Advances in Immunology Volume 49

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ADVANCES IN

Immunology

VOLUME 49

This Page Intentionally Left Blank

ADVANCES IN

Immunology EDITED BY

FRANK J. DIXON Scripps Clinic and Research Foundotion Lo jollo, California

ASSOCIATE EDITORS

K. FRANK AUSTEN LEROYE. HOOD JONATHAN W. UHR TADAMITSU KISHIMOTO FRITZMELCHERS

VOLUME 49

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

Sun Diego N e w York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1991 BYACADEMIC PRESS, INC. All Rights Reserved. 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.

Academic Press, Inc. San Diego, California 92101 United kingdom Edition plbltshed by ACADEMIC PRESS LIMITED 2428 Oval Road, London NWl 7DX

Library of Congress Catalog Card Number: 61-17057

ISBN 0-12-022449-6 (alk. paper)

PRINTED IN THE UNITED STATES OFAMERICA 9 1 9 2 9 9 9 4

9 8 7 6 5 4 3 2 1

CONTENTS

Human Immunoglobulin Heavy-Chain Variable RegionGenes: Organization, Polymorphism, and Expression

VIRGINIA PASCUAL A N D J. DONALD CAPRA I. Introduction 11. Gross Organization of the Human Ig Complex

111. V, Families N.Polymorphism of V, Gene Segments V. DSegments VI. Human JkI Segments VII. Expression of Human V, Gene Segments VIII. Regulation 1X. Conclusions References

1 3 6 21 28 33 35 52 61 64

Surface Antigens of Human Leucocytes

V. HoREJS~ I. Introduction 11. Antigen-Specific Receptors

111. MHC Glycoproteins N.Adhesion Molecules V. Receptors for Immunoglobulins (Fc-Receptors) VI. Receptors for Complement Components VII. Receptors for Lymphokines and Other Growth and Differentiation Factors VIII. Membrane Enzymes IX. Transport Proteins X. Other Interesting Molecules XI. Concluding Remarks References Note Added in Proof

75 76 80 82 89 91 94 111 114 116 118 126 147

Expression, Structure, and Function of the CD23 Antigen

G. DELESPESSE, U. SUTER,D. MOSSALAW, B. BETTLER, M. SARFATI, H. HOFSTETTER, E. KILCHERR, P. DEBRE, A N D A. DALLOUL

149 150

I. Introduction 11. Cellular Expression V

vi

CONTENTS

111. Biochemical Structure

155

Iv. Regulation of CD23 Cleavage

158 158 16'2 167 174 176 177

V. Regulation of CD23 Expression VI. Molecular Biology of FcrRII VII. Biological Activity of CD23 VIII. CD23 Expression in Various Clinical Conditions 1X. Conclusions References

Immunology and Clinical Importance of Antiphospholipid Antibodies H. PATKICK MCNEIL, COI.IN N . C I I E S TER MAN, A N D S T E V E N A. K R I L I S

I. Introduction 11. Phospholipid Biochemistry 111. Historical Background

IV. Clinical Aspects of Antiphospholipid Antibodies V. The Immunology of Antiphospholipid Antibodies VI. The Pathogenic Potential of aPL Antibodies VII. Summary and Conclusions References

193 194 198 200 224 252 257 259

AdoptiveT Cell Therapy of Tumors: Mechanisms Operative in the Recognition and Eliminationof Tumor Cells Ptiii.iP D. GREENBERG 1. Introduction 11. Principles of Adoptive Therapy with Specifically Immune T Cells 111. Mechanisms by Which T Cells Mediate Tumor Rejection IV. Recognition of Distinct Tumor Antigens by CD4+and CD8+T Cells as a Potential Basis for Selective Efficacy of a T Cell Subset V. Accessory Cell, Antigen-Presenting Cell, and Cytokine Requirements for Effective Expression of Antitumor Responses by CD4+and

28 1 286 299

CD8+T Cell Subsets VI. Concluding Statements References

324 332 335

318

The Developmentof Rational Strategies for Selectivelmmunotherapy against Autoimmune DemyelinatingDisease LAw REN CE

S'I'E IN M A N

1. Introduction 11. Multiple Epitopes of Myelin Basic Protein in Mice and Humans 111. Human and Rodent TCR Usage Restriction in T Cells Responding to Specific Epitopes of Myelin Basic Protein

rV. Possibilities for Future Immune Intervention in Multiple Sclerosis References

357 357 363 368 375

CONTENTS

vi i

The Biology of Bone Marrow Transplantation for Severe Combined Immune Deficiency

ROBERTSON PARKMAN 1. Introduction 11. Severe Combined Immune Deficiency 111. Characteristics of Stem Cell Engraftment for the Correction of SCID IV. Histocompatible Bone Marrow Transplantation V. Transplantation with Fetal Tissues VI. Bone Marrow Transplantation with T Cell-Depleted Haploidentical Bone Marrow VII. Lack of Stem Cell Engraftment VIII. Graft-versus-Host Disease I>

-2 3

+ 2 X 107CD8+~L-2/Tc+

CY

-

No 2 0 - Therapy

CY + 5 X 106CD8+1~-2/~c+ IL-2 IL-2 CY

0 , 0

+ 2 x 107CD8+1~-2/~c

20

40

+ 5 X 106CD8+1~-2/~c I

1

60

80

Days FIG. 4. Therapy of disseminated FBL-3 leukemia with an IL-2-producing, FBLspecific CD8+ cytolytic T cell clone (CD8+1L-z,Tc).On day 0, BWThy-1.1 hosts were inoculated intraperitoneally with 5 x 10" viable FBL-3 leukemia cells and were left untreated, were treated on day 5 with 180 mg/kg CY, or were treated on day 5 with CY plus 5 x lo6 or 2 x lo7 FBL-specific, IL-2-producing, cytolytic CD8+ cloned T cells either alone or followed by 5000 U IL-2 given daily intraperitoneally for 6 days.

