GC, antigen retention on FDC is important in providing C3d ligand and antigen for maintenance of longterm memory B cells. Ahearn et al. found that chimeric mice bearing Cr2-deficient FDC but Cr2+/+ B cells made an apparently normal IgG response after challenge with bacteriophage antigen; however, the antibody response decreased rapidly within two weeks after challenge (Ahearn et al., 1996). This suggests that in the absence of efficient antigen retention, the memory response is impaired. Using a similar approach of reconstituting Cr2+/+ or Cr2-/- mice with BM isolated from WT or deficient mice, Fang et al. reported a reduced secondary response to SRBC or KLH in the absence of FDC expression of CD21/CD35 in chimeric mice (Fang et al., 1998). Moreover, the reduced antibody response correlated with a reduction in size of the splenic GC. They concluded that receptor expression on both B cells and FDC was essential for a normal memory response. Although FDC express Fc receptors ( FcR), efficient localization of antigen requires complement receptors as immune complexes (i.c.) retention, and the memory response are not impaired in FcRI- and FcRIII-deficient mice (Vora et al., 1997). Deficiency in FcRIIB which is expressed primarily on B cells and FDC does not appear to alter the secondary response because deficient mice have a normal immune response to TD antigen (Takai et al., 1996). It will be important to clarify further the individual roles of complement and FcRIIB receptors in trapping and retaining antigen on FDC. Preliminary studies examining long-term memory (i.e., greater than 4 months) in chimeric mice reveal that both antibody titer and affinity are significantly reduced in the absence of CD21/CD35 expression on FDC (M. B. Fischer and M. C. Carroll, unpublished results). Combined, these findings suggest that efficient retention of antigen by FDC is dependent on CD21/CD35, and that, in the absence of complement receptors, B cell memory is impaired. In summary, both coreceptor signaling and antigen localization via CD21/ CD35 are critical in B cell immunity. Coreceptor signaling is critical in early B cell activation and survival, whereas efficient antigen localization appears to be critical for maintenance of long-term memory. IV. Negative Selection of Self-Reactive B Cells

Characterization of transgenic mice expressing immunoglobulin transgenes has confirmed earlier hypotheses proposing that self-reactive B lym-

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73

phocytes are either eliminated or inactivated (Burnet, 1959; Nossal, 1983). For example, encounter of B cells bearing self-reactive immunoglobulin transgenes specific for cell surface MHC class I proteins are eliminated within the bone marrow (Nemazee and Buerki, 1989). Likewise, in the lysozyme-double transgenic model, HEL-specific B cells are eliminated within the BM in mice expressing a membrane form of lysozyme (Hartley et al., 1991). In contrast, self-reactive B cells are not eliminated but functionally inactivated in double transgenic mice expressing a soluble form of HEL (sHEL) (Goodnow et al., 1989). In transgenic models such as anti-dsDNA or Smith antigen (Sm) in which the self-antigen is a natural one, self-reactive B cells are either eliminated (Chen et al., 1995) in the bone marrow or anergized in the periphery (Mandik-Nayak et al., 1997; Santulli-Marotto et al., 1998). In the models examining soluble selfantigens, it is not known how the antigens are localized in the bone marrow and periphery for exposure to immature (Norvell et al., 1995; Norvell and Monroe, 1996) and transitional (Carsetti et al., 1995) B cells that are sensitive to negative selection. Goodnow has proposed that the strength of signal induced via the BCR is critical in determining whether the selfreactive B cell is eliminated, anergized, or ignored (Goodnow, 1996). For example, variations in the concentration of soluble antigen in the sHEL model can lead to anergy or clonal escape (Goodnow et al., 1989). Alternatively, combinations of heavy and light chains that reduce the affinity of binding (Hartley and Goodnow, 1994) or mutations in the BCR signaling pathway which reduce signal intensity such as CD45-deficiency (Cyster et al., 1996) result in clonal escape. In contrast, increases in the sensitivity of the BCR signaling apparatus such as loss of protein tyrosine phosphatase 1C (PTPlC or SHP1) or gain of an additional CD19 signaling protein (Inaoki et al., 1997) results in elimination of B cells bearing both higher and lower affinity self-reactive receptors (Cyster and Goodnow, 1995). Given the important role of the complement system in regulating localization of antigen and coreceptor signaling,it is possible that it is also involved in induction of negative selection of self-reactive B cells. IS IMPAIRED IN Cr2-/A. NEGATIVESELECTION

MICE

In the lysozyme-anti-lysozyme tg model of peripheral tolerance, selfreactive B cells are not eliminated but anergized on encounter with a threshold level of sHEL-self-antigen, Once in the peripheral compartment, anergic B cells have a reduced expression of surface IgM, reduced life span, and failure to respond to antigen ex vivo (Goodnow et al., 1995). Given the role of complement in regulating B cell activation, it is likely that it might also play a role in regulation of self-reactive B cells. Prodeus

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et al. examined the importance of complement in the lysozyme double tg model by breeding the C r P " mice with the sHEUHEL-Ig tg mice. They found that HEL-binding B cells escaped tolerance in mice deficient in CD21/CD35. Characterization of double tg mice deficient in CD21/CD35 revealed an apparent absence of anergy, although anti-HEL antibody was not detected in the serum. By contrast to that found with C + controls, a normal number and half-life of mature HEL-specific tg B cells were identified in the complement-deficient double tg mice relative to single tg mice. Although the level of surface IgM was reduced, the Cr2-/- selfreactive B cells remained responsive to antigen ex vivo as culture with antigen-induced significant Ca2+iflux and upregulation of the activation marker B7-2 (CD86) (Prodeus et al., 1998). Deficiency in CD21/CD35 could affect the amount of self-antigen retained in the BM and peripheral lymphoid compartment. Alternatively, absence of coreceptor expression on immature B cells might raise the threshold of BCR signaling. Immature B cells express both CD19 (Inaoki et al., 1997) and CD21 (Takahashi et al., 1997); however, a role for the coreceptor in signal transduction on immature B cells has not been reported. Evidence in support of a role for complement receptors in retention of self-antigen was obtained by analyses of radiation chimeras in which expression of CD21/CD35 was differentially expressed (i.e., Cr2+ B cells and Cr2-/- FDC or vice versa). Interestingly, expression of CD21/CD35 on FDC was critical to maintenance of peripheral tolerance to sHEL (Table I) (0.Pozdnyakova, J. Gommerman, A. Prodeus, and M. C. Carroll, unpublished results). Tolerance was evaluated

TABLE I MAINTENANCE OF PERIPHERAL TOLERANCE IN THE LYSOZYME-ANTILYSOZYME DOUBLE TRANSGENIC MICE REQUIRESCD21ICD35 EXPRESSION BY FDCS AND RADIO-RESISTANT STROMAL CELLS~

BM Cr2+ CrP' Cr2+ Cr2""'

Recipient Cr2+ c1-2"~ Crgnd Cr2 +

CD21/CD35 on B cells

BM StromdFDCs

+

-

+

Tolerance

+++ +/+/-

+++

Comparison of peripheral tolerance in chimeric mice prepared by reconstituting lethally irradiated CrB+ or Cr2-I- sHEL tg mice with either Cr2+/+ or Cr2-/- HEL-Ig tg BM. Results with the mixed chimeras demonstrate that CD21/CD35 expression on B cells but not FDC is not sufficient to maintain tolerance. Tolerance is based on combined results examining life span, total number, and frequency of mature CD23+ HEL-Ig tg B cells in LNs of double tg chimeras and responsiveness of splenocytes to stimulation with HEL antigen ex oioo.

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by flow cytometric analysis of mature spleen and lymph node (LN) cells, BrdU labeling, and responsiveness to antigen ex uiuo. These results are somewhat surprising because it was assumed that expression of the coreceptor would be as important in BCR signal transduction on immature B cells as it is on mature B cells. They raised questions as to the anatomical location which is most critical for induction of anergy (i-e.,bone marrow or spleen), and the importance of the stromal cell in presentation of soluble self-antigen. The IgM receptor of circulating B cells in sHELiHEL-Ig tg mice is partially occupied with bound HEL antigen suggesting that soluble self-antigen is available in solution for uptake. Therefore, why is it necessary to localize soluble antigens on stromal cells? One possible explanation is that monomeric antigen in solution does not efficiently cross-link the BCR and induce a threshold signal. Attachment of antigens on a cell surface via complement receptors would increase the overall avidity and enhance cross-linking of BCR. Alternatively, stromal cells (and FDC) might provide an accessory signal in addition to capturing antigen. It will be important to determine the nature of the CD2UCD35-t stromal cells. I N COMPLEMENT C4 ALTERSB CELLANERGY B. DEFICIENCY

The major ligands for murine CD21/CD35 are activated products of complement C3 and C4 as discussed earlier (Kinoshita et al., 1985; Ahearn and Fearon, 1989; Kalli and Fearon, 1994; Molina et al., 1994). C 3 is the central component of complement and its activation represents an amplification step. Therefore, it might be expected that it is the major ligand for binding self-antigens. The importance in tolerance of these distinct components was examined in the sHEUHEL-Ig model using the radiation chimera approach discussed earlier (Prodeus et al., 1998). Double transgenic mice deficient in serum C3 or C4 were constructed by grafting C+ HEL-Ig BM into irradiated C3- or C4-deficient sHEL tg mice (Fig. 5).Characterization of BM, spleen, and LN cells harvested from the double and single tg mice revealed that in the absence of C4, self-reactive B cells escaped tolerance (Fig. 6) (Prodeus et al., 1998). By contrast, deficiency in serum C3 did not appear to alter induction of peripheral tolerance. A limitation with the chimeric approach is that BM-derived myeloid cells are C+; monocytes can synthesize complement components C3 and C4 within the lymphoid compartment (Fischer et al., 199813; E. Alicot, H. Jezak, L. M. Shen and M. C. Carroll, unpublished results). Thus, it is possible that low levels of C3 or C4 are synthesized by donor-derived monocytes in the chimeric animals, although the respective proteins were not detectable in the serum. The finding that C4 is critical in induction of anergy in the HEL model suggests that CD35 is the relevant complement

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FIG.5. Complement C4 is required for maintenance of peripheral B cell tolerance in lysozyme-antilysozyme double transgenic mice. Double transgenic radiation chimeras were constructed as described in Fig. 6 and splenocytes examined for responsiveness ex uiuo for upregulation of activation molecule B7-2 (CD86). Splenocytes harvested from the three groups of single and double transgenic mice were cultured with 100 ngml of HEL antigen overnight and subsequently analyzed by three-color flow cytometry for expression of HELIg, B220 and CD86. Results reveal that serum C4 but not C3 is critical to maintenance of tolerance in this tg model.

receptor for retention of self-antigens as CD21 is not thought to bind C4. It also raises the question: how is complement activated by self-antigen? It is likely that complement is activated following binding of low levels of HEL-specific IgM (released by tg B cells) to sHEL antigen. On activation, C4, like C3, forms a covalent bond with the antigen (Harrison et al., 1981). Complexes of C4b-HEL antigen presumably are retained on FDCs in the peripheral lymphoid compartment and on stromal cells within the BM via CD35. C4 protein is thought to provide an important role in clearance of immune complexes via the immune adherence receptor. Binding of C4 to immune complexes inhibits formation of nonsoluble complexes and facilitates uptake by myeloid cells via CD35 by direct binding and activation and attachment of C3b (Schifferli et al., 1986). In humans, the immune adherence receptor is expressed on red blood cells; however, its homologue in the mouse is not known as mouse RBCs do not express CD35 (Kinoshita et al., 1988). An alternative explanation for the role of C4 in tolerance is that it is critical for clearance of complexes of lysozyme and antilysozyme antibody. According to this model, complexes of sHEL-IgM are not cleared appropriately and form aggregates that induce a sufficiently strong signal

THE ROLE OF COMPLEMENT IN B CELL ACTIVATION AND TOLERANCE

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FIG.6. Construction of single and double transgenic radiation chimeras. Injected into lethally irradiated complement sufficient- or deficient-recipients were 2 X lo6bone marrow cells harvested from C+ HEL-Ig tg mice. No antigen recipients were used as controls. Mice were rested for 30 days prior to examination.

to break anergy. If this were the case, then one would expect to find an activated phenotype on analysis of the self-reactive B cells in the C4-/mice. However, analysis of splenocytes on isolation from the C4-/- double tg mice identified only background level of expression of the activation

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MICHAEL C. CARROLL

marker B7-2. Moreover, BrdU-labeling experiments indicated a low frequency of turnover of mature self-reactive B cells in the C4-/- compared to C+/+ double tg animals. Probably the strongest evidence against impaired immune clearance as an explanation is that the mixed radiation chimeras (Cr2+/+ HEL-Ig into Cr2-/- sHEL recipients) break tolerance even though they express normal levels of C4, and donor-derived myeloid cells express complement receptors CD21/CD35 (0.Pozdnyakova,J. Gommerman, A. Prodeus, and M. C. Carroll, unpublished results) (Table I). Thus, the most probable mechanism to explain the role for complement C4 and its receptor CD35 in maintenance of peripheral tolerance is that attachment of C4b to self-antigen mediates localization to BM stromal cells and FDC in the lymphoid compartment and therefore enhances presentation to immature B cells. V. Complement Deficiency and SLE

Systemic lupus erythematosus (SLE) is a common autoimmune disease which affects women nine times more frequently than men, with over 200,000 patients in the United States (Michet et al., 1979; Cortan et aZ., 1994). A hallmark of lupus is circulating autoantibodies specific for dsDNA and ribosomal nuclear proteins most probably due to disregulation of selfreactive B cells (Tan, 1982). The etiology of SLE is not known; but deficiency in complement C l q or C4 is a major predisposing factor (Agnello, 1978; Walport and Morgan, 1991). Though rare, almost all individuals which are deficient in C l q or C4 develop lupus which can be fatal. This presents somewhat of a paradox as complement is not only a mediator of inflammation but, as discussed earlier, important in enhancing B cell response to antigens. Therefore, complete deficiency in complement might be expected to protect against inflammatory disease and increase the threshold required for antigen activation of B lymphocytes. Interestingly, deficiency in C3 is not a major factor in SLE. About 25% of mice deficient in C l q spontaneously develop lupus characterized by antinuclear antibodies and glomerulonephritis (Botto et aZ., 1998). A common explanation is that deficiencyin C l q or C4 impairs the normal immune clearance function (as discussed earlier) of the classical pathway and that aggregates of selfantigen are immunogenic. Korb and Ahern (1997) found that C l q binds to DNA and nuclear antigens released by apoptotic cells (Casciola-Rosen et al., 1994) and proposed that, in absence of this clearance function, self-antigens accumulate and become immunogenic. Similarly,pre-existing natural antibody specific for self-nuclear antigens activates classical pathway complement and this leads to clearance via complement receptors (Fig.

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7). The clearance hypothesis presumes that failure to appropriately clear self-antigens results in activation of circulating self-reactive B cells in the presence of T cell help. An alternative hypothesis is that SLE results from impaired B cell tolerance and that complement is important in providing SLE autoantigens for negative selection (Carroll, 1998) (Fig. 7).According to this hypothesis, deficiency in C4 or C l q predisposes individuals to SLE because self-antigens such as dsDNA are not localized efficientlyto the BM and lymphoid compartments. Characterization of mouse models bearing transgenes specific for DNA (Chen et al., 1995; Mandik-Nayak et al., 1997) or nuclear proteins (Santulli-Marotto et al., 1998) reveal that self-reactive B cells are either deleted or anergized. In the lysozyme model of peripheral tolerance, deficiency in either CD21/CD35 or C4 results in a loss of anergy against the soluble self-antigen. Complete deficiency in CD35 in humans has not been described; however, there is a correlation between reduced levels of CD35 expression on RBCs with disease severity in lupus-prone M R U p r mice (Takahashi et al., 1997) and in lupus patients (Miyakawa et al., 1981).

FIG.7 . Role of complement in maintenance of B cell tolerance. Complement C l q and C4 are critical to retention of highly conserved self-antigens such as dsDNA by bone marrow stromal cells in order to enhance recognition by immature self-reactive B cells. Self-antigen retention is mediated by complement receptors CD21/CD35 expressed on BM stroma and FDCs.

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The Ipr mice develop a lupus-like disease characterized by glomerulonephritis and autoantibodies to dsDNA and nuclear protein (Theofilopoulos and Dixon, 1985). The lupus-prone mice bear a natural deficiency in Fas (CD95) that leads to impairment of peripheral tolerance in T and B lymphocytes (Takahashi et al., 1994). Disease severity is strain-dependent; on the C57BLJ6 background Fas-deficient mice are apparently normal and do not develop glomerulonephritis. Defects in the immune system might be expected to decrease autoimmune disease. Surprisingly, deficiency in complement C4 or the receptors CD21/CD35 exacerbates disease. Prodeus et al. reported that deficiency in C4 or CD21/CD35 in lpr mice on the C57BLJ6 X 129 background results in severe autoimmune disease characterized by a dramatic increase in autoantibody titers to dsDNA and nuclear antigens (Fig. 8) (Prodeus et al., 1998). The dramatic increase in autoantibody titers in the complement-deficient mice correlated with an increase in glomerulonephritis. Interestingly, C3-/- lpr mice appear very similar to C+ controls and did not develop elevated levels of autoantibody or significant disease in this model. This is an important finding because, as discussed earlier, deficiency in C3 is not a major predisposing factor to lupus in humans. The findings with the C4- or CD21/CD35-deficient lpr mice are consistent with a role for complement in B cell tolerance. Accordingly, in the absence of efficient localization of soluble self-antigens such as dsDNA or nuclear proteins, self-reactive B cells escape negative selection and accumulate in the periphery. The presence of self-reactive T cells in the Fas-deficient mice would provide help resulting in secretion of autoantibodies. An alternative view is based on the immune clearance function of C4 as discussed earlier. In this model, self-antigens are not sequestered efficiently and become exposed to circulating self-reactive B cells resulting in increased activation and autoantibody production. The failure to clear self-antigens could result in formation of aggregates and activation of alternative pathway leading to attachment of C3d. The presence of C3d would enhance their immunogenicity. The finding that individuals deficient in C3 are not predisposed to lupus would be consistent with this model. Evidence that C3 is not required in escape of tolerance in lpr mice comes from experiments in which double knockout mice (i.e., C3- and C4deficient) were crossed with Ipr mice. Interestingly, the mice develop an autoimmune disease similar to the C4-/- lpr mice (0.Pozdnyakova, S. Einav, and M. Carroll, unpublished results). Therefore, C3d interaction with B cell coreceptor is not required for breaking tolerance. Other evidence that is inconsistent with the immune clearance hypothesis is the finding that CD21/CD35 deficiency exacerbates disease in lpr mice. De-

THE ROLE OF COMPLEMENT IN B CELL ACTIVATION AND TOLERANCE

A

"T

B 0

0

0 0

2.51 2

1. ~ 1

0 0

0

I

13 weeks

C

81

-3 0 -

17 weeks

D l2o0.1

FIG.8. Autoimmune disease is exacerbated in mice deficient in C4 or CD21/CD35 but not C3. (A and B) Splenomegaly and lymphadenopathy are sigdcantly elevated in Cr2-/lpr mice. (C and D) Antinuclear and dsDNA autoantibodies are significantly elevated in Ipr mice deficient in complement C4 or CD21/CD35 but not C3.

spite normal levels of serum complement, the deficient mice have impaired tolerance. The clearance hypothesis and antigen localization hypotheses are not mutually exclusive. It is possible that nuclear antigens released by dying cells in the periphery such as skin are cleared by complement. However, dying cells in the BM where turnover of cells is high would

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provide sufficient self-antigen for negative selection via complement receptors.

A. ROLEOF NATURAL ANTIBODY IN NEGATIVE SELECTION OF B CELLS The formation of autoantibodies specific for only a certain set of autoantigens in SLE suggests there is a selective loss of tolerance to these particular self-antigens. Lupus autoantigens have in common that they are highly conserved among vertebrates and lower forms including microbes. For example, nucleic acid, a common lupus antigen, is found among all living organisms. Therefore, loss of tolerance to these highly conserved antigens could result in autoimmunity following a microbial infection (Pisetsky et al., 1990). A unifylng notion is that the immune system uses natural IgM and complement to recognize and induce tolerance to these particular antigens. Thus, according to this hypothesis, deficiency in either complement (Clq, C4, or CD21/CD35) or an alteration in the repertoire of natural IgM would impair negative selection leading to an accumulation of selfreactive B cells in the periphery. This hypothesis would also explain the finding that Ig deficiencies are often predisposing factors for autoimmune disease. VI. Summary

It is becoming well accepted that innate immunity serves as a natural adjuvant in enhancing and directing the adaptive immune response. In this review, I have discussed how the complement system, a major mediator of innate immunity, links the two systems. The recent availability of knockout mice bearing selective deficiencies in the critical complement proteins and receptors has allowed formal demonstration of the importance of complement in enhancement of humoral immunity. Characterization of the mice has also uncovered mechanisms for maintaining survival of activated B cells within the lymphoid compartment. For example, co-ligation of the CD21/CDlS/Tapa-l receptor with the BCR not only reduces the threshold for B cell follicular survival but provides a unique signal for survival in the germinal centers. In addition complement receptors are critical for localization of antigen and C3d ligand to FDCs for maintenance of longterm B cell memory. A surprise that has come from analysis of the deficient mice is that complement is also important in negative selection of B lymphocytes. This observation provides new insight to a long-standing enigma that the major predisposing factor in lupus is deficiency in complement C l q or C4. The seeming contradiction of dual role for complement in both B cell activation and tolerance is reconciled by the hypothesis that natural IgM provides a mechanism to selectively identify self-antigens that

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are highly conserved and cross-react with microbial ones such as DNA and nuclear proteins. Thus, the importance of complement in tolerance to self-antigens is restricted to those self-antigens that are evolutionary conserved, and they are identified by natural antibody. The future should hold further surprises as to the intricate interactions between the complement system and acquired immunity.

ACKNOWLEDGMENTS I thank present and former members of the laboratory who performed much of the work that was described in the review and especially Drs. Robert Barrington, Jennifer Gommerman, Olga Pozdnyakova, and Ming Zhang for preliminary results. I would also like to thank collaborators Drs. Garnett Kelsoe and Christopher Goodnow for discussions of the role of complement in acquired immunity.

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ADVANCES IN IMMUNOLOGY, VOL. 74

Receptor Editing in B Cells DAVID NEMAZEE The scripps Research Instirvte, la hlh, California

1. Introduction

B lymphocytes can be regulated by secondary V(D)J recombination that alters antigen receptor genes. This “receptor editing” mechanism is under the control of antigen receptor signaling itself. In immature, bone marrow B cells, antigen receptor signaling stimulates V(D)J recombination in cells whose antigen receptors bind self-antigen. Resulting rearrangements can render such cells nonautoreactive. Furthermore, V( D)J recombinase is reexpressed in germinal center cells participating in an immune response to foreign antigen. In this context, it appears that T-cell-derived stimuli activate recombinase in B cells, and that this induction is suppressed by B-cell antigen receptor cross-linking. Overall, it appears that the immune system takes advantage of its control of V(D)J recombination to modify antigen receptors such that specificity for foreign antigens improves and affinity for self is reduced. II. Receptor Selection versus Clonal Selection

The classical view of the immune system is that lymphocytes, like individuals in a natural animal population, have preexisting genetic diversity upon which selection can act (Burnet, 1959; Talmage, 1959). According to this Darwinian view, the selected growth or death of clones regulates immune specificity. We review here the evidence, derived primarily from studies of mouse B cells, that a second mechanism of immune regulation, receptor selection, can modify the lymphocyte repertoire by regulating the receptor gene diversification mechanism itself. This article first reviews essential facts about the organization of immunoglobulin genes, V( D)J recombination, and B-cell development. We then discuss how signals from the antigen receptor influence gene rearrangements during development, tolerance-induction and immunity. Receptor editing has been the subject of several brief reviews (Hertz and Nemazee, 1998; Nussenzweig, 1998; Ohmori and Hikida, 1998; Radic and Zouali, 1996; Rajewsky, 1998). 111. Antigen Receptor Gene Assembly by V(D)J Recombination

The preimmune repertoire diversity is generated during development in the primary lymphoid organs (fetal liver, bone marrow) by the V( D)J 89

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recombinase, which 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 C exons of these genes. Diversity is generated through the well-known combinatorial assembly of numerous minigene elements and by nucleotide additions and deletions at gene segment ends prior to joining (Tonegawa, 1983). V( D)J recombination is initiated by the lymphocyte-restricted RAG-1 and RAG-2 proteins (Oettinger et al., 1990; Schatz et al., 1989). RAG1/2 complexes can carry out much of the reaction in vitro (Agrawal et al., 1998; Grawunder et al., 1998; Ramsden et al., 1997; Schatz, 1997).Terminal deoxynucleotidyltransferase (TdT), which is expressed in some lymphoid cell stages, adds untemplated nucleotides to protein coding segment ends exposed by RAG activity (Gilfillan et al., 1993; Komori et al., 1993; Landau et al., 1987). A set of ubiquitously expressed proteins that repair DNA damage are required to resolve the coding join reaction (Bogue et al., 1997; Chu, 1997; Gao et al., 1998; Gu et al., 1997; Nussenmeig et al., 1996). Because the V( D)J recombination machinery randomly adds or deletes bases during joining, most V(D)J joins are out of frame and therefore fail to encode functional proteins. However, in developing fetal lymphocytes, TdT is not expressed (Bogue et al., 1992; Li et al., 1993), joins lack N-regions (Bogue et al., 1992; Feeney, 1990; Gu et al., 1990; Lafaille et al., 1989), and the recombination joins can deviate significantly from random because small sequence identities at the junctions favor precise joins in particular frames (Gu et al., 1990; Komori et al., 1996; Lafaille et al., 1989). IV. Recombination Signals and the 12/23 Rule

In order to be recognized by the recombinase, the two DNA elements to be joined must each be flanked by a recombination signal sequence (RSS) composed of a conserved heptamer motif, followed by a spacer, usually of 12 or 23 bases, and conserved nonamer motif reviewed in (Schatz et al., 1992; Tonegawa, 1983). Recombination occurs by DNA cleavage at the RSSs followed by joining of the four DNA ends. Depending on the relative transcriptional orientations of the minigene elements, rearrangement results in the excision of intervening DNA or in retention of DNA by inversion. Elements flanked by a 12 base pair spacer recombine in cis with those bearing a 23 base pair spacer (Lewis, 1994; Lewis and Wu, 1997). In some cases, this rule is apparently relaxed, possibly indicating that recombinase sequence specificity can be regulated (Chen et al., 1995; Durdik et al., 1984). Since each rearranging minigene segment carries its own RSS, variations in RSS sequence occur (Ramsden et al., 1994) and

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play a role in defining rearrangement preferences and hierarchies in loci involving many independently rearranging elements (Connor et al., 1995; Nadel et al., 1998; Ramsden and Wu, 1991; Suzuki and Shiku, 1992). V. Ig Genes of Mouse and Man

The organization of rearranging receptor gene segments has important effect on both the generation of receptor diversity and on receptor editing. We focus here on the organization of Ig genes of mouse and human (Fig. 1).In order to encode Ig heavy chains, the Ig-H locus has to assemble variable genes with D elements in addition to V and J elements and, therefore, requires two rearrangement steps to complete gene assembly, whereas Ig-L K and A genes are formed in a single rearrangement step by direct V-to-J joining. The mouse Ig-H locus is made up of -100 VH genes (Brodeur and Riblet, 1984) or more (Livant et al., 1986), 13 D elements (Feeney and Riblet, 1993; Kurosawa and Tonegawa, 1982; Wood and Tonegawa, 1983), and 4 Js (Maki et al., 1980; Sakano et al., 1980, 1981). The human VH locus has been sequenced and covers nearly a megabase in which -40

FIG. 1. Organization of the mouse and human immunoglobulin loci. Filled and open triangles represent recombination signal sequences carrying 23 or 12 base pair spacers, respectively. Constant (C) region exon structure is not shown. Small circles represent V-gene segment promoters.

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functional genes are present (Matsuda et al., 1998) upstream of 25 Ds (Corbett et al., 1997) and 6 active Js (Ravetch et al., 1981; Siebenlist et al., 1981). As in all Ig loci, the J regions are just upstream of the constant region encoding exons. The estimated combinatorial diversity of mouse and human loci is thus -6000. In both mouse and human, these elements appear to be in the same transcriptional orientations, and assembly results in deletion of intervening DNA (Brodeur et al., 1988; Corbett et al.,1997; Matsuda et al., 1998). The mouse Ig-K locus contains -140 VK gene segments spread over about 3 Mbp (Kirschbaumet al., 1996; Schupp et al., 1997) and 4 functional J elements (Max et al., 1979; Seidman et al., 1979; Tonegawa, 1983). The human K locus encodes 76 VK gene segments of which 66 represent duplicated pairs distributed in opposite orientations, and 30-50 of these are functional (Brensing-Kuppers et al., 1997; Schable and Zachau, 1993; Weichhold et al., 1993). Unlike the H-chain locus, in the K locus of mouse and humans about half of all V genes are in an inverted orientation relative to the J-C locus, which means that recombination often occurs by inversion rather than deletion (Kirschbaum et al., 1998; Lewis et al., 1982; Van Ness et al., 1982). The locus has 5 JK gene segments. The theoretical combinatorial diversity of the mouse and human K loci is 150-600. The lambda locus in the laboratory mouse is composed of two linked miniclusters spanning about 200 kb (Bernard et al., 1978; Brandle et al., 1992; Carson and Wu, 1989; Dildrop et al., 1987; Sanchez and Cazenave, 1987; Selsing and Daitch, 1995). VA2, VAx/JA2CA2 lies upstream of VAll JA3-CA3, JA1-CA1, but most rearrangements occur between V and J regions of the same cluster. All rearrangements involve DNA deletions. The recombined Vhl-JAl-CAl gene is the most common type of A rearrangement (Eisen and Reilly, 1985). Thus, combinatorial diversity is extremely limited in the mouse A locus. The human A locus has an array of -70 VA genes upstream of seven JA-CA clusters; each J-C cluster has a single J, and only three of the clusters are functional (Ignatovich et al., 1997; Kawasaki et al., 1997). Unlike in the mouse A locus, this organization allows nested recombination and receptor editing (Stiernholm and Berinstein, 1994). VI. Secondary Rearrangements

Ig gene organization manifests features that appear to be adapted to promote secondary rearrangements in the L-chain loci and to suppress them in the IgH locus. Secondary rearrangements are defined here as gene rearrangements on alleles that have already assembled variable region genes. Secondary rearrangements can replace primary rearrangements that

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are out-of-frame, salvaging function. They can also alter function by replacing functional rearrangements or inactivating loci altogether. 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 adjacent to the gene segments. The Ig-K locus appears to be particularly specialized to carry out secondary rearrangements. This locus lacks D gene segments, consequently, upon primary VKJKjoining on one allele, secondary rearrangements between remaining upstream VKSand downstream JKS can occur in a single step. ~ are preferred (Wood and In the mouse, J K ~and J K rearrangements Coleclough, 1984); consequently, primary rearrangements usually involve these segments, leaving downstream Js available for secondary rearrangements (Selsinget d.,1984).Analysis of double strand DNA breaks at RSSs is consistent with the notion that rearrangements progress with time from 5’ to 3’ JKS (Constantinescu and Schlissel, 1997). Furthermore, K loci often rearrange by inversion, retaining thereby the entire repertoire of VKSfor subsequent rearrangements. Deletional rearrangements, which occur when the V gene segments are in the same transcriptional orientation as the segments to which they rearrange, excise intervening DNA, yielding episomes that are permanently lost from the chromosome. Ig-K loci in mouse or human B cell lines can undergo two or more successive V-J recombinations on a single allele (Clarke and McCray, 1991; Feddersen and Van Ness, 1985; Feddersen et al., 1990; Huber et al., 1992; Lewis et al., 1982; Prak et al., 1994; Selsing et al., 1984; Shapiro and Weigert, 1987). Excised circular DNA from sequences intervening V/J recombination often contain previously generated VJ joins, suggesting that secondary K rearrangements are common in nontransformed cells (Harada and Yamagishi, 1991; Shimizu et al., 1991). B cells of mice lacking the K locus on one chromosome through targeted germline mutation have increased usage of downstream JKS,consistent with displacement of out-of-frame VJ joins by nested rearrangement (Prak et al., 1994). Further examples of mouse K secondary rearrangements will be cited later.

A. RS/kde A unique feature of the Ig-K loci is the RSkde element (“recombining sequence” in mouse, “kappa deleting element” in human) (Siminovitch et al., 1987).This element inactivates the Ig-K locus by deletional recombination (Moore et al., 1985). RS is found -25 kb downstream of the mouse CKexon (Muller et al., 1990).The kde is in a similar position in the human locus (Klobeck and Zachau, 1986). 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 VKSor to noncanonical

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RSS sites in the JK-CKintron (Durdik et al., 1984; Shimizu et al., 1991; Siminovitch et al., 1987).RS/kde rearrangements occur in -75% of mouse B cells that express A L-chain (Nadel et al., 1990; Retter and Nemazee, 1998) and in an even higher fraction of human A-producing cells (Hieter et al., 1981; Retter and Nemazee, 1998). The RS rearrangements also occur on the second allele of approximately 12% of mouse K-expressing cells (Dunda and Corcos, 1997). Because the RSkde encodes no protein, these joins are nonfunctional (Daitch et al., 1992; Durdik et al., 1984). It appears that the RS/kde elements have no other purpose than to inactivate K genes, often after functional recombination (Retter and Nemazee, 1998). Since RS rearrangement appears to occur concurrently with Ig-A locus recombination, it might be predicted to clear the way for A expression (Daitch et al., 1992; Muller and Reth, 1988). Thus K locus structure favors replacement reactions that allow receptor editing. In contrast to the Ig-K locus, the organization of the Ig-H locus tends 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 (Brodeur et al., 1988; Matsuda et al., 1998), forcing VDJ assembly at the Ig-H locus to occur by deletion of the intervening DNA (Brodeur et al., 1988). 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.Even though unrecombined upstream VHS and downstream JHs are typically retained, they both have recombination signal sequences carrying 23bp spacers and therefore cannot be joined together by recombinase. 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 because D regions are flanked on both sides by similar RSSs and can rearrange by inversion (Reth et al., 1986), but this apparently does not occur in humans (Corbett et al., 1997). Furthermore, VH elements can sometimes undergo secondary recombination to preformed VDJ elements through “VHreplacement” by targeting noncanonical, but conserved, RSS-like sequences that are present in the 3‘ end of the rearranged VHcoding sequence (Kleinfieldand Weigert, 1989). VH replacement will be discussed later. MI. Recombinational Accessibility

Recombinase enzyme activity is necessary, but not sufficient, for recombination because of strict control of DNA substrate “accessibility” (Alt et al., 1992; Gorman and Alt, 1998; Schlissel and Stanhope-Baker, 1997;

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Sleckman et al., 1996). Control of accessibility is a major means by which the immune system controls the order of gene element recombination duringV(D)Jjoining (e.g.,V-to-D vs. D-to-J). Accessibility has been shown to depend on cis acting DNA elements present in the vicinity of rearranging genes. These include the promoters of the V gene segments and enhancercontaining sequences present near J and C regions (Ferradini et al., 1996; Hempel et al., 1998; Hiramatsu et al., 1995; Schlissel and Stanhope-Baker, 1997; Sleckman et al., 1996). Accessibility is not understood in molecular detail but is correlated with hypomethylation and transcription (Gorman and Alt, 1998). Transcription of unrearranged V and C elements is often, but not always, correlated with the recombinational accessibility of a locus (Angelin-Duclos and Calame, 1998). Because each unrearranged V gene has its own promoter, subtle promoter sequence differences may affect relative efficiencies of rearrangements of particular V genes within a locus, such as was shown for the human lambda locus (Stiernholm and Berinstein, 1995). Similarly, in loci that undergo secondary rearrangements, such as mouse Ig-K, promoter differences may define a preferred V-gene rearrangement hierarchy (Jeong and Teale, 1988; Kalled and Brodeur, 1990; Medina and Teale, 1993; Milstein et al., 1992). It appears likely that rearrangement accessibility controls the delayed gene rearrangement, relative to K, of murine A loci (Arakawa et al., 1996). Analysis of the phylogeny of Ig genes suggests that primitive vertebrates generally have a “cluster” type gene organization. In each cluster, a single V, zero to two Ds, and a single J rearrange in cis, but not in combination with numerous other such clusters that may be present nearby on the same chromosome (Rast et al., 1995). This type of arrangement greatly limits combinatorial diversity but is retained in part in the mouse A locus (Fig. 1).To the extent that combined specificity of promoter and enhancer type elements can regulate rearrangements, cluster type gene organization, although inefficient in its ability to maximize combinatorial diversity, may provide greater control over accessibility of rearrangements. VIII. Quasi-Ordered Rearrangements

Ig rearrangements generally occur in a particular temporal sequence that is regulated by control of gene accessibility. In developing mouse B cells identified by multiparameter flow cytometry (Hardy et nl., 1991; Li et al., 1993,1996), commitment to rearrangement can be observed in early bone marrow progenitors (Hardy “fraction A ’ ) by the appearance of sterile transcription through the JH-Cplocus, which is rapidly followed by RAG gene expression (Li et al., 1996). Rearrangements initially involve joining

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of one of 13 DH elements to one of four JH elements (in “fraction B” cells), and this usually occurs on both alleles (Alt et al., 1984). Further differentiation in fraction B/C cells is associated with joining of VHgenes to previously formed DJs (Alt et al., 1984; Li et al., 1993). Cells bearing in-frame, functional IgM H chains ( pchains) are selected for clonal expansion and differentiation, whereas cells lacking p chains are eventually targeted for elimination (Ehlich et al., 1994). Cells lacking p 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 p chain based on its ability to associate with surrogate L chain components A5 (Kitamura et al., 1992) and VpreB (Kudo and Melchers, 1987) and to mediate transmembrane signals through associated Ig-dp signal transducers (Karasuyama et al., 1996; Reth and Wienands, 1997; Tsubata et al., 1991a, b). Proliferation of these (now fraction C‘) cells is largely dependent on interleukin (1L)-7 (Grabstein et al., 1993; Hardy et al., 1991; Maeurer and Lotze, 1998; Peschon et al., 1994; Saeland et al., 1991). Mutations that effect IL-7 signaling prevent this expansion and substantially reduce B-cell production (Maeurer and Lotze, 1998; Peschon et al., 1994). The proliferating cells bear distinct surface markers, lack RAG expression, and are almost totally devoid of rearrangements on the L-chain loci (Hardy et al., 1991; Li et al., 1993). It is only after a proliferative burst of approximately six cell divisions (Decker et al., 1991) that cells exit cell cycle and further differentiate to a stage called fraction D, in which recombination activity is redirected to the Ig L-chain loci (Constantinescu and Schlissel, 1997).The L-chain gene rearrangements begin at the K locus. The A loci almost always rearrange after in both mouse and human (Arakawaet al., 1996; Hieter et al., 1981). IX. Allelic Exclusion

The vast majority of mammalian B cells bear a single antibody heavy and light chain (Bernier and Cebra, 1964; Cebra et al., 1966; Kitamura and Rajewsky, 1992; Pernis et al., 1965; Tsukamoto et al., 1984; Weiler, 1965), despite the ability to produce two, one encoded by each allele. In the mouse, most cells express a single light chain (Coleclough, 1983; Takeda et al., 1993), despite the ability to express six (2 alleles X 3 isotype loci: K , AYh, AllA3). As discussed later, allelic and isotypic exclusion are insured by a variety of active processes. The 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 apT-cells allelic inclusion of TCRP chains in rare, but in these same cells coexpression of two TCRa

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chains is common (Malissen et al., 1992). Allelic inclusion is also observed in TCRG (Sleckman et al., 1998). 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. X. Mechanisms of Allelic Exclusion

A. STOCHASTIC FACTORS Several mechanisms have been proposed to explain the phenomena of allelic and isotypic exclusion in B cells. 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. There is some evidence for both of these factors playing a role in L-chain allelic and isotypic exclusion. In the mouse K-light chain locus, DNA demethylation and concomitant accessibility to recombination is, at least initially, monoallelic (Mostoslavskyet al., 1998), although this is clearly not a strict rule since about half of all B cells have rearrangements on both K alleles (Coleclough, 1983). Furthermore, most K-expressing cells lack h rearrangements, whereas virtually all h-producing cells bear K rearrangements, usually on both alleles (Alt et al., 1980; Coleclough et al., 1981; Hieter et al., 1981; Korsmeyer et al., 1982; Nadel et al., 1990). During B cell development in the bone marrow, A-bearing B cells emerge 24 hours later than K+ B cells (Arakawa et al., 1996), and analysis of hproducing hybridomas revealed that most A' cells had rearranged a single h gene, suggesting that h-gene recombination is both inefficient and time limited (Nadel et al., 1990). Even though these stochastic factors reduce allelic and isotypic inclusion, they are complemented by more active regulatory processes.

B. SELECTIVEFACTORS A second mechanism that could enforce allelic exclusion in mature cells is counter selection against double-producing cells. As originally conceived,

it was suggested that excess H-chain expression may be toxic to cells (Wabl and Steinberg, 1982). Evidence suggesting this possibility came from analysis of myeloma cells, in which L-chain loss is toxic if H chains are produced (Kohler, 1980).While this toxicity explanation is not supported by

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experiments in which double H-chain-expressing B cells were generated by transgenesis in vivo (Sonoda et al., 1997), it is nevertheless possible that 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 (Nemazee, 1996), cells bearing two different receptors would be much more likely to be autoreactive than cells with a single H/L pair. As a result, counterselection of double producers may occur as consequence of tolerance induction. This might explain the curious finding that in A5-deficient mice, which manifest poor allelic exclusion of H chains in the preB compartment (Loffert et al., 1996), allelic exclusion is restored in peripheral B cells (Kitamura et al., 1992).

c. FEEDBACK SUPPRESSION OF RECOMBINATION Although selective forces obviously play a major role in lymphocyte biology in general, there is a considerable body of evidence arguing that instructive mechanisms have a dominant role in establishing allelic exclusion. In preB cells, functional p chains actively block further H-chain rearrangements (Kitamura et al., 1992; Nussenzweig et al., 1987; Rusconi and Kohler, 1985; Sakaguchi and Melchers, 1986). This inhibition occurs through assembly of the surrogate L-chain components A5 (Sakaguchi and Melchers, 1986; Sakaguchi et al., 1986) and VpreB (Kudo and Melchers, 1987) with the membrane-bound form of p chain (Kitamura et al., 1991; Manz et al., 1988; Nussenzweig et al., 1987, 1988b), followed by signaling via the associated Ig-OJP complex (Nussenzweig et al., 1988b; Papavasiliou et al., 1995a,b). Forced expression of a heavy chain by transgenesis substantially blocks VDJ assembly on the H-chain locus (Manz et al., 1988; Nussenzweig et al., 1988a;Rusconi and Kohler, 1985;Weaver et al., 1985). Disruption of the preB cell receptor (preBCR) signaling complex inhibits both B cell development and allelic exclusion at this stage (Kitamura and Rajewsky, 1992; Loffert et al., 1996). H chains that fail to associate with surrogate L chain, such as certain chains that include VH81X sequences, are essentially absent from the peripheral B cell pool (Decker et al., 1991; Keyna et al., 1995a, b). 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 assemblyin a preBCR complex (Altet al., 1984; Hardy et al., 1991).In addition, in the mouse, otherwise nonfunctional D p chains generated from partial D-J rearrangements that involve D reading frame 2 can terminate recombination (Ehlich et al., 1994; Gu et al., 1991; Tornberg et al., 1998). Overall, there is compelling evidence for active feedback regulation of H-chain gene recombination that contributes to allelic exclusion.