ADOPTIVE T CELL THERAPY OF TUMORS

305

which the clonal progeny of a single tumor-specific Class I-restricted Tc can completely eliminate a disseminated tumor. The antitumor activity of Tc derived from the CD8' subset has been demonstrated in many other tumor models against a very wide range of tumor histologies, including leukemia, lymphoma, hepatoma, plasmacytoma, sarcoma, glioma, mastocytoma, melanoma, and carcinoma (Greenberg, 1986; Mills and North, 1983,1985; Rosenstein et al., 1984; Dailey et al., 1982; Evans, 1984; Yamasaki et al., 1984; Matis et al., 1986; North, 1984; Forman et al., 1985; Awwad and North, 1988; Ward et al., 1988; Barker and Mokyr, 1988; Yoshioka et al., 1988; De Graaf et al., 1988; Wortzel et al., 1984; Schild et al., 1987; Kast et al., 1989; Fan and Edgington, 1989). In many of these models, the therapeutic activity of CD8' T cells is dependent on the presence of CD4' TH during the generation and/or expression of the antitumor response, consistent with our finding that the availability of endogenous IL-2 limits the efficacy of purified or cloned CD8+ Tc. The extent to which IL-2 or CD4+ TH are necessary for the therapeutic efficacy of CD8+ T cells likely reflects the magnitude and duration of the antitumor response necessary to treat the host, with settings that require prolonged immune responses such as rejection of disseminated leukemias or of locally advanced and metastatic tumors requiring that helper function be provided (Greenberg, 1986; Mills and North, 1985). By contrast, rejection of pulmonary micrometastases might be achievable by delivering only CD8+ T cells directly to the lung by intravenous injection (Ward et al., 1988), although even in this setting providing the CD8+ population with IL-2 may significantly augment the antitumor effect (Shu and Rosenberg, 1985). The value of providing IL-2 even in this setting of limited disease is supported by recent studies in our laboratory examining the treatment of pulmonary micrometastases of melanoma origin with tumor-specific CD8+ Tc clones that do not make IL-2-complete tumor elimination was achieved with a lower cell dose and in a higher percentage of mice if IL-2 was administered after transfer of the CD8+ Tc clones. The importance of providing adequate amounts of IL-2 has also been demonstrated in studies examining rejection of allografts disparate from the host only in Class I antigenshelper T cell function determined if a graft could be rejected by cytolytic CD8' T cells and regulated the rate of tissue rejection (A. S. Rosenberg et al., 1986, 1989). In conclusion, these studies have demonstrated that, under appropriate settings, effector T cells derived from either the CD4+ or CD8' subset can mediate tumor rejection in the absence of a contribution by effector T cells derived from the other subset. However, in addition to

306

PHILIP D. GREENBERG

T cell subpopulations, other cells of the immune system can interact with T effector cells and contribute to the elimination of tumor cells. Therefore, in order to understand the mechanisms by which T cells mediate tumor eradication, it is necessary to determine the roles of host macrophages, B cells, and NK cells in the elimination of tumor cells in settings in which adoptively transferred T cells make an essential contribution to tumor elimination. The importance of such non-T effector cells will be discussed in the sections that follow. IN TUMOR ELIMINATION B. ROLEOF MACROPHAGES Studies in many tumor models have suggested that tumoricidal macrophages make a critical contribution to the outcome of tumor therapy (Alexander et al., 1972; Evans, 1986; Evans and Alexander, 1972a,b; Evans et al., 1972; Hibbs et al., 1972; Herberman et al., 1980; Russell et al., 1980; Mantovani et al., 1986).This antitumor activity is not mediated by normal resting macrophages, but rather requires macrophage activation that induces at least two new functions: (1) the capacity to selectively bind to transformed cells; and (2) the capacity to deliver a cytolytic signal. These functions of activated macrophages have been the subject of numerous reviews (Adams and Hamilton, 1984; Johnson et al., 1984; Drysdale et ul., 1988), and will be only briefly discussed here. Selective binding of transformed rather than normal cells by activated macrophages is an energy-dependent phenomenon accomplished by a trypsin-sensitive binding structure on the macrophage (Fidler and Schroit, 1988; Hibbs, 1974; Marino and Adams, 1982), but neither the nature of this receptor nor the recognition structure expressed on the tumor cell have been identified. Activated macrophages can mediate target lysis by several distinct but not necessarily mutually exclusive mechanisms, including: (1)the release of TNF (although soluble T N F released at a distance from the tumor can directly mediate lysis following binding to specific receptors on tumor cells, delivery of macrophage membrane-bound T N F and/or secretion of T N F directly into a macrophage-tumor microenvironment results in more efficient target destruction and the lysis of a broader range of tumor targets) (Reed and Lucas, 1975; Haranaka et al., 1986; Klostergaard, 1987; Decker et al., 1987; Feinman et al., 1987; Tsujimoto et al., 1985; Urban et al., 1986);(2) the release of IL-1, which has cytocidal activity for a range of tumor targets (Onozaki et al., 1985; Lachman et al., 1986; Ruggiero and Baglioni, 1987; Lovett et al., 1986); ( 3 )the generation of reactive oxygen intermediates, particularly hydrogen peroxide (Nathan et al., 1979a,b; Nathan and Cohn, 1981); (4) secretion of neutral proteases that have high lytic potency selec-


307

tively for neoplastic cells (Adams, 1980; Adams et al., 1980; Piessens and Sharma, 1980);and (5)a biochemical pathway leading to target cell death involving the formation of nitrites by deimination of L-arginine and resulting in inhibition of tumor cell mitochondria1 respiration and DNA synthesis (Hibbs et al., 1987a,b). Although activation of macrophages to a tumoricidal state can be induced by a variety of agents, including phorbol esters, LPS, muramyl dipeptide (MDP), and calcium ionophores (Drysdale et al., 1988),the most important agents in the context of T cell-mediated tumor elimination are lymphokines. Following antigen-specific triggering, T cells secrete a number of lymphokines that have complex effects on macrophages, including the induction of tumoricidal activity by two distinct lymphokine-dependent pathways. One of these pathways requires only a single signal, and can be induced by IL-4, GM-CSF, TNF, and possibly IL-2 (Philip, 1988; Grabstein et al., 1986; Crawford et al., 1987; Philip and Epstein, 1986). In general, macrophage killing induced by this one signal pathway proceeds via a mechanism requiring macrophage production of TNF (Decker et al., 1987; Philip, 1988; Philip and Epstein, 1986; Feinman et al., 1987). This effector mechanism will result only in the lysis of TNF-sensitive targets and, in the absence of other cooperative tumoricidal activities, can lead to selection in uiuo and in uitro of TNF-resistant tumor variants that can grow unimpeded (Feinman et al., 1987; Urban and Schreiber, 1983; Lattime and Stutman, 1989). The second pathway of macrophage activation requires two signals for induction of cytotoxicity, but such activated macrophages lyse a broader range of targets than TNF-secreting macrophages and rarely permit the outgrowth of tumor cells resistant to lysis (Fogler and Fidler, 1985). The requisite signals in this pathway are defined operationally as a first priming signal which is necessary to render the macrophage responsive to a second trigger signal, which results in target killing (Pace and Russell, 1981; Meltzer, 1981; Adams and Hamilton, 1984). The best defined priming signal, and the lymphokine most commonly discussed in the literature as macrophageactivating factor (MAF), is .)I-IFN,which at physiologic concentrations renders macrophages capable of selectively binding to transformed cells and responsive to a trigger signal for cytolysis (Pace et al., 1983; Schultz and Kleinschmidt, 1983; Svedersky et al., 1984a; Spitalny and Havell, 1984; Schreiber et al., 1985; R. D. Schreiber, 1984; Marino and Adams, 1982). Our laboratory has recently characterized a unique T cell-derived lymphokine that similarly primes macrophages for tumor cytotoxicity (Kern et al., 1989), but the biological importance of this lymphokine in T cell responses to tumors remains to be elucidated.

308

PHILIP D.GREENBERG

Following macrophage priming, a small amount of LPS endotoxin is commonly used as the second signal to trigger lytic activity in vitro, but it is assumed that this highly sensitive trigger step is more commonly mediated in vivo by cytokines released at the site of the immune response. Factors such as T N F and lymphotoxin are among the cytokines shown to have such triggering activity, and will only work as triggers if the macrophage has been previously primed (Meltzer, 1981; Krammer et al., 1985; Esparza et al., 1987; Drapier et al., 1988). These studies demonstrating that macrophages can be activated to a tumoricidal state by lymphokines expected to be produced by T cells responding to a tumor, and that macrophages can lyse tumor cells by multiple mechanisms, strongly suggest that macrophages are important effector cells during T cell-mediated tumor elimination. This conclusion is further supported by the demonstration that macrophages isolated from tumors undergoing immune-mediated regression exhibit greater cytotoxicity than macrophages isolated from progressing tumors (Russell and McIntosh, 1977). The role of tumoricidal effector macrophages would be expected to be particularly important for eliminating tumor cells in situations in which only CD4+ T cells recognize the immunogenic tumor antigen, and, as with the FBL leukemia model studied in our laboratory, the tumor cell itself cannot be directly recognized by such CD4+ T cells due to lack of expression of Class I1 molecules (Greenberg et al., 1985). Unfortunately, reagents have not been available to directly analyze the contributions of cytolytic macrophages b y selective depletion of this effector population or by interference with the cytolytic function. Disruption of macrophage function by the administration of reagents such as carageenan or silica has been shown to interfere with T cell tumor therapy (Mu16 et al., 1985), but these results cannot be interpreted as defining a role for effector macrophages, since the reagents also interfere with the accessory and antigen presentation functions of macrophages necessary for the induction and maintenance of the T cell proliferative response required for tumor elimination. Recently, monoclonal antibodies have been generated to membrane antigens that are expressed on macrophages primed with y-IFN and that correlate with tumoricidal activity (Paulnock and Lambert, 1990), but the utility of these antibodies for selectively interfering with macrophage effector functions in vivo remains to be determined. In light of these limitations, it has only been possible to indirectly assess the role of macrophages in tumor elimination mediated by tumor-specific T cells. We have examined the potential contribution of tumoricidal macrophages using two approaches: (l),determine if T