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XI. Ig-1-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 H-chain allelic exclusion. B cells from normal animals manifest substantial allelic exclusion and isotypic exclusion (i.e.,expression of K not A and vice versa) (Tsukamoto et al., 1984; Takeda et al., 1993), but sensitive techniques are able to identify double-producing cells above background (Tsukamoto et al., 1984; Giachino et al., 1995; Gollahon et al., 1988;Pauza et al., 1993). In contrast to the ability of H-chain transgenic constructs to suppress H-chain rearrangements, enforced expression of Lchain transgenes led to mixed results. Initial experiments by Storb and colleagues using the MOPC 21 K-chain transgene were consistent with a strict feedback regulation model, as hybridomas generated from these mice appeared to lack endogenous K expression when H chain was also expressed (Ritchie et al., 1984). Curiously, in this study many hybridomas lacked Hchain expression but expressed endogenous K chains (Ritchie et al., 1984). It was later found that A-chain-producing hybridomas from these mice not only coexpressed the K transgene but also often rearranged and expressed endogenous K loci (Gollahon et al., 1988). Some other transgenic mouse lines apparently failed to exclude endogenous rearrangements because of insufficient protein expression (Rusconi and Kohler, 1985), 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 K-gene transgenic mice endogenous K expression was suppressed in only a subset of B cells (Manz et al., 1988). It is unlikely, but not formally excluded, that the incomplete allelic exclusion observed in these K-transgenic mouse experiments was solely the result of defective transgene expression. Certain conventional K transgenic mice manifest excellent allelic exclusion in a substantial proportion of cells (Carmack et al., 1991; Pelanda et al., 1996). Several lines of evidence suggest that in some cells even normal Lchain gene expression is not sufficient to suppress further L-chain gene rearrangement. Several sIg+ B-cell tumor lines, and long-term IL-7dependent bone marrow B cell lines have been shown to express new receptor chains through secondary rearrangements (Berinstein et al., 1989; Hardy et al., 1986; Huber et al., 1992; Levy et al., 1989; Rolink et al., 1993; Verkoczy et al., 1995). In an analysis of episomal DNA present in mouse spleen cells, Harada and Yamagishi found that of 16 clones containing VKJK joins excised by nested K rearrangements, five were in-frame and theoretically functional (Harada and Yamagishi, 1991), suggesting that in

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these B cells the natural genes were frequently incapable of suppressing further rearrangements. Perhaps more convincingly, in mice in which functional VKJK-genes were targeted to the natural locus, endogenous K rearrangements were inhibited in some, but not all, B cells (Pelanda et al., 1996; Prak and Weigert, 1995). Again, it would appear that only a subset of cells, which varied in frequency depending upon the variable region gene used, was subject to feedback suppression. A criticism of these experiments is that even the targeted K genes may be aberrantly expressed owing to the premature juxtaposition of a VKpromoter near the CKlocus. This could conceivably target recombination to the K 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 K locus is stochastically controlled, 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 L-chain mice (Pelanda et al., 1996, 1997; Prak and Weigert, 1995). In contrast to these results, it appears that when expressed as transgenes, certain Ig-WL pairs are extremely efficient at suppressing endogenous Land H-chain protein expression and RAG expression in the bone marrow (Chen et al., 1997; Goodnow et al., 1988; Pelanda et al., 1997; Tiegs et al., 1993), 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 (Ma et al., 1992; Rolink et al., 1993). 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 later, 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. XII. Receptor Editing Monitored in Vivo in Transgenic Models of Immune Tolerance

That H+L antibody transgenes could be used to generate mice in which most B cells had a defined specificity (Rusconi and Kohler, 1985) stimulated studies analyzing immune tolerance in B and T cells (Nossal, 1994). Transgenic (Tg) mouse models using autoantibodies to HEL, MHC class I alloantigens, DNA, erythrocytes, and other antigens have been useful in

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defining a number of ways that self reactivity is controlled. In Tg 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 (Hartley et al., 1991; Nemazee and Buerki, 1989; Nemazee and Burki, 1989). 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. Tiegs et al. found that in mice transgenic for the 3-83 antibody (reactive to the anti-MHC class I alloantigens H-2Kk and H-2Kb) B cells were numerous and almost exclusively expressed the Tg-encoded antibody as a consequence of feedback suppression of recombination (Tiegs et al., 1993). 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 anti-self reactivity. The remaining B cells in the peripheral lymphoid organs retained surface expression of the 3-83 H chain, but not the Tg L chain, and an extremely high percentage of cells expressed endogenous A 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 by immature bone marrow B cells could reinduce or prolong L-chain gene rearrangements, allowing a cell to alter specificity and escape death. 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 perphery, under control of a liverspecific Kb transgene, underwent profound B-cell deletion without appreciable receptor editing. Independent evidence for receptor editing was obtained in transgenic mice carrying H + L genes of double stranded (ds) DNA antibody 3H9 (Gay et al., 1993). Spleen cells of H+L Tg mice and hybridomas generated from them were analyzed for antibody specificity and endogenous rearrangements. Even though dsDNA-specific B cells were lacking from the hybridoma sample, indicating that specific tolerance was induced, splenic B-cell numbers were surprisinglynormal, particularly in older mice. Splenic B cells retained Tg 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 Bcell specificity and extinguished dsDNA binding. Suppression of dsDNA specificity was reversible in one hybridoma, when the endogenous K 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

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phenotypic allelic exclusion, among these hybrids there was excess use of endogenous V~12/13genes associated with downstream JKS.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 V K and J usage. Radic et al. (1993b) and Prak et al. (1994) extended this work to test for tolerance-induced receptor editing in 3H9 H-chain-only Tg mice. This was possible because 3H9 H chain binds DNA in association with diverse L-chain partners (Radic et al., 1991), but tolerance regulates these cells, as spleen hybridomas from 3H9 H-chain Tg mice lack reactivity to dsDNA (Erikson et al., 1991; Radic et al., 1993). Hybridoma analysis again revealed a strikingly limited usage of V K genes in splenic B cells with excess use of V~12/13and skewing to downstream J K S , particularly J K ~ 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 K-chains that conferred autoreactivity, these results provide an explanation for skewing of JK 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 V H l l anti-DNA Hchain mice yielded similar results (Pewzner-Jung et al., 1998). Since in these studies no L-chain transgene was present, the later stages of B-cell development should have been relatively normal. And because simple apoptosis of autoreactive cells from an initially random population would be unlikely to account for the observed skewing in JK usage, these results suggested that autoreactive cells were rescued by ongoing Ig-K recombination. XIII. Analysis of Receptor Editing in Gene-Targeted Antibody Gene Mice

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 Hchain 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 failed to identify use of the conserved heptamer

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embedded in the expressed VH,but destructive rearrangements caused by D joining to other heptamer-like sites in the gene were observed (Taki et al., 1995). Another study (Chen et al., 1995) found frequent editing by D regions, but also found VH-to-VDJreplacement events that rescued Hchain function, a result that was predicted to be possible from prior work with cell lines (Kleinfield and Weigert, 1989; Reth et al., 1986). This and subsequent results from studies of other H-chain-targeted mice (Bertrand et al., 1998; Cascalho et al., 1996; Pewzner-Jung et al., 1998; Sonoda et al., 1997) 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 uiwo. But there is debate about the frequency, stage specificity, and tolerance inducibility of the VH replacement reaction (Fanning et al., 1998). In nitro analyses indicate that the embedded heptamer is extremely inefficient at targeting V(D)J recombination relative to conventional RSSs (Nadel et al., 1998). 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 VHreplacements occur at the proB stage, prior to L-chain gene rearrangements (Cascatho et al., 1996; Chen et al., 1995; Sonoda et al., 1997). This in turn suggests that tolerance signaling through intact surface immunoglobulin does not drive the VH-to-VDJreplacement response. This latter possibility 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 ~ H ~ H N K ~ anti-dsDNA antibody, or the 3H9 H chain with a different targeted Lchain ( V K ~that ) conferred reactivity to ssDNA (Chen et al., 1997). The 3 H 9 N ~ 4dsDNA reactive combination was subject to extensive K-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 K alleles. Only 4% of hybridomas lacked detectable secondary K rearrangements. L-chain editing occurred by inversional rearrangements in 57% of the hybridomas, deletional type rearrangement events occurred in 28% of the cells, and inclusional rearrangements (i.e., on the opposite allele with an unaffected targeted allele) occurred in only 11%of cells. In the 3H9H/ V K mice, ~ B cells bearing this receptor are often seen in the periphery, in an anergic state (Erikson et al., 1991). But suggestive evidence of receptor editing was also observed in the ~ H ~ H BNcells K because ~ both in conventional and ~ H ~ H gene N K targeted ~ mice a subset of B-cell hybridomas showed exaggerated usage of V~12/13along with skewed JK usage (Chen et al., 1997). In the targeted ~ H ~ H mice, N K these ~ rearrangements oc-

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curred on the untargeted allele, however. 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, in this case represented by dsDNA and ssDNA, respectively. In a second study, targeted K-H 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 (Pelanda et al., (1997).Quantitative Southern blotting assessed the extent of recombination in the K 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 K-only mice these percentages were 10 and 27%, respectively. As a control, the 3-83 K mice were bred with H-chain gene mice of an irrelevant, nonautoreactive specificity. (Unfortunately, in this study, it was not possible to compare 3/83H L targeted mice with and without antigen because the targeted H-chain gene was linked to a crossreactive 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. The RS-type recombinations inactivating the K 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 (Hardy fraction D) 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 sIgstages. This is, as one might predict, from the high frequency of nonfunctional secondary rearrangements associated with inactivation of the targeted K-gene. In both this study and that of Chen et al. (1997), it appeared that receptor editing could allow a rather efficient rescue of previously autoreactive cells because peripheral B cell numbers were relatively normal.

+

XIV. 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 (Lawton and Cooper, 1974). And early studies on B-cell tolerance to membrane MHC molecules indicated that self-reactive B-cells were absent from the peripheral lymphoid organs, but large numbers of newly formed autoreactive cells bearing a low density of sIg were retained in the bone marrow (Nemazee and Burki, 1989; Nemazee and Burki, 1989). Because these data were superficially consistent

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with the clonal selection hypothesis and classical studies on B-cell tolerance (Nossal, 1983), 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 (Raff et al., 1975; Sidman and Unanue, 1975).In these cultures, immature B cells rapidly lost sIg but were not reduced in number, as assessed by quantitation with anti-MHC class I1 antibodies, and 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. In more recent times, 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, rather than editing (Monroe, 1996; Norvell et al., 1995; Nossal, 1983). A possible resolution of the discrepancy between the two types of study is that the presence of bone marrow accessory cells is required for the editing response ( J . Monroe, personal communication). 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 cells bearing membrane bound HEL blocked developmental progression at the fraction E (immature B cell) stage but failed to cause rapid cell death. The block was reversible upon antigen removal during the first 1-2 days, but at later times B-cell death occurred. Enforced expression of Bc12 in immature autoreactive B cells was shown to further prolong survival at this developmental stage (Hartley et al., 1993) 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 (Hertz and Nemazee, 1997). In this same

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study, it was found 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 challenged with antigen. Under these conditions, autoantigen blocked developmental progression and promoted receptor editing but did not appreciably accelerate cell death (Melamed and Nemazee, 1997; Melamed et al., 1998). Consistent with this idea, it was found that Bcl-2 overexpression did not allow autoreactive receptor-bearing B cells to escape from the bone marrow but appeared to improve the efficiency of their escape by receptor editing, probably by prolonging the time window of secondary rearrangement (Lang et al., 1997). Bcl-2 overexpressing immature B cells were shown to spontaneously switch from K to A L-chain expression in vitro (Rolink et al., 1993). Somewhat analogous results were obtained in Bcl-xl-overexpressinganti-HEIJmHEL 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 (Fang et al., 1998). Curiously, however, the same Bcl-xl transgene had minimal effects in the 3-83 model of central tolerance (J. Lang and D. Nemazee, unpublished data). Overall, these data suggest that receptor editing is associated with a developmental checkpoint. The notion that through V(D)J 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 PO ulate the periphery. This idea was directly tested in both the antiH-2Kb,! and anti-DNA models by breeding H + L Tg mice to a RAG-1- or RAG-2-deficient background (Spanopoulou et al., 1994; Xu et al., 1998). B-cell development did not advance past the Hardy fraction D/E (i.e., bone marrow) stages, and B cells did not populate the peripheral lymphoid organs. The developmentally arrested RAG-deficient autoreactive cells underwent apoptosis in situ (Xu et al., 1998). In contrast, on an antigenfree (H-zd),RAG-deficient background anti-H-2KksbB 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. XV. 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, but some editing at the H-chain locus may occur as well. One indication of this is that most autoantibody Tg mice exhibit excellent H-chain exclusion, but poor Lchain allelic exclusion (Chen et al., 1997; Gay et al., 1993;Tiegs et al., 1993).

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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 K rearrangements could be induced in over two-thirds of cells upon anti-BCR treatment (Melamed and Nemazee, 1997). In these same cells, VDJ assembly on the H-chain locus was not detectable by a sensitive PCR assay. But, as discussed earlier, conventional H-chain transgenic mice do not allow an assessment of VHreplacement events and when targeted H-chain autoantibody mice have been analyzed, V,-to-VD J replacement occurred infrequently and usually included N-region additions indicative of recombination at the proB stage prior to sIgM expression (Chen et al., 1997). However, even in conventional H-chain Tg mice, allelic inclusion has been observed in a number of situations that may indicate an antigeninduced editing. For example, spontaneously activated B cells of two different autoimmune-prone mouse strains were shown to manifest poor allelic exclusion of autoantibody H-chain transgenes in two different systems (Iliev et al., 1994; Roark et al., 1995). But in these cases, the respective roles of cellular selection and recombinase induction could not be assessed, and the cells analyzed themselves produced class switched, endogenously encoded autoantibodies. A separate study by Chen, Weigert, and colleagues revealed an additional mechanism of recombination-mediated cell rescue in autoantibody Tg mice with very high affinity to dsDNA (Chen et al., 1994). In this model, a mutant 3H9 H-chain (non-site-targeted) transgene with an additional CDR2 arginine mutation conferring high affinity DNA binding was used alone or paired with a V K L-chain ~ Tg. Hybridoma analysis revealed that in these mice, B cells escaped death by modifymg their receptors in two ways. In a minority of cases, ongoing L-chain gene recombination rescued the cells, and, as was seen in other studies, escape was associated with limited VL usage and skewing to downstream JKS. Surprisingly,a larger fraction of cells had entirely eliminated the multicopy H-chain Tg from the chromosome by an unknown recombination mechanism. When the endogenous H-chain loci were made unavailable by crossing to a JH-deficient background, little B-cell escape was possible owing to the ability of this mutant H chain to confer dsDNA binding with a host of different L chains, and cells were eliminated at a fraction D/E stage. It is unclear if this type of H-chain editing has any counterpart in normal B-cell physiology. XVI. 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 are succeeded by a

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stage that is instead apoptosis-sensitive. These cells can be distinguished

by a series of surface markers (Carsetti et al., 1995) and by the ability of the latter cells to migrate to the spleen (Allman et al., 1992). 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 (Melamed et al., 1997; Rolink et al., 1993). In these cultures, IL-7 withdrawal is required for recombinase expression, possibly because cell cycle events in proliferating cells suppress recombinase expression (Lin and Desiderio, 1995). It is known that RAG2 protein is selectively degraded in cycling cells (Lin and Desiderio, 1995). Upon IL-7 withdrawal, as cells express increasing amounts of sIg, the ability to respond to antigen challenge by editing decreases and apoptosis sensitivity increases concomitantly. Since these, more developed, B cells presumably must at some point fix their receptors in order to participate in the immune response, their inability to undergo receptor editing in response to antigen alone makes sense. XVII. A Role for Receptor Editing in Receptor Diversification?

Besides providing an elegant self-tolerance mechanism, receptor editing may have an additional biological benefit. As indicated in the discussion of variable gene assembly, 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. XVIII. Editing in Mature, Antigen-Reactive B-Cells: Receptor Revision

The ability to promote receptor diversification has been invoked to explain why receptor editing is not limited to immature B cells but is also induced in mature B cells during the immune response in germinal centers (Hikida et al., 1996; Han et al., 1996, 1997a; Hikida and Ohmori, 1998; Papavasiliou et al., 1997). The first indications of this possibility were that RAG mRNA and protein were inducibly expressed in mature B cells under certain culture conditions or during a germinal center immune reaction (Han et al., 1996; Hikida et al., 1996). Histological analysis of spleens and lymph nodes of immunized mice indicated that the cell iype expressing RAG in vivo was the centrocyte (Han et al., 1996; Hikida et al., 1996,

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1997). 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 (Han et al., 199713). 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 up-regulation of RAG genes (Hikida et al., 1997, 1998; Hikida and Ohmori, 1998). Treatment of cells with a combination of bacterial lipopolysaccharides and IL-4 had a similar effect (Hikida et al., 1996, 1997). Later studies suggested that IL-7 could substitute for IL-4 and was probably the critical cytokine in vivo because antibodies to IL7R, though they permitted germinal center formation, prevented RAG expression in centrocytes (Hikida et al., 1998). To distinguish secondary Ig gene rearrangements of germinal center cells from those in immature bone marrow B cells, the alliterative term “receptor revision” has been coined (Han et al., 1996). Certain features of the receptor revision response are puzzling. 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 (Hikida et al., 1996, 1997). And in histological sections of germinal centers, RAG protein expression appeared to be primarily localized to the cytoplasm rather than the nucleus (Han et al., 1996; Hikida et al., 1996). In any case, RAG gene products induced in vivo and in vitro were nevertheless shown to cause double-stranded DNA breaks adjacent to recombination signal sequences (Papavasiliou et al., 1997) and new Lchain protein expression (Hertz et al., 1998; Hikida and Ohmori, 1998; Papavasiliou et al., 1997). Curiously, in one study, the cells undergoing these recombination reactions and receptor alterations did not manifest a loss of surface Ig expression (Papavasiliouet al., 1997). Since most receptor revision is predicted to result in out-of-frame joins, this result suggests that sIg- cells died rapidly in these cultures, which might explain why in other studies RAG expression was often found in dying cells. Perhaps most intringuingly, the cell fractions that expressed recombinase also expressed other markers characteristic of B cell precursors, including surrogate L-chain components (Meffre et al., 1998), IL-7R (Hikida et al., 1998), the surface marker GL-7 (Han et al., 1996), and, in human germinal center cells, TdT (Meffre et al., 1998). The reexpression of surrogate Lchain components is particularly interesting because of the likelihood that many editing events would initially silence L-chain production, perhaps requiring surrogate L chain to temporarily pair with H chain. Expression of surrogate L-chain components also is observed in cycling, centroblast cells (Meffre et al., 1998). These unexpected similarities between bone marrow and germinal center cells, first pointed out by Han, Kelsoe, and

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colleagues on the basis of RAG expression (Han et al., 1996), suggest that many elements of B-cell development are recapitulated during the immune response. But 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 revision in germinal center cells, and BCR stimulation under such conditions actively blocks the recombinase response (Hertz et al., 1998; Meffre et al., 1998). In one study, in vivo RAG expression appeared to be present in a subset of germinal center centrocytes distinguishable by a reduced CD45 levels and nonoptimal receptor gene usage for cognate immunogen (Han et al., 1997a). 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 revision in the diversification of the receptors of antigen-reactive cells. But, as we discuss later, there are also problems with this interpretation. Very recently, data were presented that makes two novel contributions to this area of research. Weigert and colleagues identified a clone of anti-dsDNA reactive cells, captured as hybridomas, from MRLApr mice harboring targeted L- and H-chain genes encoding anti-DNA. In this clone, the L-chain transgene 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 K-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 ediging may be specifically stimulated by the loss of BCR expression. A second recent study argues that receptor editing can occur in mouse B-1 cells (Qin et al., 1999), which are not believed to take part in germinal center reactions. IgM+B220'" peritoneal cells, particularly those from the autoimmune-prone NZB strain, were shown to express RAG mRNA and to possess double strand breaks at J K RSSs. In analysis of mice-carrying functional replacements of IgH and IgL variable genes, B-1 cells lost idiotypic determinants, which is 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. It will be important to determine the signals that stimulate editing in B-1 cells. XIX. locus Specificity of Receptor Revision

Several studies documented renewed recombination of L-chain genes in mature B cells (Han et al., 1997a; Hikidaet al., 1998; Hikida and Ohmori,

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1998; Meffre et al., 1998; Papavasiliou et al., 1997; Qin et al., 1999), and a single study provided possible evidence of rearrangements at the IgH locus (Papavasiliou et al., 1997). 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. This thus experimental system may either represent the ideal model to reveal physiologically relevant receptor reversion on the IgH-locus or 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. XX. Potential Functions of Receptor Revision

Receptor revision could in principle be advantageous in a number of circumstances, depending upon its temporal and cell lineage relationship to other forms of postimmune diversification, such as somatic point mutation. For example, point mutations that are deleterious because they introduce stop codons or disrupt antibody protein folding are unlikely to be efficiently corrected by further point mutation but could be more efficiently replaced by receptor revision. Alternatively or in addition, receptor revision may precede point mutation. One might imagine that after antigen encounter and some cell divisions, a clone of cells might be large enough to benefit from receptor revision. Revision after a proliferative burst would potentially allow the system to screen the ability of a particular promising H chain to bind to antigen in association with a random collection of L chains, some of which may bind better than the original. Receptor revision occurring on the nonexpressed allele may speed clonal evolution in another way by leading to transient L-chain allelic inclusion, which would allow the cell to retain the old L chain, while testing new LJH combinations. Further selection in conjunction with mutation or recombination could then inactivate the least helpful allele, a possibility that was demonstrated experimentally in multicopy Tg mice (Neuberger et al., 1989). H-chain revision might also occur on a second allele. This type of scenario might explain the unexpected H-chain allelic inclusion seen in autoreactive Bcells of certain H-chain transgenic mice (Roark et al., 1995). On the other hand, allelic inclusion has not typically been seen in non-Tg B cells. Taken at face value, the available evidence is not inconsistent with the occurrence of receptor revision before, during, or after somatic point mutation. Furthermore, the scope and frequency at which receptor revision occurs is still an open question.

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XXI. Problems with the Receptor Selection Concept

A. How MANYREARRANGEMENT TRIESDO B CELLSHAVE?RESULTS FROM MATHEMATICAL MODELING Are the structures of the L-chain gene loci compatible with a physiological role for receptor editing either in tolerance or immunity? In the mouse, each K locus has only four functional J regions; therefore, there exists the possibility for a maximum of eight rearrangement attempts at the K locus and four more at the A loci. In humans there are theoretically even more L-chain gene rearrangement options. Is this number of options sufficient to be of benefit and does the mode of rearrangement really take advantage of the theoretical maximum number of options? Finally, does empirical data fit with frequent occurrence of receptor editing? One problem that has been pointed out is that at least half of all B cells isolated from the mouse spleen (that predominantly represent the preimmune repertoire) have rearrangements on only one of their K alleles (Coleclough et al., 1981). A second problem is that the vast majority of mouse spleen B cells use J K or ~ J K in ~ their functional K chains (Wood and Coleclough, 1984). These data put constraints on the extent of receptor editing that could have occurred in the mouse preimmune repertoire. A crude mathematical model, based on the idea that rearrangement attempts occur relatively slowly and progressively to allow the cell the opportunity to detect L-chain function, indicates that these data are nevertheless compatible with massive tolerance-induced editing and -4 rearrangement attemptshell (Nemazee, 1998).The reason for this is that cells that are fortunate enough to generate an acceptable receptor during the first rearrangement attempts are overrepresented both for probabilistic reasons and because of a higher rate of production. For a cell to first generate an autoreactive receptor and then to correct it requires more time and is relatively less efficient than generating a nonautoreactive receptor on the first or second try. A second reason that relatively few rearrangements attempts have occurred in most mouse K + spleen cells is that they are specifically depleted of the cells that have made numerous rearrangement attempts, which are highly concentrated in A+ cells and a subpopulation preB cells. As discussed earlier, A+ cells usually have K rearrangements that are inactivated by RS/kde type recombination. We have recently found that about half such K rearrangements are in-frame and presumably functional (Retter and Nemazee, 1998). One would imagine that receptor editing would be most helpful in rescuing “problem” VDJ H genes that have intrinsic propensity to be selfreactive, allowing their participation in the repertoire paired with Lchain partners.

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B. Is RECEPTOR EDITING NEEDEDTO AVOIDWASTE? A general criticism of the receptor editing concept is that, although editing may be a thrifty way of maintaining tolerance, this occurs in the face of massive cellular waste at all levels. For example, the bone marrow of a laboratory mouse generates -5 X lo6 B cells/day but only needs to replace -lo5 cells/day, and the excess cells turn over rapidly (Osmond et al., 1994). If salvage of B cells in development is so important that receptor editing is required, why is such hematopoietic extravagance permitted? It may be that receptor editing is important either to relieve developmental bottlenecks that would result in even greater cell loss or to allow better representation of infrequently rearranging gene segments to compensate for rearrangement preferences early in development.

C. EDITING PREDICTS ALLELICINCLUSION! How can allelic exclusion be maintained if receptor editing often occurs by rearrangement on the nonexpressed allele? In immature B cells, several mechanisms may conspire to make allelic inclusion unlikely. First, receptor editing in these cells is driven by the signaling of autoreactive receptors. Such a signal is by definition dominant and can only be turned off if surface expression of the offending receptor is suppressed by inactivation of the offending L-chain gene or through phenotypic suppression by competitive binding of a new L chain to the cell's H chain. In either case, phenotypic allelic exclusion would result. Second, there is some evidence that initial Lchain gene rearrangements may be limited to a single allele (Mostoslavskyet al., 1998), and the K loci are so constructed that secondary rearrangement on the same allele silences primary rearrangement. Even though rearrangements on the h locus would be predicted to enhance the potential for Lchain isotypic inclusion, the h locus rearranges later than the K locus, and concomitantly with RS recombination, which effectively silences the K locus in most A-producing cells. If tolerance-induced receptor editing in immature B cells may be called genetically dominant, receptor editing during d p T-cell development or in the germinal center is recessive, in that it is terminated only upon positive selection by antigen of a particular subset cells with certain antigen receptor specificities. Thus, just as receptor editing as a result of negative selection is consistent with allelic exclusion, receptor editing (or receptor revision) that is suppressed by positive selection is predicted to be associated with allelic inclusion. D. RECEPTOR EDITINGIN DISEASE Receptor editing appears to be a major mechanism of B-cells tolerance to DNA, tolerance to which is often broken in lupus erythematosus (Radic

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and Weigert, 1995), raising the possibility that receptor editing defects may promote lupus disease. Several investigators have now begun to look in human patients for evidence of perturbations of receptor editing (Bensimon et al., 1994; Dorner et al., 1998; Suzuki et al., 1997). One study suggested that editing was defective in lupus patients because they failed to avoid the use of a highly cationic V K gene that contributed to DNA reactivity (Suzukiet al., 1997).This gene was associated with lupus nephritis (Suzuki et al., 1996). Another study suggested that editing might be impaired in lupus patients because their B-cells tend to lack multiple K-gene rearrangements (Bensimon et al., 1994). On the other hand, a different group found increased downstream J K usage in B cells of a lupus patient and, therefore, suggested that more editing may have occurred (Dorner et al., 1998).Overall, it is difficult at present to come to any firm conclusions regarding the relevance of receptor editing to disease etiology. XXII. Conclusion

Understanding the basic biology of the immune system is a continuing challenge. The complexity of the recognition process and the heterogeneity of antigen receptors and their ligands is enormous. The lymphocyte repertoire nevertheless must be strictly regulated to avoid self-reactivity and to promote robust responses to microbes. In this regulation, two methods are used: those that might be called “clone selective,” which regulate cell growth and death, and a second category that is “receptor selective,” in which signaling through the antigen receptor itself plays a key role in regulating whether functional receptor genes are modified or replaced. In this latter category may be included other types of somatically induced variable exon changes, such as hypermutation and gene conversion (Reynaud et al., 1985, 1995). These two processes of receptor selection and clonal selection, often working in succession, may account for the astonishing ability of the immune system to adapt to the environment.

ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health and the Arthritis Foundation. The author thanks Marc Retter, Norman Klinman, and Martin Weigert for their comments on the manuscript, and Kathy Offerding for secretarial assistance.

REFERENCES Agrawal, A., Eastman, Q . M., and Schatz, D. G. (1998).Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394,744-751. AUman, D. M., Ferguson, S. E., and Cancro, M. P. (1992). Peripheral B cell maturation. I. Immature peripheral B cells in adults are heat-stable antigenhi and exhibit unique signaling characteristics. J. Immunol. 149,2533-2540.

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ADVANCES IN IMMUNOLOGY, VOL. 74

Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection PlUS LOETSCHER, BERNHARD MOSER, AND MARC0 BAGGlOUNl

Theodor Kocher Institute, University of Bern, Bern, Swikerland

1. Introduction

Research on chemokines has progressed rapidly during the last two years, and since our most recent update (Baggiolini et al., 1997), numerous review articles and essays have appeared (Rollins, 1997; Sallusto et al., 1998a; Baggiolini, 1998; Luster, 1998; Cairns and D’Souza, 1998; Wells et al., 1998; O’Garra et al., 1998). With few exceptions (Rollins, 1997; Luster, 1998), these reviews are focused on special issues. The enormous growth of the chemokine literature makes it more and more difficult to cover the field comprehensively. We review here two research areas that have attracted unprecedented attention in a growing number of laboratoriesthe functions of chemokines and their receptors in lymphocytes and lymphoid tissues and the role of chemokines in HIV infection. II. Expanding Funciional Implications of Chemokines

Several new chemokines and new receptors have been identified (Figs. 1-3 ), and information on their binding selectivity and on the chromosomal location of their genes has been extended (Tables I and 11). Chemokine activities are primarily confined to leukocyte activation and chemotaxis which are elicited by heptahelical receptors coupled to GTP-binding proteins, but wider pathophysiological implications are apparent. Chemokines are not just mediators of leukocyte recruitment for host defense (e.g., in inflammation and immune responses against foreign material), as it was believed some years ago (Baggiolini et al., 1994). They have a major role in the regulation of the migration and recirculation of lymphocytes from and to different tissues and tissue compartments, which is required for the maturation and differentiation of immune cells and for the generation of secondary lymphoid organs (Baggiolini, 1998). Much information is still needed for the full picture, but the recent findings about new chemokines like BCA-1, SLC, ELC and their murine homologues, which are expressed constitutively in restricted lymphoid tissue areas and attract functionally defined types of lymphocytes bearing the appropriate receptors, are already viewed as paradigms for the homeostatic role of these attractants in the 127

Copyright D 2000 by Academic Press. All rights of reproduction in any form reserved.

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I

SDF-1 BCA-1 IL-8 GROa GROP GRoV NAP-2 ENA-78 GCP-2 El0 Mig I-TAC BRAK

FIG. 1. Structure similarity diagram of human CXC chemokines. Similarity scores of the proteins were determined by the average linkage cluster analysis (Sneath and Sokal, 1973).The gap penalty, window size, filtering level, and K-tuple size parameters were set at 3, 10, 2.5, and 1, respectively. Distance to the branching points indicates the percent of sequence divergence. GeneBank accession numbers for the sequences are (from top to bottom):U16752, AJ002211,Y00787, J03561, M36820, M36821, M54995, L37036, Y08770, X02530, X72755, AF030514, AF073957.

immune system. Other functions are under study. Chemokines may be involved in the turnover of short-lived leukocytes like the neutrophils by inducing their migration to sites of disposal and the egress of new cells from the bone marrow. It has been reported that chemokines have growthpromoting as well as growth-inhibiting functions in myelopoiesis (Cook, 1996;Verfaillie, 1996).As judged from gene deletion studies which revealed defects in the cardiovascular and central nervous system (Nagasawaet al., 1996a; Tachibana et al., 1998; Zou et al., 1998; Ma et al., 1998), SDF-1 appears to be involved in morphogenesis, a function that could depend on chemotactic effects on cells other than leukocytes (Baggiolini, 1998). Several chemokines were reported to have angiogenic or angiostatic activities and to elicit or inhibit chemotaxis of endothelial cells in vitro (Arenberg et al., 1997). It is still unknown, however, how these effects are mediated, and there is little information on the receptors involved (Rollins, 1997). The research concentrates largely on CXC and CC chemokines, and only a few laboratories have worked on lymphotactins, which have two instead of four conserved cysteines (Kennedy et al., 1995; Yoshida et al., 1996), and fractalkineheurotactin, a membrane-bound protein expressed in the brain, which consists of a mucin stalk bearing a chemokine-like structure with three amino acids between the first two cysteines (C&C motif) (Bacon et al., 1997; Pan et al., 1997). Receptors for lymphotactin and the chemokine moiety of fractalkineheurotactin, XCRl (Yoshidaet al., 1998a) and CSCR1 (Imai et al., 1997a),respectively, have been identified

CHEMOKINES AND RECEPTORS IN LYMPHOCYTES AND HIV INFECTION

II

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MCP-1 MCP-3 Eotaxin MCP-2 MCP-4 Eotaxin-2 I309 MlP-la MIP-1p DC-CKl HCC-1 HCC-3 HCC-2 CKpS RANTES HCC-4 TARC MDC Ltn a I Ltn p Fractalkine LARC ELC SLC TECK

FIG.2. Structure similarity diagram of human CC, C, and C&C chemokines. Similarity scores were determined as described for Fig. 1. Only the chemokine-like structure of fractalkine was used for the similarity analysis. GeneBank accession numbers for the sequences are (from top to bottom): X14768, X72308, D49372, P80075, U46767, U85768, M57502, X03754, J04130, AB000221,24927O,Z70293,Z70292,U85767, M21121, U91746, D43767, U83171, D63790, D63789, U84487, D86955, U77180, AB002409, U86358.

recently (Table I). The functions of these chemokines in physiology and pathology are still unknown, however. 111. Chemokines and Chemokine Receptors in Lymphocytes

T lymphocytes express the widest variety of chemokine receptors (Table 111).Only a few years ago, no one would have considered such a possibility because the responses of blood lymphocytes to chemokines were admittedly weak and variable (Baggiolini et al., 1994). The situation changed with the observation that monocyte chemotactic proteins, MCP-1, MCP2, MCP-3, and MCP-4, are potent attractants of T lymphocytes, natural killer, and dendritic cells (Baggiolini et al., 1997), and that the expression of chemokine receptors on lymphocytes is highly regulated. CXCRS, CCR1, CCR2, and CCR5 (Loetscher et al., 1996a,b;Bled et al., 1997),for instance, are upregulated by IL-2 and decay rapidly when IL-2 is withdrawn. Differ-

TABLE I HUMAN CHEMOKINES, CHROMOSOMAL LOCATION, AND RECEFTOR SELECTMTY Chemokine

Chromosome

IL-8 GROa CROP GROp NAP-2 ENA78 GCP-2 PF4 IPlO Mig I-TAC BCA-1 SDF-1 BRAK

cxcL8

MCP-1 MCP-2 MCP-3 MCP-4 RANTES

ccL2 CCL8 CCL7 CCLl3

CXCLl cxcL2 CXCU cxCL7 CXCL.5 CXCLG CXCLA CXCLlO CXCL9 CXCLll CXCL13 CXCLl2

ccL5

Reference

4q13-21 4q13-21 4q13-21 4q13-21 4q13-21 4q13-21 4q13-21 4q13-21 4q21.21 4q21.21 4q21.2 4q21 1Oqll.l 5q31

(Ahuja et d.,1992) (Ahuja et al., 1992) (Ahuja et d.,1992) (Ahuja et al., 1992) (Ahuja et d.,1992) (Chang et al., 1994) (Modi and Chen, 1998) (Ahuja et d.,1992) (Lee and Farber,1996) (Lee and Farber, 1996) (Erdel et d.,1998) (Gunn et d.,1998a) (Shirom et d.,1995) (Hromas et al., 1999)

17q11.2 17q11.2 17q11.2 17q11.2 17q11.2

(Naruse et al., 1996) (Van Coillie et d.,1997) (Naruse et al., 1996) (Naruse et al., 1996) (Naruse et al., 1996)

Receptor(s) CXCR1, CXCR2 cxcR2 cxcR2 CXCR2 CXCR2 CXCR2 CXCR1, CXCR2 CXCR3 CXCR3 CXCR3 CXCR5 CXCR4 CCR2 CCRl, CCR2, CCR3, CCRS CCR1, CCR2, CCRS CCR2, CCR3 CCR1, CCR3, CCR5

MIP-la MIP-la 1-309 HCC-1 HCC-2 HCCmEC/LMC DC-CKl/PARC/AMAC-1 CK@YMPIF-1 Eotaxin TARC MDC/STCP-1 LARC/MIP-3cr/Exodus-1 SLC/GCkine/Exodus-2 ELC/MIP-3P/Exodus-3 Eotaxin-Z/MPIF-2 TECK

CCL3 CCL4 CCLl CCL14 CCLl5 CCL16 CCL18 CCL23 CCLll CCL17 CCL22 CCL20 CCL21 CCL19 ccL24 CCL25

17q11.2 17q11.2 17q11.2 17q11.2 17q11.2 17q11.2 17q11.2 17q11.2 17q21.1 16q13 16q13 2q33-37 9q13 9q13 7q11.23 19q13.2

(Naruse et al., 1996) (Naruse et al., 1996) (Naruse et al., 1996) (Naruse et al., 1996) (Naruse et d., 1996) (Naruse et al., 1996) (Hieshima d al., 199%) (Nomiyama et al., 1999) (Garcia-Zepeda et al., 1997) (Nomiyama et al., 1998a) (Nomiyama et al., 1998a) (Hieshima et al., 1997a) (Nagira et d.,1997) (Yoshda et al., 1997) (Nomiyama et al., 1998b) (Nomiyama et al., 1998~)

Lymphotactin a/SC M- 1dATAC Lymphotactin bISCM-lP

XCLl xcL2

lq23 lq23

(Yoshida et al., 1996) (Yoshida et al., 1996)

XCRl XCRl

Fractalkine/neurotactin

C&CLl

16q13

(Nomiyama et d., 1998a)

CXSCR1

CCR1, CCR5 CCR5 CCR8 CCRl

CCR3 CCR4 CCR4 CCR6 CCR7 CCR7 CCR3

The new nomenclature for chernokines, which was presented at the 1999 Keystone Symposium on Chemokines and Chemokine Receptors, is shown in addition to the currently used terms. CXC, CC, XC, and CX3C refer to the subgroup of chemoknes, the letter L stands for ligand, and the number corresponds to the numbering of the respective gene.

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TABLE I1 HUMAN CHEMOKINE RECEPTORSAND CHROMOSOMAL LOCATION Receptor

Chromosome ~

Reference

~~

CXCRl CXCR2 CXCR3 CXCR4 CXCR5

2q34-35 2q34-35 Xq13 2q21 1lq23"

(Ahuja et al., 1992) (Ahuja et al., 1992) (Loetscher et al., 1998a) (Federspiel et al., 1993)

CCRl

CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9

3p21.3 3p21.3 31321.3 31324 3p21.3 6q27 17q12-21.2 3~21.3-24 3p21.3

(Samson et al., 1996a) (Samson et al., 1996a) (Samson et d.,1996a) (Samson et al., 1996a) (Samson et al., 1996a) (Liao et al., 1997a) (Schweickart et al., 1994) (Samson et al., 1996b) (Bonini et al., 1997)

XCRl

3~21.3-21.1

(Yoshida et al., 1998a)

C&CR1

3p21

(Raport et d.,1995)

ccR2

' M . Lipp, personal communication.

ent stimulatory conditions modulate the expression of other receptors (e.g., CCR3, CCR7 or CXCR4).The receptor pattern appears to be characteristic for the state of activation and differentiation of lymphocytes which can thus be recruited selectively to fulfill specific functions. A. NAIVEAND MEMORY T LYMPHOCYTES In contrast to phagocytes, T lymphocytes continually recirculate from the blood into tissues and back. Naive T lymphocytes enter secondary lymphoid tissues where they sample antigens. When they encounter an appropriate antigen, they become activated.They differentiateinto effector and memory T cells and proliferate. In contrast to their naive precursors, effector and memory T cells predominantly enter peripheral nonlymphoid tissues, which are the main sites of antigen penetration, but they retain the ability to home to secondary lymphoid tissues for subsequent rounds of activation (Butcher and Picker, 1996; Mackay, 1993).A series of steps characterizes the migration of T lymphocytes (Butcher and Picker, 1996; Springer, 1995). In microcirculation experiments, the cells are first seen to roll on the endothelial surface as a consequence of selectin-mediated interactions. Rolling is followed by firm adhesion and arrest mediated by integrins which are upregulated and activated by chemokines produced

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CXCRl cxcR2 cxcR3 CXCRS CCR6 CCR7 CCRl ccR3 ccR2 CCRS CCR4 CCRB CX,CRl ccR9 XCRl

FIG.3. Structure similarity diagram of human chemokine receptors. Similarity scores were determined as described for Fig. 1. GeneBank accession numbers for the sequences are (from top to bottom): X71635, M68932, M73969, X95876, X68149, U45984, L08176, L10918, U51241, U03905, X91492, X85740, U62556, U20350, Y12815, L36149.

locally. The arrest precedes the migration into tissues across the microvessel wall in response to a chemoattractant concentration gradient. The selectivity of lymphocyte recruitment depends mainly on the locally confined production of chemokines in the tissues and the expression of appropriate chemokine receptors on the responding cells (Baggiolini, 1998). T lymphocytes were initially considered as poor targets for chemokines, and early studies suggested that only memory and activated T cells were responsive. In vitro chemotaxis was observed in response to inflammatory chemokines, namely RANTES, MIP-la, MIP-1P, and the MCPs (Schall et al., 1990; Carr et al., 1994; Loetscher et al., 1994a; Roth et al., 1995; Uguccioni et al., 1996; Qin et al., 1996) acting via CCRl and CCRB (Loetscher et al., 1996b). Using memory, CD45RO' blood lymphocytes cultured under different stimulatory conditions, it was then shown that the expression of both receptors depends strictly on stimulation with IL2 (Loetscher et al., 199613).IL-4, IL-10, and IL-12 have similar but weaker effects, whereas IL-13, IFNg, IL-1P, and TNFa are ineffective. Other stimulatory regimens, in particular treatment with anti-CD3 alone or in combination with anti-CD28, rapidly down-regulate receptor expression and migration (Loetscher et al., 1996b). Subsequent studies showed that the expression of CXCR3, CCR5 and C&CRl is also regulated by IL-2 (Imai et al., 1997a; Loetscher et al., 1996a; Bleul et al., 1997; Loetscher et al., 1998a; Qin et al., 1998; Cole et al., 1998; Wu et al., 1997a; Trkola et al., 1996) and that CXCR3 and CCR5 are down-regulated by treatment

TABLE 111 CHEMOKINE RECEETORS IN T AND B LYMPHOCYTES Receptor

Expression"

CXCR3" CXCR4d

I L 2 activated T cells' (Thl > Th2) Resting and activated T cells B cells

CXCR5

B cells T cell subsete

CCRl" CCR2" CCR3

I L 2 activated T cells I L 2 activated T cells" Th2 cells

CCR4

Th2 cells aCDWaCD28-activated Thl cells I L 2 activated T cells (preferentially Thl cells)

CCR5b CCRQ CCR7g CCR8

Resting T cells B cells Resting and activated T cells, B cells aCD3/aCD28-activatedTh2 cells

References 1998; Cole et d.,1998 Loetscher et d.,1996a, 1998a,b; Qin et d., Bled et al., 1997; Forster et al., 1998; Bermejo et al., 1998; Mo et d., 1998; Berkowitz et al., 1998; Jourdan et al., 1998; Hori et al., 1998; Vincenti-Manzanares et d.,1998 Forster et al., 1994 Loetscher et d., 1996b Loetscher et al., 199613; Qin et al., 1996 Sallusto et d.,1997; Gerber et d.,1997; Bonecchi et al., 1998; Andrew et al., 1998 Bonecchi et al., 1998; Sallusto et al., 199813; D'Ambrosio et d.,1998; Imai etd., 1999 1997a; Bonecchi et al., 1998; Bled et al., 1997; Qin et al., 1998; Wu et d., Loetscher et al., 1998b; Sallusto et al., 1998b Baba et al., 1997; Liao et al., 1999 Willimann et al., 1998; Yoshida et al., 1998b Zineoni et al., 1998; D'Ambrosio et d.,1998

' Determined by Northern blot analpis and/or flow cytometry. Wliere apprupriate, conditions for expression are indicated.

* Phenotype: CD25+,CD%w, CD45ROt.

Down-regulated by treatment with aCD3 in the presence or absence of crCD28. Present in nave and memoly T cells. ' Phenotype: CD4+ > CDV, CD25-, CD44', CD62L-, CD45RO+. Effect of I L 2 is controversial. g Transient up-regulation by IL-2, long-lasting upregulation by PHA.

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with anti-CD3ICD28 (Loetscher et al., 1998a). After antigen recognition on antigen-presenting cells in secondary lymphoid organs, naive or memory T lymphocytes develop into effector cells (see Section 1II.B). During IL2-dependent clonal expansion, they up-regulate receptors for inflammatory chemokines and can thus be recruited to sites of immune intervention. The effect of IL-2 on CCRG expression is unclear. It was reported that IL-2 up-regulates CCRG mRNA in CD4+ and CD8' T lymphocytes (Baba et al., 1997),but these findings were not confirmed in a more recent study (Liao et al., 1999) showing that CCRG is present on resting memory blood T cells (CD45RO' and CD26') and that activation by IL-2 or anti-CD3 in vitro does not increase mRNA and surface expression. CCRG is expressed in 15-40% of CD4' and up to 14% of CD8' blood T cells, and in virtually all a4p7 memory cells and CIA+ cells (Liao et al., 1999). The CCRG selective chemokine ligand LARC was shown to induce [Ca2+Iichanges, chemotaxis and adhesion in freshly isolated blood lymphocytes (Liao et al., 1999; Hieshima et al., 1997a; Campbell et al., 1998a). These observations suggest that LARC may be important for immune surveillance in nonlymphoid tissues and at early stages of inflammation for the recruitment of T cells to sites of antigen recognition (Liao et al., 1999). It was shown that LARC is induced by TNF in endothelial cells (Hromas et al., 1997a) and is expressed in the lymph nodes and the appendix (Baba et al., 1997; Hromas et al., 1997a; Rossi et al., 1997). Cellular contact, which is required for recognition of antigen presented by dendritic cells, may alsobe influenced by chemokines. PARC or DC-CK1 (Adema et al., 1997; Hieshima et al., 1997b), a CC chemokine that is expressed constitutively by dendritic cells in the germinal centers and the T cell areas of secondary lymphoid organs, may have such a function. The receptor involved, however, is unknown. CXCR4 is expressed in naive and in memory T lymphocytes which readily respond to its unique ligand, SDF-1 (Bleul et al., 1996b, 1997; Campbell et al., 1998a; Forster et al., 1998; Bermejo et al., 1998; Mo et al., 1998; Berkowitz et al., 1998). Some reports suggested that CXCR4 is expressed preferentially in naive cells (Bleul et al., 1997; Forster et al., 1998), whereas no difference between naive and memory cells was found in other studies (Bermejo et al., 1998; Mo et al., 1998; Berkowitz et al., 1998).A considerable variability of CXCR4 surface expressionwas observed in blood lymphocytes (Forster et al., 1998; Bermejo et al., 1998; Jourdan et al., 1998; Hori et al., 1998). Bermejo et al. (1998) showed that CXCR4 is largely internalized in naive and memory T cells and is readily translocated to the surface after culturing for a few hours. Rapid receptor internalization is observed after exposure of CXCRCbearing cells to SDF-1 and activation with PMA, PHA, anti-CD3, or anti-CD3KD28 (Forster et al., 1998; Bermejo et al., 1998; Jourdan et al., 1998;Amara et al., 1997; Peacock

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and Jirik, 1999; Loetscher et al., 1994b). On the other hand, treatment with PHA and anti-CD3KD28 or exposure to IL-2 were also reported to increase expression (Bleul et al., 1997; Carroll et al., 1997). The ligandinduced internalizationof CXCR4 is independent of signalingby B.pertussis toxin-sensitive G-proteins but is prevented when the COOH-terminal receptor domain is deleted (Forster et al., 1998; Amara et al., 1997). Although CXCR4 is expressed constitutively in several types of leukocytes, up-regulation is induced by IL-4, and down-regulation,by IFNy (Jourdan et ul., 1998; Galli et al., 1998).SDF-1, the only known ligand for CXCR4, has a broad range of activities (Baggiolini,1998).Its involvement in lymphocyte migration and distribution is suggested by studies with transgenic mice expressing human CD4 and CXCR4. Overexpression of CXCR4 led to a marked decrease of circulating CD4' T cells and a concomitant accumulation in the bone marrow where high levels of SDF-1 are produced (Sawada et al., 1998). CCR7 is another receptor with homeostatic rather than inflammatory functions (Willimann et al., 1998; Gunn et al., 199813; Ngo et al., 1998; Campbell et al., 199813; Nagira et al., 1998; Yoshida et al., 199813; Kim et al., 1998~). It is present at low levels in resting naive and memory blood T lymphocytes and is up-regulated rapidly by stimulation with IL-2 and/ or PHA, which also enhances migration responses to its ligands, SLC and ELC (Willimann et al., 1998; Yoshida et al., 1998b). IL-2 has a transient effect, whereas PHA has a more protracted effect. As shown by in situ hybridization, SLC and ELC are constitutively expressed in the interfollicular, T cell-richareas of secondarylymphoid tissues, presumably by dendritic cells, but are absent in the follicles and germinal centers (Willimann et al., 1998; Gunn et al., 199813; Ngo et al., 1998; Nagira et al., 1998; Yoshida et al., 199813). In addition, murine SLC is expressed in high-endothelial venules of lymph nodes and Peyer's patches (Gunn et al., 199813). It was shown recently that SLC and ELC induce integrin-dependent adhesion of naive lymphoyctes under flow conditions by a B. pertussis toxin-sensitive pathway (Campbell et al., 1998a; Gunn et al., 199813; Pachynski et al., 1998; Tangemann et al., 1998). The rapid and transient up-regulation of CCR7 in T lymphocytes after activation suggests that SLC and ELC may regulate lymphocyte migration into and within T cell areas of lymph nodes and lymphoid organs in general (Willimann et al., 1998). CCR7 may be expressed in T lymphocytes that are engaged in antigen recognition and are exposed to IL-2. In this way antigen-specific cells may be retained in the proper activation and proliferation environment by locally produced SLC and/or ELC. Chemotactic retention will eventually cease because CCR7 is expressed transiently, and clonally expanded effector cells will then be able to re-enter the circulation.

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Recent work indicates that SLC and ELC are involved in the recruitment of naive T cells and dendritic cells to secondary lymphoid tissues (Nakano et al., 1998; Gunn et al., 1999). Nakano et al. (1998) described a mouse with defective T cell homing into lymph nodes, Peyer’s patches, and the spleen as a consequence of an autosomal recessive gene defect termed plt (paucity of lymph node T cells). The plt gene is localized in a region of mouse chromosome 4 corresponding to the p locus of human chromosome 9 ( 9 ~ 1 3 which ) harbors the SLC and ELC genes (Nagira et al., 1997; Yoshida et al., 1997). It has been shown most recently that the SLC gene maps to the plt locus and that plt mice do not express SLC and only low levels of ELC (Gunn et al., 1999). The SLC gene of these mice, however, is normal, and the lack of expression may depend on a defect of a distant regulatory region, although the study does not exclude that the plt phenotype involves more than one gene defect. There is evidence for a role of SLC in the homing of dendritic cells to secondary lymphoid organs (Gunn et al., 1999). A study in mice by Ngo et al. (1999) suggests that the expression of SLC, ELC, and BLC in the spleen depends on TNF and lymphotoxin because the production of all three chemokines is markedly decreased in mice that are deficient in TNF, TNFR1, lymphotoxin-a, or lymphotoxin-p.

B. Thl AND Th2 CELLS T lymphocyte-dependent immune responses rely on helper cells of Thl and Th2 phenotype (Mosmann and Coffmann, 1989; Romagnani, 1994; Paul and Seder, 1994;Abbas et al., 1996). Both lineages arise from common, naive precursors during antigen priming, and their development is conditioned by the cytokines that are released at the site of antigen recognition. Thl and Th2 cells are characterized by the cytokines that they produce. Thl cells secrete mainly IFNg and lymphotoxin and foster cell-mediated immunity against intracellular pathogens, whereas Th2 cells secrete IL-4 and IL-5 and regulate allergic responses and humoral immunity against parasites. In view of their distinct effector functions, it was natural to assume that Thl and Th2 cells can be recruited selectively to diseased tissues. The mechanism of this selectivity remained unclear until recently when it was realized that the two types of helper cells express different chemokine receptors and respond to distinct sets of chemokines. Helper cells of either phenotype can be generated in vitro from cord blood T cells that are stimulated with PHA and cultured in the presence of IL-12 yielding Thl cells or IL-4 yielding Th2 cells. The process is called polarization and is facilitated by the addition of neutralizing antibodies against IL4 or IL-12 to maximize the desired effect (Rogge et al., 1997). A similar type of polarization is obtained with naive blood T lymphocytes.

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It was first shown that the chemokine receptor CCR3, which is selective for the eotaxins and was originally identified on eosinophils and basophils (Ponath et al., 1996; Daugherty et al., 1996; Uguccioni et al., 1997; Forssmann et al., 1997), is expressed on a small percentage of blood T lymphocytes. Sallusto et al. (1997) showed that T cell lines generated from CCR3bearing lymphocytes produce predominantly Th2-type cytokines, and that Th2 cells obtained from cord blood by polarization express high levels of CCRS indicating that this receptor is a Th2 marker. We showed that CCRS is expressed in allergen-specific T cell clones with a Th2 phenotype, and that CCR3-positive T cells are recruited together with eosinophils to sites of allergic inflammation (Gerber et al., 1997). The levels of CCRS expression in Th2 cells and the consequent responsiveness to eotaxin were shown in several studies to be moderate and variable suggesting that CCR3bearing cells may constitute a Th2 subpopulation (Gerber et al., 1997; Bonecchi et al., 1998; Loetscher et al., 1998b;Andrew et al., 1998,Annunziat0 et al., 1998). Other studies showed that Th2 cells also express CCR4 and CCR8 (Bonecchi et al., 1998; Sallusto et al., 199813; Zingoni et al., 1998; D’Ambrosio et al., 1998; Imai et al., 1999). Activation of Th2 cells with anti-CD3 and anti-CD28 leads to a transient up-regulation of both receptors, by a process that does not involve IL-4, the cytokine that is needed for polarization toward Th2 (D’Ambrosioet al., 1998). In contrast to CCR8, which is present only on activated Th2 cells, CCR4 appears to be expressed constitutively and is also found in activated T h l cells (D’Ambrosio et al., 1998). CCR4 binds TARC and MDC (Imai et al., 199%; Godiska et al., 1997), and CCR8 binds I309 (Roos et al., 1997; Tiffanyet al., 1997).All these ligands induce [Ca2+],changes and chemotaxis in Th2 cells indicating that both receptors are functional. The transient up-regulation after T cell receptor engagement suggests that CCR4 and CCR8 are involved in the control of T cell migration at sites of antigen recognition, as formerly suggested for CCR7 (Willimann et al., 1998). According to several reports, Thl cells are characterized by the preferential expression of CCR5 (Bonecchi et al., 1998; Loetscher et al., 199813; Annunziato et al., 1998; Siveke and Hamann, 1998). It has been shown, however, that CCR5 is present in both Thl and Th2 cells obtained from cord blood by polarization (Sallusto et al., 1998b), while cloned Th2 cells are normally CCR5-negative (Loetscher et al., 1998b; Sallustoet al., 199813). Thl cells also express high levels of CXCR3, the receptor for IP10, Mig, and I-TAC (Bleul et al., 1997; Loetscheret al., 1998a; Qin et al., 1998;Cole et al., 1998).We and others have found that CXCR3 is highly expressed in both types of helper cells after expansion with IL-2 (Loetscher et al., 1998b; Andrew et al., 1998; Annunziato et al., 1998) although preferential expression in Thl cells has been reported as well (Bonecchi et al., 1998;

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Sallusto et al., 199813).The lymphocyte infiltrates in the pannus of rheumatoid arthritis joints, which are rich in Thl cells and are strongly positive for CCR5 and CXCR3, whereas CCR3 is virtually absent (Qin et al., 1998; Loetscher et al., 199813). Polarization experiments showed that transforming growth factor P (TGFP),which maintains T cells in a semi-naive state (Sad and Mosmann, 1994), enhances the expression of CCR4 and CCR7, and decreases the expression of CCRS (Sallusto et al., 1998b). Down-regulation of CCRS is also observed with INFa which induces polarization toward T h l and upregulates CCRl and CXCR3 (Sallusto et al., 199813). CD8’ T cells can differentiate into two distinct subsets of cytolytic effector cells, Tcl and Tc2, with characteristic cytokine production patterns (Sad et al., 1995). Tcl secrete cytokines that are typical for T h l cells, including INFg and IL-2, whereas Tc2 cells produce Th2-type cytokines including IL-4, IL-5, and IL-10. Polarized Tcl and Tc2 cells derived from cord blood are similar to T h l and Th2 cells in terms of pattern and regulation of chemokine receptor expression and responsiveness to chemokines. As shown by D’Ambrosio et al. (1998) transcripts for CCR5 and CXCR3 are characteristic of the Tcl and transcripts for CCR3, CCR4, and CCR8 of the Tc2 phenotype. C. B LYMPHOCYTES After maturation in the bone marrow, B lymphocytes home into secondary lymphoid tissues where they are soon released into the circulation or move through different areas while undergoing final differentiation (Goodnow and Cyster, 1997).The involvement of chemokines in the migration and recruitment of B cells has been recognized only recently. It was first observed that a heptahelical, putative chemokine receptor termed BLRl (Dobner et al., 1992) or MDR15 (Barellaet al., 1995) was expressed prominently in mature circulating B lymphocytes as well as in Burkitt’s lymphoma cells (Forster et al., 1994). Deletion of the BLRl gene in mice yielded a remarkable phentoype: The animals lacked inguinal lymph nodes and showed a defective formation of primary follicles and germinal centers in the spleen and the Peyer’s patches (Forster et al., 1996). Receptordeficient B lymphocytes enter T cell areas but do not migrate further suggesting that BLRl is needed for the development of the B cell areas in the lymphoid tissues. The ligand for BLRl was identified as a novel CXC chemokine, and the receptor was renamed CXCR5. The human ligand was termed B cell attracting chemokine 1 (BCA-1) (Legler et al., 1998) and the murine ligand B lymphocyte chemokine (BLC) (Gunn et al., 1998a). In both species, BCA-1/BLC is the only chemokine that binds to CXCR5. As shown by immunohistochemistry and in situ hybridization,

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BCA-UBLC is expressed constitutively in the follicles of Peyer’s patches, spleen, and lymph nodes, indicating that this chemokine functions as the attractant of B cells from the blood into the proper areas of the lymphoid tissues. Ngo et al. (1999) have recently shown that mice with a deletion of the TNF, TNFR1, or lymphotoxin gene have decreased BLC expression in follicular stromal cells of the spleen, which is in agreement with the role for BLC and CXCR5 in the homing of B cells. It is interesting that CXCR5 is absent in immature B cells, is expressed when maturation is completed and the cells are ready to leave the bone marrow, and is downregulated again after terminal differentiation into IgG secreting B cells (Forster et al., 1994). Analysis of the CXCR5 promoter region showed that gene expression is cooperatively regulated by the transcription factors Oct-2, Bobl, and NF-kB (Wolf et al., 1998). This conclusion is supported by the study of mice with targeted deletion of Oct-2, Bobl, or p50/p52 NF-kB, which lead to a severe impairment of CXCR5 expression (Wolf et al., 1998). In vitro assays with human blood-derived B lymphocytes show that BCA-1 is a chemoattractant of high efficacy but relatively low potency. Chemotaxis and [Caz+],changes are also observed in the murine pre-B cells 300-19 transfected with human CXCR5 (Legler et al., 1998). In addition, CXCR5 expression and responsiveness to BCA-1 was a observed in a small subset of T lymphocytes (Forster et al., 1994). Another receptor that is prominently expressed in B lymphocytes is CCR7 which was identified several years ago in cells infected with EpsteinBarr virus (EBV) and termed EBI-1 (Birkenbach et al., 1993). Resting B lymphocytes migrate in response to the two known ligands for CCR7, SLC, and ELC (Gunn et al., 1998b; Ngo et al., 1998; Nagira et al., 1998; Kim et al., 1998c; Hromas et al., 199713). CCR7 is up-regulated in B lymphocytes by EBV infection as shown initially (Birkenbach et al., 1993; Burgstahler et al., 1995; Schweickart et al., 1994) but also by stimulation with LPS or pokeweed mitogen and activation via the B cell receptor, which enhance the chemotactic responsiveness to SLC and ELC (Ngo et al., 1998; Nagira et al., 1998). A correlation between CCR7 expression and B lymphocyte migration in response to ELC was reported by Yoshida et al. (1998b). It can be assumed that B lymphocytes are attracted from the blood into secondary lymphoid tissues, across high-endothelialvenules, by SLC and ELC which are generated in the T cell zone. The observed enhancement of B lymphocyte responsiveness after activation (Ngo et al., 1998; Nagira et al., 1998) could represent a mechanism to retain antigenengaged B cells at functionally relevant sites. B lymphocytes also express CXCR4. Its ligand, SDF-1 (Tashiro et al., 1993),was originally reported to act in combination with IL-7 as a growth factor for B lymphocyte precursors in the bone marrow and was termed

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a PBSF (Nagasawa et al., 1994). Unlike CXCR5 and CCR7, CXCR4 is expressed in a variety of other cell types (Baggiolini et al., 1997; Bleul et al., 1997; Forster et al., 1998; Hori et al., 1998).The importance of CXCR4 and SDF-1 for the generation and maturation of B lymphocytes is emphasized by studies in mice with a deleted receptor or chemokine gene, which have a severely impaired lymphopoiesis and abnormally low numbers of circulating B cells (Nagasawa et al., 1996a; Tachibana et al., 1998; Zou et al., 1998; Ma et al., 1998).The deletion of either gene resulted in defective angiogenesis, incomplete heart septum closure, and disturbed neuronal development in the cerebellum, indicating that SDF-1 may affect embryonal development. SDF-1 could be involved in morphogenesis by attracting or keeping together cells that form a functional unit (Baggiolini, 1998). Such activities may be shared by other, yet unknown chemokines. SDF-1 is a strong attractant for blood B lymphocytes (Frade et al., 1997; Vicente-Manzanares et al., 1998). Work with tonsillar B cells by Bled et al. (1998) indicates that the local environment, the state of differentiation as well as B cell receptor signaling affect the expression of CXCR4 and responsiveness to SDF-1. Naive and memory B lymphocytes (CD38CD44+IgD+ and CD38-CD44+IgD- cells, respectively), which reside outside germinal centers, are CXCR4-positive and respond to SDF-1 as shown by chemotaxis and actin polymerization. Migration responses, however, are decreased after activation of the B cell receptor and CD40. Similarly, B cells within germinal centers, which undergo activation and affinity maturation in viuo, express CXCR4, but do not respond to SDF1. Responsiveness, however, is restored upon in vitro differentiation into memory cells. In the tonsils, SDF-1 is expressed by reticular cells around germinal centers (Bleul et al., 1998).The lack of responsiveness by germinal center B lymphocytes may be a mechanism to prevent their emigration before maturation is complete. CCR2 is expressed on resting and activated B lymphocytes as assessed by flow cytometry, but chemotactic responses to MCP-1 were not reproducibly observed (Frade et al., 1997; Vicente-Manzanares et al., 1998). Circulating B lymphocytes express CCR6 but do not respond to LARC (Baba et al., 1997; Liao et al., 1999). It has also been reported that B cells respond to MIP1-a (Kim et al., 1998c; Schall et al., 1993) implying that they express CCRl and/or CCR5. D. NATURAL KILLERCELLS Together with granulocytes and mononuclear phagocytes, natural killer (NK) cells are important effectors of innate immunity owing to their cytolytic activity toward target cells that are recognized as foreign or modified, and their ability to produce considerable amounts of cytokines. The NK

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cells are found primarily in the blood and the spleen but can also occur in nonlymphoid tissues including the lung, the intestinal mucosa, and the liver. As defense cells, they are recruited into target areas, in particular to sites of viral infection (Trinchieri, 1989). It is generally assumed that this process and the traffic of NK cells are regulated by chemokines, but the information about the expression of chemokine receptors and its regulation in NK cells is scarce (Maghazachi and Al-Aoukaty, 1998). It is known that many chemokines, including the MCPs, RANTES, MIPla, MIP-1P, LARC, ELC, MDC, lymphotactin, fractalkine, IPlO and SDF-1 induce NK cell migration (Imai et al., 199%; Godiska et al., 1997; Maghazachi et al., 1994; Allavena et al., 1994; Taub et al., 1995; Loetscher et al., 1996c; Bianchi et al., 1996; Maghazachi et al., 1997; Hedrick et al., 1997; Maghazachi 1997; Al-Aoukaty et al., 1998). In addition, several chemokines were shown to induce [CaZ+lichanges (Loetscher et al., 1996c; Maghazachi et al., 1997; Maghazachi, 1997), exocytosis of granzyme A, and N-acetyl-P-glucosaminidase (Loetscher et al., 1996b) and to enhance target cell lysis by blood-derived NK cells (Taub et al., 1995). As in T lymphocytes (Loetscher et al., 1996b), conditioning of NK cells with IL2 enhances the expression of CCR2 and CCR5 resulting in stronger responsiveness to MCP-1, RANTES, MIP-la, and MIP-1P (Polentarutti et al., 1997, Nieto et al., 1998). Of interest is the observation that circulating NK cells, as defined by CD16 positivity, express C&CR1 constitutively and show migration and increased adhesion in vitro after stimulation with the chemokine-like moiety of fractalkine (Imai et al., 1997a). Recent studies document the potential role of chemokines in the regulation of NK cell migration in vivo. Salazar-Mather et al. (1998) have shown that MIP-la attracts NK cells to the liver in cytomegalovirus-infectedmice. MIP-la deficiency results in a decreased inflammatory reaction and increased susceptibility to the infection. Furthermore, intraperitoneal administration of lymphotactin in mice was shown to enhance NK cell accumulation, a response that was inhibited by lymphotactin-neutralizing antibodies (Hedrick et al., 1997). E. LYMPHOCYTE PROGENITORS T lymphocytes, B lymphocytes, and NK cells are believed to originate from common progenitor cells (Shortman and Wu, 1996).Their proliferation and differentiation into mature, naive cells takes place in specific areas of the bone marrow for B lymphocytes and the thymus for T lymphocytes, whereas the site of maturation of NK cells is unknown. There is multiple evidence suggesting that chemokines regulate the migration of hematopoietic progenitor cells including those of lymphocpc lineage to the appropriate tissue compartments. The involvement of SDF-1 and CXCR4 is

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indicated by the severe impairment of lymphopoiesis in the bone marrow and the liver of mice with a deletion of the corresponding genes (Nagasawa et al., 1996a; Tachibana et al., 1998; Zou et al., 1998; Ma et al., 1998). Pro- and pre-B cells depend on close contact with bone marrow stromal cells for growth and differentiation. We studied responses of B cell precursors at different stages of maturation and found that SDF-1 is chemotactic for early precursors but not for the more mature forms (D'Apuzzo et al., 1997). Owing to this activity, SDF-1 could attract progenitor B cells into the vicinity of stromal cells from where it originates and which are the source of growth and differentiation factors (D'Apuzzo et al., 1997). SDF1is chemotactic for CD34' hematopoietic progenitors of different lineages which all express CXCR4 (Berkowitz et al., 1998; Aiuti et al., 1997; Deichmann et al., 1997; Mohle et al., 1998; Ruiz et al., 1998), whereas ELC attracts preferentially CD34+ cells that are restricted to the macrophage lineage and express CCR7 (Kim et al., 1998a). Common lymphoid progenitors from the bone marrow settle in the thymus where they mature to functionally competent T lymphoyctes. During this process, the maturing cells migrate within the thymus and home to different anatomic sites depending on their stage of differentiation (Boyd et al., 1993). The outer and the inner areas of the cortex are populated by the most immature, double-negative (CD4-CD8-) and by the more developed, double-positive (CD4+CD8+)cells, respectively, whereas mature, single-positive(CD4+or CD8') thymocytes are found in the medulla. Recent studies have shown that thymocyte subsets representing discrete stages of development differ in their expression of CXCR4, CCR4, CCR5 and CCR7, suggesting that the migration steps required for maturation are controlled by different chemokines (Berkowitz et al., 1998; Ngo et al., 1998; Moepps et al., 1997; Kim et al., 1998b; Kitchen and Zack, 1997; Dairaghi et al., 1998; Zaitseva et al., 1998; Zhang et al., 1998; PedrozaMartins et al., 1998; Campbell and Butcher 1999). Immature thymocytes of double-negative or double-positive phenotype express higher levels of CXCR4 than the mature, single-positive cells both in humans (Berkowitz et al., 1998; Kitchen and Zack, 1997; Zaitseva et al., 1998; Pedroza-Martins et al., 1998) and in mice (Moepps et al., 1997; Kim et al., 1998b) and respond to SDF-1 as shown by Zaitseva et al. (1998) for human thymocytes. Mature, single-positive thymocytes express CCR7 and respond to ELC and SLC (Ngo et al., 1998; Kim et al., 1998b). These observations indicate that SDF-1 acting via CXCR4 may be important for the distribution and retention of immature thymocytes in the cortex, while thymocyte migration within the medulla and their egress into the blood and their homing into secondary lymphoid tissues may depend on ELC and SLC acting via CCR7.

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In addition to SDF-1 and ELC, other chemokines including TECK (Vicari et al., 1997), MDC (Godiska et al., 1997; Chang et al., 1997),TARC (Imai et al., 1996) and LARC (Hieshima et al., 1997a; Hromas et al., 1997a; Rossi et al., 1997; Tanabe et al., 1997a) appear to be involved in the regulation of the intrathymic traffic of the maturing cells. TECK which attracts thymocytes, activated macrophages, and dendritic cells is of particular interest because it is mainly confined to the thymus where it is expressed by medullary dendritic cells (Vicari et al., 1997). Its receptor is still unknown, but it has been shown that thymocyte responses to TECK are inhibited by B. pertussis toxin. The present evidence suggests that TECK recruits immature thymocytes into the medulla for the final stages of maturation via a G-protein coupled receptor. Campbell and Butcher (1999) have shown most recently in mice that TECK attracts immature thymocytes, and that this activity is lost when the cells begin to express L-selectin which is important for homing into peripheral lymphoid tissues. It was also suggested that TECK may be implicated in the deletion of autoreactive cells due to its capacity to corecruit dendritic cells, macrophages, and thymocytes (Vicari et al., 1997). Thymic dendritic cells are believed to be involved in the negative selection of thymocytes (Inaba et al., 1991), and macrophages could scavenge cells undergoing apoptosis. The role of CCR5 in thymocytes is less clear. It was reported that CCR5 is expressed at low levels in most thymocytes and is down-regulated upon maturation, but no CCR5-dependent function such as [Ca2+],changes and chemotaxis has been observed (Zaitseva et al., 1998). Other studies show that CCR5 is moderately expressed in double-positive and CD8' singlepositive thymocytes (Berkowitz et d., 1998; Dairaghi et d., 1998).

N. Chemokine-Mediated Signal Transduction Signalingof chemokines via heptahelical receptors coupled to B. pertussis toxin-sensitive G-proteins has been studied most extensively in neutro-

phils and cell lines transfected with the IL-8 receptors, CXCR1, or CXCRB (Baggiolini et al., 1997). Binding of IL-8 to CXCRl initiates a cascade of events including activation of phosphatidylinositol-specific phospholipase C, protein kinase C, small GTPases such as RhoA, MAP kinases, Srcrelated tyrosine kinases, phosphatidylinositol-3-OH hnase (P13K), phospholipase D, and protein kinase B (Baggioliniet al., 1994, 1997; Baggiolini, 1998). Phospholipase C generates two second messengers, inositol-1, 4, 5-trisphosphate which induces the release of Ca2' from intracellular stores leading to a transient rise of [Ca"],, and diacylglycerol which activates protein kinase C. The [Ca"], rise is essential for granule release and the respiratory burst but is not required for the cytoskeletal rearrangements

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leading to shape change and cellular motion (Baggiolini et al., 1994). Although the basic mechansims of signaling by eukaryotic chemoattractant receptors are very similar, subtle differences may be found in relation to specific functions of chemokines. It has been shown, for instance, that superoxide production in neutrophils is induced by stimulation via CXCR1, but not via CXCR2, and that this may reflect the activation of phospholipase D which depends on ligation of CXCRl (L'Heureux et al., 1995;Jones et al., 1996). More pronounced differences in signaling may be found eventually between chemokines with homeostatic and chemokines with inflammatory activities. Experiments with HEK cells transfected with CXCR2 (Neptune and Bourne, 1997) and mouse lymphoid cells transfected with CCR2 (Arai et al., 1997) have shown that the G-protein & subunit which is released after receptor activation by IL-8 or MCP-1 mediates signals leading to chemotactic migration. The duration of the response may be modulated by regulators of G-protein signaling (RGS) which enhance the intrinsic GTPase activity of G a ,and thus induce the reassembly of Gai,and G&, (Kehrl, 1998).These novel proteins were shown to inhibit the migration of cells bearing CXCRl orCCR2inresponse to IL-8or MCP-1 (Bowmanetal., 1998)and to decrease IL-8 dependent MAP kinase activation in cells expressing CXCRl (Druey et al., 1996). Duration and extent of signaling can also be influenced byphosphorylation-dependent receptor desensitization and by receptor internalization. On phosphorylation of serines and threonines in the COOH-terminal region by receptor kinases, arrestins can bind and prevent further G-protein coupling, resulting in receptor uptake (Aramori et al., 1997; Aragay et al., 1998). The importance of the duration of signaling is indicated by experiments with rat basophilic leukemia cells transfected with wild-type or COOH-terminally truncated CXCR2 (Richardson et al., 1998). Although both receptors mediated chemotaxis and exocytosis, the truncated form was not internalized leading to protracted signaling and to responses, such as the activation of phospholipase D, that are normally not mediated by CXCR2 (Jones et al., 1996; Richardson et al., 1998). Chemokine-dependent signaling in lymphocytes is poorly understood, and the information on various transduction pathways is both intricate and fragmentary (Ward et al., 1998). Most studies were performed with RANTES for which two types of signaling and activation mechanisms were described in T lymphocytes. According to Bacon et al. (1995), RANTES signals via G-protein-coupled receptors at nanomolar concentrations, and via a pathway involving tyrosine kinases at micromolar concentrations. Phosphorylation and activation of the protein tyrosine kinases ZAP-70, p125FAK, and Pyk-Wyk-2H were described after T cell stimulation with RANTES or MIP-lP (Bacon et al., 1996; Davis et al., 1997; Dikic and

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Schlessinger, 1998; Ganju et al., 1998a). The focal adhesion kinases p125FAK and Pyk-2 were shown to convey signals to the cytoskeleton and the nucleus (Schlaepfer and Hunter, 1998). Phosphorylation of Pyk-2 was observedin the mouse pre-B lymphomacell line L1.2 transfectedwith CCR5 after stimulation with MIP-1P. This was followed by activation of the c-Jun N-terminal kinase ( JNK), stress-activated protein kinase (SAPK) and p38 MAP kinase, and the phosphorylation ofpaxillin,a major cytoskeletalcomponent of focal adhesions, and its association with Pyk-2 (Ganju et al., 1998a). The association of paxillin with p125FAK and ZAP-70 was observed after treatment of T cell clones with micromolar concentrations of RANTES (Baconetal., 1996).Furthermore,inrecentstudiesonTcelllinesandCCR5transfectedcells, RANTES and MIP-la were shown to induce tyrosine phosphorylation and activation of STAT1 and STAT3 (Wong and Fish, 1998) as well as JAKUSTAT5 (Rodriguez-Frade et al., 1999). It is well established that phosphatidylinositol 3-kinases are involved in chemokine mediated signaling, and various leukocyte responses are believed to be triggered by their lipid products (Turner et d.,1995; Thelen et al., 1995; Tilton et al., 1997; K n d et al., 1997; Ganju et al., 1998b; Turner et al., 1998). Different PI3K isoforms are activated by interaction with the GPy complex, small GTPases, Src-related tyrosine kinases, and phosphotyrosine binding to the SH2 domain of PI3K (Bokoch, 1995;Vanhaesebroeck et al., 1997). In T cells, RANTES induces PI3K activity and the selective PI3K inhibitor wortmannin blocks cytoskeletal rearrangement and chemotaxis (Turner et al., 1995). PI3K activation may be linked to the GTPase-dependent stimulation of phospholipase D (Bokoch, 1995). RANTES was recently shown to activate phospholipase D in Jurkat cells via the GTP-binding proteins ARF and RhoA (Bacon et al., 1998).Phosphorylation of Pyk-2, p42/44 MAP kinases and MEK kinases was observed in T cells (Jourdan et al., 1998; Davis et al., 1997; Popik et al., 1998) and in the CD34' progenitor CTS cell line (Dutt et al., 1998) after stimulation with SDF-1. In CTS cells (Dutt et al., 1998) and the murine pre-B cell line L1.2 (Ganju et al., 1998b) phosphorylation and association of focal adhesion components was induced by SDF-1, which also activated PISK, p42J44 MAP-kinases and MEK-kinases (Ganju et al., 1998b). Signaling by SDF-1 also appears to lead to gene expression as suggested by the activation of NF-kB, which is critically involved in HIV replication (Ganju et al., 1998b),and the up-regulation of membrane-bound TNFa in macrophages and TNFR2 in CD8+ T cells (Herbein et al., 1998) (see Section V.C). V. HIV Infection

A. HIV CORECEFTORS AND MECHANISM OF VIRAL ENTRY It had been known for about 10 years that CD8+ T cells release factors that suppress HIV infection (Walker et al., 1996) when, at the end of 1995,

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Cocchi et al. (1995) showed that the chemokines RANTES, MIP-la, and MIP-lp produced by CD8+ T cell lines have such effects. These observations suggested that chemokines may influence HIV infection and progression to AIDS. A few months later, Feng et al. (1996) reported that a putative chemokine receptor complements CD4 in a cell fusion model of T lymphocyte (T)-tropic HIV-1 infection. The coreceptor was then found to bind SDF-1 and was termed CXCR4 (Oberlin et al., 1996; Bleul et al., 1996a). SDF-1 is a potent inhibitor of infection by T-tropic HIV-1 strains in cell lines and blood lymphocytes. The HIV-suppressive factors RANTES, MIP-la, and MIP-lp have a corresponding activity but on another coreceptor, CCR5, which mediates entry of macrophage (M)tropic HIV-1 strains (Dragic et al., 1996; Deng et al., 1996; Choe et al., 1996; Doranz et al., 1996; Alkhatib et al., 1996). Meanwhile, 12 additional chemokine or putative chemokine receptors were found to act as HIV coreceptors in vitro (Table IV). Most of the alternative coreceptors are selective for HIV/SIV strains that also bind to CCR5. They appear to recognize a more restricted repertoire of viral strains than CCR5 and CXCR4, and their role in HIV/SIV infection and disease progression has not been studied in depth. Viral tropism depends on coreceptor expression. Therefore, a new, more precise classification of HIV-1 strains has been introduced based on coreceptor usage (Berger et al., 1998). Strains that bind CCR5 are termed R5, and strains that bind CXCR4 are termed X4, whereas R5X4 is used for dual-tropic strains that can use either coreceptor. The great majority of primary isolates from asymptomatic HIV-positive individuals are R5 viruses with a non-syncytmm-inducing (NSI)phenotype, whereas most X4 viruses are syncytium-inducing (SI) and are frequent in patients with AIDS (Moore, 1997; Littman, 1998). The infection of cells by HIV or SIV particles begins with the interaction of the viral envelope glycoprotein (Env) with two cell surface proteins, CD4 and a chemokine receptor (Moore, 1997;Chan and Kim, 1998; Berson and Doms, 1998; Rucker and Doms, 1998). Env consists of two subunits, the surface-bound gpl20 and the transmembrane protein gp41, which arise from the proteolytic cleavage of a precursor, gp160. The larger subunit forms a trimeric complex by binding first to CD4 and then associating with a chemokine receptor. As a consequence, gp41 is exposed in close apposition to the target cell and induces the fusion between the viral envelope and the target cell membrane. A trimeric complex also forms when soluble gp120 is added to cells expressing CD4 and an appropriate chemokine receptor (Trkola et al., 1996; Lapham et al., 1996; Ugolini et al., 1997; Wu et al., 1996). Some gp120 proteins can bind to coreceptors even in the absence of CD4. The affinity is moderate but increases considerably when soluble CD4 is supplied presumably because additional coreceptor binding epitopes of gp120 are unmasked. The interaction of gp120

TABLE IV HIV/SIV CORECEFTORS Coreceptor

Ligands

C h k i n e receptors ccR2 MCPs

CCRS

CCR8

Eotaxin, Eota~in-2, RANTES, MIP-la RANTES, MIP-a, MIP-1P 1-309

CCRS CXCR4

CC chemokines SDF-1

C&CRl

Fractalkine

CCR5

Chenwattractant receptor BLTR LTBi Orphan receptors TYMSTW unknown BONZO/ STRL33 BOB/GPR15 unknown

Viral Tropism

References

HIV-1 (R5X4) HIV-2 HIV-1 (R5, R5X4) HIV-2 HIV-1 (R5, R5X4) HIV-2, SIV HIV-1 (R5, X4) HIV-2, SIV HIV-1 (R5X4) HIV-1 (X4, R5X4) HIV-2 HIV-1 (X4, R5X4) HIV-2

Choe et al.,1996; Doranz et al.,1996

HIV-1 (X4)

Owman et al., 1998

HIV-1 (R5, X4) HIV-2, SIV

Deng et al., 1997; Liao et al., 1997b; Loetscher et al., 1997; Edinger et al., 199813

HIV-1 (R5, X4) HIV-2, SIV

Albright et al., 1999; Deng et al., 1997; Edinger et al., 1998b; Farzan et al., 1997 Edinger et al.,199813; Farzan et al., 1997 Edinger, 1998a; Albright et al., 1999; Choe et al., 1998

GPRl APJ

unknown

SIV

unknown

Chem23

unknown

HIV-1 (R5X4) SIV HIV-1 (R5X4) SIV

Choe et aL, 1996; Doranz et al., 1996; Reeves et aZ., 1997 Dragic et al., 1996; Deng et al., 1996; Choe et d.,1996; Doranz et al., 1996; Akhatib et al., 1996; Simmons et al., 1996 H o d et d.,1998; Jinno et al., 1998; Rucker et al., 1997; Albright et al., 1999 Choe et al., 1998 Feng et al.,1996; Oberlin et al., 1996; Bled et al.,1996%Reeves et d.,1997; Simmons et al., 1996 Reeves et al., 1997; Rucker et al., 1997

Samson et al.. 1998

Viral receptor US28

CC chemokines

HIV-1 (R5, X4) HIV-2

Rucker et al., 1997; Pleskoff et al., 1997

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with CD4 occurs via invariant domains while hypervariable regions (V3 and to a lesser extent V1N2 loops) determine the binding of the gpl20CD4 complex to a coreceptor (Rizzuto et al., 1998). This interaction is complex and can include multiple extracellular coreceptor domains. The NHz-terminal region appears to be essential for CCR5 but not for CXCR4 which interacts with gpl2O via the first and second extracellular loop (Moore, 1997; Berson and Doms, 1998; Rucker and Doms, 1998). Marked strain-dependent variations are observed for the binding of gpl20 to the coreceptor domains, which may explain the observed differences in the HIV-suppressive activity of RANTES, MIP-la and MIP-1P (Moore, 1997; Berson and Doms, 1998; Rucker and Doms, 1998; Trkola et al., 1998).

B. GENETIC VARIATIONS OF CORECEPTORS The importance of CCR5 for HIV infection and pathogenesis is highlighted by a defect of the coreceptor gene (Moore, 1997; Littman, 1998; Rowland-Jones, 1998; Paxton et al., 1998). Early studies showed that blood leukocytes of some individuals that remained seronegative despite highrisk sexual behavior (so-called exposed uninfected, EU, individuals) were resistant to HIV infection in vitro (Paxton et al., 1996). A defect of the CCR5 gene consisting of a 32-bp deletion and resulting in the expression of a truncated, nonfunctional coreceptor variant, CCR5(A32), was then identified (Samson et al., 1996c; Liu et al., 1996; Dean et al., 1996). This mutation is frequent in Caucasians but is not found in people from Africa or Asia, and it is estimated that it occurred only a few thousand years ago. The frequency of heterozygotes declines continuously from 16% in Finnland and northeastern Russia to a minimum of 4% in Southern Europe (Libert et al., 1998). Individuals with defective CCR5 have a normal immune defense suggesting that other chemokine receptors compensate for the defect. In several AIDS cohort studies including a large number of HIV-infected individuals, no homozygotes for CCRS(A32) were found (Samson et al., 1996c; Liu et al., 1996; Dean et al., 1996). CCRS(A32) homozygotes, on the other hand, amounted to 2-4% of all EU subjects and to as much as 25% of the EU individuals with a history of high-risk sexual behavior of more than 8 years, underscoring the protective role of the CCR5 defect (Paxton et al., 1998; Dean et al., 1996). Blood mononuclear cells from individuals homozygous for CCRS(A32) can be infected with X4 strains in witro. It is conceivable that the rare HIV-1 positive CCRS(A32) homozygotes have been infected by X4 or R5X4 viruses. Several other polymorphisms that may decrease susceptibility to HIV infection have been identified. CCR5(m303) carries a T to A point mutation of the coding sequence which introduces a premature stop codon yielding a truncated, nonfunctional CCR5 protein (Quillent et al., 1998).Individuals

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that are homozygous for this defect have not been identified thus far, but CCR5(m303) can occur together with CCR5(A32), and mononuclear cells from individuals with CCR5(A3Ym303) are resistant to infection by R5 viruses. A single-base mutation in the coding sequence of the CCR2 gene which causes a Val to Ile substitution at position 64, CCU(V64I) was reported to correlate with a delay in the onset of AIDS in infected individuals (Smith et al., 1997).This defect, however, does not protect against HIV infection and does not appear to affect disease progression (Michael et al., 1997; Mummidi et al., 1998). Polymorphisms of the CXCR4 gene have not been reported. Such genotypes may be difficult to find since deletion of the CXCR4 gene was shown to be lethal in mice (Nagasawa et al., 1996a; Tachibana et al., 1998; Zou et al., 1998; Ma et al., 1998). An allelic variation of the SDF-1 gene termed SDF1-3'A with a G to A substitution at position 801 in the 3'-untranslated region, was nonetheless described and reported to delay the onset of AIDS in HIV-1 infected individuals (Winkler et al., 1998). This conclusion, however, has recently been questioned (Mummidi et al., 1998; Van Rij et al., 1998). SIGNALING C. CORECEFTOR HIV-1 entry does not appear to require G-protein-dependent signaling by the coreceptors, as shown in fusion or viral infection assays performed with target cells containing inactivated G-proteins (obtained by B. pertussis toxin treatment or mutagenesis) or COOH-terminally modified chemokine receptors that do not couple to G-proteins (Amara et al., 1997; Alkhatib et al., 1997; Cocchi et al., 1996; Doranz et al., 1997b). Some soluble HIV1/SIV Env proteins, however, were shown to interact with coreceptors and to induce chemokine-like responses like Ca" mobilization and chemotaxis in CD4' T cells (Weissman et al., 1997), macrophages (Herbein et al., 1998), and a neuronal cell line (Hesselgesser et al., 1998). G-proteindependent activation of the tyrosine kinase Pyk2, that can occur after binding of chemokines to their receptors (Davis et al., 1997; Ganju et al., 1998a,b), was observed after mixing coreceptor bearing cells with soluble or cell-associated Env proteins from R5 or X4 strains (Davis et al., 1997). Exposure to HIV-1 particles, gpl20, or HIV-suppressive chemokines was shown to signal for up-regulation of TNFR2 in CD8' T cells and of membrane-bound TNFa in macrophages which were then able to kill the CD8' T cells in cocultures (Herbein et al., 1998). On the other hand, treatment of CD4' T cells with soluble X4 Env proteins induced Fad FasL-independent apoptosis which could be inhibited by SDF-1 (Berndt et al., 1998). HIV coreceptors appear to be involved in neuronal apoptosis and may thus contribute to AIDS dementia (Price et al., 1988; Fauci, 1996). Neurons express several functional chemokine receptors, including

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CXCR4 and CCR5, which were reported to trigger cell death after binding gp120 proteins (Hesselgesser et al., 1997, 1998; Meucci et al., 1998). I N HIV PATHOGENESIS D. CHEMOKINES The recognition of the essential function of coreceptors for HIV-1, HIV2, and SIV entry into CD4-bearing cells highlighted the importance of chemokines for HIV pathogenesis. CCR5 and CXCR4, the major coreceptors, are widely, but differentially expressed in blood and tissue cells (Table V). Today, a large body of evidence supports the role of chemokines as HIV-suppressive factors and indicates that the effects that have been characterized in vitro are apt to retard disease progression. In some cases, however, chemokines have also been shown to enhance rather than inhibit HIV infection.

1. HZV Suppression Chemokines can block HIV entry and replication in CD4+ cells by competing with viral gpl20 for binding to the coreceptor and by inducing receptor down-regulation as a consequence of ligand-dependent internalization. Studies with laboratory-adapted strains have shown that chemokines and the binding epitopes of gpl2O recognize overlapping but not identical domains of the coreceptor (Moore, 1997; Berson and Doms, 1998). Although particular regions of the V3 loop determine coreceptor selectivity and viral tropism, many different primary HIV-1, HIV-2, and SIV isolates can share the same coreceptor (Moore, 1997; Berson and Doms, 1998). An alternative way by which chemokines can interfere with viral entry is coreceptor internalization, a rapid, B. pertussis toxin-insensitive process that depends on the phosphorylation of serine and threonine residues within the COOH-terminal receptor domain by receptor kinases (Bermejo et al., 1998; Amara et al., 1997; Aramori et al., 1997; Tarasova et al., 1998; Signoret et al., 1997; Haribabu et al., 1997). As shown by Amara et al. (1997), in the presence of excess ligand CXCR4 is rapidly down-regulated to a steady-state surface expression of about 20% of the total cellular content. That coreceptor internalization can contribute significantly to HIV suppression was shown by comparing the infectability of cells expressing wild-type CXCR4 or a mutant with COOH-terminal deletion that is not internalized (Amara et al., 1997). A third type of anti-HIV activity was observed with RANTES which enhances the cytotoxicity of MHC class I restricted, CD8+ T cells obtained from HIV- l-infected individuals toward target cells expressing epitopes of the HIV-lUIproteins Gag, Pol, Env, and Nef (Hadida et al., 1998). The increased HIV-specific cytotoxicity was observed in patient-derived cyto-

TABLE V EXPRESSION OF CCR5 AND CXCR4 IN BLOODAND TISSUE CELLS ~~

CCR5 mRNA"

CXCR4

Proteinb

Blood cells/precursors Neutrophils Monocytes

r

i?!

T lymphocytesd

Protein

+ (+)"

(+)"

+

+ +

+

+

+

+

+

+

+

+

+ + +

+

B lymphocytes

Megakaryocyted Platelets CD34' cells pro-/pre-B cells Thymocytes

mRNA

(+)"

(+)"

+ +

References Wu et d.,1997a; Forster et al., 1998; Loetscher et d., 199413; Bonecchi et d.,1999 Bleul et d.,1997; Wu et al., 1997a; Forster et al., 1998; Hori et d.,1998; Loetscher et d.,1994b; Wang et al., 1998c; Naifet al., 1998; Penton-Rol et al., 1998; Ostrowski et al., 1998 Bled et d.,1997; Loetscher et d.,1994b, 1998a,b; Qin et d., 1998; Wu et d.,1997a; Forster et al., 1998; Bermejo et al., 1998; Mo et d.,1998; Berkowitz et d.,1998; Jourdan et al., 1998; Hori et al., 1998; Carroll et ul., 1997; Bonecchi et al., 1998; SaUusto et al., 1998b; Zhang et d.,1998; Ostrowski et d.,1998; Wang et d.,1998a 1997a; Forster et al., 1998; Bled et d.,1997, 1998; Wu et d., Hori et d.,1998; Loetscher et al., 1994b; Vicenti-Manzanares etd., 1998 Hamada et al., 1998; Wang et d.,1998b Berkowitz et d.,1998; Deichmann et al., 1997; Mohle et d., 1998; Ruiz et al., 1998 D'Apuzzo et d.,1997 Berkowitz et al., 1998; Moepps et al., 1997; Kitchen and Zack, 1997; Dairaghi et d.,1998; Zaitseva et al., 1998; Zhang et al., 1998; Pedroza-Martinset d.,1998

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Wu et al., 1997a; B a n g et al., 1998; Wang et al., 1998c; Naif et al., 1998; Vallat et al., 1998 Sozzani et al., 1997, 1998; Rubbert et al., 1998; Sallusto et al., 1998c; Granelli-Piperno et al., 1996; Zoeteweij et d.,1998; Ayehunie et d.,1997 Albright et al., 1999; Vallat et d.,1998; Sanders et d.,1998; He et d.,1997; Ghorpade et al., 1998; Lavi et al., 1998 Meucci et al., 1998; Hesselgesser et d.,1997; Vallat et d.,1998; Sanders et d.,1998; Lavi et al., 1998 Sanders et al., 1998; Tanabe et al., 1997a Tachibana et al., 1998; Edinger et al., 1997; Gupta et al., 1998; Volin et al., 1998; Feil and Augustin, 1998 Moepps et d.,1997; Nagasawa et al., 199613 Moepps et al., 1997 Moepps et al., 1997; Nagasawa et al., 1996b Moepps et al., 1997; Nagasawa et al., 1996b

CCR5/CXCR4 mRNA expression detected by Northern blot, RT-PCR or in situ hybridization. CCR5KXCR4 protein determined by flow cytometry or immunobistochemistry. Freshly isolated blood monocytes express CCR5 at very low levels; short-term culture (e.g., adherence to plastic) upregulates CCRS expression. CCR5-positive T cells are activated effector/memory cells which produce Thl cytokines. CXCR4 is present in all T cells, most prominently in resting naii.e and memory cells. Low to undetectable expression of CCR.

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toxic T lymphocyte lines as well as in freshly isolated blood mononuclear cells and found to depend on RANTES acting via CCR3. Several recent papers provide strong evidence for a protective role of RANTES, MIP-la, and MIP-1P in HIV-infected individuals. Enhanced in vitro production of CC chemokines was observed in CD8+ T cells obtained from hemophiliacs who were not infected despite repeated administration of Factor VIII preparations contaminated with HIV-1 (Zagury et al., 1998). Similarly, enhanced production of RANTES, MIP-la, and MIP-10 and reduced infectivity of CD4' T cells were found to distinguish cells from EU individuals from those of healthy controls (Paxton et al., 1998), and from infected individuals with nonprogressor phenotype from those of patients with AIDS. (Saha et al., 1998). Protection by CC chemokines is also suggested by a study including 245 HIV-infected individuals that revealed an inverse correlation between CC chemokine production and disease progression (Ullum et al., 1998). Despite the undisputable beneficial effects of CCR5 and CXCR4 binding chemokines, other factors that are present in the culture supernatants of activated blood mononuclear cells may contribute to HIV suppression (Lacey et al., 1997; Paliard et al., 1996; Rubbert et al., 1997; Moriuchi et al., 1996; Lunardi-Iskandar et al., 1998; Ohashi et al., 1998; Barker et al., 1998; Lee et al., 1998; Pal et al., 1997). Most chemokines bind to soluble or surface-bound sulfated glycosaminoglycans like heparan sulfate and heparin via cationic motifs in their COOH-terminal domain (Graham et al., 1996; Crump et al., 1997; Bums et al., 1998) while retaining full biological activity (Webb et al., 1993; Tanaka et al., 1993; Rot, 1992). CD8' T cells from HIV-infected individuals were shown to release RANTES and MIP-la in a complex with sulfated glycosaminoglycans, and such complexes were more potent than the free chemokines as inhibitors of HIV infection of macrophages (Wagner et al., 1998). RANTES, MIP-la, and MIP-1P have only weak HIV suppressive activity on macrophages (Dragic et al., Moriuchi et al., 1996; Wagner et al., 1998; Oravecz et al., 1997; Simmons et al., 1997) which may reflect the absence of appropriate cell surface glycosaminoglycans. The binding of RANTES to monocytes and macrophages was shown to be dependent on chondroitin sulfate and the suppression of HIV infection in T cells on heparan sulfate which facilitates the high-affinity binding of RANTES (Orvecz et al., 1997). The importance of such interactions is also supported by the finding that RANTES loses its HIV suppressive activity and the ability to elicit Ca2+mobilization when its glycosaminoglycan binding site is masked with an antibody (Burns et al., 1998). In addition, enzymatic removal of cellular glycosaminoglycans markedly reduced RANTESdependent Ca2+mobilization.

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2. Znfection Enhancement Several studies of HIV infection in vitro show that chemokines can have enhancing rather than inhibitory effects (Schmidtmayerova et al., 1996; Margolis et al., 1998; Gordon et al., 1999; Dolei et al., 1998; Kelly et al., 1998; Kinter et al., 1998; Moriuchi et al., 1998). Cell stimulation by chemokines induces G-protein dependent and independent signaling events that elicit rapid and transient responses and occasionally long-lasting effects involving gene expression (see Section IV). Enhancement of HIV infection by chemokines may be related to either type of signaling responses and may even be mediated by receptor-independent events. B. pertussis toxin-sensitive, CC chemokine-mediated stimulation of in vitro infection with X4 viruses was reported by several laboratories (Dolei et al., 1998; Kelly et al., 1998; Kinter et al., 1998; Moriuchi et al., 1998). As shown by Kinter et al. (1998) enhancement was induced by RANTES, MIP-la, MIP-10, and MCP-1 (i.e., by chemokines that do not interact with coreceptors recognized by X4 viruses). In CD4' T cells, the effect was seen only at low viral doses and depended on the type of HIV-1 strain used. In addition, considerable variation between cells from different donors was observed. Work by Kelly et al. (1998), on the other hand, shows that pretreatment of monocytes and macrophages with RANTES, MIP-la, or MIP-lP for 48 h enhanced infection, while addition of the same chemokines together with the inoculum or after infection was inhibitory. Using HeLa and GHOST cell lines expressing CD4 together with CXCR4 or CCR5, Gordon et al. (1999) showed that RANTES markedly enhanced the infection by X4 and R 5 viruses as well as the infection by a murine leukemia and avesicular stomatitis virus, which are both unrelated to HIV. RANTES was added before infection and was effective at concentrations above 100 nM. It could not be substituted by MIP-la or MIP10 even at high concentrations, and the effect did not involve CCR5. It is not known whether enhancement of infectivity is receptor mediated. Sulfated glycosaminoglycans on the cell surface could be the RANTES binding sites, as also shown for IPlO in several cell lines (Luster et al., 1995) and IL-8 in endothelial cells (Rot, 1992). At high concentrations, RANTES was reported to induce proliferation and cytokine production in T lymphocytes, which may explain the observed enhancing effect on viral infectivity (Bacon et al., 1995, 1996, 1998).