309

cells responding to stimulation with tumor cells produce cytokines that can render macrophages tumoricidal; and (2), determine if macrophages in a diffusion chamber can be induced by T cells to eliminate viable tumor cells in uiuo. Macrophage-mediated killing of FBL, which is resistant to direct lysis by TNF, requires macrophage activation by the two-signal pathway. Therefore, the ability of FBL-specific T cells to produce MAF was defined functionally as the ability of culture supernatants obtained 20 hours after stimulation with FBL to prime a macrophage monolayer for lysis of FBL tumor cells in the presence of a trigger signal provided by 100 ng of LPS. By this assay, unfractionated splenic T cells immune to FBL were found to produce adequate amounts of MAF for the supernatants to prime macrophages for tumoricidal activity. Purified FBL-reactive CD4+ T cells produced similar amounts of MAF, but purified CD8+ T cells produced comparatively small amounts of MAF following stimulation with FBL. These results suggested that only the CD4' T cells might efficiently activate macrophages to participate in tumor elimination. However, previous studies had demonstrated that the in uiuo efficacy of the CD8+ subset is limited by the availability of exogenous IL-2 (Greenberg, 1986), which, in addition to promoting T cell growth, increases lymphokine secretion by CD8+ T cells (Kelso et al., 1982).Therefore, the ability of CD8' T cells to produce MAF in the presence of exogenous IL-2 was examined. Consistent with the requirement for IL-2 for optimal in uiuo efficacy and activity, stimulation of CD8+ T cells with FBL in the presence of IL-2 resulted in the production of MAF at levels similar to those detected following stimulation of CD4+ T cells. These results affirm that tumoricidal macrophages can be induced during tumorspecific T cell responses, and suggest that the CD8+ T cell subset as well as the CD4' T cell subset can produce M A F to induce this effector mechanism. Studies to define the nature of the MAF responsible for this lytic activity have demonstrated that the production by both CD4+ and CD8+ FBL-specific T cells of 7-IFN, a lymphokine known to prime tumoricidal macrophages (Schreiber et al., 1985),correlates with MAF activity. We have recently explored the ability of these T cell subsets to activate macrophages in viuo. Double diffusion chambers were implanted into the peritoneal cavities of mice, with the first chamber containing responder T cells, macrophage accessory cells, and irradiated FBL stimulator cells, and the second chamber containing potential effector macrophages and labeled viable tumor cells. Mice were sacrificed 3 days after chamber implantation to evaluate the antitumor response (Fig. 5). No killing was evident in the absence of specific

310

PHILIP D.GREENBERG

Chamber Containing Responding T Cells FB L- Pr I mad

J s i R U Q c Stlmuletor CD4*

T cells

Admln. of IL-2

(B6)x (FBL),

-

(FBL),

+

FIG. 5. In oioo activation of M@s for tumor cell lysis by purified CD4+ and CD8' FBL-immune T cell subsets. lo7 CD4+ or CD8' responder T cells from FBL-primed mice were placed with macrophage accessory cells and lo5 irradiated FBL stimulator tumor cells or B6 spleen cells into the upper chambers of Millipore double diffusion chambers. 2 x lo6 thioglycollate-induced peritoneal exudate cells plus 2 x 10' [3H]uridine-labeled RBL tumor cells were placed in the lower chamber. The chambers were sterilely implanted into the peritoneal cavities of B6 mice. Selected mice received 3 daily intraperitoneal injections of 2.5 x lo3 units of recombinant IL-2. The chambers were harvested after 72 hours and percent tumor lysis determined as a reflection of the amount of label lost from the chamber.

stimulation in the first chamber or if no effector macrophages were added to the second chamber. However, specific stimulation with FBL of either CD4+ or CD8+ T cells in the first chamber induced the production of a diffusible MAF that activated macrophages in the second chamber to kill viable tumor cells. Although exogenous IL-2 was not essential for MAF production by either subset, the administration of exogenous IL-2 increased the magnitude of tumor killing by both subsets, particularly the CD8+ T cell subset, which initially produced lower levels of MAF. In these in vivo experiments, no exogenous LPS trigger signal was added, implying that endogenous cytokines produced during the in vivo response of both CD4+ and CD8+ T cells can provide macrophages with both the priming and triggering