E. CORECEPTOR-BASED THERAPEUTICAL APPROACHES The discovery of HIV coreceptors and the HIV suppressing activities of chemokines and chemokine receptor antagonists has opened new therapeutic perspectives as recently reviewed by Cairns and D'Souza (1998). Infec-

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tion depends on coreceptor availability which can be influenced by downregulating expression, enhancing internalization, or blocking with agonists, antagonists, or antibodies (Cairns and D'Souza, 1998; Baggiolini and Moser, 1997). The naturally occurring chemokines that compete for gpl20 binding are not regarded as promising potential therapeutic agents mainly because of their activating effects on leukocytes. For this reason, we considered chemokine receptor antagonists and found that NHz-terminallytruncated RANTES analogs (Gong et al., 1996), RANTES(8-68), and RANTES (9-68)prevent the infection by R5 viruses in uitro (Arenzana-Seisdedos et al., 1996).These results, which were fully confirmed by subsequent work (Ylisastiguiet al., 1998), were the first demonstration that the leukocyteactivating effects of chemokines are not required for HIV suppression. Inhibition of infection in vitro was also observed with a RANTES analog, AOP-RANTES, obtained by NHz-terminal elongation with aminoowentane (Simmons et al., 1997). AOP-RANTES was later found to be an agonist rather than antagonist as claimed in the original report (RodriguezFrade et al., 1999; Mack et al., 1998),which may limit its usefulness. Mack et al. (1998) reported that AOP-RANTES is more potent than RANTES in down-regulating surface expression of CCR5 since it apparently inhibits receptor recycling after internalization. Although chemokine-derived antagonists may turn out to be therapeutically useful, low molecular weight compounds are preferred because of easier handling and better bioavailability. Several structurally unrelated compounds were found to inhibit the replication of X4 HIV-1 strains. AMD3100, a heterocyclic substance belonging to the bicyclams which were known for their anti-HIV properties, was reported to compete for SDF1 binding to CXCR4, to prevent SDF-1 dependent Caz+mobilization at 100 ng/ml, and to inhibit infection by X4 viruses with an ICmof 1-10 ng/ ml (Schols et al., 1997; Donzella et al., 1998). AMD3100 is not toxic in mice, but its bioavailability is poor (Donzellaet al., 1998).T22, a peptide of 18 amino acids that was isolated from American horseshoe crab hemocytes, blocks CXCR4 and prevents infection of CD4' target cells by X4 viruses (Murakami et al., 1997).A similar activity was reported for ALX40-4C ( N acetyl-nona-D-arginineamide)which is also selective for CXCR4 and blocks SDF-1 induced Ca" mobilization (Doranz et al., 1997a). NSC 651016, an analog of distamycin which inhibits HIV infection in uitro, is the only substance reported so far that interferes with infection by X4 and R5 viruses (Howard et al., 1998a,b). NSC 651016 competes for chemokine binding to CCR1, CCR3, CCR5, and CXCR4 and is effective as an inhibitor of Ca" mobilization and chemotaxis, and inhibits replication of HIV-1, HIV-2 and SIV isolates (Howard et al., 1998a,b).

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Theoretically, monoclonal antibodies that block ligand binding to coreceptors represent an alternative anti-HIV approach. Several coreceptor antibodies are indeed effective in vitro, like 12G5 (Endres et al., 1996; McKnight et al., 1997), 2Bll (Forster et al., 1998), IVR7, and TSH123 (Hori et al., 1998) which all recognize CXCR4 and prevent infection by X4 viruses, and 2D7 (Wu et al., 1997a,b) which recognizes CCR5 and prevents infection by R5 viruses. The antibodies, however, are mainly useful for experimental studies and for monitoring coreceptor expression. Several genetic approaches have been designed to decrease coreceptor availability. The transfer of genes encoding chemokines (so-called intrakines) that are modified in order to be retained in the endoplasmatic reticulum has been used successfully to trap newly synthesized CXCR4 or CCR5 which are rapidly degraded rather than being translocated to the cell surface (Yang et al., 1997; Chen et al., 1997). These studies show that blood lymphocytes expressing SDF-1 and RANTES intrakines cannot be infected by X4 and R5 viruses in vitro (Yang et al., 1997; Chen et al., 1997). Similar effects may be obtained by transfer of vectors encoding chemokine receptor targeted intrabodies, ribozymes, and anti-sense RNA (Cairns and D’Souza, 1998). Application of live, but attenuated retrovirus particles which recognize and kill HIV-l-infected target cells is an attractive alternative strategy. It was shown that retroviral particles expressing human CD4 and either CCR5 or CXCR4 are highly selective for cells expressing HIV Env proteins (Endres et al., 1997). A novel therapeutic approach using noninfectious retroviruses with cytophatic activity can be developed on the basis of this principle. ACKNOWLEDGMENTS We thank Dr. Beatrice Dewald for critical reading of the manuscript. This work was supported by Grant 31-55996.98 to M.B., B.M. and P.L. from the Swiss National Science Foundation. B.M. is recipient of a career development award from the Prof. Max Cloetta Foundation.

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ADVANCES IN IMMUNOLOGY, VOL 74

Escape of Human Solid Tumors from T-cell Recognition: Molecular Mechanisms and Functional Significance FRANCESCO M. MARINCOLA; ELIZABETHM. JAFFEE,~DANIEL J. HICKLIN,+AND SOLDANOFERRONE~ 'SurIlery Bmnch, Division of Clinical Sciences, Nation01 Cancer lnstiw ond HLA Iahomtoty, Depomnent of Tmnsfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Morykmd; fDepahent of Oncdogy and Immunology, The Johns Hopkins University School of Medicine, 6altimom, Maryland; beparlrnent of Immunology, ImClone System Inc., New York, New York; kepadment of /mmunobgy, Roswell Pork Concer Institute, Buffalo, New York

1. Introduction

It has been known for some time that malignant transformation of human cells may be associated with the appearance of tumor associated antigens (TAA). Decades of research have been aimed at the identification of TAA that can serve as targets for the immunotherapy of malignant diseases. However, the identification of TAA and their molecular characterization have occurred only in recent years. In the late 1970s the development of the hybridoma technology provided probes to identify and characterize TAA defined by antibodies (Reisfeld and Cheresh, 1987; Herlyn and Koprowski, 1988; Graf and Ferrone, 1989). More recently, the dramatic progress in our understanding of the molecular basis of target cell recognition by cytotoxic T lymphocytes (CTL) has provided the background to design effective strategies to identify TAA recognized by CTL on tumor cells. The extensive application of these strategies by a number of investigators has resulted in the identification of various families of TAA on various types of solid tumors (Rosenberg, 1997; Boon et al., 1997; Old and Chen, 1998). Molecular characterization of these TAA has demonstrated a broad spectrum of protein products of normal or mutated genes expressed variably in tumor cells. The TAA grouped according to similar biologic characteristics are shown in Table IA-E. Mouse tumor models have played an important role in elucidating the mechanisms by which the immune system interacts with tumor cells and eradicates cancer. These studies have shown that CD8' T cells are critical effectors that have the capability of recognizing TAA and eradicating tumors in vivo (Restifo and Wunderlich, 1996b). The CTL recognize 8-10 amino acid peptide fragments derived mostly from cellular proteins. These proteins are degraded in the cytoplasm by proteasomes into short peptides, which are then transported into the endoplasmic reticulum where they complex with newly assembled Major Histocompatibility Complex (MHC) 181 AU

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TABLE IA TUMOR SPECIFIC ANTIGENSOR TUMOR DIFFERENTIATION ANTIGENS Antigen

Residues

Restriction Element

Epitope

Reference

MART-l/MelanA (118 aa)

24-34 27-35 32-40

HLA-B"4501 HLA-A"0201 HLA-A"0201

AEEAAGIGILTV AAGIGILTV ILTVILGVL

Schneider et d.,1998 Coulie et d.,1994; Kawakami et d.,1994a Caste& et d.,1995

17-25 154-162 209-217 280-288 457-466 476-485 614-622 619-627 639-647

HLA-A"0301 HLA-A'0201 HLA-A"0201 HLA-A"0201 HLA-A"0201 HLA-A"0201 HLA-A"0301 HLA-A'0201 HLA-A"0201 HLA-A"2402

ALLAVGATK KTWGQYWQV ITDQVPFSV YLEPGPVTA LLDGTATLRL VLYRYGSFSV LNRRRLMK RLMKQDFSV RLPRIFCSC VYFFLPDHL"

Skipper et d.,1996 Kawakami et d.,1995 Kawakami et d.,1995 Coxetd., 1994 Kawakami et d.,1995 Kawakami et d.,1995 Kawakami et d.,1998 Kawakami et d.,1998 Kawakami et d.,1998 Robbins et d.,1997

gp100/Pme117 (661 aa)

Tyosinase (530 aa)

HLA-A"0201 HLA-A"0101 HLA-B"4403 HLA-A"2202 HLA-A"0201

MLLAVLYLL S S DYVIPIGTY SYEIWRDIDF AFLPWHRLF YMNGTMSQV

Brichard et al., 1993; Wolfel et al., 1994 Kawakami et al., 1998 Brichard et al., 1996 Kang et al., 1995 Visseren et d.,1995

HLA-A"31""

MSLQRQFLR~

Wang et al., 1996

180-188 197-205 197-205

HLA-A"0201 HLA-A"3101 HLA-A*33" HLA-A"6801

SWDFFVWL) LLGPGTPYR LLGPGTPYR EVISCKLIKR

Parkhurst et al., 1998 Wang et al., 1996 wang et d.,1996 Lupetti et al., 1998

n.r

HLA-A2?/AlI

QDLTMKYQIF"

Mono et d.,1994

HLA-A"0201 HLA-A"0201 HLA-A~O~O~

TILLGIFFL FLALIICNA AIIDPLNA

Salazar-Onfray et al., 1997 Salazar-Onfray et d.,1997 Salazar-Onfray et al., 1997

1-9 146-156 192-200 206-214 369-377

gp75 (TRP1) TRP-2 (519 aa)

M 14 MClR

a

244-252 283-291 291-299

This epitope spans an intronic region of an incompletely spliced gp 100 gene transcript.

* This epitope results from an alternative open reading frame of a nonmutated gene. For this reason no residue location is given. Abbreuiations: MART-1: melanoma antigen recognized by T cells; MClR: melanocyte stimulating hormone receptor; TRP: tyrosinase related protein; n.r. = not reported.

TABLE IB SILENTGENEPRODUCTS OR CANCER TESTISANTIGENS REACTIVATED Antigen

Residue

Restriction Element

MAGE-1(30%)

161-169 230-238

HLA-A"0101 HLA-C'1601

EADPTGHSY SAYGEPRKL

van der Bruggen et d.,1991 van der Bruggen et d.,199413

MAGE-3 (70%)

168-176 271-279 167-176

HLA-A"0101 HLA-A'0201 HLA-B"4403

EVDPIGHLY FLWGPRALV MEVDPIGHLY

van der Bruggen et al., 1994a

BAGE (20%)

2-10

HLA-C"1601

AARAVFLAL

Boel et al., 1995

GAGE-1 (4%)

n.a.

HLA-CwG

YRPRPRRY

1995 Van den Eynde et d.,

HLA-A"2402

LYVDSLFFL

Ikeda et al., 1997

10-18

HLA-A24

AYGLDFYIL

Robbins et d., 1995

53-62

HLA-A'0201 HLA-A"31

(Q)SLLMWITQC(FL) ASGPGGGAF'R

Jager et al., 1998 Wang et al., 1998

571-579

HLA-A"0201

YLSGANLNL

Tsang et al., 1995

PRAME (30-80%)

P15

1051-1257

NY-ESO (30%) CEA

Epitope

Reference

Gaugler et al., 1994 Fleischhauer et al., 1996

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TABLE IC MUTATEDGENEPRODUCTS Antigen

Restriction Element

Epitope

Reference ~

p-Catenin

HLA-A'2402

SYLDSGIHF

Rubinfeld et al., 1997

NA17-A

HLA-A'0201

VLPDVFIRCV"

Guillow et al., 1996

CDK-4

HLA-A"0201

ACDPHSGHFV

Wolfel et al., 1995

MUM

HLA-B044"

EEKLIWLF

Coulie et al., 1995

aa 170 (Arg to Ile)

Brandle et al., 1996

FPSDSWCYF

Mandruzzato et al., 1997

HLA-A2 mutant CASP-8

HLA-B"3505

This epitope spans an intronic region. Abbreviations: NA17: N-Acetylglucosaminy-L-Transferase-V (also called GnT-V).

TABLE ID ONCOGENE PRODUCTS WILD-TYPEOR MUTATED Restriction Element

Epitope

Reference

HER2heu

HLA-A"0201 HLA-A'0201

IISAWGIL KIFGSLAFL

Peoples et al., 1995 Fisk et al., 1995

P53

HLA-A"0201

LLGRNSFEV

Gnjatic et al., 1998

TABLE IE VIRALGENEPRODUCTS

HPV-16 E7

Site

Restriction Element

EDitoDe

Reference

11-20 82-90

HLA-A"0201 HLA-A"0201 H LA-A"O2O 1

YMLDLQPETT LLMCTLGIV TLGIVCPI

Ressing et al., 1995 Ressing et al., 1995 Ressing et al., 1995

86-93

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MARINCOLA d a1

class I molecules. These peptide/MHC class I antigen complexes are then transported to the cell surface for presentation to CD8' T cells (Germain, 1996). This pathway was first characterized for viral proteins synthesized by infected host cells (Germain, 1996). Studies describing the isolation of MHC class I restricted TAA have confirmed that the mechanism of TAA presentation is similar to that of viral proteins (Lurquin et al., 1989; Huang et al., 1996). This finding has been paralleled by similar results obtained with the characterization of HLA (i.e., Human Leukocyte Antigen) class I associated human TAA (Rosenberg, 1997; Boon et al., 1997). Malignant melanoma has been the most extensively studied human tumor system, because of the greater availability of TAA-specific T cells that can be isolated from patients with this disease. Several CTL-defined melanoma-associated antigens (MAA) have been identified. They fall into four categories: (i)Tissue-specificantigens that are expressed by melanoma cells and normal melanocytes (Brichard et al., 1993; Cox et al., 1994; Coulie et al., 1994; Kawakami et al., 199413; Morioka et al., 1994; Wolfel et al., 1994; Kang et al., 1995; Kawakami et al., 1995;CasteUi et al., 1995;Visseren et al., 1995; Brichard et al., 1996; Skipper et al., 1996; Wang et al., 1996; Robbins et al., 1997; Kawakami et al., 1998; Schneider et al., 1998). These antigens are also referred to as tumor differentiation antigens (TDA); (ii) Proteins that are not expressed in any normal adult tissues, except for the testes, and appear to be derived from the reactivation of fetal oncogenes (van der Bruggen et al., 1991, 1994a,b; Gaugler et al., 1994; Boel et al., 1995; Robbins et al., 1995; Fleischhauer et al., 1996; Ikeda et al., 1997; Jager et al., 1998). These antigens are generally expressed by more than one tumor type and are called tumor-specific antigens (TSA) or cancertestis antigens; (iii) Intron encoded antigens that are present in a low percentage of tumors and are not usually expressed in normal tissues (Coulie et al., 1995; Guilloux et al., 1996; Robbins et al., 1997; Lupetti et al., 1998); and (iv) Antigens that are products of mutated genes, have a restricted distribution in tumors, and are not expressed in normal tissue (Wolfel et al., 1995; Guilloux et al., 1996; Rubinfeld et al., 1997). Much to tumor immunologists' surprise, most MAA have been found to be shared among over 50% of patients with melanoma. It had been previously predicted that TAA would be unique new-antigens encoded by mutated genes in tumor cells. Expression of shared CTL defined MAA in a large percentage of patients with melanoma has facilitated their use as immunogens to implement active specific immunotherapy in clinical trials. Some of the identified human MAA have already undergone testing in patients. Because of the rapid accrual of patients in these vaccination protocols, a large amount of data has been rapidly accumulated on the immune and clinical responses these MAA elicit (Marchand et al., 1995; McRae et al., 1995;

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Cormier et al., 1997; Nestle et al., 1998; Rosenberg et al., 1998). These studies have shown that in the majority of patients immunization with MAA does not yield the clinical responses predicted by murine models. Furthermore, progression or recurrence of disease has been observed in spite of the induction or persistence of MAA-specific CTL responses. Although the clinical results have been disappointing, the utilization of defined MAA or peptides derived from their sequence for vaccination of patients with advanced melanoma has yielded an unprecedented opportunity to analyze the dynamics of in vivo interactions between the host immune system and tumor cells in humans. Furthermore, the utilization of well-defined immunogens, particularly in patients with melanoma, has provided the opportunity to analyze in detail the molecular mechanisms utilized by tumor cells to escape from CTL recognition. Therefore, this review will emphasize the discussion of results obtained in melanoma. Understanding the mechanisms by which tumor cells expressing TAA can escape immune recognition by TAA-specific CTL is critical for the design of effective vaccine strategies against cancer. Figure 1A describes sequential steps in the pathway of HLA class I associated antigen presentation which, if altered, may lead to tumor escape from immune recognition. Furthermore, other biological behaviors of tumor cells that could potentially lead to altered T cell function at tumor sites are suggested, including secretion of immune suppressive cytokines or surface expression of apoptotic signals. These mechanisms fall into four broad categories: (i) inadequate antigen presentation by tumor cells resulting in their poor sensitivity to lysis by CTL; (ii) inhibitory signals provided by the tumor microenvironment; (iii) inability of TAA-specific CTL to localize at a tumor site; and (iv) inability of tumor microenvironment to sustain T cell function in vivo. Figure 1B describes CTUtumor cell interactions in the context of other immune cells including helper T cells and antigen presenting cells (APC). It is suggested that the immune reaction of the host against tumor cells starts with incorporation of TAA shed by cancer cells into APC, which can present HLA class I1 antigen associated epitopes to helper T cells. These in turn may secrete stimulatory factors, which enhance a TAA-specific CTL response (Yewdell and Bennink, 1990) or, as most recently suggested, activate directly antigen presenting cells by CD40-ligand-CD40 interactions (Ridgeet al., 1998).This response is expected to control tumor growth unless tumor cells utilize mechanism to escape from immune recognition. In this review, following a brief description of the local and systemic mechanisms utilized by tumor cells to escape from immune recognition, we will focus on factors affecting the microenvironment where tumor cell-host immune system interactions are likely to occur. We will not review systemic factors, which may lead to tumor cell escape since they

b-

W

FIG.1. (a) Molecular basis of the presentation of intracellular antigen derived peptides to CD8+ CTL.(b) Binary logic of antigen presentation to T cells through the HLA class I antigen dependent and the HLA class I1 antigen dependent pathway (Yewdell and Bennink, 1990).

189

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have already been elegantly discussed by others (Toes et al., 1997). We will discuss factors affecting tumor cell-host interactions at a tumor site, individually and in combination, in an attempt to explain the puzzling clinical observation of tumor cell survival in spite of an easily identifiable TAA-specificCTL response. We will emphasize results obtained in patients with malignant melanoma, since this type of human tumor has been most extensively investigated. II. Tumor Cell-Escape Mechanisms

Host immune surveillancewas suggested a long time ago to play a crucial role in the control of tumor growth (Thomas, 1959; Burnet, 1970) on the assumption that tumor cells are seen as foreign by the host. As tumors arise after embryogenesis, negative thymic selection of T cells, based on self-discrimination, does not occur. Newly arising tumors are eliminated as they originate, and only the few lacking “neo-antigens” can escape immune surveillance and become clinically detectable. This theory, therefore, postulated that most newly arising cancer cells are destroyed by a vigorous immune system. Only occasionally tumor cells, which do not express neo-antigens, escape immune surveillance. These cells divide undisturbed to develop into observable tumor masses. The original concept of immune surveillanceis no longer accepted since the molecular characterization of TAA has shown that, for the most part, they are nonmutated self-molecules (Table I). Furthermore, at this point the role of immune suppression in the development of various cancers has not been conclusively established. Therefore, complex interactions between host and tumor cells that could lead to tumor rejection through breaks of self-tolerance similar to those which develop in auto-immune pathology need to be considered (Marincola, 1997). Thus, because most TAA are represented by nonmutated self-molecules and TAA-specific CTL responses have been identified in patients, the growth of tumors in spite of this immune response represents a phenomenon of peripheral tolerance. Admittedly, this view assumes that a host TAA-specific CTL response plays a predominant role in the control of tumor growth. Such a predominant role is still controversial, and other immune mechanisms are likely to participate in the control of tumor growth (Sogn, 1998). However, a discussion of the role of T cell and/or other immune mechanisms on the control of tumor growth is beyond the purpose of this review. Tumor cell-escape mechanisms will be analyzed here with the assumption that the TAA-specific CTL responses identified in humans may have a direct impact on the natural history of cancer. As previously discussed, MAA-specific CTL can readily be detected among tumor infiltrating lymphocytes (TIL) and in peripheral blood mono-

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nuclear cells (PBMC) (Salgalleret al., 1995; Rivoltini et al., 199513; Marincola et al., 1996b; D’Souza et al., 1998). Furthermore, T-cell responses against “self’ MAA can be strongly enhanced in vivo by antigen-specific stimuli (Salgalleret al., 1996; Cormier et al., 1997; Rosenberg et al., 1998). Yet in the majority of cases, the T cell reactivity observed in vitro does not correlate with effective immune responses in vivo as is apparent by the persistence or growth of tumors. The paradoxical lack of effectiveness of MAA-specific T cells in vivo may reflect either the inability of the host immune system to mount an efficient immune response and/or the inability of fully activated TAA-specific CTL to recognize neoplastic cells in the tumor microenvironment. Ineffective immune responses, whether due to systemic or local factors, could explain the continued growth of tumors both in untreated patients and in patients treated with active-specific immunotherapy. Table I1 lists the multiple mechanisms, which can lead to an ineffective TAA-specific CTL response. Many of these mechanisms have been suggested by results obtained in preclinical models (Moskophidis et al., 1993; Alexander-Miller et al., 1996; Effros et al., 1996; Toes et al., 1996a, 1997; Effros and Pawelec, 1997; Lauritzsen et nl., 1998; Van Parijs and Abbas, 1998). Whether all these mechanisms play a role in humans remains to be determined. However, it is now possible to address this question because of the identification of a number of human TAA and the understanding of the molecular basis of their recognition by CTL. Useful in this regard will be patients immunized with well-defined TAA, who should provide the opportunity to monitor the immune response to well-characterized moieties and to evaluate the clinical significance of these responses. For example epitope-specific vaccines allow for restricted analyses of systemic immune responses in the context of a single HLA class I antigen restricted TABLE I1 MECHANISMS UNDERLYING TUMOR CELLESCAPE FROM POTENTIAL HOSTIMMUNE RECOGNITION Insufficient Systemic Immune Response Immune tolerance: Peptide induced Clonal exhaustion Replicative senescence and telomeres shortening Fas/FasL induced apoptosis Immune suppression: Immune suppressive cytokines T cell 5 chain down-regulation

Insufficient Local Immune Response Lack of tumor localization of CTL Paracrine immune suppression by tumors Fas/FasL interactions Inadequacy of tumor cells as targets: TAA loss or down-regulation TAP loss or down-regulation HLA class I antigen loss or downregulation

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TAA. Consequently monitoring of relevant peptide/HLA class I antigen expression at tumor site can be enormously simplified by limiting the analysis to the peptide/HLA class I allele complex targeted by the vaccination. These type of studies will simplify the analysis of an immune response to a specific peptide/HLA class I antigen targeted by the vaccination. Information from these studies may lead to the identification of clinically relevant immunological principles and mechanisms that can be further tested in animal models and/or in multicentric investigations involving large patient populations. There are currently strict limits in our ability to investigate the interaction between tumor cells and host immune system with existing methodologies. In particular, difficulties are often encountered when attempting to combine information derived from functional tests performed in vitro on cell lines with descriptive data obtained from the analysis of surgical specimens. Tumor cell-host immune system interactions in situ have traditionally been studied by immunohistochemical (IHC) analysis of formalin-fbed or frozen surgically removed tumor specimens. More recently this type of analysis has been combined with the molecular analysis of TAA or HLA class I antigen expression at the DNA and/or mRNA levels. An obvious limitation of these types of analysis is the inability to perform functional studies involving assessment of T cell recognition of tumor cells. To overcome this limitation, some have suggested the utilization of freshly isolated cells derived from in vitro digestion of tumor specimens for functional studies. However, this approach is also limited by the extensive contamination of freshly isolated tumor cells with other cell types. Furthermore, experimental results are often confounded by the alteration of tumor cells which have undergone enzymatic or mechanical treatments. Therefore, established cell lines derived from resected lesions are more frequently utilized in vitro to assess the functional significance of TAA or HLA class I antigen expression. At present, it is not clear whether such cell lines are representative of the in vivo situation. Due to the genetic instability of neoplasms, early passage cells are extremely heterogeneous (Lengauer et al., 1998). However, after a few passages in vitro, oligoclonal populations of tumor cells emerge. This change may reflect a preferential expansion of tumor cells more suited for in vitro culture and eliminate a large component of the original cell population. For this reason, results of experiments performed with established cell lines are often looked upon with skepticism.Nevertheless, experiments performed with cell lines have established important principles of T cell recognition of tumor cells. Furthermore, expanded cell lines have been used for analysis of T-cell-tumor interactions in vivo through engraftment into animal model systems (Williams et d.,1996).

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Excisional biopsy of tumors and subsequent expansion of TIL and tumor cells obtained from these samples has proved to be a useful tool for the analysis of tumor-host interactions. (Pandolfi et al., 1991). With this strategy, models can be developed for the characterization in vitro of CTL/ tumor cell interactions at a given time point in the course of the disease. However, an excised lesion is rarely representative of all metastasic lesions in a patient, since synchronous metastases are often heterogeneous in the expression of TAA and HLA class I antigens (Cormier et al., 1998b). Furthermore, the removal of the tumor excludes comparative studies of the same lesion at different points in time in relation to the natural progression of the disease or in response to immune pressure. To overcome these limitations, some investigators have utilized technology such as fine needle aspiration (FNA) which provides the opportunity to evaluate the expression of TAA, HLA class I antigens, and/or other markers in malignant lesions at various time points during the evolution of the disease (Marincola et al., 1996a).Characterization of tumor cells removed at different time points from a single lesion can eliminate the confounding results obtained by analyzing distinct lesions removed at different time points. Furthermore, the ability to expand TIL and autologous tumor cells from FNA permits the analysis of CTL localization and function at a tumor site (Lee et al., 1998). For the moment, however, FNA suffers from its own limitations because expansion of cells is quite laborious and the number of cells obtained is extremely small. Furthermore, FNA does not allow a pathological assessment of the lesion. In conclusion, no “ideal” method is available for the analysis of tumor-host interactions. It is likely that a combination of both in vitro and in vivo analyses described earlier will continue to be used in the future. Our working hypothesis, derived from the analysis of the immune and clinical responses of patients with melanoma immunized with MAA, is that the natural history of metastases is predominantly influenced by local factors (Fig. 2). Within each tumor, a balance is struck between the host recognition of cancer cells and the ability of cancer cells to avoid it. Two lines of evidence support this hypothesis. First, the ease with which MAAspecific CTL can be detected among PBMC and TIL suggests that the host immune system can be sensitized against self-molecules. Most MAA are targets of TIL normally populating melanoma metastases. Immunization with MAA-derived peptides has also shown that MAA-specific CTL can be activated and expanded in viva (Salgaller et al., 1996; Cormier et al., 1997; Rosenberg et al., 1998). Taken together these findings suggest that specific immune tolerance to MAA caused by clonal exhaustion and replicative senescence may not play a predominant role in human melanoma as it does in murine models (Moskophidis et al., 1993; Effros et al.,

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MARINCOLA et al. Mixed Response Phenomenon Detection of CTL

CTL Localization

L

\

FIG.2. Role of systemic and local mechanisms leading to tumor tolerance.

1996;Toes et al., 199613; Effros and Pawelec, 1997; Lauritzsen et al., 1998). It has been suggested that alterations in T cell receptor (TCR) function and signal transduction in vivo may lead to central immunosuppression of patients with melanoma (Zea et al., 1995; Wojtowicz-Praga, 1997). However, to our knowledge, there is no convincing evidence that patients with melanoma are more susceptible to viral or bacterial infections than normal individuals. On the contrary, in vitro analysis of Flu-specific CTL reactivity detected no difference between patients with melanoma and healthy controls (Marincola et al., 1996b). Furthermore, MAA-specific CTL reactivity in patients with melanoma was found to be higher than in nontumor bearing individuals (Marincola et al., 199613; D’Souza et al., 1998). Thus immunosuppression secondary to the tumor bearing status does not appear to play an obvious role in the immunobiology of melanoma. The second line of evidence is represented by the phenomenon of a “mixed response.” A mixed response occurs rather frequently in patients with metastases, although its actual frequency has never been documented. Mixed responses are characterized by the different behavior of synchronous metastases in response to T cell-based immunotherapy. Although some metastases decrease in size or disappear in response to immune manipulation, some continue their growth unaffected by therapy. As a patient’s immune response at a single time point is a constant, mixed responses are likely to reflect the major role played by the interactions between tumor cells and immune cells at each tumor site. As we will discuss later, differences in response of individual metastases could be due to heterogeneity in target molecules (i-e.,TAA and/or HLA class I antigens or apoptotic

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signal expression), paracrine secretion, and other factors that might affect localization and/or function of TAA-specific CTL at a tumor site. An additional possible mechanism that could affect the variable behavior of synchronous metastases is the localization of TAA-specific CTL at a tumor site. Earlier studies have shown that localization of "'Indium-labeled adoptively transferred TIL at tumor site is a prerequisite for a clinical response to occur, although it is not sufficient to predict it (Pockaj et al., 1994). Indeed, in several patients the localization of TIL at a tumor site did not lead to tumor regression. This discrepancy suggests that other factors within the tumor microenvironment allow the defiant survival of tumor cells. Therefore, tumor response to immune pressure appears to be highly influenced by local factors that could either affect sensitivity of tumor cells to CTL or decrease their cytolytic activity. 111. Inadequacy of Tumor Cells as Targets

The recognition of tumor cells by CTL requires a sufficient presentation to TCR of TAA derived peptides in association with the restricting HLA class I allele. Defects in either of these two sets of molecules as well as those involved in generation of peptides from TAA and transport through the tumor cell can provide tumor cells with an escape mechanism from CTL recognition. We discuss each set of molecules first individually and then their interactions since they will determine the final outcome of CTL activation and effector function.

A. TAA Loss OR DOWN-REGULATION Alterations in TAA expression are one of the mechanisms by which tumor cells may escape CTL recognition in viva Changes in TAA expression range from down-modulation to total loss (Table 111).In the early studies, abnormalities in TAA expression were identified in murine tumor models. More recently, the characterization of human TAA, in particular MAA, TABLE 111 MECHANISMS OF TUMOR CELLIMMUNE POTENTIAL ESCAPE DUETO ALTERATIONSIN TAA EXPRESSION TAA loss (total or partial) TAA down-regulation Down-regulation of CTL activity by epitope mimics Disruption of CTL defined epitopes on TAA Masking of cryptic antigens by immunodominant TAA

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has broadened our understanding of aberrations leading to defects in TAA expression. 1. Frequency of TAA Loss or Down-Regulation In animal models, it is commonly observed that immunogenic tumors that elicit TAA-specific CTL eventually grow and kill their host. It is possible that some tumors grow rapidly and outpace the immune system. However, this mechanism of tumor escape from immune control does not explain the observation that some tumors begin to grow in uiuo, become dormant for a period of time, and subsequently progress and metastasize. Using the murine mastocytoma P815 tumor model, Biddison and Palmer (Biddison and Palmer, 1977; Uyttenhove et al., 1983) presented the first evidence that tumor escape may be due to the emergence of stable antigenloss variants. Tumor cells of the subline P815-Y that were collected from the peritoneal cavity of tumor bearing animals a few days after the partial rejection phase were less sensitive to CTL lysis than the original tumor cells. The molecular basis for this finding was subsequently elucidated by Boon and his colleagues (Van den Eynde et al., 1991; Boon et al., 1992; Lethe et al., 1992). These authors identified the gene PlA, which is silent in normal tissues with the exception of testes and encodes the mastocytoma’s tumor rejection antigen P815AB. This antigen is composed of two distinct CTL epitopes. Because of deletions within the P1A gene, both epitopes are undectable in P815 mastocytoma cells that escape tumor rejection in uiuo. This finding provided convincingevidence that tumor cells can escape from CTL recognition because of the development of variants that no longer express the rejection antigen(s) recognized by TAA-specific CTL. Most of what we know about alterations in antigen expression in human tumors comes from studies performed with melanoma cells. Analysis of melanoma cell lines has shown that heterogeneity in MAA expression (de Vries et al., 1997; Cormier et al., 1999) is independent of HLA class I antigen expression and that the degree of heterogeneity vanes among different types of MAA. These findings are not unique to CTL defined M U ; similar results were obtained when the expression of antibody defined MAA was investigated in melanoma cell lines and tumor lesions (Albino et al., 1981; Burchiel et al., 1982; Natali et al., 1983, 1985; Cillo et al., 1984). Early studies utilizing reverse transcriptase polymerase chain reaction (RT-PCR) showed that, among members of the MAGE family, MAGE-1 was not expressed in a significant proportion of melanoma lesions, whereas MAGE-3 had a wide distribution in melanoma and in other malignancies (van der Bruggen et al., 1991). Differences in degree of expression among members of the MAGE family and other cancer testis antigens appear to reflect different susceptibility to a genome-wide demethylation

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process associated with tumor progression (De Smet et al., 1996). Two lines of evidence support the role of demethylation in the expression of cancer testis antigens. First, the higher frequency of cancer testis antigen expression in metastatic than in primary lesions parallels the higher degree of demethylation of genes encountered in metastases than in primary lesions (Bedford and van Helden, 1987; Liteplo and Kerbel, 1987). Second, treatment with demethylating agents such as 5-Aza-2’-deoxycytidine can induce expression of MAGE-1 in cell lines that do not express this TSA (Weber et al., 1994; De Smet et al., 1996).5-Aza-2‘-deoxycytidineinduced expression of members of MAGE-1 and other cancer testing antigens in a high percentage of melanoma and lung carcinoma cell lines and could induce recognition by TAA-specific, HLA class I antigen-restricted CTL (Bahler et al., 1987; Weber et al., 1994). The effect of 5-Aza-2’deoxycytidine is not restricted to cancer testis antigens because this demethylating agent can induce prolonged HLA class I antigen and costimulatory molecule up-regulation (Coral et al., 1999). Therefore, this drug may have therapeutic applications for T cell based treatment of patients with melanoma. Nevertheless, enhancement by demethylating agents of the recognition of tumor target cells by CTL has not been explored as an adjuvant treatment in clinical settings due to the effects of these agents on fibroblasts and activated lymphocytes (Weber et al., 1994; De Smet et al., 1996). The identification of MART-l/MelanA (Kawakami et al., 199413; Coulie et al., 1994) and gp 100/Pme117 (Cox et al., 1994; Kawakami et al., 1995) and characterization of their tissue distribution utilizing RT-PCR suggested that TDA are expressed by the majority of melanoma lesions. In the earlier studies MART-l/MelanA mRNA was detected in all 26 cell lines tested and in most of the melanoma lesions analyzed (Coulie et al., 1994). Although informative, these studies based on the detection of MAA mRNA provide no information about the heterogeneity of MAA protein expression among different melanoma cells within a lesion. Furthermore, detection of MAA mRNA does not guarantee expression of the corresponding protein as mutations resulting in alternative open reading frames, truncation or alteration of the translation process may cause lack of expression or expression of abnormal gene products. As a matter of fact, MAA mRNA could be detected in 75% of melanoma lesions that were not stained by anti-MAA mAb in IHC reactions (Cormier et al., 199813).Furthermore, trace amounts of gp 100/Pme117 mRNA have been reported to be detectable by RTPCR even in nonmelanocytic tumors and normal tissues, which do not express TDA-derived proteins (Brouwenstijn et al., 1997; de Vries et al., 1997). Notably, in these cases, the amplified gp 100/Pme117 RT-PCR product from nonmelanocytic cell lines has the wild-type nucleotide se-

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quence suggesting that trace amounts of gp 100/Pme117 mRNA may be found in cells of nonmelanocyhc lineage. The reasons for the lack of translation of these genes are presently poorly understood. Whatever the cause, the discrepancy between transcription and translation of genes encoding MAA provides a mechanism for the lack of susceptibility to lysis by MAA-specific CTL of melanoma cells with detectable MAA mRNA by RT-PCR (Cormier et al., 199813). The development of mAb specific for gplOOiFme117, MART-l/MelanA and tyrosinase (Vennegoor et al., 1985, 1988; Scott-Coombes et al., 1993; Wolfel et al., 1994; Chen et al., 1995; Kang et al., 1995; Brichard et al., 1996;Kawakami et al., 1997; Busam et al., 199813)has facilitated the analysis of their distribution in cell lines and in surgically removed lesions. These studies have revealed that gplOO/Pme117,MART-l/MelanA, and tyrosinase expression is not as ubiquitous as previously suggested by molecular methods (Marincola et al., 1996a). Most primary melanoma lesions express TDA (de Vries et al., 1997; Busam et al., 1998a);however, metastases are quite heterogeneous in their expression (Scheibenbogen et al., 1996; de Vries et al., 1997; Cormier et al., 1998b; Riker et al., 1999). In about 10-2096 of metastatic lesions, gp100/Pme117 and MART-l/MelanA have not been detected, whereas tyrosinase appears to be less frequently lost (Chen et al., 1995; Cormier et al., 1998a).Although the loss of either gp100/Pme117 or MART-l/MelanA occurs relatively frequently in melanoma metastases, the combined loss of the two MAA occurs less frequently (Cormier et al., 1998b; Riker et al., 1999). Loss of gp100/Pme117 is more common than that of MART-UMelanA. Overall, MART-l/MelanA and gp100/Pme117 have not been detected in about 10 and 20%,respectively, of the melanoma metastases analyzed (Scheibenbogen et al., 1996; Cormier et al., 1998b; Riker et al., 1999). The reason(s) for the more frequent gp100/Pme117 loss is(are) not known. One possibility is that gp100/Pme117 is subjected in vivo to stronger immune selection than MART-l/MelanA because it expresses a higher number of epitopes recognized by CTL (Table IA). Furthermore, the lower frequency of gp100/Pme117 than of MART-1/ MelanA detection in melanoma metastases may reflect at least in part a bias related to the preferential immunization of patients with gp100/Pme117 at the Surgery Branch of the National Cancer Institute, Bethesda, MD, where the study was performed (Cormier et al., 1998b). About 25% of synchronous metastases display significant differences in the expression of CTL-defined MAA (Scheibenbogen et al., 1996; Cormier et al., 1998b; Riker et al., 1999). This finding parallels heterogeneity in the expression of antibody defined MAA in autologous metastases in patients with melanoma (Natali et d., 1983, 1985).Antigenic heterogeneity of synchronous metastases could account for differences in their growth

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pattern during disease progression in untreated patients or in patients immunized with MAA-derived peptides. The variability in MAA expression may also reflect clonal differences or may reflect dynamic differences within a cell population due to temporary shutdown of protein synthesis during various phases of the cell cycle. The latter hypothesis, however, is not supported by observations performed in clonal cell lines. When cell lines are separately analyzed, independently from their cell cycle, they demonstrate a relatively homogeneous pattern of TAA expression (Marincola d al., 1996a;Cormier d al., 1999). Intralesional heterogeneity of MAA expression among subclonal populations caused by the genetic instability of cancer cells may explain selection based on elimination of target cells expressing higher levels of the TAA and therefore tumor progression after temporary regression or stabilization. The MAA may also be lost or down-regulated concomitantly to HLA class I antigens. Of 155metastases, IHC did not detect MART-l/MelanA, gplOOA'me117, and HLA-A2 antigens in 8, 21 and 6%, respectively. Furthermore, neither a single MAA nor HLA-A2 antigens were detected in 10-25% of the metastatic lesions. Thus, as many as one in four metastases may be poor targets for recognition by MAA-specific, HLA-A*0201 restricted CTL. These findings point out the necessity of studying both MAA and HLA class I molecule expression in malignant lesions (Cormier et al., 199810). 2. Functional Signifkance of TAA Loss or Down-Regulation The IHC staining of metastatic lesions has shown variations not only in the percentage of malignant cells expressing TAA but also in the level of their expression as measured by the intensity of staining (Chen et al., 1995; de Vries et al., 1997; Cormier et al., 1998b). These findings have been corroborated by testing cell lines by FACS analysis (Cormier et al., 1999) and real time quantitative RT-PCR (Riker et al., 1999). Variations in the level of TAA expression may explain the coexistence of TAA-specific TIL and malignant cells in lesions expressing the target TAA. In the normal environment, productive engagements between TCRs and peptide/HLA class I antigen complexes may proceed to a point of balance between avidity for and availability of epitope. At this point, T cells fail to destroy the remaining malignant cells efficiently. The size of metastases is determined at that point predominantly by the growth rate of cancer cells. Yet, MAA-specific CTL could still populate metastases because MAA shed by dying cancer cells could be up-taken and processed by intratumoral APC or by APC in the draining lymph nodes. Because of their superior antigen presenting capability, APC could perpetuate the presence of TIL within the tumor.

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In vitro analysis of 25 melanoma cell lines with a heterogeneous expression of gp100/Pmell7 and MART-l/MelanA, but with a similar expression of the restricting HLA class I element, has shown a correlation between level of expression of a TAA on targets and their recognition by CTL (Yoshino et al., 1994; Marincola et al., 1996a). In this panel of cell lines the level of a TAA expression was closely associated with the extent of lysis by MAA-specific CTL (Cormier et al., 1999). Such correlation was independent of structural abnormalities of HLA class I antigens and/or antigen processing machinery as up-regulation of endogenous MAA expression by infection with MART-1 containing vectors equalized sensitivity of all cell lines to CTL. Along the same line, a relationship was found between level of MAGE-1 mRNA measured by quantitative PCR in a panel of cell lines and the extent of their lysis by MACE-1 specific CTL (Lethe et al., 1997). It is noteworthy that 10% of the mRNA present in a reference cell line well recognized by CTL was the minimal threshold of expression required for CTL lysis. Whether cell lines are truly representative of the in vivo cell population from which they originated is a frequent question in tumor immunology. In an attempt to correlate the results of in vivo and in vitro studies, the level of gp100/Pme117 and MART-l/MelanA expression in cytospins from FNA of 15 melanoma metastases was compared with their expression in cell lines derived from the same FNA. The results of the analysis of these two types of substrates were found to be highly correlated (Riker et al., 1999).Cell lines derived from MAA negative lesions or from lesions characterized by a small percentage of cells expressing MAA did not express that MAA. Conversely, lesions characterized by high expression of MAA by IHC yielded predominantly MAA positive cell lines. Occasionally, cell lines with low or no expression of the MAA were generated from lesions with predominantly MAA-expressing cells, suggesting that, in heterogeneous cell populations anaplastic, MAA negative cells may benefit from a survival advantage in vitro. Escape from immune recognition could be functionally defined, in part, by inability of target cells to be recognized and killed by specific CTL. Others have suggested that IHC and/or FACS may not be sensitive enough to detect levels of expression of MAA which are low, yet remain above the threshold for CTL recognition. However, in the early passage cell lines derived from the FNA previously described, a strong correlation was noted between the expression of MAA, measured by FACS analysis, and recognition by CTL. Thus, expression of MAA documented in melanoma lesions by IHC paralleled the level of expression documented by FACS in the corresponding cell lines. In turn MAA expression in cell lines correlated strongly with CTL recognition in vitro. These studies, combined with the

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variability of MAA expression noted in vivo, suggest that reduction in MAA expression may act as a gradual mechanism of tumor escape from CTL recognition. MAA loss as a potential mechanism utilized by tumor cells to escape from host immune recognition has been suggested by several in vivo observations. First, MAA down-regulation has been noted in residual tumors following T cell-based immunotherapy (Jager et al., 1996; Scheibenbogen et al., 1996). Second, although the HLA class I antigen used as the restriction element was expressed, the MAA used as the target of active specific immunotherapy was not detected in some metastases removed during rapid progression of the disease from a patient with recurrent melanoma after a complete response to T cell-based immunotherapy. Conversely, the restricting HLA class I allele was not detected in other synchronous metastases that expressed the MAA used as a target. In either case the metastases were insensitive to the MAA-specific CTL response elicited by the vaccination. Finally, IHC staining of metastases from patients with melanoma treated with a gp100/pme117derived peptide showed down-regulation of this MAA in 29% of 155 lesions obtained following immunization but in only 18% of 175 lesions obtained before immunization. This difference was selective for gp100/Pme117, since MART-l/MelanA down-regulation was detected with similar frequency in the two groups of lesions (Riker et al., 1999).These results are compatible with the possibility that gp100/Pme117is susceptible to immune pressure. A similar conclusion was reached by comparing the expression of Erb-2, which contains an HLA-A"0201 associated epitope in breast carcinoma lesions from HLAA"0201 positive and HLA-A"0201 negative patients (Nistico' et al., 1997). The proto-oncogene down-regulation was more marked in HLA-A"0201 positive than in HLA-A"0201 negative breast carcinoma lesions. This difference may reflect a less marked immune selection against this TAA in lesions expressing HLA class I alloantigens other than HLA-A"0201. All of these studies provide circumstantial but not conclusive evidence for preferential TAA loss in response to TAA-specific immune pressure. The resulting TAA loss could render tumor cells insensitive to immune recognition. Among the mechanisms that could lead to altered expression of TAA associated CTL epitopes are transforming gene product mutations. Several of them have been described for types of human tumors that are associated with viral transformation. They include cervical cancer, hepatoma, Hodgkin's lymphoma, and leukemia associated with infections by human papilloma virus, hepatitis B virus, Epstein Barr virus, and HTLV MI viruses, respectively. These transforming viruses induce the expression of specific antigens that provide targets for CTL immune responses. The latter have the ability to modulate tumor cell growth in vivo and to abrogate cellular

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transformation in vitro. Although the previously mentioned virus induced antigens elicit CTL in patients during the course of the disease or, in some cases, following active specific immunotherapy, the cancer progresses in most patients. Tumor progression in these cases may be explained by a weak CTL response, by TAA loss or down-regulation or by loss of CTL defined epitopes because of mutations in the transforming gene products. The latter mechanism is suggested by findings in an experimental model of a viral oncogene transformed tumor. The simian virus 40 (SV40) large tumor (T) antigen is a 94-kD protein that is sufficient to induce transformation of mammalian cells to malignancy. This antigen represents a tumor rejection antigen that can be recognized by CTL (Tanaka et al., 1988; Levine, 1989; Tevethia, 1990). Transformed murine tumors in H-2b mice express four H-2Db-restricted antigenic epitopes (Tanaka et al., 1988,1989) and one H-2Kb-restrictedantigenic epitope (Tanakaet al., 1988)that are the targets of T-antigen-specific CTL. Analysis of SV4O-transformedC57BW6 primary kidney cells (K-0) that had become resistant to T-antigen specific CTL after coculture with the five T-antigen-specific CTL populations identified changes in the primary amino acid sequence of the T antigen. These changes did not affect the antigen expression but disrupted the epitopes recognized by CTL. These findings suggest an additional mechanism by which tumor cells can escape T cell recognition provided that the disruption in the epitopes recognized by T-antigen-specific CTL are an in vivo phenomenon and do not represent an in vitro artifact. 3. CTL Activity Reduction by TAA Epitope Mimics Host-tumor cell interactions may be affected by environmental factors. Among those that may shape the TCR repertoire after birth are repeated encounters with degenerate epitopes from endogenous or exogenous sources able to interact with the TCR recognizing a TAA. This welldescribed phenomenon, which is referred to as molecular mimicry (Wucherpfennig and Strominger, 1995), underlies interactions of environmental pathogens or self-antigens with MAA-specific CTL (Loftus et al., 1996). A database search for potential peptides analogs of the immunodominant MART-1 epitope MART-127-35 (see Table IA) identified epitope mimicry sequences in proteins of viral, bacterial, and human (self) origin that could react with MART-l/MelanA-specific CTL. Therefore, analog peptides could shape TCR repertoire by direct interactions during thymic selection (Bevan, 1997) or by repeated encounters in the periphery during mature life (Matsushita et al., 1997; Bevan, 1997). Although an individual TCR can engage different, though biochemically related, peptide sequences, the result of the various TCR-epitope interactions can yield remarkably different outcomes on a particular T cell ranging from fully agonist to

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antagonist (Lyonset al., 1996).Loftus et al. (1998)identified partial agonists of MART-127.35, which could partially tolerize MART-1 specific CTL. These in vitro experiments suggested that encounters with partial agonist or antagonist peptides may hamper CTL responses to their natural ligands and, in the case of TAA-specific CTL, be responsible for impaired antiTAA CTL responses. Most of the MART-lZ7.%analogs so far identified are peptide sequences from proteins produced by human pathogens or expressed by human normal tissues. Therefore, it could be postulated that such analog peptides could be commonly present in the organism and be responsible for decreased function of TAA-specific CTL. However, it is not known whether these analog peptides are present in the general circulation or in the tumor microenvironment in concentrations sufficient to modulate the activity of MART-l-specific CTL. Therefore, the clinical significance of the in vitro findings we have discussed remains to be determined. 4. Masking of Subdominant TAA by Zmmunodominant TAA As discussed earlier, experimental and human tumors express multiple CTL defined tumor-specific epitopes that may be lost following coculture with TAA-specific CTL. Schreiber and colleagues (Urban et al., 1984,1986) have demonstrated that there is a hierarchy in the recognition pattern of TAA expressed by tumor cells that are the targets of TAA-specific CTL. In a series of studies, recognition of a second TAA occurred only after the expression of the first TAA had been lost. This finding suggeststhat an immunodominant TAA expressed by a tumor cell may mask recognition of other TAA, preventing immune attack on the tumor cell variants that have lost the immunodominant TAA. This phenomenon of sequential antigen recognition is not unique of tumor cells, since it has also been described for multiple minor histocompatibility antigens expressed by a single cell (Wettstein and Bailey, 1982).Three murine model systems suggest that immunodominant TAA may inhibit in vivo the development of immune responses toward subdominant TAA. In one of them, tumor cells with a selective loss of a MHC class I allele which presents an immunodominant TAA failed to elicit a CTL immune response to other TAA presented by the expressed MHC class I alleles. The variant cells continued to induce a CTL immune response to the immunodominant TAA, although the latter could not serve as a target (Seung et al., 1993). In the other two models (Dudley and Roopenian, 1996; Van Waes et al., 1996)followingimmunizations with an immunodominant MHC class I alloantigen or TAA loss variants naive mice developed a subdominant TAA-specific CTL response restricted by the expressed MHC class I alleles. However no response to subdominant TAA was detected when mice were immunizedwith a mixture ofparental and loss variant tumor cells. Therefore

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in the mouse models analyzed, at least two mechanisms (i.e.,cross presentation by professional antigen presenting cells of immunodominant TAA expressed by MHC class I alloantigen loss variants and TAA presentation by parental tumor cells underlie the lack of MHC class I antigen restricted CTL response to subdominant TAA). The same mechanisms could account for the ability of HLA class I allospecificity loss human tumor cells to escape from CTL mediated recognition, since they are mixed with parental cells in viva Patients bearing a specific HLA class I allospecificity associated with a dominant immune reaction toward a TAA may have reduced CTL reactivity restricted to a subdominant epitope/HLA class I antigen combination. In humans, TAA-specific CTL reactivity toward a specific epitope/HLA class I alloantigencombination has never been demonstrated to be dependent upon the other HLA class I alloantigens present in the patient’s phenotype. However, if the hypothesis that immunodominant epitope/HLA class I antigen combinations have inhibitory effects on the development of CTL responses toward subdominant epitope/HLA class I antigen combinations is correct, loss of a dominant HLA class I alloantigen may provide tumor cells with a significantadvantage in their progression. Thus, these masked T cell epitopes may have important implicationsfor the future design of more effective cancer vaccines because they may provide a means to overcome the problem of antigen loss variants.

B. HLA CLASSI ANTIGEN Loss OR DOWN-REGULATION Altered MHC class I antigen expression in tumors was first reported over a quarter of a century ago (Hellstrom, 1960; Klein et al., 1960; Moller and Moller, 1962). Selective loss of MHC class I alleles was subsequently described in several mouse tumors including the TL leukemia cell line (Cikes, 1971a),the MCA induced tumors (Haywood and McKhann, 1971), the murine leukemia virus induced tumors YCAB and YAC (Cikes, 1971a; Cikes and Friberg, 1971b; Cikes et al., 1973), the Gardner lymphoma and MCG4 cell lines (Garrido et al., 1976, 1979) and the P815 mastocytoma (Wolf et al., 1977). It was soon realized that this phenotypic alterations of MHC class I antigen expression may permit tumor cells to avoid or to survive attack by the immune system. This notion was supported by the enhanced growth of mouse tumor cells which had down-regulated MHC class I antigen expression following transfection with antisense DNA (Hui, 1989) and by the loss of tumorigenicity of aggressive MHC class I-negative mouse tumor cells following transfection with MHC class I genes (Hui et al., 1984). The latter results presumably reflect the induction of TAAspecific, MHC class I antigen restricted CTL by the transfectants in the host. Similar findings have been reported in several other tumor systems such as T10 sarcoma, 3LL Lewis lung carcinoma, and B16 melanoma

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(Tanaka et al., 1985; Wallich et al., 1985; Bahler et al., 1987; Plaksin et al., 1988; Porgador et al., 1989). The metastatic capacity of tumor cells was also shown to be associated with altered patterns of MHC class I gene expression (De Baetselier et al., 1980; Eisenbach et al., 1983, 1984). The metastatic competence of cloned cell populations of mouse 3LL Lewis lung carcinoma cells was correlated with the level of H-2Kbantigen downregulation (Eisenbach et al., 1983, 1984). In some cases, the metastatic phenotype of tumor cells could be converted to a nonmetastatic phenotype by transfection of syngeneic MHC class I molecules (Wallich et al., 1985; Plaksin et al., 1988; Feldman and Eisenbach, 1991). Interestingly, in the AKR leukemia, 3LL lung carcinoma, and B16 melanoma models, preimmunization with MHC class I antigen positive cells decreased the tumorigenicity and metastatic spread in animals challenged with parental MHC class I negative cells (Hui et al., 1984; Plaksin et al., 1988; Porgador et al., 1989). These results suggested that the low level of MHC class I antigen expression on these cells was adequate for susceptibility to lysis by MHC class I antigen restricted CTL, but was not sufficient to elicit a primary immune response. In spite of the relevance of these intriguing results for the development of immunotherapeutic approaches to cancer, interest in abnormal MHC class I antigen expression by tumor cells waned in the 1980s. The loss of interest reflects, at least in part, the skepticism generated in the field by the realization that alien histocompatibility antigens described in a number of mouse tumors and of human cell lines (Bortin and Truitt, 1980, 1981; Pierotti and Permiani, 1984)were essentiallytechnical artifacts. Specifically the detection of alien histocompatibility antigens was caused by crosscontamination of strains and cell lines in mice and by contamination of anti-HLA class I antibodies and alloantiserawith anti-HLA class I1 antibodies and by the unexpected expression of HLA class I1 antigens by tumors of nonlymphoid origin in humans. Furthermore, conflicting data regarding the clinical significance of HLA class I antigen down-regulation in human tumors (Durrant et al., 1987; van den Ingh et al., 1987) had a negative impact on the development of this line of research. In retrospect, these conflicting results are not surprising given the diverse patient populations investigated, the different role played by immunological events in the pathogenesis and clinical course of the various types of cancer, and the different characteristics of the mAb used by the various investigators. Renewed interest in MHC class I antigen loss in tumors took place in the early 1990s with the realization of the crucial role played by MHC class I antigens in the recognition of tumor cells by CTL (Rosenberg et al., 1988; Wolfel et al., 1989; Crowley et al., 1991) and the emphasis on the use of T cell based immunotherapy for the treatment of human cancer (Boon et al., 1997; Rosenberg, 1997).

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Early studies of HLA class I antigen expression in human tumor specimens were limited by tlie lack of appropriate reagents and technical difficulties associated with the detection of MHC class I antigens in surgically removed tumor specimens. However, over the past two decades the availability of HLA-specific mAb suitable for IHC staining and technical advancements in IHC staining techniques have allowed extensive analysis of HLA class I antigen expression in cryopreserved tumors. Furthermore, the use of mAb recognizing monomorphic, locus-specific, and allele-specific determinants has identified distinct phenotypes of malignant cells with abnormalities in HLA class I antigen expression (Ferrone and Marincola, 1995; Garrido et al., 1997). In recent years, these studies have benefited from HLA and Cancer Workshops (Gamdo et al., 1997), which have contributed to standardize reagents and methodology and have facilitated the comparison of results obtained by different laboratories. Analysis of surgically removed tumor specimens by IHC staining allows one to examine the pattern of HLA class I antigen expression within an entire, heterogeneous tumor cell population and to compare these patterns to surrounding normal tissue expression. The latter is usually used as a reference to evaluate the level of HLA class I antigen expression in malignant cells. Furthermore, this type of analysis eliminates potential artifacts that may result through selection of tumor cell populations during establishment of cultured cell lines in uitro. Although the level of surface expression of MHC class I antigens on tumor cells has been extensively studied (Momburg et al., 1986, 1989; D’Alessandro et al., 1987; Holzmann et al., 1987; Natali et al., 1989; Mechtersheimer et al., 1990; Cordon-Cardo et al., 1991; D’Urso et al., 1991; Ruiter et al., 1991; Ruiz-Cabello et al., 1991; Gattoni-Celli et al., 1992; Wang et al., 1993), these analyses have been performed mostly with mAb recognizing monomorphic determinants. Recently, however, mAbs recognizing determinants restricted to the gene products of HLA-A and HLA-B loci or polymorphic determinants defining HLA class I allospecificities (Garrido et al., 1997) have been utilized to analyze malignant cells. The IHC staining of malignant lesions has detected distinct phenotypes of altered HLA class I antigen expression on tumor cells. They include (i) total HLA class I antigen loss or down-regulation and (ii) selective loss or down-regulation of HLA class I alleles (Table IV). The latter phenotype includes loss of a haplotype, down-regulation of the gene products of HLA-A or HLA-B loci and HLA class I allospecificity loss or down-regulation. These abnormal phenotypes may be present individually or in combination in a given tumor cell population resulting in a complex phenotype. Furthermore, many of these phenotypes may be represented within a given malignant lesion resulting in a heterogeneous pattern of HLA class I antigen expression. As it will be discussed later,

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TABLE IV AL,TEREDHLA CLASSI PHENOTYPES DESCRIBED IN TUMOR CELLS

Phenotype 1. Total loss or down-regulation a. Loss b. Down-regulation 2. Selective loss or down-regulation a. Loss of haplotype b. HLA-A or -B down-regulation c. Loss of allele d. Down-regulation of' alleles 3. Complex phenotype

Mechanism Mutations in the Pzm gene Altered transcriptional regulation Genomic loss Altered locus specific transcriptional regulation Mutation of heavy chain gene ? Combination of mechanisms described above

cell lines established from various tumors have facilitated the analysis of these phenotypes at the molecular level and the characterization of their functional relevance. HLA class I antigens are lost or down-regulated in many types of human tumors. The most common types of solid tumors for which more than 100 surgically removed primary lesions have been analyzed include melanoma and carcinomas of the breast, cervix, colon, head and neck squamous cell, kidney and prostate (Table V ) . Frequency of altered HLA class I antigen expression in human tumors is summarized from these studies as loss or down-regulation of monomorphic determinants or selective downregulation of one or more allospecificity(ies).The frequency of total HLA class I antigen loss and down-regulation in primary lesions, which has been determined by IHC staining with mAb to monomorphic determinants ranges from 16 to 50% among the various types of tumors tested. The frequency of selective HLA class I allospecificity loss and down-regulation, which has been assessed by staining lesions with mAb to polymorphic determinants of HLA class I antigens, ranges from 4 to 35% among the types of tumors analyzed. It is significantly higher in cervical carcinoma, prostate carcinoma, and melanoma than in head and neck squamous cell, breast, lung, and colon carcinoma. Whether any particular HLA class I allospecificity is preferentially down-regulated in malignant lesions and/or in any type of cancer remains to be determined. At present, the number of lesions and allospecificities tested is still too small to draw conclusions given the degree of polymorphism in the population for HLA class I antigens. It should be noted that the figures shown in Table V are likely to be an underestimate of the frequency of selective HLA class I allospecificity down-regulation, since the limited number of mAb to polymorphic determinants of HLA class I antigens suitable for IHC staining of tissues has

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TABLE V FREQUENCY OF HLA c u s s 1 ANTIGENLoss OR DOWN-REGUMTION IN HUMAN CANCERS Cancer Type Head and neck" Breast Lung" Renal"' Colone CervicalJ Prostat& Melanomah

# Lesions Total Loss (%) Locus or Allelic Loss (%) Combined (%) 381

524

24 43

794

27 16 27

488 118 569

29 50 16

366 264

9

11 4 16 16 26 20 35

33 54

31

32 43

55 70 51

Esteban et al., 1989, 1990, 1996; Lopez-Nevot et al., 1989; Ruiz-Cabello et al., 1989; Houck et al., 1990; Lai et al., 1990; Mattijssen et al., 1991; Vora et al., 1997. Whitwell et al.,1984; Perez et al., 1986; Zuk and Walker, 1987; Natali et al., 1989; Ruiz-Cabello et al., 1989; Wintzer et al., 1990; Cordon-Cardo et al., 1991; Goepel et al., 1991; Pantel et al., 1991; Solana et al., 1992; Maiorana et al., 1995; Cabrera et al., 1996. Natali et al., 1989; Redondo et al., 1991a,b; Passlick et al., 1994, 1996. Natali et al., 1989; Ruiz-Cabello et al., 1989; Cordon-Cardo et al., 1991; Buszello and Ackermann, 1992, 1994; Ohmori et al., 1995; Gastl et al., 1996; Luboldt et al., 1996. van den Ingh et al.,1987; Flees et al., 1988; Lopez-Nevot et al., 1989; Mornburg et al., 1989; Natali et al., 1989; Ruiz-Cabello et al., 1989; Smith et al., 1989: McDougall et al., 1990; Cordon-Cardo et al., 1991; Goepel et al., 1991; Moller et al., 1991; Pantel et al., 1991; Kaklamanis et al., 1992; Cabrera et al., 1998. 'Natali et al., 1989; Connor and Stem, 1990; Torres et al., 1993; Cromme et al., 1993a,b; Honma et al., 1994; Hilders et a!., 1994, 1995; Keating et al., 1995; van Driel et al., 1996. 6 Natali et al., 1989; Sharpe et al., 1994; Blades et al., 1995; Bander et al., 1997. Ferrone and Marincola. 1995.

restricted the analysis to only a few HLA class I allospecificities. An additional contributing factor to an underestimation of selective HLA class I allospecificity down-regulation in tumors is the difficulty in adequately assessing the heterogeneity of HLA class I antigen expression in malignant lesions with the currently available IHC staining techniques. HLA class I antigen loss has been described in other types of human cancers. However, the number of lesions tested is too small to be a representative sample. They include stomach (Lopez-Nevot et al., 1989),pancreatic (Torres et al., 1996; Ruiz-Cabello et al., 1989),bladder (Cordon-Cardo et a!., 1991), germ cell (Klein et al., 1990) and basal cell (Ruiz-Cabello et al., 1989) carcinomas. It is also worth noting that in some tumors such as liposarcomas, meningiomas, and embryonal carcinomas, cells acquire rather than lose HLA class I antigen expression during the process of malignant transformation (Natali et al., 1984).

1. Molecular Basis of Altered HLA Class I Antigen Expression in Tumor Cells Characterization of the molecular basis of altered HLA class I antigen expression in tumor cells has contributed invaluable information regarding

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the genesis of these defects and how the immune system interacts with malignant cells. Crucial to the success of these studies has been the isolation and establishment of cell lines from surgically removed lesions with altered HLA class I antigen expression. As described previously, tumors frequently have a heterogeneous pattern of HLA class I antigen expression, and several of the pheno-es may be represented within a given tumor cell population. Cell lines representing each of the previously mentioned phenotypes have been established and successfully utilized to characterize the molecular basis of HLA class I antigen defects. Several distinct molecular mechanisms that underlie the various phenotypes of HLA class I antigen down-regulation found in tumor cells have been identified.

2. Total HLA Class Z Antigen Loss or Down-Regulation Total HLA class I antigen loss in tumor cells is caused by mutations in the Pzm gene that result in loss of functional Pzm expression. Mutations in the P2m gene have been described in colon and lung carcinoma and in malignant melanoma (D’Urso et al., 1991; Wang et al., 1993; Bicknell et al., 1994; Chen et al., 1996; Hicklin et al., 1997, 1998; Benitez et al., 1998). Transfection of &m-deficient cell lines with a wild-type P2m gene has restored cell surface expression and function of HLA class I antigens demonstrating that the mutations in the P2m gene are entirely responsible for total HLA class I antigen loss in these cell lines (D’Urso et al., 1991; Wang et al., 1993; Bicknell et al., 1994; Hicklin et al., 1998). This finding illustrates the critical role of P2m in proper expression of HLA class I allospecificities.The P2m mRNA is usually present in this phenotype, but functional P2m protein and cell surface HLA class I molecules are not expressed. Abnormalities ranging from point mutations to large deletions within the P2m gene have been described in several colon and melanoma cell lines and in one lung carcinoma cell line (Fig. 3). In the majority of cases, these mutations (frameshifts, early stop codons, deletions) affect P2m expression at the posttranscriptional level thus explaining the lack of functional protein in spite of the apparently normal mRNA expression. Of interest are several reports demonstrating a mutation “hot spot” within the Pzm gene that frequently is responsible for total HLA class I antigen loss in colon carcinoma and melanoma cells. This hot spot is located in an 8-bp CT repeat region in exon 1of the Pzm gene where dinucleotide CT deletions have been identified in several cell lines (Bicknell et al., 1994; Chen et al., 1996; Hicklin et al., 1998). This type of mutation may reflect the genetic instability or mutator phenotype in tumor cells, which display a high mutation rate and accumulation of mutations during tumor progression (Branch et al., 1995; Bicknell et al., 1996; Lengauer et al., 1998). The 8-bp CT repeat region of the Pzm gene is a likely target for mutation in tumor cells that exhibit the mutant phenotype because such nucleotide

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1. Large deletion

2. ATG point mutation 3. CT deletion

5’ untranslated region -t Inhon 1 Exon 1

Transcription

FO-1

Melanoma

D’Urso et al. 1991

Translation

H2009

Lung

Chen et al. 1996

Exon 1

Frameshill

LB1622-MEL 1074mel LOVO H63O

HCT-15

Melanoma Melanoma Colon Colon Colon Melanoma Melanoma Melanoma Melanoma Melanoma Colon Colon

Benitez et al. 1998 Liang et al. I999 Bicknell et al. 1994 Bicknell et al. 1994 Bicknell et al. 1994 Hicklin et al. 1998 Perez-Burkhardtet al. 1996 Liang et al. 1999 Liang et al. 1999 Liang et al. 1999 Bicknell et al. 1994 Gattoni-Celli et al. 1992

Me18105

Melanoma

Hicklin et al. 1998

sw48

4. TCTT deletion 5. C -t Apoint mutation A -t 0 point mutation 6. G - t Apoint mutation 7. Singlebase deletion 8. C -tGpoint mutation 9. C -+Gpoint mutation 10. 14-bp deletion

Exon 1 Inhon 1- splice acceptor site

Frameshift Frameshill

Me1386 M-34 1106mel 1180mel 1259mel HRA19

Exon 2

Frameshift

C84

Colon

Bicknell et al. 1994

Exon 2

Frameshift

SK-MEL-33

Melanoma

Wang et al. 19%

Exon 2

Early stop codon

BB74-MEL

Melanoma

Benitez et al. 1998

Exon 2

Early stop codon

1174mel

Melanoma

Liang et al. 1999

Exon 2

Frameshift

Me9923

Melanoma

Hicklin, 1998

FIG.3. Map of described mutations leading to &-p loss in malignant cells.

elements are more susceptible to defective DNA repair mechanisms. In fact, analysis of 37 colorectal carcinoma cell lines showed that only those cell lines with a mutator phenotype also failed to express functional Pzm (Branch et al., 1995). Only one type of &m gene mutation is usually found in tumor cells with total HLA class I antigen loss. The lack of &m protein synthesis in these cells is likely to reflect the loss of the wild-type &m allele, since identical &m gene mutations are not likely to occur independently in both alleles in a cell. These findings are consistent with the loss of heterozygosity frequently found in tumor cells because of their genetic instability (Lengauer et d., 1998). Possible mechanisms responsible for the loss of heterozygosity for the wild-type Pzm allele include loss of one copy of chromosome

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15 where the Pzm gene is located (Goodfellow et al., 1975) or loss of the wild-type Pzm allele because of a mitotic recombination event. Pzm loss and gene mutations found in tumor cell lines are not an in vitru artifact because the same mutations have been also found in situ in the tumor lesions from which the cell lines were derived (Wang et al., 1993; Bicknell et al., 1996; Hicklin et al., 1998). In a recent study, loss of Pzm expression was found by IHC staining of a melanoma lesion from which a cell line with a Pzm gene mutation was derived (Benitez et al., 1998). Furthermore, loss of Pzm expression has been observed by IHC staining of HLA class I antigen negative tumor specimens (Ruiter et al., 1984; Takata et al., 1989; Cabrera et al., 1991). These data provide important in vivu evidence that HLA class I antigen loss, especially in colon carcinoma and malignant melanoma, is associated with mutations in the Pzm gene. It is conceivable that total HLA class I antigen loss may also result from large deletions or rearrangements within the MHC region, or loss of both copies of chromosome 6 where the MHC region is located (Francke and Pellegrino, 1977). Yet, no such molecular defects in tumor cells have been described in the literature. It is interesting to note that lack of Pzm gene transcription due to defects in regulatory mechanisms has not been described in tumor cells to date. It is worthwhile testing tumor cells with Pzm loss for these defects because similar regulatory mechanisms control Pzm and HLA class I heavy chain gene expression (Gobin et al., 1998b). In contrast to total loss, HLA class I antigen down-regulation on tumor cells that are composed of all alleles reflects defects in the regulatory mechanisms responsible for the expression of HLA class I heavy chains. The complex mechanisms controlling HLA class I gene expression and how these mechanisms are altered in tumor cells is still under investigation. However, various studies have shown that defects in transcriptional regulation of HLA class I gene expression result in down-regulation of mRNA levels and their expression on the cell surface (Doyle et al., 1985; Blanchet et al., 1991; Ruiz-Cabello et al., 1991). Tumor cell lines with HLA class I antigen down-regulation were shown to have altered binding of regulatory factors to HLA class I heavy chain gene enhancer elements (Henseling et al., 1990; Blanchet et al., 1991, 1992). Specifically, differential binding of the NF-KB and K B F l transcription factors to the enhancer A element were implicated in these studies. Furthermore, studies in mouse tumor cell lines have demonstrated that an excess of the p50 subunit (p50-p50 homodimers) over the p65 subunit of NF-KB is responsible for downregulation of MHC class I antigen expression in these cells (Plaksin et al., 1993).

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Another proposed mechanism of HLA class I antigen down-regulation implicates the oncogene product N-myc. A correlation exists between HLA class I antigen down-regulation and elevated expression of the oncogene product N-myc in neuroblastoma cell lines (Bernards et al., 1986). Moreover, transfection of the N-myc gene into cells expressing normal levels of HLA class I antigens leads to their down-regulation. This abnormality is mediated by defective binding of NF-kB at the enhancer A element of the MHC class I heavy chain promoter (Lenardo et al., 1989). This mechanism was further defined showing that N-myc inhibits p50 subunit activity by reducing its expression and inhibiting NF-KB binding activity (Van’t Veer et al., 1993). 3. Selective HLA Class I Allospeci,ficity Loss or Down-Regulation Abnormalities in HLA class I antigens may selectively affect only some of the HLAclass I allospecificitiesencoded in a malignant cell. These abnormalities include loss of doantigens encoded in a haplotype, down-regulation of the gene products of HLA-A or HLA-B loci and loss or down-regulation of a single allospecificity. Loss of an HLA class I haplotype has been described in malignant melanoma and pancreatic carcinoma (Marincola et al., 1994a; Torres et al., 1996).This defect arises through mechanisms of defective chromosomal segregation, nondysjunction, or mitotic recombination that cause loss of variable portions of genomic DNAin the short arm of chromosome 6. Selective down-regulation of the gene products of the HLA-A or HLAB locus has been found frequently in tumor cell lines (Marincola et al., 1994a; Hicklin et al., 1998) and in surgically removed lesions by IHC staining with locus-specific mAb (Ferron et al., 1989; Lopez-Nevot et al., 1989; Ruiz-Cabello et al., 1991;Kim et al., 1996). The frequency of HLA-B antigen down-regulation is higher than that of HLA-A antigens in malignant lesions (Marincola et al., 1994a). No information is available about the selective down-regulation of the gene products of the HLA-C locus because mAb recognizing determinants restricted to HLA-C antigens are not available. The differential staining of malignant cells by mAb recognizing determinants restricted to the gene products of the HLA-A or HLA-B loci is not likely to reflect differences in the characteristics of the mAb used in the IHC assays since they stained the surrounding normal cells to a similar extent. Down-regulation of the gene products of one HLA class I locus is often reversible in vitro by incubation of cell lines with interferon-y (IFNy ) (Marincola et al., 1994a). The precise molecular mechanisms leading to down-regulation of the gene products of HLA-A and HLA-B loci are not known. It is likely that a common pathway is responsible for HLA class I locus-specific downregulation in tumor cells as most HLA class I alloantigens within the same

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locus behave similarly within a cell line; i.e., they are simultaneously downor up-regulated ( Marincola et al., 1994b).Analysis of nucleotide sequences within the promoter of HLA-A and -B genes suggests that locus-specific down-regulation takes place through cis-acting regulatory mechanisms. The enhancer A element contains several locus-specific residues, suggesting locus-specific regulation of enhancer A-driven transcription of HLA class I genes (Girdlestone et al., 1993; Gobin et al., 1998a). This possibility is supported by the finding that NF-KB can activate HLA-A enhancer A to higher levels than HLA-B enhancer A (Girdlestone et al., 1993; Gobin et nl., 1998a). Moreover, different nuclear factors have been shown to interact with HLA-A and -B enhancer A (Soong and Hui, 1992; Girdlestone et al., 1993; Gobin et al., 1998a). There are also differences in the upregulation of the gene products of HLA class I loci in response to IFNy. Two locus-specific residues within the interferon stimulating responder element of the HLA class I heavy chain promoter are responsible for the higher induction of HLA-B than HLA-A antigen expression in cells treated with IFN-y (Hakem et al., 1991; Girdlestone et al., 1993). Differential expression of HLA-A and -B antigens attributed to regulatory mechanisms has been described in colorectal carcinoma cell lines. In these studies, differences in binding of locus-specific transcription factors to the kB2 site within the enhancer A element were observed (Soong and Hui, 1992). In human melanoma cell lines, a correlation has been described between HLA-B antigen down-regulation and elevated c-myc expression (Versteeg et al., 1988, 1989a,b). Transfection of c-myc into high HLA-B antigen expressing melanoma cell lines leads to HLA-B antigen down-regulation (Peltenburg and Schrier, 1994). This evidence was taken to suggest that HLA class I locus-specific down-regulation is somehow related to the oncogenic process. However, HLA-B antigen down-regulation by c-myc may not explain this phenotype in all melanoma cells, since HLA-B antigens are down-regulated in normal epithelial melanocytes with the same frequency as in melanoma cells (Marincola et al., 1994a). Furthermore, HLAB antigen down-regulation has been found in other tumor cells with no association to c-myc activation (Feltner et al., 1989; Wolfel et al., 1989; Redondo et al., 1991b). Selective loss of a HLA class I alloantigen has been clearly documented by IHC staining of surgically removed malignant lesions and by analysis of cell lines with a variety of techniques (Browning et al., 1993; Rivoltini et al., 1995a; Koopman et al., 1998; Wang et al., 1999). This abnormality is not unique of malignant cells because it has been described also in normal cells, although with markedly lower frequency (Lienert et al., 1996; Laforet et al., 1997; Magor et al., 1997; Mine et al., 1997). The molecular defects underlying the selective loss of a HLA class I allospecificity in

2 14

MARINCOLA et d.

tumor cells have been characterized in only a few cases. The lesions identified include loss of a HLA class I heavy chain allele gene in a colon carcinoma cell line (Browning et al., 1993) and a mutation at the 5' donor site of intron 2 of the lost HLA class I allospecificity which results in two aberrant transcripts in a melanoma cell line (Wang et al., 1999). In other colon carcinoma cell lines, the molecular lesion(s) underlying the selective loss of a HLA class I allospecificity has not been characterized yet. The phenotype is compatible with a mutation in the HLA class I gene itself or in the upstream promoter region because the HLA class I alloantigenspecific mRNA was detected in the cell lines. Furthermore, the expression of the missing HLA class I allospecificitywas restored followingtransfection of the cell line with the wild-type gene (Browning et al., 1993). Mutations in the genes encoding the HLA class I heavy chain have also been suggested to underlie the selective loss of HLA class I allospecificities in two cervical carcinoma cell lines (Koopman et al., 1998). A less extensively studied abnormality has been the selective downregulation of one or more of the HLA class I allospecificities expressed by a tumor cell. This phenotype has been analyzed only on cell lines, since the availableIHC methodologyis not suitable to measure the expressionof HLA class I alloantigen in tissues. Marked variation in the level of HLA-A2 allospecificity was identified in clones isolated from a bulk melanoma cell line (Rivoltiniet al., 1995a).The level of HLA-A"0201 antigens correlated closely with the susceptibilityof the clones to lysis by HLA-A"0201 restricted, MAA specific CTL. These results have been confirmed by a recent analysis of a panel of melanoma cell lines, which has shown marked variations in the expression of HLA-A2 antigen among them (Cormieret al., 1999).The mechanism(s) underlying the variability of alloantigen expression noted in cell lines has not beeninvestigated. Furthermore, it is not known whether the naturally occurring variation in HLA class I alloantigen expression noted in cell lines corresponds to similar variability in uiuo. C. DEFECTS IN HLA CLASS I-DEPENDENT ANTIGENPROCESSING HLA class I antigen down-regulation is frequently associated with defects in antigen processing. Correct assembly of HLA class I molecules and efficient presentation of antigenic peptides is dependent on the generation of peptides by the proteasome complex and transport of these peptides into the endoplasmic reticulum where they are assembled with HLA class I heavy chains and &m. Tumor cells may alter expression of components of the HLA class I antigen processing pathway leading to abnormal processing and presentation of TAA. In these tumor cells, the population of HLA class I antigens is likely to be different from that of cells with normal HLA class I-dependent antigen processing. However, in contrast to defects in

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Pzm, those in antigen processing may not cause a discernible loss of HLA class I antigen expression because peptides may be generated by alternative proteasome complexes (York and Rock, 1996) and supplied through TAPindependent mechanisms (Wei and Cresswell, 1992). Several studies have demonstrated abnormal expression of the proteasome subunits LMP2 and LMP7 and/or the transporter subunits TAPl and TAP2 in cell lines of hepatocellular, lung, prostate, and renal carcinomas; lymphoma; malignant melanoma; and neuroblastoma (Seigler et al., 1971; Restifo et al., 1993; Ferrone and Marincola, 1995; Rowe et al., 1995; Sanda et al., 1995; Alpan et al., 1996; Kurokohchi et al., 1996; Seliger et al., 1996a,b; Singal et al., 1996; Johnsen et al., 1998; White, 1998). Simultaneous down-regulation of multiple components (i.e.,LMP2, LMP7, TAP1, and/or TAPB) has been detected in several cell lines. Functionally, altered expression of LMP or TAP subunits causes defective processing and presentation of antigenic peptides to specific CTL (Restifo et al., 1993; Rowe et al., 1995; Sanda et al., 1995; White, 1998). The LMP and/or TAP gene transfection of these cell lines restores HLA class I presentation of antigens and tumor cell recognition by antigen-specific CTL. Although a number of molecules involved in the antigen processing machinery have been recently identified (Pamer and Cresswell, 1998), the availability of antibodies to study these molecules is limited. This accounts at least in part for the limited information about the expression of components of the antigen processing machinery in surgically removed malignant lesions. Only a small number of lesions from various human cancers have been tested for the expression of components of the antigen processing machinery and the analysis has been primarily restricted to the expression of the TAPl subunit of the TAP transporter. The TAP1 down-regulation has been observed in head and neck squamous cell, breast, lung, colon, and cervical carcinomas and in melanomas (Table VI). The frequency of TAPl down-regulation vanes among different types of tumors ranging from 6% in head and neck carcinoma to 56% in malignant melanoma. TAPl is down-regulated more frequently in metastatic than in primary lesions of breast carcinoma, cervical carcinoma, and malignant melanoma. The expression of TAP2 and of the proteasome subunits LMP2 and LMP7 has been analyzed only in breast carcinoma and in melanoma lesions. In both types of malignant lesions, TAP2 down-regulation parallels that of TAP1. In all the breast carcinoma lesions tested, LMP2 and LMP7 have been detected. The intensity of staining varied markedly among the lesions with no relationship to tumor grading. In both primary and metastatic melanoma lesions, LMP2 is expressed less frequently than LMP7. Significantly, TAP down-regulation in malignant lesions is usually associated with the synchronous HLA class I down-regulation (Cromme et al., 1994a,b;

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TABLE VI FREQUENCY OF TAP Loss OR DOWN-REGULATION IN HUMAN CANCERS Primary Lesions

Metastases

Cancer Type

# Lesions

(%)

#Lesions

(%)

Head and neck" Breastb Lung" Colond Cenical" Melanomaf

34 116 93 81 196 60

9 29 21 16 38 53

n.d. 63 n.d. n.d. 20 38

n.d. 42

n.d. n.d. 85 83

Vora et al., 1997. Kaklamanis et d., 1995; Vitale et al.,1998. Korkolopoulou et d.,1996. Kaklamanis et al., 1994. Cromme et al., 1994; Keating et al., 1995. 'Kageshita et al., 1999. n.d. = not determined

'

Kaklamanis et al., 1995; Korkolopoulou et al., 1996; Vitale et al., 1998; Kageshita et al., 1999).Whether synchronous TAP and HLA class I downregulation within a lesion occurs in the same tumor cells and is due to related or independent mechanisms is not known at the present time. Little information is available about the molecular basis of LMP and/or TAP down-regulation in malignant cells. Synchronous loss of LMP and TAP subunits in some tumor cell lines suggests that mechanisms of gene regulation are defective in these cells because it is unlikely that mutations in each gene are the basis for this phenotype. In support of this hypothesis, this phenotype can be corrected by treatment with IFN-y (Restifo et al., 1993;Seligeret al., 1996a;Johnsen et al., 1998;White, 1998).The molecular basis for these regulatory defects has not yet been identified. Selective down-regulation of individual LMP or TAP subunits has been described in some tumor cell lines. These defects can be attributed to either regulatory mechanisms or mutations in the LMP or TAP subunit genes. Evidence for regulatory mechanisms responsible for selective LMP or TAP loss stems from the described reversal of this deficiency following treatment of cells with IFN-y (Singal et al., 1996; Johnson et al., 1998).To date only one study has described a genetic mutation in tumor cells involving an antigen processing molecule. Chen et al. (1996) have identified a point mutation in the TAPl gene in a lung carcinoma cell line. This mutation introduces a premature stop codon which results in a nonfunctional TAPl protein.

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1. Functional Signijkance of HLA Class Z Antigen Loss or DownRegulation and Antigen Processing Machiney Abnormalities

It was established several years ago that the expression of MHC class I antigens is necessary for tumor recognition by CTL (Rosenberg et al., 1988; Wolfel et al., 1989; Crowley et al., 1991). The molecular basis of this phenomenon was provided by the crystallization of HLA class I antigens (Bjorkman et al., 1987) and of the TCR-peptide/HLA class I antigen complex (Garboczi et al., 1996) that illustrates the interaction between CTL and peptides bound to HLA class I molecules. Thus, altered HLA class I antigen expression by tumor cells will presumably lead to loss of recognition by TAA-specific CTL and render these cells resistant to CTL lysis (Wolfel et al., 1989; Ellison et al., 1990; Lehmann et al., 1995; Rivoltini et al., 1995a; Wang et al., 1996). The extent of tumor cell resistance to CTL lysis depends largely on the phenotype of HLA class I antigen downregulation. As described earlier, these phenotypes range from complete loss to single losses of a HLA class I restriction element. During the course of malignant disease, tumor cell escape from immune destruction depends on the complex interplay between reactive CTL and the HLA class I phenotype of tumor cells. Aside from HLA class I antigen total loss, quantitative differences in the level of HLA class I antigen expression on target cells might also determine the extent of CTL-mediated lysis versus tumor cell resistance. The majority of studies which have analyzed the role of HLA class I antigens in the recognition of tumor cells by TAA-specific CTL has evaluated the effect of HLA class I antigen expression on the extent of lysis in terms of an all or none phenomenon. The effect of reduced levels of the restricting HLA class I allele on the interaction of tumor cells with cognate CTL has been investigated only to a limited extent, although HLA class I antigens are frequently down-regulated in tumor cells. The limited information on this topic is likely to reflect the general assumption that a few (1-5) peptide/ MHC class I antigen complexes on a target cell are sufficient for recognition by CTL (Christinck et al., 1991; Brower et al., 1994; Sykulev et al., 1996; Porgador et al., 199%). As a consequence, one may be inclined to dismiss the possibility that down-regulation of the restricting HLA class I allele may provide tumor cells with an escape mechanism from CTL recognition. However, TAA-derived peptides are likely to compete with thousands of other intracellular peptides for a specific MHC class I allele (Hunt et al., 1992). Therefore, not all possible endogenous epitopes can be presented with a density sufficient for recognition by CTL (Carbone et al., 1988), especially in those cases where the restricting HLA class I allele is downregulated. However, it should be taken into account that high avidity TCW

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MARINCOLA et ~ l .

ligand interactions in the context of self-recognition are eliminated during thymic selection (Chen et al., 1994). Because many TAA are self-antigens, assuming their expression in the thymus during fetal maturation, TCR activation may require a threshold of antigen density higher than that for non-self-antigens (Brower et al., 1994; Sykulev et al., 1996). Recent studies have shown that the level of HLA class I allele expression may vary considerably among tumor cells (Cormier et al., 1999).Because of the highly competitive characteristics of peptide/MHC class I antigen binding (Schild et al., 1990; Foa et al., 1992), variations in MHC class I molecule expression may significantly affect recognition of self-antigenic peptides by MHC class I antigen restricted CTL. This variability may be particularly important for poorly immunogenic human tumors in which a combination of a low level of TAA-derived peptides, low affinity of peptide for HLA class I allele, and HLA class I allele down-regulation may switch on and off the capability of a TAA-specific CTL to recognize tumor cells. When HLA class I allele availability is sufficient to present one or more antigen-derived peptides on the cell surface, a target cell is expected to be lysed through a single or through serial TCR engagement as an all-ornone phenomenon (Valitutti et al., 1995; Viola and Lanzavecchia, 1996). However, if a heterogeneous cell population rather than a single cell is considered, a proportional relationship is expected to exist between epitope density and extent of CTL-mediated lysis of target cells since the epitope density on some cells is below and on others above the threshold required for recognition of targets by CTL. Furthermore, the statistical probability of productive encounters between TCR and epitopes increases with their density. These mechanism(s) account for the relationship found between level of the restricting HLA class I allele and extent of CTL-mediated lysis, when the antigenic peptide is not a limiting variable (Rivoltini et at., 1995a). Loss of HLA class I antigen expression may also have positive effects on effector cell recognition whether the effector cells are CTL or natural killer (NK) cells. According to the “missing self’ hypothesis, HLA class I antigen down-regulation on tumor cells should make these cells more susceptible to NK cell mediated lysis (Ljunggren and Karre, 1985). Such phenomenon is related to the down-regulation of NK cell function by the interaction of HLA class I antigens on target cells with two distinct families of NK cell inhibitory receptors (Lopez-Botet et al., 1996). The first family of receptors belongs to the immunoglobulin (Ig) superfamily of type I glycoproteins and is referred to as killer inhibitory receptors (KIR). The KIR family of receptors contains either 2 (p50, p58) or 3 (p70) Ig-like domains in their extracellular region and specifically recognize groups of HLA-A, -B, and -C alleles. The second family of inhibitory receptors

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includes type I1 molecules characterized by a C-type lectin domain. They are expressed as heterodimers (CD94/NKG2) composed of a CD94 glycoprotein that is disulfide bonded to a member of the NKG2 family of proteins. The CD94NKG2 is a specific receptor for HLA-E antigens that bind leader peptides derived from HLA-A, -B, -C, and -G polypeptides (Braud et al., 1997; Lanier, 1998). The CD94NKG2 receptor functions to detect the overall amount of HLA class I antigen expression on target cells. Natural killer cells monitor HLA class I antigen expression on target cells through these inhibitory receptors and eliminate those with HLA class I antigen down-regulation. Target-cell lysis depends on the degree of MHC class I antigen down-regulation and on the inhibitory receptor repertoire within a NK cell population because subsets of NK cells express different inhibitory receptors with distinct specificity. Increased susceptibility of tumor cells with MHC class I antigen down-regulation to NK cellmediated lysis has been demonstrated in several studies (Ljunggren and Karre, 1985; Maio et al., 1991; Schrier and Peltenburg, 1993). Most studies analyzing this phenomenon have been performed using cell lines with total MHC class I antigen loss (Ljunggren and Karre, 1985; Ferrone and Marincola, 1995; Porgador et al., 1997a). Restoration of MHC class I antigen expression in these cells leads to loss of NK cell-mediated lysis. In addition, it has been shown that NK cells can lyse not only tumor cells with total HLC class I antigen loss, but also those with HLA class I antigen down-regulation (Pende et al., 1998). Nevertheless, other studies have clearly shown that some tumor cell lines are resistant to NK cell-mediated lysis despite total HLA class I antigen down-regulation (Pena et al., 1990; Ferrone, unpublished results; Porgador et nl., 1997a). Furthermore, it is evident that HLA class I antigen negative tumor cells are able to grow and metastasize in patients without susceptibility to NK cell-mediated attack. Therefore, the role of N K cells in immune surveillance of human tumors is still unclear. Expression of nonclassical HLA class I antigens may play a role in NK cell recognition of tumor cells. A recent study (Paul et al., 1998) described inhibition of NK cell-mediated lysis by HLA-G expression on melanoma cells. In this study, HLA-G expression protected melanoma cell lines from NK cell lysis. In addition, a higher level of HLA-G expression was found in metastatic melanoma lesions than in normal skin leading the authors to suggest that melanoma tumors expressing HLA-G are protected from NK cell destruction. However, there is contradictory information regarding NK cell recognition of the nonclassical HLA-G class I molecule. Some studies have shown that the KIR p58 and p70 interact with HLA-G (Pazmany et al., 1996; Munz et al., 1997), whereas other studies have demonstrated that the CD94NKG2 inhibitory receptor interacts with HLA-G

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(Pende et al., 1997; Perez-Villar et al., 1997).Moreover, it has been shown that multiple receptors from both the KIR family and the CD94NKG2 family and possibly an unkhown receptor can interact with HLA-G molecules (Mandelboimet al., 1997).A recent study (Allanet al., 1999)suggests that HLA-G does not interact with members of the KIR family or CD94 NKG2 family but rather with members of the Ig-like transcript (ILT) family of proteins. Therefore, clarification of these results is needed before a conclusion can be drawn regarding the role of HLA-G expression on tumor cells. Furthermore, there is insufficient information about the expression of nonclassical HLA class I antigens such as HLA-E and -G on malignant cells. The expression of HLA-E in tumor cells could be of particular interest for CTL mediated tumor recognition, since expression of HLA-E is dependent upon binding of peptides derived from the signal sequence of most HLA-A, -B, -C, and -G molecules (Braud et al., 1998). Therefore, modulation of HLA-E expression could also regulate tumor cell recognition by NK cells. Inhibitory HLA class I antigen receptors are expressed on T cells as well as NK cells (Lanier and Phillips, 1996; Moretta et al., 1997). The interaction between KIR on a T cell and HLA class I molecules on a target cell can inhibit T cell effector functions mediated by the TCR complex. The presence of KIR on TAA-specific CTL clones may have important implications for recognition of tumor cells with altered HLA class I antigen expression. This notion is supported by recent findings of inhibitory receptors on TAA-specific CTL (Ikeda et al., 1997).HLA class I antigen expression and autologous CTL reactivity was examined using two melanoma cell lines established from metastases that had been removed 5 years apart from a patient. The cell line MEL.A derived from an early lesion expressed all HLA class I allospecificitiespresent in the patient’s phenotype and was recognized by several autologous TAA-specific CTL clones. In contrast, the MEL.B cell line derived from a metastasis removed 5 years later lost expression of all HLA class I molecules except for HLA-A24 and was resistant to all CTL clones generated against MEL.A cells. Autologous CTL clones were subsequently generated against the MEL.B cell line that recognized the HLA-A24 restricted tumor antigen PRAME. Remarkably, these CTL clones were unable to lyse the MEL.A cell line. Analysis of the HLA-A24 restricted CTL clones revealed the presence of an inhibitory receptor that interacted with HLA-Cw7 molecules on the MEL.A cell line. This important finding suggests that TAA-specific CTL may be present in some cancer patients but are unable to attack tumor cells due to the presence of inhibitory receptors. Yet, these findings also suggest that selective loss of a HLA class I allele may be detrimental to tumor cells. Selective loss of a particular HLA class I allele interactingwith an inhibitory receptor

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could deprive tumor cells of an escape mechanism because it eliminates a negative signal that prevents the activation of cytotoxicity by CTL.

2. Immune Selection of Tumor Cells with Abnormal HLA Class I Antigen Expression Molecular defects that alter the expression of HLA class I antigens in tumor cells are a consequence of the genetic instability of malignant cells. Several explanations for HLA class I antigen loss in tumors have been proposed. As described earlier, MHC class I antigen loss may be associated with particular oncogenes that down-regulate their transcription such as that described for the c-myc oncogene family (Schrier and Peltenburg, 1993). Also, it has been proposed that altered MHC class I antigen expression in some tumors reflects the state of differentiation (or dedifferentiation) of these cells or a selective growth advantage rather than selection of a particular tumor cell population (Ottesen et al., 1987; Tomita et al., 1990a,b; Kageshita et al., 1999). Even though there are several possible explanations for how HLA class I antigen negative cells originate in tumors, it is generally accepted that these variants are selected in vivo through a process of immune selection. The HLA class I antigen-restricted recognition of tumor cells by T lymphocytes applies selective pressure to the tumor cell population, and those cells expressing the restricting HLA class I allele will be readily eliminated. During this process, tumor cells with altered HLA class I antigen expression may escape from this immune attack because of the loss of the restricting HLA class I element. Support for the immune selection or escape theory comes from the higher frequency of HLA class I antigen loss in metastases than in primary lesions (CordonCardo et al., 1991; Cromme et al., 1994b; Hilders et al., 1995; van Driel et al., 1996; Kageshita et al., 1999) and from the loss of HLA class I alleles which is increasingly found in a large percentage of tumors (Table V). Additional convincing evidence in support of the immune selection theory stems form the results of serial studies which have correlated changes in the expression of HLA class I antigens in metastatic lesions with emergence of TAA-specific CTL. In a study by Lehman et al. (1995), the melanoma cell line LB33-MEL.A established from a lesion of a patient following surgery was found to express all alleles present in the patient’s HLA class I phenotype. Several CTL clones were generated by stimulating the patient’s peripheral blood lymphocytes with LB33-MEL.A in vitro. The CTL clones were found to recognize at least five distinct HLA class I antigen restricted TAA. Five years later, following several rounds of treatment with cytokines, autologous melanoma cells and chemotherapy the cell line LB33-MEL.B was established from a recurrent metastatic lesion. This cell line had lost all but one HLA class I allele and was resistant to

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LB33-MEL.A cell line. These results are compatible with the possibility that immune selection by TAA-specific CTL responses in these patients resulted in loss of several HLA class I alleles. One question that arises from these findings is whether alterations of MHC class I antigens on tumor cells is an early or late event in the disease process. Current evidence suggests that defects in HLA class I antigen expression and immune selection of these clones can occur at different stages of tumor progression (Fig. 4). The HLA class I antigen negative clones may develop during the course of malignant disease or late in tumor all CTL clones elicited with the

FIG.4. Appearance of HLA class I antigen abnormalities in malignant cells at an early or at a late stage of the disease.