311

signals essential to induce tumoricidal activity. Studies in Hamaoka’s laboratory using two non-cross-reactive tumors have demonstrated with diffusion chambers that the in uiuo production of MAF requires tumor-specific T cells, but that the tumor target need not express the stimulating antigen to be lysed by the activated macrophages (Sakamoto et al., 1986). Their results suggest that the activation of tumoricidal macrophages by T cells, including the CD8+ T cell subset, may provide a means to eliminate antigen-loss variant tumor cells (Sakamoto et al., 1988). Studies in two other tumor systems have provided additional circumstantial evidence that macrophages may contribute to the therapeutic efficacy of CD8+ T cells. In a model similar to our adoptive chemoimmunotherapy model, the efficacy of adoptive therapy with a CD8+ T cell clone was markedly enhanced by the concurrent administration of recombinant y-IFN. Analysis of the effects of the administered y-IFN demonstrated potential activation of tumoricidal macrophages, but no change in other effector mechanisms such as direct tumor lysis by CD8+ T cells (De Graaf et al., 1988). Studies in the second model employed a sponge matrix to isolate and analyze the cells involved in the eradication of tumor, and found that tumorspecific CD8+ T cells induced an inflammatory response by recruiting macrophages to participate in the antitumor response at the site of the tumor (Zangemeister-Wittke et al., 1989). Studies examining infectious agents rather than tumor cells as the replicating antigen have provided more definitive evidence that CD8+ T cells utilize macrophages to mediate effector functions. Virus-specific CD8+ T cells, independent of CD4+ T cells, can induce extensive inflammation at the site of a virus-specific T cell response (Doherty et al., 1990). This secretion of factors by antigen-specific CD8+ T cells that attract macrophages to the site of an ongoing immune response represents an important mechanism for recruiting potential effector macrophages. Moreover, CD8+ T cells specific for intracellular bacterial antigens have been shown to activate macrophages for microbicidal activity, an effector function with many similarities to tumoricidal activity (Mielke et al., 1989). Thus, the cumulative indirect evidence strongly suggests that tumoricidal macrophages represent an important in viuo participant in tumor elimination promoted by both subsets of tumor-specific T cells. Indeed, during elimination of Class 11- tumors by CD4+ T cells, as in our model with disseminated FBL leukemia, it is likely that macrophages are the major effector cell responsible for tumor lysis. Studies demonstrating that CD8+ T cells can also induce tumoricidal macro-

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PHILIP D. GREENBERG

phages in vivo have raised the question of whether such macrophage effector cell activity is only complimentary to direct lysis by CD8+ T cells or if it is mandatory for complete tumor elimination. As new reagents that specifically interfere with the cytolytic activity of macrophages become available, further analyses will be necessary to precisely define the role of effector macrophages in tumor elimination. However, the recruitment of this population to the effector response has the intrinsic advantage of immediately broadening the scope of the T cell response. Macrophages, unlike antigen-specific T cells, do not recognize unique tumor antigens but rather more generally bind to transformed cells, and thus have the capacity to eliminate cells in a tumor population that have lost expression of the tumor antigen or become deficient in MHC antigen expression.

C. ROLEOF B CELLSIN TUMOR ELIMINATION B cells and their antibody products unquestionably have an important role in the field of tumor immunology. With the development of monoclonal antibody technology, a whole discipline has evolved with the goals of generating monoclonal antibodies to membrane antigens expressed on tumor cells, and of modifying these antibodies, such as by conjugation with toxins or radionuclides, to render them more effective in the treatment of tumors (reviewed in Dillman, 1989; Schlom, 1986; Badger and Bernstein, 1986). A description of these therapeutic approaches devoted to enhancing antibody antitumor activity is outside the scope of'this review, which will focus on only the potential contributions of B cell responses to T cell-mediated tumor rejection. The prerequisites for an effective antibody response to a tumor include that the tumor express an antigen capable of being recognized by B cells, that this tumor antigen also have an epitope presentable to CD4' T cells in the context of Class I1 molecules, and that a CD4+ T cell response, particularly of the T H 2 subtype producing IL-4, IL-5, and IL-6 be elicited (Mosmann and Coffmann, 1989a; Killar et al., 1987; Boom et al., 1988).The most direct mechanism by which tumorreactive antibodies can enhance tumor eradication is by lysing tumor cells in a complement-dependent fashion or by inducing apoptosis (Trauth et al., 1989). A second tumoricidal mechanism is antibodydependent cellular cytotoxicity (ADCC), which appears to be the major effector pathway by which therapy with unmodified monoclonal antibodies results in tumor eradication (Denkers et al., 1985).Lysis by ADCC proceeds via binding of the Ig variable region to the tumor target and of the Fc portion to an Fc receptor (FcR)-bearing effector


'

313

cell, with subsequent signal transduction through the FcR and activation of the cytolytic mechanism of the effector cell. Although FcR+ macrophages and NK cells are the major ADCC effector cells, B cells can also function in ADCC to mediate tumor lysis (Padmanabham et al., 1988;Lopez et al., 1989). T cell responses to tumor promote ADCC both by the secretion of lymphokines that provide help for B cell antibody responses, and by secretion of lymphokines that activate ADCC effector cells (Morgan et al., 1989; Basham e t al., 1988; Eisenthal et al., 1988; Ostensen et al., 1987; Kushner and Cheung, 1989). The remaining and most complex mechanism by which antibody responses can participate in T cell antitumor responses is through a regulatory circuit defined as an idiotype network. Idiotypes are unique sequences in the variable regions of B cell and T cell receptors that are involved in antigen binding, and anti-idiotypes represent antibodies that bind to this idiotypic region of the receptor. The binding region of anti-idiotypic antibodies that recognize the same antigen-specific receptor can have many different molecular configurations, with one form being an internal image of the antigenic epitope normally recognized by the receptor. Such an internal image anti-idiotype may uniquely stimulate or inhibit antigen-specific responses by providing the antigenic epitope in a new context distinct from native antigen. The complexity of idiotype network regulation precludes a meaningful discussion of this phenomenon here, and the reader is referred to discussions of regulatory anti-idiotypes as naturally occurring events during tumor growth (Raychaudhuri et al., 1987; H. Schreiber, 1984; Kennedy et al., 1987), and of the use of anti-idiotypes to positively regulate tumor-specific T cell and B cell immunity (Nepom and Hellstrom, 1987). Our model for adoptive T cell therapy of disseminated FBL leukemia represents an appropriate model for examining the contribution of B cells to the outcome of tumor therapy. FBL is a Friend MuLVinduced leukemia, and immunization of B6 mice with FBL induces a strong antibody response that, cross-reacts with other tumors transformed with Friend or the closely related Rauscher and Moloney retroviruses (Glynn et al., 1968). Although we have never pursued monoclonal antibody therapy of FBL, infusion of FBL-specific polyclonal antibodies has had little effect in tumor therapy (Fefer, 1969). Therapy of related Moloney and Rauscher virus-transformed tumors with monoclonal antibodies has been examined in other laboratories and results have confirmed the potential in vivo contribution of an ADCC mechanism; however, effective immunotherapy with antibody

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PHILIP D. GREENBERG

in these models ultimately required a tumor-specific T cell response (Kennel et al., 1985; Berends et al., 1989).Additionally, administration of a monoclonal antibody to Moloney virus-transformed cells has been shown to elicit a multilevel anti-idiotypic network (Powell et al., 1988). Thus, studies in the FBL and related retroviral tumor models suggest that all aspects of B cell and antibody function are potentially operative in response to these tumors. To determine if the B cell response makes a significant contribution to tumor elimination following therapy with T cells, we employed a strategy similar to the one used to evaluate the contribution to tumor elimination of T cell subsets. Briefly, mice were rendered B cell deficient by chronic treatment from birth with high doses of rabbit antimouse IgM (anti-p)antibody using a well-characterized regimen (Hayglass et al., 1986). These anti-p treated mice were demonstrated to have no IgM' cells or detectable serum immunoglobin and to be unresponsive to the B cell mitogen LPS, but to retain normal T cell function. The efficacy of adoptive chemoimmunotherapy of disseminated FBL with purified T cells was then compared in B-deficient and normal control tumor-bearing mice (Fig. 6). Mice depleted of B cells

100 a

6

(B deficient): CY + 5.0 x 10 B6 aFBLT cells 80-

0

>

*E a

*

(Control): LT CY +0.5 X 10 B ~ ~ F BCeIIS

1 No 60-

40-

8

20

-

Therapy

I

6

(B deficient): CY +0.5x10 BGaF,,T

cells

--

(B deficient): (Control): CY Alone -& C C Y Alone

n-

I

0

20

40

60

80

Days FIG.6. Efficacy of adoptive T cell therapy of disseminated FBL in host mice depleted of B cells. B6 host mice continuously treated from birth either with normal rabbit immunoglobulin (control) or with rabbit anti-p antibody to deplete B cells (B deficient) were inoculated with 5 x 10"FBL tumor cells on day 0 and received either no therapy, 180 nig/kg cyclophosphamide (CY) on day 5, or CY plus 5 x lo6 or 0.5 x 10" purified B6-immune T cells (>97% Thy-1.2+).


315

exhibited a therapeutic response similar to normal host mice to both a high dose of adoptively transferred T cells, that cured 100% of mice, and a low dose that cured only a fraction of recipients. These results, which demonstrated that host B cells do not make a requisite contribution to T cell-mediated tumor eradication, do not imply that there are no circumstances in which antibody responses could augment the therapeutic antitumor responses following adoptive T cell therapy, or that the antibody response could not be specifically manipulated to enhance tumor elimination. However, considering that FBL expresses multiple epitopes to which antibodies can be generated, the data argues that the endogenous antibody response to most tumors will not generally be a major factor in determining if an ef‘fector T cell response is curative.

D. ROLEOF NK CELLS NK cells represent a cell population that was originally characterized on the basis of an in vitro defined function-the capacity to “

naturally” mediate spontaneous and nonadaptive non-MHC restricted cytolysis of a wide variety of targets, including some normal fresh cells, many cultured cell lines, immature hematopoietic cells, and tumor cells. The lytic activity for tumor cells received a great deal of attention, and investigators enthusiastically endowed NK cells with significant biological qualities previously reserved for T cells, such as immunologic surveillance against spontaneously arising tumors. This focus on tumor reactivity delayed identification of many of the diverse biological activities of NK cells, such as regulation of hematopoiesis and natural resistance to microbial infections, which may be more significant in the evolution of this effector population. A discussion of the biology of NK cells is beyond the scope of this chapter, but has been the subject of a recent extensive review (Trinchieri, 1989), and this section will be limited to evaluating the role of NK cells in tumor rejection responses for which there is a T cell component. Although NK cells do not represent an entirely homogeneous population, an operational definition has been provided by the Fifth International Workshop on Natural Killer Cells (Fitzgerald-Bocarsly et al., 1988):NK cells are large granular lymphocytes that express no C D 3 molecules or T cell receptor chains, express CD16 and NK1.1/2.1 (or Leu-19 in humans), and mediate cytolysis of targets even in the absence of MHC expression. By distinction, cells bearing a/3 or y6 T cell heterodimeric receptors that demonstrate non-MHC restricted lytic activity following activation by IL-2 or by other means are defined as cells of T cell origin that functionally are NK-like. The mechanism by