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progression and originate in metastases. This possibility is supported by the analysis of early primary lesions compared to metastatic lesions from the same patient. The IHC staining of archival tumor sections has demonstrated total or selective HLA class I antigen loss in lesions from advanced stages of disease compared to lesions with normal expression early on in the disease (Cordon-Cardo et al., 1991; Pantel et al., 1991; Cromme et al., 199413; Hilders et al., 1995; van Driel et al., 1996; Kageshita et al., 1999). In a few cases, progressive HLA class I antigen loss could be shown in tumor cell lines and/or lesions isolated from early and late stage of the disease (Lehmann et al., 1995; Restifo et al., 1996a; Cormier et al., 1998b). As will be described later, analysis of tumors from patients undergoing vaccination with TAA also supports the notion that HLA class I antigen negative clones can develop at later stages of disease progression under the influence of immunoselection. Alternatively, defects in HLA class I antigen expression may occur at an early stage in tumor development where HLA class I antigen negative clones are generated and selected by the host immune system. These HLA class I antigen negative clones may gain the capacity to disseminate and form distant metastases that are predominantly HLA class I antigen negative. Data to support this possibility are not obtained easily because tumor cell lines required to characterize defects at an early stage are rarely established from both a primary and autologous metastatic lesions. However, serial analysis of few patients’ malignant lesions (or metastases) has demonstrated HLA class I antigen loss at an early stage in disease progression. Melanoma cell Me9923P, originated from a primary lesion, and the autologous cell line Me9923 derived from a metastasis, were found to have total HLA class I antigen loss (Hicklin et al., 1998). Characterization of the molecular basis for this defect showed that Me9923P cells carry the same Pzm gene mutation as the autologous cell line Me9923. These findings suggested that the Pzm gene mutation occurred at an early stage in melanoma progression. 3. Clinical Relevance of Altered HLA Class 1 Antigen Expression and Antigen Processing Defects

Several lines of evidence suggest that, in at least some types of tumors, altered HLA class I antigen expression and antigen processing defects have clinical relevance because they are associated with histopathological characteristics of primary lesions and with the clinical course of the disease. As already mentioned, in various types of tumors TAP and/or HLA class I antigen down-regulation is more frequent in metastases than in primary lesions (Cordon-Cardo et al., 1991; Pantel et al., 1991; Cromme et al., 1994b; Hilders et al., 1995; van Driel et al., 1996; Kageshita et al., 1999). The HLA class I antigen and TAP expression in primary melanoma lesions

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has been shown to decline with local tumor progression, as determined by Clark level and Breslow thickness (Brocker et al., 1985a; Kageshita et al., 1999). Furthermore, a relationship has been found between HLA class I antigen and/or TAP down-regulation and disease progression in patients with cervical carcinoma. These abnormalities were found in malignant lesions but were not detected in normal cervical tissues and in premalignant cervical intraepithelial neoplasia (Hilders et al., 1994). The HLA-A locus antigen and HLA-A2 loss in primary cervical carcinoma lesions was also correlated with the presence of tumor-positive lymph nodes and number of lymph nodes involved (van Driel et al., 1996). In contrast, there are conflicting results in the literature about the association of HLA class I antigen and/or TAP down-regulation with tumor grading and with disease progression in breast (Kaklamanis et al., 1995; Vitale et al., 1998) and in colon (Durrant et al., 1987; van den Ingh et al., 1987; Kaklamanis et al., 1994) carcinomas, respectively. Furthermore, no association was found between HLA class I antigen and TAP down-regulation and progressive disease in head and neck squamous cell carcinoma and in lung and ovarian carcinoma (Kabawat et al., 1983; Mattijssen et al., 1991; Korkolopoulou et al., 1996). Similar results have been obtained when the relationship between the level of TAP and HLA class I antigens in malignant lesions has been correlated with the clinical course of the disease. An early study showed that HLA class I antigen down-regulation in loco-regional metastases of patients with melanoma was associated with a poor prognosis especially when the level of HLA class I1 antigens was high (van Duinen et al., 1988). These findings have been recently paralleled by an association of HLA class I antigen and TAP down-regulation in primary melanoma lesions with disease-free interval and survival (Kageshita et al., 1999) (Fig. 5). However, conflicting results have been obtained in patients with breast carcinoma. An association was found between HLA class I antigen loss in primary breast carcinoma lesions and survival in one study but not in another one (Wintzer et al., 1990; Concha et al., 1991). Furthermore, recent studies have found no correlation between TAP down-regulation and survival in patients with breast carcinoma, with lung carcinoma, or with colon carcinoma (Kaklamaniset al., 1994; Korkolopoulou et al., 1996; Vitale et al., 1998). These contradictory results from various types of cancer may reflect differences in the role played by immunological events in the clinical course of each type of cancer, the selection of patients included in the studies, and/or technical artifacts. It remains to be determined whether altered HLA class I antigen expression and antigen processing defects can serve as prognostic factors in human cancers.

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The HLA class I antigen down-regulation in metastatic lesions appears to have a negative impact on the outcome of T cell based immunotherapy in patients with melanoma. The recent use of this type of immunotherapy for treatment of patients with melanoma has allowed the serial analysis of HLA class I antigen expression in lesions and tumor cell lines throughout the course of therapy. Lack of HLA class I antigen expression due to loss of Pzm expression was noted in melanoma cell lines established from patients who had recurrence of disease after an initially favorable clinical response to T cell based immunotherapy or cytokine gene therapy (Restifo et al., 1996a).These investigators suggested that lack of HLA class I antigen in melanoma cells in these patients may have been responsible for the recurrence of their disease and poor response to immunotherapy. Additional studies of patients with melanoma immunized with MARTl.MelanA, tyrosinase, or MAGE-1-derived peptides have found HLA class I antigen loss or down-regulation in metastases following immunotherapy (Jager et al., 1997; Benitez et al., 1998; Cormier et al., 1998b). Thus, there is circumstantial evidence that HLA class I antigens targeted by MAAspecific CTL may become down-regulated in response to immunotherapy and that this abnormality has an adverse effect on the outcome of this

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therapeutic strategy. However, it is noteworthy that there is no conclusive evidence of a causal relationship between HLA class I antigen loss and progression of disease. A definitive study in this regard is quite complex to perform and will require a large population of genetically related (i.e., the same HLA genotype) and similarly treated patients to be followed prospectively. It is only by providing accurate clinical correlation to the description of various defects of HLA class I antigen expression that a final judgment can be made. IV. Potential Role of HLA Class I Polymorphism in Tumor Cell Escape

As previously discussed, many MAA contain multiple peptide sequences that can function as T cell epitopes in the context of the same or different HLA class I alleles (Table IA). Furthermore, bulk TIL populations include subpopulations of T cells specific for more than one MAA. Thus, the host immune response against cancer cells is potentially broad. Contrasting this potential wealth of T c e l h m o r cell interactions is, however, the practical observation that most often MAA immunodominance is restricted to few HLA class I alleles and TIL recognize predominantly one MAA epitope for each tumor. This phenomenon, which is referred to as epitope immunodominance, reflects discrepancies between the number of peptides within an antigenic protein that could bind to a particular HLA class I allele and the number of epitopes actually recognized in a CTL response to that protein (Kim et al., 1997). A more generally used terminology describes antigenic dominance whereby CTL can be elicited with greater frequency against a particular molecule relative to other molecules. Although the antigenic pluripotentiality of a molecule in the context of several HLA class I alleles is an appealing concept, there is no convincing evidence that it regularly occurs. Most MAA preferentially function as immunogens in association with particular restriction elements. For example, MART-1/ MelanA is considered immunodominant among HLA-A"0201 allele associated MAA because it is recognized most frequently by T cells elicited from tumor deposits (Kawakami et al., 1994b), from melanoma-invaded lymph nodes (Labarriere et al., 1998), and from PBMC stimulated in vitro with melanoma cells (Stevens et al., 1995). Of the potential peptide sequences included in the MART-1 molecule, MART-& is immunodominant (Kawakami et al., 1994b; Rivoltini et al., 1995b). Analysis of T cell induction in association with various HLA-A alleles of a given patient's phenotype revealed that MART-l/MelanA could not efficiently immunize in association with HLA-A alleles other than A"0201 (Bettinotti et al., 1998). Clonal analysis of MART-l/MelanA-specific CTL confirmed that MARTl/MelanA immunodominance is strongly restricted to the epitope resulting

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from the MART-127.35peptide/HLA-A"0201 complex. Thus, autologous induction of MART-l/MelanA-specific CTL by whole antigen processing and presentation is restricted to a unique HLA class I allele/ligand combination and is excluded by minimal changes in HLA class I allele structure. Immunodominance in relation to HLA class I phenotype may have important implications in relation to escape mechanisms and has to be taken into account in the design of vaccination strategies. In this regard, whole TAAs appear to be preferable to TAA-derived peptides as immunogens. Whole TAAs do not require epitope identification and allow presentation of multiple epitopes in association with a single restriction element, as in the case of gp100Pme117 in the context of HLA-A"0201 allele (Table IA). Furthermore, whole antigens include peptide sequences functioning as epitopes in association with multiple HLA class I alleles. This in turn potentially broadens the range of specificity of CTL response elicited by the vaccine, which could overcome limitations imposed by HLA class I allele, haplotype, or locus-specific down-regulation in tumor cells. These advantages outset the potential practical benefits of peptide based vaccinations. Although peptide vaccines are easy to administer and inexpensive and can induce in uivo sensitization of T cells (Vitielloet al., 1995; Salgaller et al., 1996; Corinier et al., 1997), they require knowledge of the epitopic determinant for each HLA class I allele, depend on the stability of the peptidelHLA complex in uiuo, and restrict their effect on a single epitope/ HLA class I allele combination (van der Burg et al., 1996). Immunodominance can be classified on the basis of clinical correlates suggesting in uiuo effectiveness of TAA-specific CTL (Kim et al., 1997). According to these criteria, gp100Pme117 appears to be recognized less frequently than MART-1 by HLA-A"0201 restricted TIL but is the determinant most commonly recognized by TIL associated with regression of metastases (Kawakami et al., 1994a, 1995).The last criterion has the advantage of taking into account clinical relevance of immunodominance; however, it suffers from the limitation of discounting the complex interactions occurring between TIL and tumor cells in the tumor microenvironment. The observed overall stringency of TIL specificity in the context of different HLA class I alleles emphasizes the importance of the patient's HLA class I phenotype when evaluating tumor cell escape mechanims. One might predict that different T M H L A class I allele combinations play a distinct role in the control of tumor cell growth. Furthermore, a patient's HLA class I phenotype is expected to influence the extent of MAA down-regulation. For example, HLA-A"0201 positive patients might lose expression of MART-1 more frequently than patients who express different HLA-A alleles. Finally, subdominant antigedepitope combinations could become important when the pressure posed on dominant

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epitopes by CTL selects variant tumor cells, which do not express dominant TAA or have lost the restriction element appropriate for a particular TAA. However it is noteworthy that a correlation between immunogenic potential of TAA and selective loss of HLA class I alleles or TAA has been difficult to document. Although loss of HLA class I alleles or MAA specific to a particular T M H L A vaccination have been occasionallyreported in limited patient populations (Lehmann et al., 1995; Jager et al., 1996, 1997; Ikeda et al., 1997; Cormier et al., 1998b; Lee et al., 1998), such a correlation has not been documented conclusively in studies with large patient populations. We are aware of only two exceptions. In one study, a higher frequency of Erb2-neu loss was noted in HLA-A2 positive patients with breast carcinoma than in patients who did not express this HLA class I allospecificity (Nistico' et al., 1997). Interestingly, in this study, the host immunological pressure had a more powerful repercussion on the expression of TAA than of that of the associated HLA class I allospecificity. Similarly, as already discussed, gp100/Pme117 was down-regulated in metastases from HLAA*0201 patients immunized against this MAA more than in metastases from patients who did not express HLA-A"0201 antigens. In addition also in this study gp100/Pme117 was down-regulated more frequently than HLA-A*0201 (Riker et al., 1999). Finally it is important to point out that HLA class I antigen polymorphism needs to be analyzed at the molecular level. For example, at least 30 subtypes of HLA class I alleles serologically defined as HLA-A2 have been described. The variant subtypes deviate from the canonical HLA-A"0201 motif by one to several residues and such amino acid substitutions have strong functional implications. Del Guercio et al. (1995) suggested that although variant residues in the HLA-A2 molecules lie in functional domains, many of these allomorphs share a common peptide-binding motif. Evidence that shared epitopes can serve as immunogens across this superfamily of HLA-A2 alleles is, however, controversial. Analysis of binding and presentation of peptides derived from MART-l/MelanA and gplOO/ Pmell7 by closely related subtypes (Rivoltini et al., 1996) suggested that peptide binding to the HLA class I allele and conformation of the peptide/ HLA class I complex can be altered even by single residue changes in the HLA class I heavy chain. Furthermore, immunogenicity of a particular peptide could not be simply predicted by structural relatedness among HLA class I alleles (Bettinotti et al., 1998). Therefore, a strong association exists between HLA phenotype and immunogenic potential of various MAA. This association needs to be taken into account when escape mechanisms utilized by tumor cells are studied, particularly if HLA associations with disease progression and prognosis are evaluated in the context of tumor escape.

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V. Inhibitory Signals Provided by the Tumor Microenvironment A. IMMUNOREGULATORY CYTOKINES

So far this review has focused on mechanisms of tumor escape from immune recognition that are consequence of the direct interaction of the TCR with the TAA derived peptide bound to the relevant MHC class I allele. However, recent data obtained in murine and human systems suggest that other variables can independently affect tumor-host interactions. Within the microenvironment, host defenses against the tumor are controlled by immunological mediators, particularly cytokines. Secreted cytokines can be both stimulatory and inhibitory. Because most tumors are not eradicated by the host immune defenses, it is likely that immune suppressive cytokines most often predominate. In fact, tumor cells, as well as normal cells, in the tumor microenvironment can spontaneously release immunosuppressive cytokines that inhibit host immune function (Chouaib et al., 1997). Although, immune suppressive cytokines may play a major role in inducing tumor tolerance by the host immune system, it is possible that lack of adequate immune stimulation in the tumor microenvironment may be as important. In regressing lesions, tumor cells secrete factors that make them more susceptible to recognition. There are many possible mechanisms by which cytokines can modulate local interactions between CTL and tumor cells. They include modulation of (i) immune regulatory cytokine production, (ii)adhesion or costimulatory molecule expression on periturnoral endothelium and on tumor cell targets, (iii) differentiation and activation of professional APC, (iv) factors involved in tumor angiogenesis, (v) growth-factor receptor expression by tumor cells, which results in a direct alteration of tumor growth, (vi) receptors expressed by tumor cells that are involved in mediating tumor cell lysis, (vii) T cell response such as shifting T h l to Th2 cellular responses, and (viii) activation of TAA-specific CTL. Table VII lists cytokines believed to be involved in these mechanisms. Evidence from investigations in mouse tumor models and in patients with malignancies has demonstrated that cytokines in the tumor microenvironment can alter tumor cells ability to serve as targets for CTL. For example, cytokines can alter the expression of adhesion molecules on the periturnoral endothelium (Yoong et al., 1998) and on target tumor cells (Vanky et al., 1990; Cao et al., 1997; Lefor and Fabian, 1998). These adhesion molecules facilitate trafficking of T cells to the tumor site and reinforce the physical interaction of T cells with tumor cells. Expression of adhesion molecules such as ELAM-1, ICAM-1 and VCAM-1, may influence tumor progression (Johnson, 1991;Wimmenauer et al., 1997; Maurer et al., 1998; Terol et al., 1998). Furthermore, various malignant cell lines

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TABLE VII ON TUMOR CELLITCELLINTERACTIONS EFFECTOF CYTOKINES Modulating Role

Cytokine Implicated

Inhibition of immune regulatoly cytokine production Modulation of receptors required for T cell activation Down-regulation of TH1 cellular responses Tumor target resistance to CTL lysis Inhibition of CTL lybc function Modulation of tumor growth Inhibition of professional APCs differentiation Induction of angiogenesis

TGF-P, IL-10 TGF-0, I L l O TGF-P, IL-10 IL-10 TGF-P, IL-10 IL-6, IL-10 VEGF VEGF

or tissues including melanoma have heterogeneous and often decreased expression of adhesion molecules (Mortarini et al., 1990; Braendstrup et al., 1996; Budinskyet al., 1997).Interestingly ICAM-1, which is an adhesion molecule known to reinforce CTL/tumor interactions, can be induced or enhanced in normal and malignant cells in vitro and in vivo by IFN-y (Temponi et al., 1988; Maio et al., 1989a,b; Mortarini et al., 1990; Kitsuki et al., 1996), interleukin (1L)-10 (Yue et al., 1997) and tumor necrosis factor-a (TNF-a) (Temponi et nl., 1988; Maio et al., 1989a,b; Mortarini et al., 1990; Krutmann et al., 1990; Lo et al., 1992; Kilgore et al., 1995; Krunkosky et al., 1996; Budinsky et al., 1997). IFN-y and TNF-a, which are secreted by activated CTL, enhance shedding of soluble ICAM-1 by malignant cells (Temponi et al., 1988). The latter can inhibit the lysis of target cells by CTL and NK cells (Becker et al., 1991; Altomonte et al., 1993) and has been postulated as an additional tumor escape mechanism. Whether changes in expression of adhesion molecules affect the interaction of tumor cells with effector cells remains to be determined because there is conflicting information regarding adhesion molecules and lytic activity of CTL and lymphokine activated killer (LAK) cells (Vanky et al., 1990; Webb et al., 1991; Akella and Hall, 1992; Gwin et al., 1996; Cao et al., 1997; Lefor and Fabian, 1998). Cytokines or other factors expressed by tumor cells may have direct immunologic effects on T cells (O’Sullivan et al., 1996; Chouaib et al., 1997). Recently IL-6 has been shown to be expressed specifically in the tumor microenvironment in 83% of patients with colorectd carcinoma (Piancatelli et al., 1999). At present it is not clear whether tumor or T cells produce this cytokine,which is known to have multiple effects on T cell functions (Brakenhoff, 1995) and may play a significant role in modulating T cell reactivity at tumor site. IL-12 has been shown to cause immune suppression in mice (Kobish et al., 1998); however, this effect is transient

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and counterbalanced by the ability of IL-12 to induce protective artitumor immunity. Originally named cytokine synthesis inhibitory factor, IL-10 has pleiotropic effects on T-cell functions. They include suppression of granulocyte macrophage-colony stimulating factor (GM-CSF), IFN-y, and IL-2 production by T helper cells (Fiorentino et al., 1989; Vieira et al., 1991) and inhibition of T cell proliferation (Taga and Tosato, 1992). Furthermore, IL-10 acts on T cells by promoting Th-2 type immune responses (Clerici and Shearer, 1994). Also, IL-10 can affect the ability of cancer cells to interact with the host immune system by down-regulating the expression of adhesion molecules and/or HLA class I and class I1 antigens. Finally, IL-10 may be a growth factor for human melanoma cells (Yue et al., 1997). Although immune cells represent the major source of IL-10 (Fiorentino et al., 1989), this cytokine can also be secreted by melanoma cell lines, and can be found in tissue samples from patients with metastatic melanoma (Dummer et al., 1996). Furthermore, elevated serum levels of IL-10 have been found in patients with metastatic melanoma suggesting that this cytokine may play a role in the down-modulation of anti-TAA immune response in vivo systemically or at tumor site (Dummer et al., 1995). Among multiple suppressors of CTL function, transforming growth factor-@ (TGF-P) was suggested by Wojtowicz-Praga (1997) to play a predominant role. Characterized by high homology and similar biological properties, TGF-@is a family of distinct homodimeric growth factors. In situ expression of the various isoforms of TGF (TGF-P1,-@2,and -P3) is common in tumors and correlates with progression of melanoma (Schmid et al., 1995;Van Belle et al., 1996; Moretti et al., 1997)and other skin tumors (Schmid & al., 1996). Particularly TGF-@2and TGF-@3are expressed in melanoma lesions but are not detectable in melanocytes. Furthermore, the level of TGF-@ tends to increase with tumor progression because it is lower in thin primary melanoma lesions than in thick primary melanoma lesions and in metastases (Moretti et al., 1997). It is believed that the presence of TGF-j3 in situ is due to the paracrine secretion of this cytokine by tumor cells, as occurs in cell lines from melanoma metastases (Rodeck et aZ., 1991). Other types of malignant lesions (Gorsch et al., 1992) have been shown to abundantly secrete this cytokine in uitro. The biological effects of TGF isoforms are generally considered to be similar and can either inhibit or stimulate cell replication (Sporn and Roberts, 1992). Indeed, TGF-@ has been implicated as a potential factor in mammary tumorigenesis (Wakefield et al., 1995). In melanoma, however, a direct inhibition of cell proliferation by TGF-@ is considered unlikely (Moretti et al., 1997). Results in murine models suggest that TGF-@can modulate melanoma cell growth by causing immunosuppression in the host

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(Wojtowicz-Praga et al., 1996). For this reason the term “tumor induced immune suppression” was suggested ( Wojtowicz-Praga,1997).This mechanism is supported by two lines of evidence. First, the tumor burden was reduced in mice injected with B16 melanoma cells following the systemic administration of anti-TGF-/3 antibodies in combination with IL-2 (Wojtowicz-Praga et al., 1996). Second, expression of an anti-sense TGF-P1 transgene reduced tumorigenicity of EMT6 mammary tumor cells (Park et al., 1997). However, these studies did not determine whether TGFP acted by inducing immunosuppression at the tumor site or systemic immunosuppression of the host because T cell function was not evaluated. At this time, it is not clear whether the immunosuppressive effects of TGFP putatively produced by tumor cells are only in the tumor microenvironment or affect the whole host immune system. A correlation was found in patients with melanoma between plasma levels of TGF-P and disease progression (Wojtowicz-Praga, 1997). This association, however, may simply reflect the higher tumor burden in patients with progressive disease. If so, TGF-P may only represent a marker of tumor progression. It is also noteworthy that in vitro TGF-/3 is a powerful inhibitor of NK cell function (Bellone et al., 1995) and totally abrogates CTL function ( Wojtowicz-Praga et al., 1996). Both TGF-/3 and IL-10 have been shown to act synergistically to induce immune privilege in the eye by down-regulating antigen-specific Th-1 cell responses (D’Orazio and Niederkorn, 1998). According to this model, TGF-P, secreted preferentially by ocular cells, induces release of IL-10 and suppresses release of IL-12 by APC turning the immune response from a Th-1 toward a Th-2-like response. As tumor cells may secrete TGF-P, such interactions among them, APC, and T cells may play a similar role by conferring privileged status to tumor tissues. Recently, differential expression of lymphoattractant C-X-C chemokines has been proposed as a relevant variable for the recruitment of specific T cells at site of pathology (Farber, 1997; Sgadari et al., 1997; Piali et al., 1998; Tensen et al., 1998). However, evidence that these chemokines play a significant role in most cancers, including melanoma, is to our knowledge not available. Cytokines secreted by normal and malignant cells in the microenvironment can also facilitate tumor growth. One example is IL-6 that can have a direct effect on tumor growth and progression (Klein et al., 1989). In addition, some cytokines can act as stimuli for induction of angiogenesis factors that are required for generating a blood supply by the developing tumor. The growth advantage of a well-vascularized tumor, however, may be counteracted by its greater accessibility to infiltration by immune cells including CTL. Interactions between the vascular endothelium within tumors and cytokines released at tumor site may also affect the outcome of the TAA-

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specific CTL response. Swelling, tenderness, and other inflammatory signs precede the disappearance of tumor masses and characterize tumor regression when it occurs. This extraordinary behavior is difficult to consider the sole effect of CTL mediated lysis of tumor cells as lysis induces apoptosis, which per se is not inflammatory. An alternative hypothesis suggests that CTL could act like "smart bombs." The CTL are capable of selectively recognizing a target tissue and act in situ not only by killing target cells but also by delivering inflammatory substances such as IFN-7 and TNF(Y (Fig. 6). Some tumors may be more sensitive than others to inflammatory signals produced by activated CTL. This difference may explain why tumors respond with similar frequency to various biological agents and why therapy with CTL, BCG, IL-2, IFN, and the like may all, in the end, result in a low but similar percentage of tumor regressions. In all cases, the therapeutic agent would behave as a trigger of inflammation, but tumor regression would depend upon the sensitivity of tumor cells to the cytotoxic substances produced by the inflammatory process. This hypothesis could also explain

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why melanoma and renal cell cancer have similar sensitivity to treatment with biological agents such as IL-2 and IFN-.)I,although they are characterized by different immunogenic potential. In melanoma multiple MAA have been identified and it is easy to derive CTL, whereas the contrary is true for renal cell cancer. It is possible that, although immunologically different, they have the same frequency of lesions sensitive to general inflammatory stimuli. If this hypothesis is correct, the molecule responsible for modulation of tumor sensitivity remains to be identified. Among possible candidates, TNF-a could play a significant role. The antitumor effects of TNF-a could be explained either by a direct induction of apoptosis following contact of CTL with tumor cells (Wang et al., 1996a) or by indirect effects on tumor vasculature (Nawroth et al., 1988). As TNF-a is a cytokine commonly secreted by activated CTL, one might postulate that factors modulating its antitumor effects could play a significant role in determining the sensitivity of a tumor to TAA-specific CTL. Recently, Wu et al. (1998) have shown that melanoma cell lines produce a cytokine called Endothelial-Monocyte Activating Polypeptide I1 (EMAPII). Melanoma cell lines secreting higher amounts of EMAPII appeared to be more sensitive in vivo to the antineoplastic effects of TNF-a probably in relation to the ability of EMAPII to induce tissue factor production by tumor vascular endothelial cells. At present, however, the frequency of EMAPII expression in specimens from various tumor types including melanoma and the correlation between this parameter and the clinical course of the disease remain to be determined. B. SURFACEEXPRESSION OF APOPTOTIC SIGNALS Recently, it has been reported that high levels of Fas ligand ( FasL) are expressed in a high percentage of melanoma cell lines and of surgically removed melanoma lesions (Hahne et al., 1996). Furthermore, high levels of FasL were also reported to be present in sera from patients with melanoma as a bioproduct of tumor derived membrane bound FasL (Hahne et al., 1996).These findings suggested a novel mechanism utilized by tumor cells to escape from T cell recognition. Through interaction with Fas on the surface of TIL, FasL could counterattack and extinguish TAA-specific CTL at a tumor site. Direct contact between TAA-specific CTL and target cells would enhance Fas/FasL interactions and therefore would result in a selective loss of MAA-specific CTL. Furthermore, it has been recently suggested that continuos interactions of TIL with TAA expressing tumor cells may enhance their sensitivity to FasL mediated apoptosis by enhancing their level of expression of Fas relative to PBMC (Car& et al., 1998). The model hypothesizes a continuous accrual of TAA-specific CTL at a tumor site, partial killing of tumor cells by these activated CTL and feedback

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killing of these effector cells. The balance of surviving cancer cells and/or CTL will eventually determine the final outcome of the TAA-specific CTL response (i.e., either regression or progression of the lesion). This model was accepted originally with enthusiasm because it provides a mechanism for the lack of tumor regressions in spite of the presence of TAA-specific CTL in malignant lesions. Additional credence to this model was provided by the reported detection of FasL in a number of cell lines utilizing FACS analysis with mAb and PCR and in a variety of surgically removed malignant lesions utilizing IHC with mAb. The tumors reported to express FasL include, besides melanoma (Hahne et al., 1996), astrocytoma (Saas et al., 1997), glioblastoma (Grata et al., 1997) and esophageal (Bennett et al., 1998), lung (Niehans et al., 1997), hepatocellular (Strand et aE., 1996), and colon (O’Connell et al., 1996; Shiraki et al., 1997) carcinoma. Upon scrutiny, however, it was realized that this model is not supported by the available experimental data. Among them is the general observation that TIL and CTL are usually expanded and produced in vitro through repeated stimulations with autologous freshly isolated tumor cells or cell lines. In most tumor immunologists’ experience, the interaction of TAAspecific CTL with tumor cells stimulates CTL proliferation rather than causing their death. In agreement with these findings is the report by Rivoltini et al. (1998) that TIL isolated from surgically removed melanoma lesions are insensitive to FasL. Furthermore, in murine models, the implantation of FasL transduced tumor cell lines did not lead to immune evasion. As a matter of fact, FasL expressing tumor cells did not grow in the recipient animals although the wild-type parental cell lines could induce tumors (Arai et al., 1997; Seino et al., 1997). Unexpectedly, FasL transfected tumor cells rather than promoting death of the TAA-specific CTL appeared to directly or indirectly cause mass suicide through FasUFas interactions among tumor cells or between tumor cells and by-stander inflammatory cells. Subsequent studies have not reproduced the results of early investigations since FasL has been detected in very few, if any of the surgically removed malignant lesions. Furthermore, in melanoma cell lines FasL could be detected only if the cell lines were pretreated with metalloprotease inhibitors (Rivoltiniet al., 1998). The conflicting information in the literature is likely to reflect the limited specificity of the anti-FasL antibodies used in IHC and FACS analyses and the contamination of tumor cells with other types of cells in studies, which have utilized RT-PCR. These possibilities are supported by the low percentage of cell lines found to be positive for FasL in recent screenings of a large panel of melanoma cell lines (Shiraki et al., 1997; Chappell et al., 1998) with multiple methods including RT-PCR, FACS and functional assays. One study included cell

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lines established from melanoma metastases, which had not responded to active specific immunotherapy with MAA derived peptides (Riker et al., 1999). Some of the cell lines analyzed were noted to lack expression of the MAA targeted by the immune response elicited by the vaccination suggesting that the loss of expression of MAA rather than FasL was the reason for tumor escape in these patients. Therefore, the available information indicates that the expression of FasL in malignant lesions is probably minimal. Furthermore, the role of the Fas/FasL pathway in melanoma cell induced apoptosis of TAA-specific T cells is probably limited as recognized also by the investigators who had originally proposed a role for tumor cell derived FasL in the escape of tumor cells from T cell recognition (Rimoldi et al.,1998). Zaks et al., (1999) have recently proposed an alternative and intriguing mechanism, which envisions a role of Fas/FasL interactions in the escape of tumor cells from T cells recognition. These authors have suggested that Fas/FasL interactions may occur between CTL since these cells express FasL upon activation with antigen, TCR cross-linking with antibody or stimulation with calcium ionophores. Therefore, CTL may not be able to efficiently attack tumor targets because, upon engagement of their TCR with the appropriate peptide/MHC class I allele complex, they are activated and up-regulate expression of FasL. This model envisions that Fas/FasL interactions occur either within the same T cell (suicide) or between closely proliferating activated T cells and cause their apoptotic death. Such an effect would be specific for areas within the tumor microenvironment that are rich in activated T cells (Zaks et al., 1999). VI. Inadequate lmmunogenicity of the Tumor Microenvironment

Inadequacy of tumor cells as targets for activated CTL may not be the only explanation for lack of tumor rejection by the immune system. Various levels of T cell receptor engagement may elicit different responses by CTL (Valitutti et al., 1996). For instance, epitope density required to stimulate target cell killing by CTL is 10- to 10,000-fold less than that required for induction of IFN-.)I and IL-2 by the same CTL (Gervois et al., 1996). Thus, the requirements to induce and sustain a CTL response in target organs are likely to be higher than those necessary for the execution of the effector response by the same CTL. The TIL able to recognize autologous tumor cells originated from the same metastasis can routinely be expanded in uitro. Yet the lesions from which TIUtumor cell pairs originate grow paradoxically unaffected by the presence of the putative ongoing immune reaction. Therefore, interactions of TIL with tumor cells observed in vitro are not sufficient to explain their ineffectiveness in vivo. Fuchs

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and Matzinger (1996) proposed an alternative model to explain the coexistence of effector and target cells in tissues without the development of blatant autoimmunity or, in the case of tumor immunity, tumor rejection. In antithesis with the theory that immunologic recognition of cancer cells reflects the capacity of the immune system to discriminate between self and nonself molecules, this model envisions that the immune system starts an immune response by detecting tissue distress (danger).It is possible that cancer cells cannot effectivelyprovide danger signals, and consequently the default response of T cells in the tumor microenvironment is turned off. This model best fits with the characteristics of MAA and with the indolent immune response to them in patients with melanoma. Indeed most MAA are nonmutated self-molecules. Furthermore, self-MAA-specific CTL are easily generated in vitro from PBMC and TIL of patients with melanoma but do not affect significantly tumor cell growth in vivo. This discrepancy may reflect the lack in vivo of a second signal, which in vitro is provided in addition to a primary specific stimulus to induce MAA-specific CTL. The most obvious example is the in vitro expansion of TIL, which requires incubation with tumor cells (providing signal 1)and IL-2 (providing signal 2). Therefore, a likely explanation for the in vivo failure of expansion and activation of CTL is lack or low level of second signals in the tumor microenvironment. The mechanism of tumor escape discussed in this section, therefore, deviates from traditional ones. Rather than suggesting that the interaction between tumor cells and host immune system is hampered by the lack of one or more components directly affecting TCWpeptidelHLA class I allele interactions (signal one), it suggests that tumor cells, being healthy growing cells, do not elicit the distress signals needed for APC activation. Therefore, the immune response is blocked by lack of signal two. This view is consistent with the low reactivity of the immune system toward self-antigens. This parallels the lack of autoimmune processes in the majority of individuals. Thus, the majority of tumors do not “escape” immune recognition but simply survive in a favorable environment without triggering a response. In circumstances where some cells undergo damage, irrespective of an immunization protocol, the host immune response switches from selftolerance to self-recognition; “tumor autoimmunity” occurs, and malignant lesions regress. It is not clear how the balance between tolerance and tumor autoimmunity is maintained in patients with malignant diseases. Availability of HLA class I antigen restricted TAA epitopes, while adequate to allow recognition and lysis of tumor cells by activated CTL, may not be sufficient to induce CTL activation and proliferation in the absence of a second stimulus provided either by costimulatory molecules or by stimulatory cytokines

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(Matzinger, 1994). Because the requirements for secretion by CTL of IL-2 or other cytokines promoting their activation and proliferation are relatively high (Gervoiset al., 1996;Valitutti et al., 1996),CD4+ T cells may have to provide additional stimulation which could lead to amplification of CTL responses and to tumor rejection. The various mechanisms by which antigen-specific stimulation of T cell responses could affect host immune recognition of tumor cell growth are summarized in Table VIII. Tumor cells, in particular melanoma cells, express HLA class I1 antigens (Pellegrino et al., 1982) and may directly elicit T helper cell responses. Furthermore, APC could present TAA shed by tumor cells to CD4+ T cells, which could in turn release immune stimulatory cytokines. Finally, HLA class I1 antigen dependent interactions of activated T helper cells with APC could lead to their activation and maturation. As APC can present exogenous antigens to HLA class I antigen restricted CD8+ T cells, they could induce activation of effector CTL by providing the necessary costimnlation. The suggested inability to initiate and sustain CTL responses because of lack of appropriate antigen presentation could represent an escape mechanism utilized by tumor cells expressing CTL-defined epitopes. A. HLA CLASSI1 ANTIGENEXPRESSION BY TUMOR CELLS Priming, activation, and proliferation of CTL depends upon presence of “help” in the form of cytokines produced by Th-1 helper cells (Ridge et al., 1998). Melanoma cells and, although with lower frequency, other types of tumor cells of nonlymphoid origin express HLA class I1 antigens or may acquire them following exposure to IFN-y in a high percentage of cases (Pellegrino et al., 1982).Furthermore, HLA class I1 antigen associated MAA have been identified (Topalianet al., 1994),and there is evidence that malignant cells can present endogenous antigens in association with HLA class I1 antigens (Akporiaye and Panelli, 1996; Brady et al., 1996; Panelli et al., 1996;Armstrong et al., 1998).However, it is not clear whether this pathway of T cell activation plays a significant role in the control of TABLE VIII ANTIGEN-SPECIFIC INDUCTION OF T CELLRESPONSES Antigen Presenting Cell Tumor cell Tumor cell APC APC

HLA Antigen Utilized class class class class

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tumor growth by TAA-specific CTL responses. This finding is supported by the correlation reported in melanoma between expression of MHC class I1 antigens in metastases and their response to T cell based immunotherapy (Rubin et al., 1989). Furthermore, CD4+ T cell infiltrates are commonly observed in regressing metastatic lesions. These findings are, however, in discrepancy with the difficulties to expand MAA-specific, HLA class I1 antigen restricted CD4+ T cells in vitro. As already mentioned, these difficulties are not encountered when expanding MAA-specific, HLA class I antigen restricted CTL. Thus, it is not known whether HLA class I1 antigen expression by tumor cells plays a significant role in the modulation of host immune recognition of tumor cells. Furthermore, it is not known whether lack of HLA class I1 antigen expression by tumor cells provides them with an escape mechanism from immune recognition. This possibility is suggested by the lower expression of HLA class I1 antigens in primary than in metastatic lesions (Natali et al., 1983; Ruiter et al., 1984; Brocker et al., 1985) and by the association of HLA class I1 antigen expression in primary lesions with histopathological and clinical markers of poor prognosis (Brocker et al., 1984; van Duinen et al., 1988). It has been recently suggested that CD4+ T cells play a role in anti-tumor immune responses not only by modulating T and B cell responses but also by influencing directly the function of APC through CD40-CD40L interactions (discussed later in this section) or by activating macrophages through the production of activator cytokines (Hung et al., 1998). Activation of macrophages leads in turn to production of superoxide and nitric oxide that contribute to killing of tumor cells. Thus, variability in HLA class I antigen expression by tumor cells may have effects beyond the classical control of CTL responses mediated by CD4+ T cells.

B. THEROLE OF APC IN THE TUMOR MICROENVIRONMENT It is often assumed that TAA shed by cancer cells are incorporated through the exogenous pathway of antigen presentation into lysosomes by APC, cleaved into peptides and bound to HLA class I1 molecules (Fig. 1B). These TAA are then presented on the APC surface for recruitment of CD4+ helper T cells, which in turn release stimulatory cytokines. The latter promote the recruitment and expansion of TAA-specific CTL in situ. The requirement for CD4f T cell help in maintaining effective CTL responses has been well documented particularly in murine viral and tumor models (Yewdelland Bennink, 1990; Hung et al., 1998; Kalams and Walker, 1998; Zajac et nl., 1998). However, there is no direct evidence in humans that APC can present efficiently TAA shed by tumor cells in uivo. This evidence is available in murine models in which APC can present endocytosed antigens in association with MHC class I and class I1 molecules. If

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adequate amount of antigen is provided, APC can induce concomitant activation of CD8+ and CD4+ Tcells (Kurts et al., 1997,1998).High doses of antigen are necessary for efficient cross-presentation as it is generally achieved in response to viral infections. If the amount of antigen incorporated by APC is not sufficient, no CD4+ T cell activation occurs. As a result, tolerance is induced, since encounters between APC and antigenspecific CD8+ T cells cause their peripheral deletion. Whether the efficiency of TAA incorporation by APC in the tumor microenvironment is adequate for cross-presentation to helper and cytotoxic T cell populations and whether an inadequate TAA incorporation by APC determines tumor cell escape from immune recognition is not known. Results of studies in model systems suggest that viral antigens can be efficiently presented by dendritic cells to CD8+ T cells (Bhardwaj et al., 1994). However, TAA may behave differently. Most tumor cells die without causing inflammation probably through an apoptotic pathway. Human CD14, which is abundantly present on the surface of phagocytes but downregulated in IL-4GM-CSF activated dendritic cells, mediates recognition and phagocytosis of apoptotic cells (Devitt et al., 1998). When apoptotic cells are incorporated through this pathway by APC, no inflammatory processes occur. For instance, contrary to LPS mediated cross-linking of CD14, incorporation of apoptotic material does not stimulate secretion of TNF-a by APC. Therefore, macrophages infiltrating tumors may only be scavengers for senescent and damaged cancer cells, and tumor cells would simply go “unnoticed.” Activated IWGM-CSF induced dendritic cells, but not monocytes can incorporate an antigen from apoptotic cells and present it in association with HLA class I antigens to CD8+ T cells (Albert et al., 1998). As a consequence, powerful CTL responses can be induced. It is possible, therefore, that a second signal is required in the tumor microenvironment to turn macrophages into efficient APC. How this step would occur in vivo is unknown. For instance, either CD4+ or CD8+ T cells could release large amounts of GM-CSF and/or TNF-a required for activation and maturation of APC. Furthermore, T helper cells can produce other cytokines including IL-4. Alternatively, monocytes themselves could produce cytokines like TNF-a that could enhance their antigen presenting potential (Sallusto and Lanzavecchia, 1994). Therefore, defects in the events triggering TAA-specific CTL and/or in the steps required for APC maturation may result in tumor cell escape from immune recognition. Efficient incorporation of TAA by APC and their proper maturation could lead to efficient cross-presentation of TAA. Yet, questions remain about the ability of APC to recruit simultaneously helper and effector T cells. A cytotoxic-helper immune reaction specific against the same TAA assumes a temporarily close contact of CD4+ and CD8+ T cells so that the IL-2 dependent CTL could benefit from cytokines secreted by the

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helper cells. This requirement is not likely to be easily fulfilled in tumors because TAA-specific CD4+ T cells are rare and their identification has been elusive. Were tumor reactive CD4+ T cells easily identified, one could postulate that the first event in tumor rejection is the arrival at tumor site of CD4+ T cells. Because of the presence of the appropriate APCI TAA complex, the helper T cell population would expand. Consequently, the tumor would become populated with TAA-specific helper T cells, which although unable to recognize tumor cells specifically could produce IL-2 (or other cytokine) for proliferation of TAA-specific, HLA class I antigen restricted CTL. The difficulty encountered in identifylng TAAspecific CD4+ helper cells suggests that tumor cells may escape immune recognition because of the low chances of productive encounters among the various immune cell populations. Environmental immunogens coincidentally expressed at the tumor site or the draining lymph nodes might stimulate proliferation of CD4+ T cells. It is possible that non-TAA-specific CD4+ helper cells are all that is needed for induction of TAA-specific CD8+ T cell responses. These helper cells would promote proliferation and activation of dormant TAAspecific CD8+ T cells (Fuchs and Matzinger, 1996). This model could explain the occasional disappearance of tumors following viral infections or following the administration of general immune stimulators such as systemic treatment with IL-2 or other cytokines. The coincidental nature of such events could offer a rational for the rarity of spontaneous tumor regressions.

C. APCIHELPER T CELLINTERACTIONS IN THE TUMOR MICROENVIRONMENT Finally the role of helper T cells is not limited to direct activation of CD8+ T cells through cytokine production. CD4+ T cells can directly activate mature APC through CD40/CD40L interactions (Ridge et al., 1998). In this case, no temporal requirements for close contact between CD8+ and CD4-f T cells is necessary. The activated APC then acts as a temporal bridge between a helper and a killer cell (Ridge et al., 1998). One CD4+ T cell could activate more than one DC, which in turn could activate more than one CTL leading to exponential expansion of the immune response. In this case, the presence of a helper epitope in the tumor microenvironment could be all that is required to initiate effective anticancer responses. Yet, in most situations, tumors might not provide these helper molecules. VII. Escaping Escape Mechanisms: Possible Immunological Alternatives

The field of tumor escape has a short history since for many years tumor immunologists have directed their efforts toward the identification of TAA

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and the development of strategies aimed at inducing and/or enhancing TAA-specific CTL reactivity. As a result, a number of immunological approaches have been developed which are very effective in eliminating tumors in mice. Established immunogenic tumors can be cured by a variety of immunological maneuvers, which induce a TAA specific CTL response. Poorly immunogenic tumors can be controlled or prevented in tumor challenge experiments where animals are immunized before implantation of tumor cells. These findings underline the need for prior exposure of T cells to the relevant TAA to successfully achieve control of tumor growth. The reason(s) underlying this finding is (are) not known. Experiments in mice have also shown that the ability of the host immune system to cope with tumors is negatively influenced by their size. Large tumors may exhaust the host immune competence by altering its metabolism or by producing immune suppression. Large cell populations of rapidly dividing cells in established tumors could overwhelm the replicative capability of TAA-specific CTL. Lastly, the extensive number of cell divisions occurring in large lesions may statistically increase chances for the development of tumor cell variants. In challenge experiments animals are injected with homogeneous tumor cell populations sensitive to the already ongoing immune reaction. Much to tumor immunologists disappointment, however, the strategies which have been effective in controlling tumor growth in mice have failed in patients with malignancies. This discrepancy is likely to reflect the diversity of human immune systems and the heterogeneity of human tumors, which have enhanced the effects of tumor escape mechanisms on the interactions of tumor cells with host immune system. If correct, this hypothesis emphasizes the need to define the molecular basis of escape mechanisms utilized by tumor cells. These studies should take advantage of new technologies, which allow one to analyze the dynamic evaluation and molecular basis of interactions between host immune system and tumor cells. Even though traditional molecular and functional studies performed in vitro will continue to suggest putative mechanisms for tumor cell escape from TAA-specific CTL recognition, the demonstration of their clinical relevance will depend upon prospective analysis of tumor lesions as allowed, for instance, by FNA. This new technology will need to be combined with the adoption of other technologies recently described, which allow for accurate analysis of small sample sizes. For instance, the ability to select distinct cell populations from a tumor specimens by microdissection allows for the analysis of gene expression by various cell types populating tumors (Bonner et al., 1997; Peterson et al., 1998). The ability to discriminate gene expression by distinct cell populations may in turn provide insights about the way various such cells interact within the

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tumor microenvironment. The utilization of epitope/HLA class I complex tetramers (Altman et al., 1996) for the ex vivo staining of antigen-specific CTL could allow selection of CTL specific for a particular TAA epitope (Romero et al., 1998). TAA-specific CTL identified by this method can be sorted from fresh tumor preparations and the level of expression of genes related to their status of activation such as cytokines could be analyzed using accurate and sensitive methodologies such as Taqman-based realtime RT-PCR (Kruse et al., 1997) or intracellular FACS analysis (Kern et al., 1998). This information might yield important information about the status of activation of CTL in wiwo and allow comparisons with data regarding requirements for CTL activation derived from in vitro testing of TAA-specific CTL. Finally, development of cDNA libraries from FNA of metastatic lesions will allow one to determine the expression patterns of thousands of genes in a single experiment (Duggan et al., 1999). This methodology combined with knowledge of the natural history of distinct lesions will allow for the collection of clinically interesting cDNA libraries whose gene expression could be compared. Hopefully such strategy might help identifylng, among the many possible variables potentially regulating tumor cell response to immunotherapy, those most frequently correlated with favorable prognostic factors. The data we have reviewed in the previous sections suggest that tumor cells can utilize multiple mechanisms to escape from T cell recognition. Therefore, it is not surprising that the success rate of T cell based immunotherapy of malignant diseases has been relatively low, especially since patients enrolled in clinical trials are not selected and monitored for the expression of markers which play a role in tumor cell escape. Nevertheless, clinical responses have been observed in some of the patients treated with T cell based immunotherapy providing evidence for the validity of this approach for treatment of cancer. Whether these clinical responses reflect the fortuitous circumstance that patient immune system is fully functional and tumor cells have not utilized immune escape mechanisms remains to be determined. If this is the case, the clinical responses, which have been described, emphasize the need to develop criteria to select patients to be treated with T cell based immunotherapy and to design strategies to identify potential escape mechanisms that can evolve over the course of therapy. Investigations of patients immunized with well-defined TAA have provided suggestive evidence that loss of the TAA targeted by the immune response is frequently associated with recurrence of the disease. Besides lending support to the role of a TAA-specific CTL response in the control of tumor growth, this evidence questions the current strategy to use one TAA to implement active specific immunotherapy in patients with malignant diseases. In view of the low frequency of the loss of more than one

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type of TAA in malignant cells, cocktails of distinct TAA to immunize patients are likely to improve the efficacy of active specific immunotherapy, also because they minimize the negative impact of antigenic heterogeneity of tumor cells on the outcome of immunotherapy of malignant diseases. Furthermore, the recurrence of disease associated with the loss of the TAA targeted by T cell based immunotherapy questions the validity of the currently used criteria to select TAA to be used for immunotherapy of malignant disease. At present, this selection relies on the in vitro recognition of the TAA by CTL and on its restricted tissue distribution. Little, if any, attention is paid to the role of the identified TAA in the economy of malignant cells. The clinical data we have summarized suggest that TAA related to the neoplastic process may be more effective targets of immunotherapy since they may be linked more tightly to cancer cell survival and be, as a consequence, more resistant to immune selection. HLA class I antigen loss or down-regulation represents an additional defect, which provides malignant cells with an escape mechanism from T cell recognition. The characterization of the molecular lesions and functional significance of abnormalities in HLA class I antigen expression suggests strategies to counteract their negative effects on host immune systedtumor cell interactions. The HLA class I antigen down-regulation caused by defects in the regulatory mechanisms, which control the expression of these antigens will benefit by combining administration of TAA with that of cytokine(s) which enhance(s) HLA class I antigen expression. In contrast, selective loss of HLA class I antigen expression caused by structural defects in the corresponding genes will require the change of the TAA used as an immunogen to a different one, which utilizes the restricting elements remaining in the tumor cell HLA class I phenotype. Alternatively, one may want to take advantage of the increased susceptibility of tumor cells with HLA class I antigen loss or down-regulation to NK cell mediated lysis. Therefore, strategies have to be designed, which utilize NK cells in addition or in place of TAA-specific CTL. The many mechanisms utilized by malignant cells to escape from T cell recognition question the validity of the major, if not exclusive emphasis on T cell based immunotherapy, which has dominated the immunological treatment of malignant diseases in recent years. As recently pointed out (Sogn, 1998), “T cell chauvinism” must be avoided. The use of antibody based immunotherapy should be reevaluated, since several lines of evidence in animal model systems suggest that anti-TAA antibodies can control tumor growth (Livingston, 1998). Furthermore, induction of anti-TAA antibodies in patients with solid tumors following immunizations with purified TAA (Livingston et al., 1994) or with anti-idiotypic antibodies, that bear the internal image of TAA, has been found to be associated with

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a statistically significant survival prolongation (Mittelman et al., 1992). Lastly testing with recombinant cDNA expression libraries constructed from tumor cells has identified high titer antibodies to cancer-specific proteins in sera of patients with malignancies. Although its clinical significance has not been assessed yet, this humoral immunity may lend to the identification of new antigens to be used as targets for immunotherapy. At present it appears that combining T cell and antibody based immunotherapy has the potential to counteract the multiple mechanisms utilized by tumor cells to escape the host’s immune system because the two types of immunotherapy have complementary advantages and different limitations (von Mehren and Weiner, 1996; Abken et al., 1998). The methodology to combine these two types of immunotherapy has been developed and has been shown to be effective in the control of tumor growth in animal model systems (Abken et al., 1998; von Mehren and Weiner, 1996). Clinical trials are required to assess its clinical significance. Lastly, the negative impact on the outcome of T cell based immunotherapy of the multiple escape mechanisms utilized by malignant cells stresses the need to carefully select and monitor patients to be treated with T cell based immunotherapy. Assays for this purpose have to document the expression of the antigens of interest at the protein level in malignant lesions, since there is convincingevidence that mutations in genes encoding TAA or HLA class I antigens inhibit their translocation but do not affect their transcription. ACKNOWLEDGMENTS This work was supported by the PHS grants CA37959, CA51814, and CA67108 awarded by the National Cancer Institute, DHHS. The authors thank Dr. Polly Matzinger for reviewing the manuscript and helpful suggestions.