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PHILIP D. GREENBERG

which NK cells recognize, bind, and selectively lyse targets has been a source of much investigation, but very little clarity, particularly with regard to the receptor on the NK effector cell and the recognition structure on transformed target cells. However, much like tumoricidal macrophages, NK cells show preferential binding of transformed cells, and similar to CD8+Tc, cytolysis proceeds via delivery of cytolytic granules (Herberman et al., 1986). Interest in NK cells as potential effector cells for tumor therapy increased dramatically with the demonstrations that the cytolytic activity of NK cells could be markedly enhanced by exposure to T cellderived lymphokines. Culture of peripheral blood lymphocytes with high concentrations of IL-2 was found to induce non-MHC restricted cytotoxic cells, termed LAK cells, that lyse NK-resistant targets, including many fresh tumor cells (Grimm et al., 1982, 1983). Moreover, the administration of in uitro generated autologous LAK cells into patients with solid tumors has been shown to induce at least partial regressions in a fraction of treated patients (S. A. Rosenberg et al., 1987). Although there was initially some disagreement about the phenotypes of the LAK'cell precursor and effector, it is now generally agreed that LAK cells represent activated NK cells (Phillips et al., 1987), and that the responsiveness to IL-2 results from expression of the intermediate-affinity, signal-transducing, p70 chain of the IL-2 receptor (Sharon et al., 1986; Kehri et al., 1988).The administration to patients of high-dose IL-2 and LAK cells represents a novel approach for the use of immunologically nonspecific effector cells to treat malignant disease, and has been extensively reviewed (S. A. Rosenberg and Lotze, 1986; S. A. Rosenberg et al., 1987). However, it has remained unclear whether conditions ever exist in uiuo, even in the microenvironment of an ongoing T cell immune response, for the induction and functional expression of LAK effector cells. The marked antitumor activity of NK cells pharmacologically activated to express the broad cytolytic reactivity of LAK cells highlights the potential contributions to T cell-mediated tumor eradication of NK cells activated b y T cell-derived lymphokines, even if such cells are less proficient killers of tumor cells than are fully activated LAK cells. y I F N can increase the cytolytic reactivity of NK cells (Perussia et d., 1980), and NK cells activated by IFN can lyse fresh tumor target cells, which are relatively resistant to lysis by unstimulated NK cells (Vanky et al., 1980). Moreover, IFN and IL-2 synergize in their enhancing effect on NK cell cytotoxicity (Svedersky et al., 1984b; Brunda et al., 1986). Thus, even if NK cells cannot be fully differentiated to LAK cells in uiuo under physiologic conditions, the milieu established b y

317


an ongoing T cell response should be sufficient for activating NK cells at a tumor site to express enhanced tumoricidal activity. Therefore, we have examined if NK cells contribute to the eradication of disseminated FBL leukemia in our adoptive therapy model. B6 mice bearing disseminated FBL were depleted of NK cells, prior to therapy with cyclophosphamide and adoptively transferred T cells, by administration of monoclonal aNK1.l antibody (Koo and Peppard, 1984; Koo et al., 1986). The regimen of a N K l . l employed was demonstrated to efficiently eliminate NKcells from B6 mice (Koo et al., 1986; Peace and Cheever, 1989). The therapeutic efficacy of adoptive therapy with either a high or low dose of transferred T cells was not significantly altered in mice lacking NK function (Fig. 7). These results suggest that NK cells do not make a significant or essential contribution in our model to elimination of disseminated tumor cells. Although these results could be interpreted as evidence that NK cells are not an important effector cell in T cell-mediated tumor elimination, recent studies defining target recognition patterns of NK cells have implied that NK cells may be important effector cells in certain settings. In particular, reduced expression of Class I molecules on

100

11

80-

-

g601

73

a40

8

20

0

-

No Therapy

\

CY Alone-

\,

I

6

cY+5.0X10 B ~ ~ F B L

-

-:%

6

cY+1.0X10 B ~ ~ F B L

]-Y+

I

-6

1.Ox10 B6aFBL +aNK1.1 I

1

FIG. 7. Efficacy of adoptive T cell therapy of disseminated FBL in host mice depleted of NK cells. B6 host mice were inoculated on day 0 with 5 x lo6 FBL tumor cells, and received either no Mab or were depleted of NK cells by administration of aNK1.1 Mab on day 5 prior to therapy and on day 12 one week after adoptive therapy. On day 5, cohorts of mice received either no therapy, cyclophosphamide alone (CY),or CY plus 5 X 10" or 1 X 10" immune T cells from FBL-primed B6 mice.

318

PHILIP D. CREENBERG

target cells has been shown to correlate with increased sensitivity to lysis by NKcells (Harel-Bellam et al., 1986; O h l h et al., 1989;Karre et al., 1986; Ljunggren et al., 1988; Storkus et al., l987,1989a,b; Tanaka et al., 1988).This enhancement in the lytic activity of NK effector cells may have evolved as a means for NK cells to serve as a first line of defense for elimination of virally infected cells that commonly exhibit decreased Class I antigen expression Andersson et al., 1985;Jennings et al., 1985; Hecht and Summers, 1972), but could have significant impact on tumor rejection. Class I deficient tumor cells are commonly observed with human tumors and in many murine tumor models (reviewed in Tanaka et al., 1988).Such tumor cell variants are no longer targets for CD8' Tc effector cells, by virtue of a failure to express Class I molecules, but can be demonstrated to be rejected by NK effector cells (Karre et al., 1986; Ljunggren et al., 1988; Tanaka et al., 1988; Kawano et al., 1986).Thus, in situations in which Class I-loss variants exist amongst a heterogeneous tumor population, NK effector cells could play an important role in promoting complete tumor elimination. We have never detected the outgrowth of Class I-loss variants in the FBL tumor model, and thus this model may not be entirely adequate for making general conclusions about the contributions of NK cells to rejection of other tumors. Further studies examining the role of NK cells in rejection of tumor masses known to contain a subpopulation of Class I-deficient tumor cells are needed to better elucidate this potentially important function for NK effector cells activated by an ongoing immune T cell response to the tumor. IV. Recognition of Distinct Tumor Antigens by CD4+ and CD8+ T Cells as a Potential Basis for Selective Efficacy of a T Cell Subset