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ADVANCES IN IMMUNOLOGY, VOL 74

The Host Response to leishmania Infection WERNER SOLBACH AND TA&

IASKAY

Insthk, fw Medico1 Microbiology and Hygiene, Universify of lwbeck, luebeck, Gemony

I. Introduction

Leishmaniasis is caused by infection with protozoan parasites of the genus Leishmania. Leishmaniasis is not a single disease but constitutes a variety of syndromes ranging from local, self-healing skin ulcers (cutaneous leishmaniasis,CL) to a severe and life-threatening systemic disease (visceral leishmaniasis, VL;reviewed in Pearson and de Queiroz, 1996). Leishmaniases often represent zoonotic infections of stray and domestic dogs, rodents, hyraxes, or slothes with variable penetration to man. There are at least 30 species of Leishmania, of which 12 named and several unnamed species infect man (Lainson and Shaw, 1987). The type of disease that evolves depends both on the species of the parasites and on the host’s immune status at the time of infection. For example, Leishmania (L.) tropica in most cases has been associated with CL but also can cause visceralizing disease as became overt in some American military personel who served in Operation Desert Storm (Magill et al., 1993). Leishmanial disease currently affects some 12 million people, mostly children and young adults, in 88 countries on all continents except Australia. Exposed to the risk of infection are 350 million people, and the annual incidence is about 2 million new cases (WHO, 1998). Recently, there has been an increase in overlapping of visceral leishmaniasis and HIV infection due to the spread of the AIDS pandemic. In southern Europe, 25-70% of adult VL cases are related to HIV infection and 1.5-9.5% of AIDS cases suffer from newly acquired or reactivated VL (Albrecht, 1997; Alvar et al., 1997; Desjeux, 1996). Deadly epidemics currently occur in Sudan with > 100,000 deaths over the past 5 years and in Eastern India (State of Bihar), where > 200,000 people are believed to have contracted the disease since 1994 (WHO, 1998). Leishmaniasis is diagnosed by direct demonstration of the parasites (microscopy, culture, DNA or RNA analysis) in appropriately selected material (skin, spleen, bone marrow, other suspected sites) and/or by immunodiagnosis (Bryceson, 1996). For checking the cellular immune response, leishmanin (a suspension of killed parasites derived from culture) is inoculated into the volar surface of the forearm and the area of inflamma275

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tion is measured 48-72 hours later. Antibody detection is done by ELISA, immunofluorescence or agglutination assays (reviewed in Berman, 1997). Although localized single skin ulcers usually heal spontaneously and do not require treatment or are treated with topical agents or physical interventions (intralesional injection of antimicrobials, temperature), involvementof internal organs requires antimicrobialswhich include pentavalent antimonials, Pentamidine, liposomal Amphotericin B, Paromomycin, Interferon-?, and others (for details see Berman, 1997). Murine experimental leishmaniasis is a useful model to study disease pathogenesis, which in its various aspects has given important insights not only for the understanding of human leishmaniases but also for other diseases caused by intracellularly living microorganisms like tuberculosis, listeriosis, brucellosis, and toxoplasmosis. In addition, data obtained from murine experimental leishmaniasis research has tremendously contributed to our current understanding of interconnections between important determinants of the innate and the acquired immune response. In this model, in its basic form, inbred mice of various genetic backgrounds are injected, usually in or underneath the skin of the footpad with L. major promastigotes. Most strains of mice, such as C57BLi6, CBA/ J, C3H, or B10D2, effectively resist the infection with clinical, albeit no parasitological, cure within a few months (Handman et al., 1979). Thus, such mice can be regarded as the model for localized leishmanial disease. By contrast, BALB/c mice and all mice with immunodeficiencies related to the T cell immune response fail to control parasite multiplication, and dissemination to visceral organs leads to death (Howard et al., 1980; Howard et al., 1982; Mitchell et al., 1980). Therefore, these animals can serve as model for studying systemic leishmaniasis. Resistance and susceptibility are closely associated with the development of T cell responses of the T h l or Th2 type, respectively. This review focuses on the relationship of the parasite with its mammalian host cells and the mechanism by which this relationship determines the development of the ensuing immune response and disease. We shall concentrate on the “decision-making” first hours to days of the infectious process. Issues regarding vaccination strategies will not be covered and have been reviewed elsewhere (Cox, 1997; Hommel et al., 1995; Modabber, 1995). Most of the data cited stem from experimental work using infection of inbred mice with various species of Leishmania. It is important to bear in mind that observations made with one parasite species in mice cannot necessarily be extrapolated to other Leishmania species or to the situation in man.

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II. The Parasite

Leishmania are transmitted between long-lived vertebrate hosts by shortlived phlebotomine sand flies (Phlebotomus spp., Lutxomyia spp. and Psychodopygus s p p . ) and have a cycle of development in each. In the sand fly and in culture medium, they are in the promastigote form (1.5-3 p m X 10-20 pm) with an anterior flagellum and in the vertebrate host they reside intra- and extracellularly as oval, nonmotile cells with only a very short flagellum and a maximum diameter of 2.5 X 6.8 pm, which are called amastigotes. Multiplication of each form is by binary fission (Bryceson, 1996). A. LIFECYCLE

1. In the Sand Fly After ingestion of a blood meal from an infected host, the parasites initially reside within the peritrophic membrane inside the sand fly’s midgut. Amastigotes are then released from the macrophages of the blood meal and differentiate into the promastigote stage. They now synthesize an increasingly dense coat of a glycocalix, which is composed of a variety of glycoconjugates that are bound to the surface of the parasite by a glycophosphatidylinositol (GPI) anchor (McConville and Ralton, 1997; Turco and Descoteaux, 1992). Underneath the glycocalix, a dense layer of low molecular-weight glycoinositol-phospholipids (GIPLs) is found, which is thought to have barrier functions. Among the glycoconjugates,a lipophosphoglycan (LPG), which contains a repeating polymer of disaccharidephosphate repeating units, is the most abundant with 1-5 X lo6molecules/ parasite (Beverley and Turco, 1998; McConville and Ralton, 1997). Following rupture of the peritrophic membrane after about 2 days, the promastigotes attach to the midgut wall through specific binding of LPG and rapidly divide. After a further 4-7 days, the parasites cease dividing and differentiate into infective metacyclic promastigotes, which have a structurally altered LPG that is incapable of binding to the midgut wall (Sacks et al., 1995). They migrate to the foregut and esophagus, where they are suspended in the sand fly’s saliva and are ready to be inoculated during the next blood meal. This process involves enzymatic damage of the insect cardic valve that normally prevents reflux from the gut to the pharynx (Schlein et al., 1992). In the sand fly’s gut, the saliva probably promotes survival and development of the promastigotes, since feeding of Phlebotomus argentipes with L. donovani suspended in serum containing antisaliva antibodies led to death of a significant number of vectors and in the survivors to an inhibition of promastigote development and a concomitant reduction of migration to the foregut (Ghosh and Mukhopadhyay, 1998).

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2. In the Mammalian Host a. Breaching the Skin. In order to get into their host, Leishmania take advantage of the feeding habits of the vector. The sand fly rips up the epidermis and eventually gains access to dermal capillaries. During this process, parasites are regurgitated into the bite wound. By using forcedfeeding techniques with L. m j o r infected Phlebotomus papatasi sand flies, it was shown that the majority (approx. 75%) egested less than 100 parasites, approx. 20% egested 100-1000 parasites and the remainder egested 10003000 parasites each (Warburg and Schlein, 1986). Thus, under natural conditions, sand flies transmit very low numbers of promastigotes, which are able to induce disease. Under experimental conditions, when promastigotes usually are suspended in saline and inoculated by syringe into the skin of inbred mouse strains, this low number of parasites will never cause disease even in mouse strains like BALB/c, which are extremely susceptible to L. major infection when 105-107parasites are inoculated. The experimental injection of such mice with low numbers of parasites (100-500) rather results in stable and protective immunity to the disease (Bretscher et al., 1992; Doherty and Coffman, 1996; Menon and Bretscher, 1998).

b. Sand Fly Saliva as Immunomodulator. As they ingest their blood meal, the sand flies continuously salivate. The saliva serves the parasite as an important adjunct to establish a successful infection. In experimental infections, salivary gland lysates injected together with L. major or L. braziliensis into various strains of mice resulted in exacerbation of both the size of the lesion and the number of recoverable parasites even when low inocula (lo2-lo4 promastigotes) were used for infection (Belkaid et al., 1998b; Lima and Titus, 1996; Titus and Ribeiro, 1988). Treatment with both, a neutralizing anti-interleukin-4 antibody or an antisaliva antibody abrogated the effects of the saliva (Belkaid et al., 1998b; Lima and Titus, 1996). Interestingly, the disease-promoting effect of saliva was seen neither in IL-4 deficient nor in SCID mice (Belkaid et al., 1998b). L. m j o r infection together with salivary gland lysates from P. papatasii showed an up-regulation of the Th2-response and down-regulation of the Thl response. Interestingly, the saliva contents induced elevated IL-4 mRNA expression even in the absence of infection (Mbow et al., 1998). Histologically, in the murine BALB/c model with L. braziliensis infection, the progressing lesions developing after injection of parasites together with saliva showed poorly organized accumulations of heavily parasitized epitheloid cells with persistent neutrophils and eosinophils, whereas the saliva-free lesions initially progressed to small organized granulomas but eventually resolved completely (Donnelly et al., 1998). Moreover, saliva

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ingredients are able to inhibit oxidative metabolic processes and antigen presentation by macrophages in vitro (Lerner et al., 1991; Theodos et al., 1991). Numerous attempts to identify the pharmacologically active principle have led to the description of a 599 base pair-encoded protein maxadilan, originally isolated from salivary glands of Lutzomyia longipalpis (Lerner and Shoemaker, 1992; Yoshida et al., 1996) and a protein phosphatase inhibitor as obtained from salivary gland lysates of Phlebotomus pupatasii (Waitumbi and Warburg, 1998). Maxadilan has been shown to be the most potent vasodilator known (Jackson et al., 1996) and also to inhibit the production of TNF-a, while concomitantly increasing the serum levels of IL-1 and IL-10 in a lethal LPS shock model (Bozza et al., 1998). Whether any of these identified components or a hitherto unrecognized molecule or a combined action of factors serves to aggravate disease in Leishmania infection remains to be determined. Also, it has to be kept in mind, that not all salivas are equal, since salivas from different sand fly species or from the same species in different geographical areas greatly vary in their composition (Warburg and Waitumbi, 1997). Hence, available evidence strongly suggests that the parasites take advantage of saliva in order to establish infection. c. Extracellular L$e in the Tissue. Immediately after transmission, but before entry into their host cells, extracellular promastigotes are exposed for a short while to potentially toxic fluid and serum components including factors of the complement system. Probably most of the parasites are rapidly killed by complement factors (Sacks and Perkins, 1984);a sufficient number, however, will survive, since they are relatively resistant to complement-mediated lysis. This is possibly achieved by spontaneous shedding of C5b-C9 complexes from the parasite surface (Puentes et al., 1990), which in turn is associated with the length of the lipophosphoglycan molecules in the glycocalix of the promastigotes (McConville and Ralton, 1997). Infective Leishmania also express a seridthreonin protein kinase (cf., LPK-1) which has been shown to inactivate C3, C5, and C9 by phosphorylation (Hermoso et al., 1991; Li et al., 1996), thus avoiding the lytic complement attack. While subverting harmful effects of the complement system, Leishmania at the same time depend on the opsonic complement factors by means of the metzincin zinc proteinase gp63 (leishmanolysin) (Schlagenhauf et ul., 1998). Gain- or loss-of-function genetic studies showed that gp63, on one hand, did not bind appreciable amounts of the terminal complement components but, on the other hand, rapidly converted C3b to iC3b, thus facilitating the uptake of the parasites by cells

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expressing the iC3b-receptor CR3 (CD llb/CDlS) (Brittingham et al., 1995; Mosser and Brittingham, 1997). d. Adherence and Invasion. Leishmania that escape the lytic attack in the extracellular environment rapidly adhere to resident or recruited cells of the monocyte/macrophage lineage including dendritic cells and Langerhans cells (Blank et al., 1993; Locksley et al., 1988; Moll et al., 1995). Few in vivo and in vitro studies suggest that also human granulocytes are wellsuited target cells (Dominguez and Torano, 1999; Palma and Saravia, 1997; Pearson and Steigbigel, 1981; Sunderkotter et al., 1993; Laskay et al., unpublished observation). The binding of the parasites to the cell surface can occur through numerous receptors, including the mannose-fucose receptor, the fibronectin receptor, and the receptor for C-reactive protein (reviewed in Bogdan and Rollinghoff, 1998). Under physiological conditions, the most important receptors are the complement receptors type I (CR1, CD35) and type I11 (CR3, CDllbICD18) (Sutterwala et al., 1996). These receptors bind to complement components attached to plasma membrane molecules of the parasite (Rosenthal et al., 1996). Complement-dependent adhesion is followed by the internalization of the promastigotes either by “coiling phagocytosis” (i.e., by wrapping with multiple layers of unilateral pseudopods of the phagocytic cells; Rittig et al., 199813)or by conventional “zipperlike” interactions (reviewed in Rittig et al., 1998a). Within minutes after phagocytosis, Leishmania are located in phagosomal compartments that are limited by a membrane originating from the host cell plasmalemma. These phagosomes then undergo remodeling via maturation and fusion with endocytic organelles and form a parasitophorous vacuole (PV) containing lysosomal hydrolases, cathepsins and 6glucuronidase (Lang et al., 1994; Russell and Talamas-Rohana, 1991). Inside the PV, the promastigotes transform into nonmotile amastigotes. This requires 2-5 days, depending on the Leishmania species studied. Amastigotes do not synthesize LPG but do contain several glycoconjugates related to LPG, including proteophosphoglycan, acid phosphatase, and GIPLs (Beverley and Turco, 1998; McConville and Ralton, 1997).Although all Leishmania PV have many common features, they are not identical morphologically. For example, PV that contain L. amaxonensis or L. m x i cana harbor numerous parasites within one large vacuole, whereas macrophages that are infected with L. donovani or L. major have many individual small PV with only one or few parasites inside each (Antoine et al., 1998). The functional consequences of these differences are largely unknown. A recent study suggests that a proteophosphoglycan secreted by intracellular L. mexicana amastigotes may be one of the factors involved in the morpho-

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genesis of the large PV (Ilg et al., 1995; Peters d al., 1997). Interestingly, in striking contrast to our knowledge of the immune responses to L. major, the characteristics of the PV of this species are the least investigated. From in vitro studies with L. donovani,L. amazonensis, and L. mexicana, it appears that in less than 30 min. after parasite ingestion, the vacuoles are acidified and reach pH5. The pH is maintained by a H+-ATPase of host cell origin and possibly by a P-type H+-ATPaselocated in the parasite plasmalemma that extrudes protons from the parasite cytosol into the lumen of the PV (Zilberstein and Shapira, 1994). In addition, the PV acquire the lysosomal glycoproteins, macrosialin, and lysosomal-associated membrane proteins LAMP-1 and LAMP-2 within 2 hours. The acquisition of acidic hydrolases and major histocompatibility complex (MHC) class I1 molecules occurs within the next 5-24 hours (Lang et al., 1994). The early events in PV formation are partially regulated by the parasites themselves. For example, LPG has been shown to transiently inhibit the fusion of the nascent promastigote-containing phagosomes with late endocytic compartments (Desjardins and Descoteaux, 1997). This temporary fusion block can be viewed to allow time for the promastigotes to begin to differentiate into the amastigote stage, which is much more adapted to the enzymes and the acidic pH of the PV. The adaptation process is linked to the expression in the amastigote plasma membrane of proton pumps that are involved in the capture of metabolites and metabolite transporters and are optimally active at acidic pH (Saar et al., 1998; Zilberstein and Shapira, 1994). The proteolytic activity of gp63 within the PV is best at neutral or acidic pH and is thought to protect the parasite from intraphagolysosomal degradation (llg et al., 1993; Medina-Acosta et al., 1989; Seay et al., 1996). A number of other molecules are synthesized by Leishmania amastigotes, the function of which is hitherto largely unknown (Ilg et al., 1998; Ismail et al., 1994; Stierhof et al., 1998). 111. The Host's Innate Response

A. INFLAMMATION

Immediately after the injection of the parasites, a local inflammatory process is initiated, which involves local accumulation of cells to clear damaged tissue and to initiate wound healing. Initially, the wound is infiltrated by neutrophilic and eosinophilic granulocytes, followed by a wave of inflammatory macrophages, which within a few day predominate the lesion. Lymphocytes are hardly detected at this early stage of infection (Beil et al., 1992; Sunderkotter et al., 1993). Using the subcutaneous air pouch inoculation technique of the parasites, we could demonstrate ample numbers of locally accumulating natural killer (NK) cells (Laskay et al.,

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1998). By the time macrophages appear, extracellular promastigotes are mostly dead or have invaded resident cells which they may use as “safe targets” (Greil et al., 1988; Mirkovich et al., 1986). VERSUSCONTAINMENT OF THE PARASITE-THE B. DISSEMINATION ROLEOF N K CELLSON THE HOST’SSITE After the infectious process is initiated and amastigotes have formed, it is thought that they start to divide within the PV until it ruptures and leads the host cell to release the amastigotes which are then taken up by neighboring competent cells. Alternatively, it can be proposed, that amastigotes are released by fusion of the PVs with the plasma membrane, thus leaving the host cell intact. Evidence for the latter mechanism may be that infection with L. donovani inhibits apoptosis of macrophages (Moore and Matlashewski, 1994) and of granulocytes (T. Laskay, H. Laufs, and W. Solbach, unpublished). Irrespective of the way in which amastigotes infect neighboring competent host cells, there is a striking difference in the kinetics of parasite dissemination in viuo. When BALB/c mice were infected into the dermis of the hind footpad with 2 X lo6 culture-derived L. major promastigotes, after 2-5 hours the parasites were found in the draining lymph node and within 10-24 hours could be detected in the para-aortic lymph nodes, the spleen, the liver, the bone marrow, and, occasionally, the kidney. In similarly infected other mouse strains with a curative phenotype like C57BL/6, CBA/J, and C3H/ HeJ, the parasites remained localized in the footpad and in the draining popliteal lymph node for 5 days or more without evidence of dissemination (Laskay et al., 1995). Early parasite dissemination was dependent on the infectious dose. Low numbers (2 X lo3)of L. major parasites were retained in lymph nodes of BALB/c mice and, expectedly, did not lead to overt disease. Also, dissemination was species-dependent, An inoculum of up to 2 X 10’ promastigotes of L. aethiopica did not disseminate beyond the draining lymph node (T. Laskay and W. Solbach, unpublished observation). Interestingly, this Leishmania species is not pathogenic for mice. Microscopical analysis of thoracic duct lymph obtained from BALB/c mice 3 hours after infection (Ionac et al., 1997) suggested that most of the parasites disseminated as intracellular forms (largely promastigote-like), but occasionally extracellular promastigotes were also observed (W. Solbach and T. Laskay, unpublished observation). Dissemination did not occur in susceptible SCID mice which lack functional T and B cells, but have a relatively high potential of functional NK cells (Kumar et al., 1989), thus suggesting a decisive role for NK cells in regulating the dissemination of parasites. Indeed, depletion of NK cells by antibody in resistant C57BL/6 mice led to rapid parasite dissemination.

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Analysis of the underlying mechanism disclosed that NK cells rapidly produced interferon-y ( IFN-y) after activation by both parasite antigens and IL-12, which in turn led to parasite containment (Laskay et al., 1995). In addition to the role of IL-12, NK cells, and IFN-y for regulating parasite dissemination and containment, more recent data point to a critical role for IFN-d/3 and inducible nitric oxide synthase (NOS2).L. major-infected, genotypically resistant F8-crosses between 129/SvEvand C57BIJ6 parental strains contain the parasites like other resistant strains. Littermates with a disrupted NOS2 gene (NOS2-’-) or C57BL/6 mice treated with antiIFN-y antibody or mice with a disrupted gene for IFN-y, however, permitted rapid spreading of the parasites and developed disease, even with 500-2000 parasites (Diefenbach et al., 1998; Laskay et al., 1995). One day after infection, NOS2-/- animals showed only very little increase in IFNy mRNA expression at the site of infection (footpad) and the draining LN when compared to their NOS2+/+littermates (Diefenbach et al., 1998). As the early production of IFN-y in L. major-infected mice is predominantly due to NK cells (Laskay et al., 1993; Reiner et al., 1994b; Scharton and Scott, 1993), these data suggest that NOS2 activity is important for NK cell-mediated parasite containment. NOS2 expression and parasite containment are also dependent on IFN-dfi, since treatment of NOS2+’+ animals with an anti I F N - d p antibody led to rapid parasite dissemination and reduced the number of NOS2-positive cells at the dermal site of infection by more than 90%. Likewise, treatment of NOS2-/- or IFN-y-’mice did not prevent early parasite spreading (Diefenbach et al., 1998). Taken collectively, these data suggest that the early containment is orchestrated by the coordinated action of IFN-d/3, IFN-y, IL-12, NOS2, and NK cells, which are active at the site of infection and the draining lymph node. In vivo depletion of NK cells using the NK cell-specific anti-NK1.l monoclonal antibody (Laskay et al., 1993) or the less specific anti-asialoGM1 antiserum (Scharton and Scott, 1993) further underline the importance of NK cells in the early defense to L. major. Enhanced disease, as measured by parasite number and lesion development, was observed in N K cell-depleted mice. In the L. major infection model a delicate balance between stimulatory and inhibitory lymphokines appear to regulate the early activation of NK cells. Rapid Thl cell development and resistance to infection in mice that develop an early NK cell response after infection was reported (SchartonKersten and Scott, 1995). IL-12 is likely to play a key role in NK cell activation, since in vivo neutralization of IL-12 eliminated the early NK cell response in L. major-infected resistant C3WHeN mice (SchartonKersten et al., 1995a). In susceptible BALB/c mice the simultaneous early production of IL-12 and cytokines that inhibit IL-12 function, such as

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TGF-0, IL-4 and IL-10, was observed. In the absence of increased IL12 production by infected macrophages as shown after infection with L. donovani, IL-12 produced by dendritic cells could provide the microenvironment for initial NK cell activation (Gorak et al., 1998). Even though NK cells play a major role in the early control of parasites, NK cells done could not sustain control of L. major in the absence of CD4+T cell-derived IFN-y and Thl development was unaffected by the presence or absence of IFN-y from non-T-cells (Wakil et al., 1998). The discrepancy of these data to those showing the role of NK cell-derived IFN-y remains to be clarified. However, these data point toward contributions of NK cells in the early stage of infection that are independent of IFN-7. Like in the murine model, NK cell-mediated mechanisms are involved in the early defense to Leishmania infection also in humans. In vitro stimulation of blood lymphocytes from normal, nonexposed individuals with L. aethiopica antigens showed proliferation and IFN-y production (Akuffo et al., 1993; Laskay et al., 1991). The main activated cell type was found to be CD3-, CD16+, CD56' NK cells. However, in patients with ongoing L. aethiopica infection CD4+ T cells were the prominent responding cells indicating that the NK cell-mediated response is restricted to the early stage of infection in naive individuals. Further studies in an endemic area of L. aethiopica infection demonstrated again that Leishmania antigens induced proliferation of CD4+ cells in patients with active disease. However, similarly to naive individuals, healed patients also showed an NK cell response, accompanied with proliferation of CD8' cells (Maasho et al., 1998). These data suggest that NK cells, in addition to the early response in naive individuals, may play a role also in the maintenance of the healing process. THE LYMPH NODESHAPETHE INITIAL C. CELLSINVADING IMMUNE RESPONSE For full appreciation of the findings mentioned earlier, it has to be considered that in the draining LN dramatic changes occur during the first 16-24 hours as far as the cellular composition and the functional performance is concerned. Infection in the footpad of different strains of mice results in a 5- to 20-fold increase in the number of LN cells within 24-48 hours with a relative decrease of CD4' cells and an increase of B cells. Based on theoretical considerations, it is likely that &IS increase in cell number is not due to in situ proliferation after parasite entry but rather the consequence of influxing cells from the peripheral blood. In peripheral lymph nodes, cellular influx is predominantly mediated by transmigration of vascular cells through high endothelial venules (HEV; reviewed in Butcher and Picker, 1996; Girard and Springer, 1995). One of

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the components in this process is the attachment of lymphocytes via binding of their L-selectin (CD62L) to the CD34 molecule on HEV. In vivo blocking of this interaction by antibody to L-selectin in L. major-infected BALB/c mice completely prevented the increase in lymph node cells, and the animals were phenotypically resistant. The remaining cells were relatively enriched for activated cells (CD25' and CD69') and cells with memory phenotype (CD45RBd""CD44'gh) (Laskay et al., 1997). Moreover, the cells produced significantly more IFN-y than those obtained from mice without CD62L-CD34 blockade. When parasite spreading was analyzed, it became apparent that, in contrast to the control animals, the parasites were contained in the draining lymph node and did not disseminate beyond (T. Laskay, unpublished). Thus, in addition to the residing cells and cytolcines in the lymph node as considered before, regulation of parasite spreading is also governed by cells from the peripheral blood, which immigrate into the peripheral lymph node very early after infection with L. mjor. Although we do not understand how the various components of the innate immune system act upon Leishmania in the peripheral lymph node, it is clear from these experiments that whenever the parasites can be contained at the site of infection and in the draining lymph node within the first hours of infection, the animal will heal the infection. On the other hand, early dissemination does not necessarily reflect progression of the disease with eventual death.

N. The Host's Adaptive Immune Response A. ANTIGENPRESENTATION The establishment of a protective anti-Leishmania immune response requires the presentation of appropriate antigens by antigen-presenting cells, the induction and expansion of CD4' Thl-lymphocytes, and the activation of macrophages for efficient killing of the parasites. On the other hand, it is to the advantage of the parasite to evade effector functions of the cell in order to survive and multiply in this intracellular site. Therefore, an important task of intracellular Leishmania parasites is to avoid the stimulation of CD4' cells. One possible way is the interference with antigen-presentation by the host cells. Leishmania infection was indeed found to avoid the induction of antigen presentation. After in vitro infection with L. mjor, macrophages from BALB/c mice had greatly reduced capacity to present both L. m j o r derived and unrelated antigens such as OVA or P-galactosidase (Fruth et al., 1993). These data suggest that Leishmania infection interferes with the intracellular loading of MHC-I1 molecules with antigenic peptides.

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Similar conclusions can be drawn from experiments with murine macrophages infected with L. amazonensis amastigotes (Prina et al., 1993). There is a significant difference in antigen-presentation capacity of macrophages infected with promastigotes or amastigotes. Macrophages infected with promastigotes were able to present endogenous parasite molecules to CD4’ T-cells. However, the capacity of the infected cells to present parasite-antigen rapidly declined after the first day of infection. In contrast, macrophages infected with amastigotes did not present endogenously synthesized parasite molecules to CD4’ cells (Kima et al., 1996). In another study, macrophages infected in vitro with promastigotes were fully competent to activate T cells reactive to an antigen designated “Leishmania homolog of receptors for activated C kinase” (LACK) (Mougneau et al., 1995) in the first 6 h after infection, but not so after 2 days of infection. Macrophages infected with amastigotes were unable to stimulate LACKspecific T cells (Prina et al., 1996). These data suggest that during the transformation process from the promastigote stage to the intracellular amastigote stage, parasite antigens are sequestered from the MHC class I1 pathway of antigen presentation which then can result avoidance of T cell activation. Analysis of the intracellular localization of MHC-I1 molecules provided insights into the events which take place in infected cells. After infection of macrophages with Leishmania a redistribution of intracellular MHC I1 was observed, resulting in an accumulation of these molecules in the parasitophorous vacuole. The MHC I1 molecules were concentrated at the attachment site between amastigotes and the PV in macrophages infected with L. amazonensis or L. mexicana, thus suggesting specific interactions between parasites and MHC class I1 molecules in the PV (reviewed in Antoine et al., 1998). The MHC I1 molecules in PV are loaded with antigenic peptides since they do not contain the invariant chain (li) transiently associated with newly synthesized MHC I1 molecules. It is not yet known whether some of these antigenic peptides are of parasite origin and whether these parasite antigens are presented on the macrophage surface to CD4+ cells. However, macrophages infected with L. mexicana overexpressing the parasite acid phosphatase have been shown to activate acid phosphatase-specific CD4’ cells (Wolfram et al., 1995). Recently, it has been reported that amastigotes of L. amazonensis, L. m i c a n a , and L. major internalize MHC I1 molecules, which requires attachment between amastigotes and the PV membrane. The uptake of molecules from the PV membrane seems to be specific for MHC I1 molecules, as other PV molecules such as rab7p, macrosialin, or LAMP glycoproteins are not internalized (de Souza-Lea0 et al., 1995).

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B. COSTIMULATORY MOLECULES In addition to the antigen-specific interaction between antigenpresenting cells (APC) and T cells, the latter require costimulatory molecules for activation. Modulation of costimulatory signals leads to significant modification of antileishmanial T cell responses (reviewed in Kaye, 1995). Infection of macrophages with L. donovani both in vivo and in vitro failed to trigger expression of B7-1 (CD80) (Murphy et al., 1997). Moreover, upon infection of peritoneal macrophages with L. donovani, CD80 expression was decreased in BALB/c mice, but there was no change in C57BL/ 6 mice. The change in CD80 expression on the surface of BALB/c macrophages resulted in the inhibition of DTH-mediating functions of Th cells. After elimination of the parasites, however, CD80 was re-expressed, which paralleled the induction of a protective T cell-mediated immune response (Saha et al., 1995). The biologcal relevance of costimulatory molecules in the development of the resistant or susceptible phenotype in mice was substantiated by studies in which the expression of costimulatory molecules were manipulated experimentally. The studies revealed differences between usage of CD80 (B7-1) or CD86 (B7-2) relevant for the development of a T h l or Th2 response, respectively. Moreover, the significance of CD80/CD86 molecules appears to be different in the early and later course of infection. Prolonged treatment of both C57BL/6 and BALB/c mice with antibody to CD86 but not to CD80 decreased parasite burden and decreased the production of Th2 cytokines (Brown et al., 1 9 9 6 ~ )Sustained . blockade of B7-2 following infection with L. donovani resulted in enhanced T h l and Th2 cytokine responses and in a significant decrease in liver parasite burden. The action of anti-B7-2 mAb did not interfere with the early T cell activation, since the treatment on day 3 postinfection was as effective as beginning it the day of infection (Murphy et al., 1997). Both, CD80 and CD86 interact with CD28 and with the structurally homologue CTLA-4 (CD152) expressed on T cells. The binding affinity of CTLA-4 to B7 molecules, however, is significantly higher than that of the CD28 molecule. In contrast to CD28, CTLA-4 was suggested to play a role in the negative regulation of T cell activation (Krummel and Allison, 1995; Walunas et al., 1994). Experiments using knockout mice indicated that costimulation through CD28 plays only a limited role in the development of Thl or Th2 response to Leishmania. The CD28-deficient BALB/ c mice and their wild-type littermates were equally susceptible to L. major infection. SimilarlyCD28-deficient C57BL/6 mice retained their resistance to the infection (Brown et al., 199613). Treatment of mice with anti-CTLA4 Fab-fragments ameliorated the disease in BALBlc animals but had no

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effect on the course of infection in C57BL/6 mice, suggesting that CTLA-4 plays a significant role in the modulation of the immune response mainly in susceptible mice. Since the same effect was also observed in CD28deficient mice, it is likely that the effect of CTLA-4 on the course of infection is not dependent on CD28-mediated preactivation. The observed amelioration of the disease was found to be associated with an increase in the number of cells secreting IFN-y as well as with an increased parasitespecific DTH response (Sahaet al., 1998).Although a single dose of CTLA4-lg was protective, prolonged treatment abolished the capacity of BALB/ c mice to resist the infection, suggesting that costimulation through this pathway is required at later stages of the immune response (Corry et al., 1994). CTLA-4 engagement is likely to play a major role in the T cell unresponsiveness characteristic for chronic visceral Leishmania infection. Blockade of CTLA-4 in vitro restored the response to parasitic antigens. The production of IL-12 and IFN-y but also of IL-4 was enhanced (Gomes et al., 1998).L. infantum-infection of canine monocyte-derived macrophages led to a decreased expression of B7 molecules and induced a reduced antigenspecific T cell proliferation. Compensation for the decreased expression of B7 molecules by the addition of B7-l-transfected cells resulted in the restoration of cell proliferative response and IFN-y production by a Leishmania-specificT cell line (Pinelliet al., 1999).These data suggest that CTLA-4 engagement by its ligands plays an important role in maintaining Leishmania-induced unresponsiveness in CD4' cells. CTLA-4 plays a significant role in regulating the defense not only to L. major but also to L. donovani. A single dose of anti-CTLA-4 mAb injected on day 1 significantly decreased the parasite burden in infected BALBlc mice. The increased antileishmanial resistance was associated with increased cytokine production (IFN-y and IL-4) and a more rapid acquisition of a tissue granulomatous response (Murphy et al., 1998).

c. INDUCTION OF CD4'

THflH2 LYMPHOCYTES Analysis of mRNA transcription for various cytokines revealed that weeks after infection, nonhealing BALBlc mice infected with L. major in their draining lymph node cells contained elevated transcripts for IL-4, but not for IFN-y. In contrast, resistant C57BL/6 mice expressed transcripts for IFN-y, but only transiently for IL-4 (Locksley et al., 1987). In BALB/c mice, the expression of IL-4 mRNA remained elevated over time, whereas in C57BL/6 mice the IL-4 response returned to background levels after the initial phase of the infection (Heinzel et al., 1989). In addition, mice made phenotypically resistant by previous treatment with anti CD4antibodies (Tituset al., 1985) showed the same mRNA profile as genotypi-

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cally resistant C57BL/6 mice (Locksley et al., 1987). These findings were confirmed by studies demonstrating differences in cytokine production by parasite-specific T cell lines that were able or not to transfer resistance (Scott et al., 1990). These and other studies (reviewed in Reiner and Locksley, 1995) emphasized the ability of CD4’ T cells to shape the immune response and the phenotype of the murine disease. In the following years, numerous studies sought to c l a r i the ~ role of cytokines, especially IL-4 and, later, IL-12 for the development of a protective or nonprotective antileishmanial T cell response. The first category of research focussed on the question whether distinct parasitic antigens were able to direct T h l or Th2 responses in susceptible BALBlc mice, raising hopes that Thl-specific epitopes could be identified as potential vaccine candidates. Initial studies used peptide epitopes from metzincin zinc proteinase gp63 (see earlier discussion) and demonstrated protection in BALB/c mice when administered with adjuvant (Jardim et al., 1990). When the peptide was given without adjuvant, however, the disease was exacerbated. Subsequent studies confirmed the protective ability of the gp63 epitope, but only in resistant CBA mice, whereas no immunogenicity could be demonstrated in BALB/c mice (Yang et al., 1991).Whether the limited ability of gp63 in inducing a protective immune response is due to the finding that the molecule is able to cleave CD4 structures on human T cells (Hey et al., 1994) or whether its efficacy is restricted to the presence of appropriate adjuvants remains to be established. In this context, it is interesting, that in a recent study it was shown that 30% of BALB/c mice recovered from L. major infection, when they were vaccinated intradermally with plasmid DNA expressing gp63 and that dendritic cells from immunized mice were able to transfer protection (Walker et al., 1998).Vaccination studies using other peptides from various Leishmania constituents also showed protection but also exacerbation after subsequent challenge infection (reviewed in Bogdan and Rollinghoff, 1996). Another approach to identify antigens which might induce either a protective or a nonprotective T cell immune response was to clone the epitope which was recognized by a protective T h l cell clone that was derived from the spleen of a BALB/c mice after vaccination with a soluble extract from L. major promastigotes. This T cell clone used a V4,8 T cell antigen receptor (TCR) which was shown previously to prevail early after infection with L. major (Reiner et al., 1993). After construction of a cDNA library obtained from L. major promastigotes and expression in E. coli, a 24-kDa protein (p24) was identified sharing homology with intracellular receptors for activated protein kinase C (RACK) and was designated “Leishmania homologue of receptors for activated C kinase” (LACK)

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(Mougneau et al., 1995). LACK was found also in L. donovani, L. a m z o nensis, and L. chagasi promastigotes and amastigotes. The immunodominant epitope was primarily associated with amino acids 158-173, which reacted with T cells bearing the V88,4 TCR. Using T cell clones, it was shown that macrophages infected with promastigotes, but not with amastigotes, were efficiently able to present the LACK antigen (Prina et al., 1996).Twofold immunization with p24 conferred protection to subsequent infection with L. major in 72% of the animals, but only if administered together with IL-12 as adjuvant. A similar level of protection was achieved when DNA encoding the LACK antigen was used for immunization (Gurunathan et al., 1997, 1998). In vitro studies showed that lymph node cells from mice immunized with p24 alone primarily secreted the Th2 type cytokines IL-4 and IL-5, but no IFN-7 (Julia et al., 1996). Interestingly, infected BALB/c mice made tolerant to LACK by a transgenic approach had a reduced Th2-type response as compared to the nontransgenic littermates. Unfortunately, no data are available on the development of disease in these mice (Julia et al., 1996). Taken together, although the available studies suggest that some antigens with Thl- or Th2-type preference might exist, the bulk of experimental data suggests that most naive T cells have the potential to develop in either subset of mature T cells and that the cytokine milieu which the T cells face at the time of the first encounter with antigen is the most critical variable. D. CYTOKINE DETERMINANTS THAT SHAPE THE DEVELOPMENT OF TH CELLS Therefore, the second direction of research during the last decade was the clarification of the critical cytokine determinants that enable the anti leishmanial immune response to develop in either a protective or a diseaseexacerbating direction. Justification for this research came from early data demonstrating that both phenotypical disease susceptibility and resistance to L. major-infection were mediated by different types of T lymphocytes, whereas B cells and antibodies were of minor importance (Handman et al., 1979; Howard et ul., 1980, 1982; Mitchell, 1983). The other set of data suggested that most naive T cells have the potential to mature into either subset of T helper cells (Thl and Th2, respectively) and that critical cytokines faced by the T cells at the time of priming mediate this differentiation (Mosmann and Coffman, 1989). IL-4 was identified to have an important role for mediating Th2 development (Seder et al., 1992), while IL12 (reviewed in Romani et al., 1997; Trinchieri, 1995) and IFN-.)I were described as decisive for T h l development and that both sets of cytokines are reciprocally active (Hsieh et al., 1995; Tanaka et al., 1993).

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The strength of the murine model of experimental infection with Leishmania came from early observations that a number of immunomodulatory or pharmacological interventions could change the phenotype of highly susceptible BALB/c mice into a resistant one and that the immunity induced was sustainable and could be transferred to naive recipients (reviewed in Milon et al., 1995; Reiner and Locksley, 1995). The unifymg immunological effect able to induce resistance has been the successful attenuation of IL-4 expression in the draining lymph node of infected mice during the first 24 hours of infection. Therefore, in the following section, we shall focus on the role of IL-4 in Leishmania infection. O N I ~ - 1 2RESPONSIVENESS E. IL-4 AND ITS IMPACT

In the L. major mouse model, IL-4 has been regarded as counterprotective cytokine, since the development of Th2 cells was clearly associated with aggravation of the disease. Neutralization of IL-4 by antibody or soluble IL-4 receptor-conferred protection, and macrophage inhibitory functions of IL-4 were more prominent than its stimulatory effects (reviewed in Bogdan and Rollinghoff, 1996). More recently, using susceptible BALB/c mice with a deleted IL-4 gene (IL-4-’-), it was shown that they were resistant to infection with L. major (Kopf et al., 1996), although another report came to different conclusions with data that were similar in principle but not identical (Kropf et al., 1997; Noben-Trauth et al., 1996, discussed in Etges and Muller, 1998). The importance of IL-4 apparently varies with different Leishmania species, since infection of IL-4-’mice with L. mexicana led to an attenuated disease progression, while they were unable to control infection with L. donovani (Satoskar et al., 1995). As mentioned before, modulation of L. major disease in susceptible BALB/c mice requires intervention either before or around the time of infection, thus pointing toward critical checkpoints operating at this early stage of the disease. With respect to the role of IL-4 in mediating both susceptibility and Th2 cell differentiation, it was shown that, in contrast to resistant C57BL/6 animals, BALB/c mice exhibited a burst of IL-4 mRNA in CD4’ T cells in the draining lymph node as early as 16 hours after subcutaneous infection with L. major promastigotes (Launois et al., 1995). The CD4’ T cells did not express the NK1.l antigen. Such cells had been shown under different conditions to be a major source of early IL-4 production (Yoshimoto et al., 1995), but were not required for the development of progressive disease in BALB/c mice infected with L. major (Brown et al., 1996a;von der Weid et al., 1996).Instead, they were confined to a CD4+ T cell-subset with a highly restricted T cell receptor usage ( Vp4Va8) and could be activated with recombinant LACK antigen from L. major (Launois et al., 1997a). It is not clear at present whether LACK is

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the antigen from Leishmania that is solely responsible for the early IL-4 response or whether other antigens are recognized. The early peak in IL-4 mRNA expression plays an essential role for the development of the second wave of IL-4 mRNA which can be observed from day 5 onward and which reflects the differentiation of parasite-specific CD4' T cells toward the Th2 functional phenotype. Both, the early and second wave of IL-4 mRNA expression could be prevented when the mice were given 1 mg of IL-12 intraperitoneally 16 hours before parasite infection. In contrast and so far unexplained as to its reason, similar treatment with 1 mg anti-IL-4 antibody had an effect solely on the second wave of IL-4 mRNA expression (Launois et al., 199%). The time window when exogenous IL-12 was effective was very narrow, since administration of IL-12 later than 48 hours after infection had no influence on the increase of the second IL-4 peak and on the development of Th2 cells. Further analysis revealed that the early IL-4 mRNA burst rapidly induced a state of unresponsiveness of parasite-specific CD4' T cells to IL-12, which then could not be longer active to induce a Thl cell response (Launois et al., 199%). Experiments to identify the molecular mechanism underlying the state of IL-12 unresponsiveness due to IL-4 signaling disclosed a rapid down-regulation of the P2-chain of the IL-12 receptor (Himmelrich et al., 1998). These data thus led to the hypothesis that the genetically determined susceptibility to infection with L. major is primarily based on an early upregulation of IL-4 production, which subsequently induces IL-12 unresponsiveness. On the other hand, it may be possible that unresponsiveness to IL-12 or maintenance of IL-12 responsiveness in resistant strains of mice is a genetic trait. IL-12 binds to a receptor complex on the surface of cells composed of at least two gpl30-related molecules, IL-12Rp1and IL-12Rp2,Binding of IL-12 initiates the phosphorylation of two members of the Janus family of kinases, Jak2 and Tyk2. Signalling is propagated through the tyrosine phosphorylation of members of the signal transducers and activators of transcription (STAT) family of proteins. These proteins, after homo- and heterodimerization, translocate to the nucleus, where they activate a specific genetic program by binding to DNA recognition sites located in promoter regions of IL-12 activated genes (reviewed in Gorham et al., 1997).To test the hypothesis that the genetic control of Thl or Th2 cell development may be mediated by intrinsic genetic differences in IL-12 responsiveness, a number of elegant studies have been performed by using CD4' T cells from aP-TCR transgenic mice that reacted to a peptide derived from chicken ovalbumin protein in the context of MHC-I1 lad.

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The transgenes were bred onto BALB/c and B10.D2 mice, both of which have the H2d/dhaplotype; however, they differ in their susceptibility to L. major infection, since B10.D2 mice are resistant. Upon specific priming in vitro, IL-12 or IL-4 expectedly induced strongly polarized Thl or Th2 cells, respectively, in T cells from both backgrounds. However, when the cells were primed without the addition of exogenous cytokines (i.e., under “neutral” conditions), T cells derived from a BALB/c background mouse developed toward Th2 cells, since they made significantly more IL-4 and less IFN-y upon secondary stimulation than T cells derived from a B 10.D2 background mouse (Guler et al., 1997). Based on these data, it was hypothesized that BALB/c mice inherently develop a Th2 cell response because they lose their ability to respond to IL-12. More recent studies using the BALB/c and B10.D2 TCR transgenic mice suggest, however, that IL-12 responsiveness is also controlled by exogenous factors since the addition of antitransforming growth factor P (TGF-P)-antibodies maintained IL12 responsiveness in T cells from BALB/c mice, whereas low doses of exogenously added TGF-P during primary activation of B10.D2 T cells inhibited the development of IL-12 responsiveness (Gorham et al., 1998). In this context, it is worth mentioning that cultured liver granuloma cells from BALB/c mice infected with visceralizing L. chagusi parasites were inhibited to produce IFN-7; neutralizing TGF-P resulted in partial restoration of the IFN--yproduction. Conversely, C3H/HeJ mice, which are genetically resistant to L. chagasi infection, could be rendered partially susceptible by administration of a recombinant adenovirus expressing TGF-P (Wilson et al., 1998). It is very possible that, in this setting, TGF-P may have suppressed IL-12 responsiveness with subsequent inability to produce IFN-y. Also, the well-known susceptibility-increasing potential of TGF-P in both human and experimental leishmaniasis (Barral et al., 1995; Rodrigues et al., 1998)may have one reason in the induction of IL-12 unresponsiveness, but also other reasons like suppression of nitric oxide production (Li et al., 1999; Vodovotz et al., 1993) are likely. The common denominator is that IFN-y-driven processes are suppressed. Crossing studies between BALB/c and B10.D2 TCR transgenic mice and sequence length polymorphism genotyping disclosed a gene in the middle portion of chromosome 11,which was called Tpml (T cell phenotype modifier l).This gene was associated significantly with the measured phenotypes of T cells (IL-12 responsiveness vs. unresponsiveness), but also other genes may contribute to IL-12 responsiveness, as suspected by other back-crosses from the parental strains of mice (Gorham et al., 1996b). The molecular identity of Tmpl has not been determined so far. Taken together, the central decisive element for the development of susceptibility or resistance after L. major infection appears to be whether

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CD4+ T cells from an infected animal remain responsive to IL-12 by maintaining the expression of the PZchain of the IL-12 receptor for more than about 16-48 hours after infection. If so, they will develop a protective Thl response, whereas in the case that their T cells become unresponsive to IL-12, the mice will develop a Th2-polarized immune response and will be susceptible. It remains to be determined as to what extent IL-12 responsiveness is a genetic trait in the IL-12 signalling pathway or is based upon regulatory elements including IL-4. ON EARLY IL-4 PRODUCTION F. I ~ - 1 2AND ITSIMPACT As mentioned before, the extent of IL-12 responsiveness is a critical determinant for the development of resistance and susceptibility to L. major infection. This implies that IL-12 itself is essential for the development of a curative immune response through its capacity to induce Thl cell development (Romani et al., 1997; Trinchieri, 1995). IL-12 expression is influenced profoundly by Leishmania parasites. Peripheral blood mononuclear cells from patients with Kala Azar did not secrete IL-12 after stimulation with leishmanial antigens in vitro (Ghalib et al., 1995). Experiments using human peripheral mononuclear cells (PBMNC) showed that the ability of L. major promastigotes to induce the production of IL-12 is dependent on their developmental stage. Although noninfective promastigotes taken from the logarithmic phase of the culture were good inducers of IL-12 production as well as of IFN-y, TNF-a, and IL-10, infective parasites from the metacyclic phase were poor inducers of IL-12 (Sartori et al., 1997). Both stages of parasites were inhibitory for IL-12 production induced by Staphylococcus aureus (Sartori et al., 1997) or IFN-y/LPS (Carrera et al., 1996). IL-12 secretion was also suppressed after infection of macrophages with amastigotesof L. mxicana; the parasites also inhibited IL-12 secretion inducible by phagocytosis of latex beads, CD 40 crosslinking, or cognate interaction with Thl cells (Weinheber et al., 1998). Intracellular staining for cytokines and parasites in inflammatory macrophages obtained from granulomas induced by polyacrylamide microbeads showed that IL-12 expression was inhibited by parasite infection since virtually every infected cell lost its ability to produce IL-12 in response to IFN-y/LPS. This effect was restricted to IL-12, as the TNF-a response remained unimpaired. On the other hand, it was not parasite-specific, since it was seen with a number of Leishmania species tested, including L. major, L. donovani, L. tropica, L. amazonesis, and L. braziliensis (Belkaid et al., 1998a). Possibly, the suppression of IL-12 production by L. major is regulated by the extent of NOS2 expression, since infected peritoneal macrophages from NOS2-deficient mice infected 15 days previously produced signifi-

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cantly more IL-12 upon stimulation with soluble leishmanial antigen than the wild-type littermates. Cultured J774 cells produced significantly more IL-12 in response to IFN-y/LPS in the presence of the NOS2 inhibitor L-N‘-monomethyl-arginine ( L-NMMA). Conversely, IL-12 production was inhibited in the presence of the NO donor S-Nitroso-N-acetylpenicillamine (SNAP) (Huang et al., 1998a). This is in contrast to a study, in which NOS activitywas studied in the early phase of infection (i.e., during the first 5 days of L. major infection). For this time point, Diefenbach et al. (1998) demonstrated a 30-fold reduction of the baseline expression of IL-12 p40 mRNA in NOS2-’- mice (of different origin than those published by Huang et al.) in the skin and draining lymph node. Taken together, these findings point toward a dual role of NO, depending on the stage of infection. Although the molecules involved in parasite-induced suppression of IL-12 have not been determined, structures related to surface LPG (see earlier discussion) may be good candidates (Liew et al., 1997). In vivo suppression of the production of IL-12 by visceralizing species of Leishmania (L. donovani, L. infanturn, and L. chagasi) may contribute to the delayed onset of cell-mediated control mechanisms and the profound immune suppression during the acute stage of visceral human leishmaniasis (Kemp et al., 1996). As mentioned before, L. major promastigotes evade induction of IL-12 during invasion of macrophages from both resistant and susceptible mice during the first 4-7 days of infection (Reiner et al., 1994a). On the other hand, the ability of anti-IL-12 antibody treatment to abrogate healing of resistant mice suggests that endogenous IL-12 is also crucial for effective control of infection during later stages (Heinzel et al., 1995; Sypek et al., 1993). This was confirmed by infecting with L. major genetically resistant 129/ Sv/Ev mice with homologous disruption of the genes coding for either the p35 subunit or the p40 subunit of IL-12. They developed large, progressive lesions and a polarized Th2 response with low mRNA for IFN-y and high IL-4 mRNA in the draining lymph nodes (Mattner et al., 1996). As mentioned previously, BALB/c mice can contain parasite spreading and mount a protective T h l response, when inoculated with low ( lo2)numbers of parasites. When, however, BALB/c IL-l2p40-’- mice were infected with as low as 100 L. m j o r promastigotes, they developed progressive disease and mounted a strong Th2 response (Mattner et al., 1997). Thus IL-12 is indispensable for protective immunity against L. major infection, presumably through induction of NK cell activity and IFN-7 production required for containing parasite spreading (Laskay et al., 1995; Scharton-Kersten et al., 1995b). The protective effect of exogenous IL-12 in BALB/c mice correlated directly with its capacity to suppress IL-4 transcription and

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protein production (Heinzel et al., 1995; Sypek et al., 1993). The IL-4 suppressing activity of IL-12 was independent of IFN-y, since in L. majorinfected mice with a deleted IFN-y gene, IL-12 was still able to inhibit IL-4 mRNA induction (Wang et al., 1994b). Also, concomitant treatment of resistant mice with anti-IL-4 antibodies blocked the exacerbation of disease otherwise achievable with anti-IL-12 or anti-IFN-y treatment (Heinzel et al., 1995).These findings indicate that IL-12 confers protection to L. major infection by suppression of both, IL-4-production and induction of IFN-y-signaling by NK cells. One of the questions to be answered is why, in susceptible animals, IL-12 does not result in impeding of the early IL-4 mRNA peak that is responsible for the development of the diseasepromoting Th2 cells as discussed in Section E. Given the importance of early IL-12 production for the development of a curative immune response, the question remains: what are the cells and the sequence of events that lead to the release of IL-12? As mentioned previously, macrophages fail to produce IL-12 following infection with Leishmania and become refractory to normal IL-12 inducing stimuli; in situ immunostaining for IL-12 p40 protein revealed that not macrophages but dendritic cells (DC) in the skin are the critical source of early IL-12 production (Gorak et al., 1998).Whether IL-12 production by DC is under control of chemokines like macrophage inhibitory protein-la! (MIP-la) or macrophage chemoattractant protein-1 (MCP-l), the occurence of which has been shown in skin lesions of patients with American cutaneous leishmaniasis (Ritter et al., 1996), is an attractive hypothesis but remains to be shown. Also, it is not known, which parasite structures induce IL-12 production. One candidate may be a recombinant protein (LeIF),originally cloned from a L. braxiliensis gene homologous to the eukaryotic ribosomal protein elF4A. LeIF was a potent inducer of IL-12 in cultured human PBMNC and DC from both patients and uninfected individuals as well as from naive SCID mice (Probst et al., 1997; Skeiky et al., 1998). In this context, it is most interesting that synthetic small unmethylated CpG oligonucleotides (CpG-ODN) were extremely effective as adjuvant in inducing a strong IL-12 and subsequent CD4' TH1 cell-mediated response and were able to induce complete protection in susceptible BALB/c mice infected with L. major. An ongoing Th2 T cell response could be redirected to a curative Thl response when the adjuvant was given as long as 20 days after the infection (Zimmermann et al., 1998). CpG-ODN, after they have been taken up by endocytosis and mature in an endosomal compartment (Hacker et al., 1998), are able to activate and mobilize DC in the skin, to convert them into professional antigen-presenting cells, and to induce the production of large amounts of IL-12 (Jakob et al., 1998; Sparwasser et al., 1998). These data underline the importance of IL-12

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for the development of a protective antileishmanial immune response and open new prospects for consideration of protective vaccines. EFFECTOR MECHANISMS G. ANTILEISHMANIAL The production of superoxide ( 02-) and nitric oxide (NO) are the two major effector mechanisms of eliminating Leishmania parasites (Bogdan, 1997,1998; Murray and Nathan, 1999).Both, neutrophils and macrophages produce superoxide using the NADPH oxidase pathway. Although superoxide production is the major antileishmanial effector mechanism of neutrophils, this pathway may not be necessary for the killing of L. m j o r by murine macrophages (Assreuy et al., 1994). Leishmania apparently have developed mechanisms to evade killing by 02-,since parasite-infected human and murine macrophages have been found to have a markedly decreased capacity to produce oxigen radicals after stimulation with phorbol esthers (Buchmuller-Rouiller and Mauel, 1987; Passwell et al., 1994). The LPG of Leishmania has been shown to mediate this activity by inhibiting the activation of protein kinase C. Leishmania gp63 was also shown to be involved in the Leishmnia-induced suppression of oxidative burst in human monocytes and neutrophils in vitro (Sorensen et al., 1994). Recently, a novel pathway of protection against oxidative damage of L. m j o r has been described. This involves the L. m j o r tryparedoxin peroxidase (TryP), a 22-kDa protein with two conserved cystein-containing domains that defines it as a 2-Cys peroxidoxin. Recombinant TryP was shown to catabolize hydrogen-peroxide utilizing a three-protein peroxidase system (Levick et al., 1998). The most important pathway mediating parasite destruction in macrophages is their activation by T cell-derived cytokines to produce NO (reviewed in Bogdan, 1997). Among macrophage-activating cytokines, IFNy is the one most important. Otherwise resistant mice rendered genetically deficient for IFN-y-production (Wang et al., 1994a) or lacking IFN-yreceptor-expression (Swihart et al., 1995)succumbed to fatal infection of L. major. The importance of IFN-y in the defense against human Leishmnia infections is underlined by successful therapy trials with IFN-y applied in combination with or without pentavalent antimony (Murray, 1994, and references therein). The role of tumor necrosis factor (TNF) and its receptors for control of L. m j o r infection is less clear. Undoubtedly, TNF participates in the induction of NO production and macrophage activation leading to parasite elimination. Mice lacking the p55 and/or the p75 receptor for TNF (TNFRp55-’-, TNFRp75-’-, and TNFRp55p75-’-, respectively) developed much larger lesions than control animals and failed to resolve these lesions. Surprisingly, however, they were able to eliminate the parasites within

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the lesions concomitant with the development of a T h l response. IFNy activation of macrophages from TNFRp75-’- animals resulted in NO

production and parasite killing (Nashleanas et al., 1998;Vieira et al., 1996). These results show that the TNFRp75 plays no essential role in murine L. major infection and that a mechanism exists by which macrophages can be primed in vivo to produce NO and kill L. major in the absence of signaling through either of the TNF receptors. For activation of macrophages to mount an antiparasitic effector function, T cells were shown to contribute through CD40-CD40L interaction. CD40-deficiency in resistant mice led to susceptibility to L. major (Kamanaka et al., 1996). Similarly, CD40L deficiency in infected mice with a resistant genetic background resulted in ulcerating lesions and an impaired Thl-response (Campbell et al., 1996). The defect in Thl-development was the consequence of the inability of CD40L-deficient T cells to induce IL12 from macrophages. Exogenous IL-12 and administration of recombinant CD40L prevented at least in part the disease progression. CD40L deficiency led to the development of progressive ulcerative lesions also in mice infected with L. amazonensis (Soong et al., 1996). The susceptibility was associated with decreased levels of IFN-y and NO production. The killing of L. major by IFN-y-treated murine macrophages is attributable to NO and the enzyme producing it (Ding et al., 1988; Green et al., 1990). Although the molecular mechanism of the action of NO on Leishmania is unknown so far, early data suggested that NO is directly cytotoxic to L. major (Liew et al., 1990). In mice, resistance to Leishmania was clearly associated with the expression of iNOS and clearly required the continuous presence of NOS2 activity (Evans et al., 1993; Stenger et al., 1994). The NO-pathway appears to be a common mechanism of Leishmania killing since not only murine but also human monocytes/macrophages were able to control L. major in a NO-dependent manner (Vouldoukis et al., 1995). Leishmania utilizes several different ways to prevent T cell-mediated induction of NOS2 (see Section IV.F), which results in the decreased production of NO. Moreover, the parasites are able to directly interfere with the NO-production of their host cells. LPG and GIPLs (glycolipids related to LPG) from L. major as well as intact L. major promastigotes strongly suppressed NOS2-activity when the interaction between the macrophages and Leishmania preceeded the stimulation of macrophages by IFN-y (Proudfoot et al., 1996). When, however, Leishmania and IFN-y were added simultaneously to macrophages, GIPLs and LPG synergized with IFN-y resulting in an increased production of NO (Proudfoot et al., 1995). Therefore, Leishmania inhibit NOS2 activity in the early stages of infection (i.e.,when the parasites enter macrophages before the production

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of T cell-derived macrophage-activating cytokines, such as IFN-y). In later stages of infection, however, Leishmania results in increased NOS2 production. In accordance with this presumption, high NOS2 mRNA levels were found in chronic, nonhealing lesions of mice infected with L. major (Nabors et al., 1995). Similarly, progressive infection correlated with increasing systemic synthesis of NO (Evans et al., 1996) and high urinary levels of nitritehitrate during the late stages of progressive infection (Evans et al., 1993). Therefore, local production of NO is a crucial mechanism for the elimination and control of parasites, but only if it occurs before the parasite burden becomes too high. From then on, elevated production of NO aggravates the inflammatory process (Giorgio et al., 1998). Recently, two reports highlighted the Fas-FasL interaction as a pathway important for the development of Thl-mediated resistance to Leishmania infection (Conceicao-Silva et al., 1998; Huang et al., 199813). Activated Thl, but not Th2 cells were shown to induce apoptotic death in target cells expressing the Fas-protein ( J u et al., 1994). MRLllpr mice, which have a single gene mutation (lpr) of thefm apoptosis gene were highly susceptible to infection with L. major (Huang et al., 199813)despite elevated IL-12 production, strong T h l response and high-level NO production. Also, resistant C57BU6 mice deficient in either Fas (Zpr) or functional FasL (gld ) failed to heal L. m j o r lesions (Conceicao-Silvaet al., 1998). Reconstitution of the Fas-FasL cytotoxic pathway in gld mice allowed the resolution of the lesions. Macrophages infected with L. major in vitro upregulated Fas in response to IFN-.)I and became susceptible to CD4' T cell-induced apoptotic death. Thus, it can be speculated that Fas-induced apoptotic death of infected macrophages limits the number of host cells at the site of infection which are required for amastigote replication. Furthermore, release of amastigotes into the extracellular milieu could also contribute to their destruction. Possibly, the NO-mediated and Fasmediated pathways of resistance to L. major have a common regulation, since NO has been shown to induce macrophage apoptosis (Sarih et al., 1993). V. Persistence

Available data suggest that after infection with Leishmania, the parasites persist probably life-long in the mammalian host. After experimental subcutaneous infection with L. major, most inbred mouse strains are resistant, since the skin lesion at the inoculation site of the parasite heals spontaneously over a period of several weeks to months. After healing, the animals are clinically healthy showing no signs of the infection. Associated with the healing process, the cured mice show a strong, long-lasting cellular

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immunity to the parasites. In spite of the clinical healing, however, L. major persist in various organs, possibly life-long. Parasites could be detected by both PCR and culture in the draining LN, spleen, and bone marrow as long as 1year after healing (Aebischer et al., 1993). The persistence was not due to loss of virulence since the parasite recovered from the healed animals were fully virulent and induced fatal disease in BALB/c mice. Persistence is not restricted to the infection of C57BIJ6 mice with L. major, since L. aethiopica parasites could also be recovered from BALB/ c mice several months after infection. Notably, the animals developed only a small cutaneous lesion with spontaneous healing (Akuffo et al., 1990). Persistence of Leishmania was also found after clinical cure of human infections. By PCR technique and in vitro culture, parasites were detected in scars of 50%of patients with treated and healed L. (Viannia)braziliensis infection between 5 and 11 years after successful therapy (Schubach et al., 1998a,b). These data suggest that, once infected, the mammalian host probably will harbor the parasites life-long. Prevention of parasite replication above a certain threshold and thereby preventing exacerbation of the disease is an active process. CD4’T cell-mediated activation of NOS2 was shown to be the component resulting in the control of parasites. Life-long and continuous expression of NOS2 at the initial site of infection and in the draining LN was associated with the persistence of L. major in resistant mice. Pharmacological inhibition of NOS2 activity by feeding L - N ~ iminoethyl-lysine (L-NIL) in the drinking water caused clinical recrudescence of the disease (Stenger et al., 1996). Failure to maintain NOS2 activity is therefore the possible mechanism of reactivation of the disease. It may well be also the mechanism underlying endogenous reactivation of latent Leishmania infection in AIDS patients (Kubar et al., 1998). L. major was found to persist in both macrophages and dendritic cells but not in granulocytes or endothelial cells. Importantly, 60-70% of persisting parasites were found in cells without detectable expression of NOS2 (Stenger et al., 1996). L. major was suggested to evade being killed by hiding in “safe target” cells, which are unable to eliminate the intracellular parasites (Mirkovich et al., 1986). These “safe target” cells may be the cells incapable of expressing NOS2, as has been discussed for fibroblasts and more distant cells such as bone marrow macrophages (Bogdan and Rollinghoff, 1998; Leclercq et al., 1996). VI. Genetic Background of Resistance and Suceptibility

The study of genetic elements determining susceptibility or resistance to Leishmania is complicated by the finding that different host genes recognize and control infection with taxonomically distinct Leishmania

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species. The application of gene knockout technology demonstrated the role of the “natural-resistance-associated macrophage protein” (Nrampl) gene (formerly ZtylLshlBcg) to regulate resistance to L. donovani (Vidal et al., 1995).This gene confers resistance also to other intracellular pathogens such as s. typhimurium (Zty),and Mycobacterium bovis (Bcg) (Vidal et al., 1993). Nrampl has multiple pleiotropic effects on macrophage function (reviewed in Blackwell, 1996) including the production of TNF-a, IL-10, and MHC-I1 molecules, all of which affect macrophage activation, antigen processing, and presentation (Lang et al., 1997). Nrampl regulates, and is regulated by, cellular iron levels (Atkinson et al., 1997), and it was suggested that Nrampl regulates the intraphagosomal replication of intracellular pathogens by controlling divalent cation concentration at this site (Govoni and Gros, 1998). Although Nrampl appears to play only a minor role in the control of L. major, the gene product was shown to be localized in the membrane of the parasitophorous vacuole in macrophages infected with L. major (Searle et al., 1998). In contrast to its role in the defense to L. donovani in mice, no evidence for linkage between NRAMPl and susceptibility to human visceral leishmaniasis could be demonstrated (Blackwell et al., 1997). Available data clearly indicate that the resistance/susceptibility to L. major is controlled by several genes. As mentioned before (see Section IV.E), a locus on chromosome 11 has been indentified. It controls the maintenance of IL-12 responsiveness and, therefore, the subsequent Thl/ Th2 response (Gorham et al., 1996a). However, other genetic data argue against this simple model. Although the chromosome 11 seems to have some effect on L. major susceptibility, responsiveness to a variety of cytokines is also under the control of this gene region (Demant et al., 1996). Zn vivo studies using intercrosses between resistant C57BLJ6 and susceptible BALB/c mice revealed linkage to a chromosome 9 locus (D9Mit67D9Mit71) and to a region including the H2 locus of chromosome 17, named lmr2 and lmrl, respectively (Roberts et al., 1997). The resistant phenotype was analyzed in vivo in another study with backcrossing resistant B10.D2 mice onto susceptible BALB/c mice for five generations. Loci on chromosomes 6,7,10,11,15, and 16 were found to be associated with resistance demonstrating the multigenic nature of this phenotype. Not all of the six loci were necessary for controlling resistance, and no locus was required singularily. Several immunologically relevant candidate genes are present within the resistance loci. On chromosome 11, the genes for the leukemia-inhibiting factor, oncostatin M, the insulinlike growth factor binding proteins 1and 3, and the IL-12 p40 subunit are located. The transcription factor interferon regulatory factor-1 (IRF-1) gene is also located on chromosome 11. IRF-1 was shown to be a determining factor that con-

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tributes to a T h l response after L. major infection. Using knockout mice technology, protection to L. major was shown to depend on IRF-1 on a gene dose-dependent manner (Lohoff et al., 1997). This is in line with earlier observations that IRF-1 is required in NO-synthase induction in macrophages (Kamijo et al., 1994). On the loci of other chromosomes, genes for the natural killer cell-associated antigen 1, IFN-y, stat-6, Eae2, IL-7 receptor, leukemia inhibitory factor receptor, complement receptors 6 and 7, IFN-y receptor @chain, and the gene for IFN-a receptor are found (Beebe et al., 1997). Their single or combined contribution to the development of resistance or susceptibility to L. major infection remains to be determined. VII. Concluding Remarks

Since the description of the leishmaniases and their life cycle (Adler and Theodor, 1927; Carini, 1911; Cunningham, 1885; Donovan, 1903; Leishman, 1903; Vianna, 1912) and the establishment of an experimental mouse model (Howard, 1985, and references therein), parasitologists and immunologists have collected a wealth of information with regard to the development of the parasite, details of its transmission, and its epidemiology. Clinical and experimental immunologistshave contributed enormously to our current understanding of this infection by creating a detailed picture of the natural immunopathogenesis, including the description of numerous mechanisms by which the parasites can subvert the immune response (reviewed in Bogdan and Rollinghoff, 1998; Solbach and Laskay, 1996). The currently prevailing picture suggests that the cytokine milieu encountered by the parasite during the first 5-36 hours after infection of the mammalian host is the most important element decisive for one or the other form of the disease that develops during the next weeks to years. This implies that the area of the infected skin site and the lymph node draining the infectious site are the organs that serve as critical checkpoints. The early local cytokine milieu is composed by genetic and, thus, individually different default settings. Superimposed on this background component is the ability of the inoculating suspension of parasites to very quickly induce regulatory events that lead to substantial imbalances of the background homeostasis, the extent of which is largely influenced by the number of infecting parasites. Research has taught that keratinocytes, dendritic cells, natural killer cells, macrophages, CD4+ T cells, and granulocytes contribute initially to the reshaping of the tissue cytokine mixture by secreting chemokines (MIP-la, MCP-l), interleukins (IL-12, IL-4, IL-2, IL-1, IL-lo), or interferons (IFN-dfi, IFN-y), as well as cytokines like Transforming Growth Factor-/3 and molecules like nitric oxide. The production of these components, the cross-talk between them, and the cells

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responding to them are tightly interconnected. The result of production and responsiveness is a complex milieu of humoral factors which decides whether in the time initially after infection the parasites will be contained at the primary complex (i.e., the skin and the draining lymph node) or will disseminate into distant organs like the spleen and the liver. In addition, the cytokine milieu governs whether specifically activated naive CD4' T h o cells, which are either present in the lymph node or being recruited from the bloodstream, will develop into either protective T h l cells or diseasepromoting Th2 cells. The CD4+T cells will then, via their cytokine secretion potential or their apoptosis-inducing capacity, retalk to parasite-containing macrophages to induce, for example, NOS2 activation required for elimination of the parasites. Although this picture suggests coherency, researchers still have to fill a number of white spots. Many questions must be answered; for example, what are the genes from L. major that cause the typical disease in inbred mouse strains, given that a similar infection with L. donovani has a completely different outcome and L. aethiopica infection in mice leads hardly to any disease? What is the precise mechanism that allows L. major to disseminate rapidly in BALB/c mice but not in other mouse strains? How do chemokines contribute to the dissemination phenomenon as well as the recruitment of CD4' T cells and other cells to the primary complex? Why and how are most, but never all, parasites eliminated from the mammalian host? What is the molecular nature of the immunomodulating capacity of sand fly saliva? What are the intracellular signaling events that are switched on or off by which components of the parasite? How is the exact interplay between the mixture of cytokines and the cross-talk of the cytokines to their respective receptors regulated? How precisely is nitric oxide involved in early regulatory events and in the late antiparasitic effector scenario? Since most of the techniques to answer these questions are available, the contributions made by Leishmania researchers will be substantial for the understanding of the leishmaniases as well as for insights into crucial aspects of normal immune physiology in the future as they have been in the past.

ACKNOWLEDGMENTS The preparation of this review and the conduct of some of the studies cited were supported by the Deutsche Forschungsgemeinschaft (projects SFB 367 (B10) and So 220/5-1). The authors acknowledge thankfully helpful comments from members of the laboratories where the work has been done and from numerous colleagues in the field. We apologize to those researchers who believe that they have not been cited appropriately.

REFERENCES Adler, S., and Theodor, 0. (1927). The transmission of Leishmania tropica from artificially infected sandflies to man. Ann. Trop. Med. Parasitol. 21, 89-110.

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INDEX

A Adaptive immune response, leishmaniasis, 285-299 antigen presentation, 285-286 antileishmanial effector mechanisms, 297-299 CD4+ T helper lymphocyte induction, 288-290, 302 co-stimulatory molecules, 287-288 cytokine determinants, 290-291 interleukin-4 role, 291-297 interleukin-12 role, 291-297 T helper cell development, 290-291 Allelic exclusion, B cell receptor editing, 96-100 immunoglobulin-L chain, 99-100 inclusion problem, 113 mechanisms, 97-98 feedback suppression of recombination, 98 selective factors, 97-98 stochastic factors, 97 Antibodies, see .specijc types Antigen-presenting cells, tumors, T-cell recognition escape helper T cell interactions, 241 microenvironment association, 239-241 Antigens, suppressor T cell factor relationship, see Suppressor-T cells, antigen-specific factors Apoptosis, T cells, tumor recognition escape, microenvironmental inhibitory signals, 234-236

B B cell receptor B cell activation, 68, 73, 82 editing, see B cells, receptor editing 319

B cells activation and tolerance, complement role, 61-83 C3, 61-65 activation mechanisms, 63-64 C3d, 61-63 humoral immunity, 64-65 innate recognition, 63-64 source, 64-65 complement deficiency, 78-82 humoral immunity enhancement, 65-72 antigen retention on follicular dendritic cells, 69-72 B cell co-receptor signaling, 69-72 CD21/CD35 role, 64, 67-69, 82 classical pathway, 65-67 overview, 61, 82-83 self-reactive B cells, negative selection B cell anergy alterations, 74-78 C4 deficiency effects, 74-78 Cr2-/- mouse impairment, 73-74 natural antibody role, 82 systemic lupus erythematosus induction, 78-82 chemokine relationship, 139-144 glycosylation inhibiting factor effects, 50-51 receptor editing, 89-114 allelic exclusions, 96-100 feedback suppression of recombination, 98 immunoglobulin-L chain, 99-100 mechanisms, 97-98 selective factors, 97-98 stochastic factors, 97 antigen-reactive B cells, 108-110 antigen receptor gene assembly, 89-90 clond versus receptor selection, 89

320

INDEX

developmental stage specificity, 107-108 editing analysis, 102-104 gene-targeted antibody gene mouse, 102-104 immunoglobulin genes, 91-92 in uiuo monitoring, 100-102 locus specificity, 106-107, 110-111 mature B cells, 108-110 overview, 89, 114 rearrangement possibilities, 112 quasi-ordered rearrangements, 95-96 secondary rearrangements, 92-94 receptor diversification, 108 receptor revision, 108-111 receptor selection concept problems, 112-1 14 allelic inclusion, 113 disease applications, 113-114 need, 113 rearrangement possibilities, 112 receptor versus clonal selection, 89 recombinational accessibility, 94-95 recombination signals, 90-91 RSkde element role, 93-94 12/23 rule, 90-91 tolerance developmental block association, 104-106 transgenic models, 100-102 V(D)J recombination role, 89-90

C Cancer, see Tumors Carbohydrate recognition domains, innate immune system role, 63-64 CD4' chemokine relationship, 137-139 leishmaniasis adaptive immune response antigen presentation, 285-286 elimination, 300 induction, 288-290,302 development cytokine role, 290-291 interleukin-4 role, 291-294 tumors, T-cell recognition escape, antigenpresenting cell interactions, 241

CD8+,see also Cytotoxic T lymphocytes HIV suppression, 146-147, 151 tumor-associated antigen identification, 181, 186 CD19, complement enhancement, 67-68, 70, 82 CD21 B cell anergy alterations, 74-78 complement enhancement, 64, 67-69, 82 self-reactive B cells, negative selection, 73-74 systemic lupus erythematosus induction role, 78-82 CD28, leishmaniasis, adaptive immune response role, 287-288 CD35 B cell anergy alterations, 74-78 complement enhancement, 64, 67-69, 82 self-reactive B cells, negative selection, 73-74 systemic lupus erythematosus induction role, 78-82 CD80, leishmaniasis, adaptive immune response role, 287-288 CD86, leishmaniasis, adaptive immune response role, 287-288 Cell-mediated immunity, see also Humoral immunity; Innate immune system leishmaniasis, 285-299 antigen presentation, 285-286 antileishmanial effector mechanisms, 297-299 CD4+ T helper lymphocyte induction, 288-290,302 co-stimulatory molecules, 287-288 cytokine determinants, 290-291 interleukin-4 role, 291-297 interleukin-12 role, 291-297 T helper cell development, 290-291 Chemokines, 127-158 functional implications, 127-129 HIV infection, 146-158 coreceptors description, 146-149 signaling mechanisms, 150-151 therapy, 155-158 variation, 149-150 pathogenesis, 151-155 HIV suppression, 151-154

321

INDEX

infection enhancement, 155 viral entry mechanisms, 146-149 overview, 127 receptors in lymphocytes, 129-144 B lymphocytes, 139-141 lymphocyte progenitors, 142-144 memory T lymphocytes, 132-137 naive T lymphocytes, 132-137 natural killer cells, 141-142 T helper cells, 137-139 signal transduction mediation, 144-146 Complement, B cell activation and tolerance, 61-83 C3, 61-65 activation mechanisms, 63-64 C3d, 61-63 humoral immunity, 64-65 innate recognition, 63-64 source, 64-65 complement deficiency, 78-82 humoral immunity enhancement, 65-72 antigen retention on follicular dendritic cells, 69-72 B cell co-receptor signaling, 69-72 CD21ICD35 role, 64,67-69, 82 classical pathway, 65-67 overview, 61, 82-83 self-reactive B cells, negative selection B cell anergy alterations, 74-78 C4 deficiency effects, 74-78 Cr2-/- mouse impairment, 73-74 natural antibody role, 82 systemic lupus erythematosus induction, 78-82 Cytokines leishmaniasis, adaptive immune response, T helper cell development role, 290-291 tumors, T-cell recognition escape immunoregulation, 229-234 microenvironmental inhibitory signals, 229-236 Cytotoxic T lymphocytes HIV suppression, 146-147, 151 human leukocyte antigen class I polymorphism, 226-228 immunoregulatory cytokines, 229-234 target cell recognition studies, 181, 186-190 tumor-associated antigen identification, 181, 186

tumor cells as targets, 195-226 activity reduction, tumor-associated antigen epitope mimics, 202-203 down regulation, 195-204 masking, 203-204 processing machinery abnormalities, 217-221 tumor-escape mechanisms, 190-195 tumor microenvironment, immunogenicity inadequacy, 236-241

G L-Glutamic acid60-L-alanine30-L-tyrosine10, suppressor-T cell interactions, 2-3, 6, 8, 11 Clycosylation inhibiting factor, suppressor-T cell subunit, 33-54 antigen-primed B cells, 50-51 antigen-primed T cells, 48-50 cellular mechanisms, 5, 40-45 I-J determinant association, 39-40 immunoregulation mechanisms, 48-5 1 overview, 51-54 structural basis of bioactivity, 34-39 target cells, 45-48 T cell receptor derivative formation, 40-45

H Helper cells, see T helper cells HIV infection, chemoldne role, 146-158 coreceptors description, 146-149 signaling mechanisms, 150-151 therapy, 155-158 variation, 149-150 pathogenesis, 151-155 HIV suppression, 151-154 infection enhancement, 155 viral entry mechanisms, 146-149 Human leukocyte antigen class I, tumors, T-cell recognition escape polymorphism role, 226-228 tumor cells as targets loss or down regulation, 204-214 processing defects, 214-226

322

INDEX

Human leukocyte antigen class 11, tumors, Tcell recognition escape, immunogenicity inadequacy, 238-239 Humoral immunity, see also Cell-mediated immunity; Innate immune system complement source, 64-65 enhancement, 65-72 antigen retention on follicular dendritic cells, 69-72 B cell co-receptor signaling, 69-72 C D 2 K D 3 5 role, 64, 67-69, 82 classical pathway, 65-6i

I I-J determinants, antigen-specific suppressorT cells glycosylation inhibiting factor bioactivity association, 39-40 historical perspectives biochemical characteristics, 11-12 cell properties, 4-7 factor properties, 7-8 I-J paradox, 14-17 suppressor T cell cascade, 8-11, 54 Immune system, see Adaptive immune response; Humoral immunity; Innate immune system Immune tolerance, see Tolerance Immunoglobulin genes, B cell receptor editing L-chain allelic exclusion, 99-100 locus specificity, 106-107, 110-111 organization, 91-92.95 quasi-ordered rearrangement, 95-96 rearrangement possibilities, 112 RSkde element, 93-94 secondary rearrangement, 92-94 Immunoglobulin-L chain, B cell receptor editing, allelic exclusion, 99-100 Immunoglobulin M monoclonal anti-suppressor T cell factor antibodies, 12-14 self-antigen identification, 64, 83 Inflammation, leishmaniasis, host innate response, 281-282

Innate immune system, see also Cellmediated immunity; Humoral immunity B cell activation and tolerance, complement role, 61-83 C3, 61-65 activation mechanisms, 63-64 C3d, 61-63 humoral immunity, 64-65 innate recognition, 63-64 source, 64-65 complement deficiency, 78-82 humoral immunity enhancement, 65-72 antigen retention on follicular dendritic cells, 69-72 B cell co-receptor signaling, 69-72 CD21/CD35 role, 64,67-69, 82 classical pathway, 65-67 overview, 61, 82-83 self-reactive B cells, negative selection B cell anergy alterations, 74-78 C4 deficiency effects, 74-78 Cr2-/- mouse impairment, 73-74 natural antibody role, 82 systemic lupus erythematosus induction, 78-82 leishmaniasis inflammation, 281-282 lymph node invasion, 284-285 natural killer cell role, 282-284 parasite dissemination versus containment, 282-284 Interleukin-4, leishmaniasis, adaptive immune response role, 291-297 Interleukin-7, B cell receptor specificity role, 108 Interleukin-12, leishmaniasis, adaptive immune response role, 291-297

Keyhole limpet hemocyanin antigen-specific suppressor-T cell generation, 1-5, 7 B cell immunity enhancement, 64 Killer cells, see Cytotoxic T lymphocytes; Natural killer cells

323

INDEX

L Leishmaniasis, 275-303 host response, 281-285 adaptive immune response, 285-299 antigen presentation, 285-286 antileishmanial effector mechanisms, 297-299 CD4+ T helper lymphocyte induction, 288-290, 302 co-stimulatory molecules, 287-288 cytokine deterniinants, 290-291 interleukin-4 role, 291-297 interleukin-12 role, 291-297 T helper cell development, 290-291 innate response inflammation, 281-282 lymph node invasion, 284-285 natural killer cell role, 282-284 parasite dissemination versus containment, 282-284 Leishmania life cycle, 277-281 mammalian host, 278-281 sand fly host, 277-278 overview, 275-276, 302-303 persistence, 299-300 resistance genetics, 300-302 susceptibility, 300-302 Lutsomyia spp., Leishmania host role, 277-281 Lymph nodes, leishmaniasis, host response, 284-285

M Monoclonal antibodies, anti-suppressor T cell factor antibodies, 12-14

N Natural killer cells chemokine relationship, 141-144 leishmaniasis, 282-284 Negative selection, self-reactive B cells B cell anergy alterations, 74-78 C4 deficiency effects, 74-78 Cr2-/- inouse impairment, 73-74 natural antibody role, 82

Nitric oxide, Leishmania elimination mechanisms. 297-300

P Phlebotomus spp., Leishmania host role, 277-281 Psychodopygus spp., Leishmania host role, 277-281

Recombination, see V(D)J recombination RSkde element, B cell receptor edlting, 93-94 12/23 rule, B cell receptor editing, recombination signals, 90-91

Signal transduction, chemokine role, 144-146 Superoxide, Leishmania elimination mechanisms, 297-299 Suppressor-T cells, antigen-specific factors, 1-54 controversial issues, 17-33 biochemical identification, 26-33 cell identity, 17-18 nominal antigen-binding, 18-22 T cell receptor gene requirement, 23-26 T cell receptor relationship, 23-33 glycosylation inhibiting factor role, 33-51 antigen-primed B cells, 50-51 antigen-primed T cells, 48-50 cellular mechanisms, 5, 40-45 I-J determinant association, 39-40 immunoregulation mechanisms, 48-51 structural basis of bioactivity, 34-39 target cells, 45-48 T cell receptor derivative formation, 40-45 historical perspectives, 1-17 activity, 4-7 biochemical characteristics, 11-12 cell properties, 3-4 factor properties, 7-8 generation conditions, 1-3

324

INDEX

I-J paradox, 14-17 monoclond anti-suppressor T cell factors, 12-14 suppressor T cell cascade, 8-11, 54 overview, 1, 51-54 Systemic lupus erythematosus induction, complement deficiency role, 78-82 receptor editing role, 113-114

T T cell receptor, antigen-specific suppressor-T cell factor relationships controversial issues biochemical identification, 26-33 cell identity, 17-18 factor relationship, 23-33 gene requirement, 23-26 nominal antigen-binding, 18-22 glycosylation inhibiting factor role, 40-45 overview, 1, 51-54 T cells, see also Cytotoxic T lymphocytes; T helper cells chemokine relationship memory T lymphocytes, 132-137 naive T lymphocytes, 132-137 progenitors, 142-144 receptors, 129-132 T helper cells, 137-139 suppressor T cells, see Suppressor-T cells tumor recognition escape, 181-245 human leukocyte antigen I, polymorphism, 226-228 immunogenicity inadequacy, 236-241 antigen-presenting cell-helper T cell interactions, 241 antigen-presenting cell role, 239-241 human leukocyte antigen 11 expression, 238-239 immunological alternatives, 241-245 mechanisms, 190-195 microenvironmental inhibitory signals, 229-236 apoptitic signal surface expression, 234-236 cytokines, 229-236

overview, 181-190 tumor cells as targets, 195-226 cytotoxic T lymphocyte activity reduction, 202-203 down regulation, 195-204,204-214 human leukocyte antigen I role, 204-214, 214-226 processing defects, 214-226 tumor-associated antigen loss, 195-204 T helper cells chemokine relationship, 137-139 leishmaniasis adaptive immune response antigen presentation, 285-286 elimination, 300 induction, 288-290,302 development cytokine role, 290-291 interleukin-4 role, 291-294 tumors, T-cell recognition escape, antigenpresenting cell interactions, 241 Thymus-dependent antigen, humoral immunity enhancement, 65-67 Tolerance B cell receptor editing developmental block association, 104-106 disease association, 113-114 transgenic models, 100-102 complement role, see Complement Transgenics, immune tolerance models, in oiuo, B cell receptor editing, 100-102 Tumor-associated antigen, tumors escape from T-cell recognition down regulation, 195-204 loss, 195-204 masking, 203-204 mechanisms, 190-195 mimics, 202-203 overview, 181, 186 Tumor necrosis factor, Ldshmunia elimination mechanisms, 297-299 Tumors, T-cell recognition escape, 181-245 human leukocyte antigen I, polymorphism, 226-228 immunogenicity inadequacy, 236-241 antigen-presenting cell-helper T cell interactions, 241 antigen-presenting cell role, 239-241

INDEX

human leukocyte antigen 11 expression, 238-239 immunological alternatives, 241-245 mechanisms, 190-195 microenvironmental inhibitory signals, 229-236 apoptitic signal surface expression, 234-236 cytokines, 229-236 overview, 181-190 tumor cells as targets, 195-226 cytotoxic T lymphocyte activity reduction, 202-203 down-regulation. 195-204 human leukocyte antigen I loss or down regulation, 204-214 processing defects, 214-226 tumor-associated antigen loss, 195-204 masking, 203-204 mimics, 202-203

v V(D)] recombination, B cell receptor editing, 89-114 allelic exclusions, 96-100 feedback suppression of recombination, 98 immunoglobulin-L chain, 99-100 mechanisms, 97-98

325 selective factors, 97-98 stochastic factors, 97 antigen-reactive B cells, 108-110 antigen receptor gene assembly, 89-90 clonal versus receptor selection, 89 developmental stage specificity, 107-108 editing analysis, 102-104 gene-targeted antibody gene mouse, 102-104 immunoglobulin genes, 91-92 in oioo monitoring, 100-102 locus specificity, 106-107, 110-111 mature B cells, 108-110 overview, 89, 114 quasi-ordered rearrangements, 95-96 receptor diversification, 108 receptor revision, 108-111 receptor selection concept problems, 112-114 dlelic inclusion, 113 disease applications, 113-114 need, 113 rearrangement possibilities, 112 receptor versus clonal selection, 89 recombinational accessibility, 94-95 recombination signals, 90-91 12/23 rule, 90-91 secondary rearrangements, 92-94 RS/kde element, 93-94 tolerance developmental block association, 104-106 transgenic models, 100-102

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CONTENTS OF RECENT VOLUMES

Volume 70

Current Insights into the “Antiphospholipid Syndrome: Clinical, Immunolo&cal, and Molecular Aspects DAVIDA. GNDIAH, ANDREJ SALI, YONGHUASHENG, EDWARD 1. VICTORIA, DAVIDM. MARQUIS, STEPHEN M. COUTTS, AND STEVEN A. KRILIS

Biology of the Interleukin-2 Receptor BRADH. NELSON A N D DENNIS M. WILLEHFORD Interleukin-12: A Cytokine at the Interface of Inflammation and Immunity GIORCIO THINCHIERI

INDEX

Recent Progress on the Regulation of Apoptosis by Bcl-2 Family Members ANDYJ. MI”, RACHEL E. SWAIN, AVERIL MA, A N D CRAIGB. THOMPSON Interleukin-18: A Novel Cytoldne That Augments Both Innate and Acquired Immunity HARUKI OKAMURA, HIROKO TSUTSUI, SHIN-ICHIROKASHWAMURA, TOMOHIRO YOSHIMOTO,AND KEN11 NAKANISHI

Volume 71 crply6 Lineage Commitment in the Thymus

of Normal and Genetically Manipulated Mice HANSJBRGFEHLING, SUSAN GILFILLAN, A N D RHODRICEREDIG Immunoregulatory Functions of y6 T Cells WILL1 BORN,CAROL CADY, JESSICA JONESCARSON, AKIKOMUKASA, MICHAELLAHN, AND REBECCAO’BRIEN

CD4+ T-cell Induction and Effector STATs as Mediators of Cytokine-Induced Functions: A Comparison of Immunity Responses against Soluble Antigens and Viral Infections TIMOTHY HOEYAND MICHAEL J. GRUSBY ANNETTEOXENIUS, ROLF M. ZINKERNAGEL. AND HANS HENCARTNER CD95(APO-l/Fas)-Mediated Apoptosis: Live and Let Die Current Views in Intracellular Transport: PETERH. KRAMMER Insights from Studies in Immunology VICTORW. Hsu A N D PETERJ. PETERS A CXC Chemokine SDF-1RBSF: A Ligand for a HIV Coreceptor, CXCR4 TAKASHI NACASAWA, KAZLINOBU Phylogenetic Emergence and Molecular TACHIBANA, AND KENJIKAWABATA Evolution of the Immunoglobulin Family SAMUEL F. JOHNJ. MARCHALONIS, T Lymphocyte Tolerance: From Thymic SCHLUTER, RALPHM. BERNSTEIN, Deletion to Peripheral Control Mechanisms SHANXIANG SHEN,AND ALLENB. BRIGI-ITA STOCKINCER EDMUNDSON 327

328

CONTENTS OF RECENT VOLUMES

INDEX Confrontation between Intracellular Bacteria and the Immune System ULRICHE. SCHAIBLE, HELENL. COLLINS, AND STEFAN H. E. KAUFMANN

Volume 73

INDEX

Volume 72 The Function of Small GTPases in Signaling by Immune Recognition and Other Leukocyte Receptors AMNONALTMANAND MARCEL DECKERT

Mechanisms of Exogenous Antigen Presentation by MHC Class I Molecules in Vitro and in Viuo: Implications for Generating CD8+ T Cell Responses to Infectious Agents, Tumors, Transplants, and Vaccines JONATHAN W. YEWDELL, CHRISTOPHER C. NORBURY, AND JACK R. BENNINK

Signal Transduction Pathways That Regulate Function of the CD3 Subunits of the the Fate of B Lymphocytes Pre-TCR and TCR Complexes during ANDREWCRAXTON, KEVINOTIPOBY, AIMIN T Cell Development JIANG, AND EDWARD A. CLARK BERNARDMALISSEN,LAURENCE ARDOUIN, SHIH-YAO LIN,ANNEGILLET, AND Oral Tolerance: Mechanisms and Therapeutic MARIEMALISSEN Applications ANAFARM AND HOWARD L.WEINER Inhibitory Pathways Triggered by ITIMContaining Receptors SILVIABOLLANDAND JEFFREY V. RAVETCH Caspases and Cytokines: Roles in Inflammation and Autoimmunity JOHNC. REED ATM in Lymphoid Development and Tumorigenesis YANG Xu Comparison of Intact Antibody Structures and the Implications for Effector Function LISAJ. HARRIS, STEVEN B. LARSON, AND ALEXANDERMCPHERSON

T Cell Dynamnics in HIV-1 Infection DAWN R. CLARK, ROBJ. DE BOER, &TJA c.WOLTHERS, AND FRANK MIEDEMA Bacterial CpG DNA Activates Immune Cells to Signal Infectious Danger HERMANN WAGNER

Lymphocyte Trafficking and Regional Immunity EUGENE C. BUTCHER, MARNAWILLIAMS, Neutrophil-Derived Proteins: Selling KENNETHYOUNGMAN, LUSIJAHROTT, AND Cytobnes by the Pound MICHAEL BRISKIN MARCOANTONIOCASSATELLA Dendritic Cells DIANA BELL,JAMES W. YOUNG, AND JACQUES BANCHEREAU Integrins in the Immune System YOJI SHIMIZU, DAVIDM. ROSE,AND MARKH. GINSBERG

Murine Models of Thymic Lymphomas: Premalignant Scenarios Amenable to Prophylactic Therapy EITANYEFENOF INDEX

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ISBN 0-12-022474-7