The discussion thus far has focused on the nature of the effector cell and effector mechanisms operative in the eradication of established tumors, and has revealed that each T cell subset, independent of the other subset, can mediate directly and/or indirectly by the recruitment of other non-T effector cells the complete elimination of a tumor. However, analyses of the T cells participating in tumor rejection have detected disparate effector T cell requirements in different tumor models. Rejection of some tumors results primarily from the activity of Class I-restricted CD8' T cells (Dailey et al., 1982; Mills and North, 1983; De Graaf et al., 1988; Greenberg, 1986;Kast et al., 1989;Ward et al., 1988; Wortzel et al., 1984; Schild et al., 1987; Barker and Mokyr, 1988), whereas Class II-restricted noncytolytic CD4' T cells are responsible for rejection of other tumors (Greenberg et al., 1985; Fujiwara et al., 1984; Forni et al., 1985; Fernandez-Cruz et al., 1980).


319

Although the observed differences in the effector T cell subset required for tumor eradication could reflect susceptibility by the tumor to the effector mechanism mediated by one subset and resistance to the effector mechanism mediated by the other subset, most studies examining tumor lysis would not support this conclusion. The major effector mechanism by which noncytolytic CD4+ T cells mediate tumor rejection appears to reflect the activation of macrophages to lyse tumor cells, and most tumors remain susceptable to lysis by tumoricidal macrophages even after extensive attempts in vitro to generate resistant variants (Fogler and Fidler, 1985). Similarly, resistance of tumor cells to direct cytolysis by CD8+ Tc is a very rare event, unless variants lacking Class I gene expression have been generated, and, as discussed in the previous section, CD8+ T c recognizing Class I + tumor cells can secrete lymphokines that activate macrophages and/or NK cells to lyse Class I- targets. Thus, rather than presume that rejection of an individual tumor is dependent upon the activity of a unique effector mechanism, alternative explanations for the preferential activity of a T cell subset in the elimination of particular tumors need to be explored. Since tumor eradication requires a prolonged in vivo response, effective tumor therapy must include not only an efferent tumoricidal component but also an afferent component resulting in in vivo proliferation and expansion of the potential effector population. Therefore, the selective therapeutic efficacy of a particular T cell subset could reflect preferential activation of that subset in response to the tumor. For example, if the tumor antigens expressed by a tumor failed to elicit a broad repertoire of T cell responses, but rather were recognized predominantly and/or more efficiently by a single T cell subset, then cells from that T cell subset would likely appear to be more effective in therapy. Presentation of an antigen for activation of MHC-restricted T cells requires that the antigen be processed to a peptide that can bind to the appropriate MHC molecule to form an immunogenic complex (Unanue and Allen, 1987; Berzofsky et al., 1988; Watts et al., 1984; Buus et al., 1986; Maryanski et al., 1988; Gotch et al. 1988). The intracellular processing pathways that result in degradation of a protein to a peptide and introduction of this peptide into the binding region of the MHC molecules are with rare exception separate for Class I and Class I1 molecules (Germain, 1986). Presentation of antigen with Class I1 molecules generally requires that exogenous antigen enter a “professional” antigen-presenting cell b y phagocytosis, pinocytosis, or receptor-mediated endocytosis and be degraded to peptide fragments by acid-dependent endosomal proteases (Shimonkevitz et

320

PHILIP D. GREENBERG

al., 1983; Buus et al., 1987; McCoy and Schwartz, 1988), whereas presentation of antigen with Class I molecules generally requires that endogenously synthesized cytosolic proteins be degraded and translocated into the endoplasmic reticulum (Braciale et al., 1987; Morrison et al., 1986; Yewdell and Bennink, 1989; Nuchtern et al., 1989).The presence of these two largely distinct pathways, one of which usually requires the presence of an APC other than the tumor cell itself, makes it possible that a particular protein might more efficiently be processed in one pathway and thus predominantly presented with a single MHC molecule. Therefore, as a model system, we have examined if the specificity of the CD4' and CD8' T cell responses to FBL reflects recognition of the same tumor antigen(s) or if the observed therapeutic efficacy of both subsets in this tumor model results from expression by FBL of more than one tumor antigen, each of which is preferentially recognized by a single T cell subset. Our studies on the specificity of T cell responses to FBL have focused on the recognition of Friend murine leukemia virus (F-MuLV) retroviral glycoprotein antigens, since these represent the most strongly immunogenic determinants on this Friend retrovirustransformed erythroleukemia (Friend, 1957; Nowinski et al., 1978; Plata and Lilly, 1979; Chesebro et al., 1981).The immune responses to the products of the two major F-MuLV genes have been examined: the glycoprotein encoded by the gag region of the F-MuLV genome, and the envelope glycoprotein encoded by the enu gene (Klarnet et al., 1989a). A preliminary analysis of FBL-specific responses by the CD4+ and CD8+ T cell subsets suggested that these should be informative FBL antigens to examine, since a mutant Friend MuLV-transformed tumor line that expressed enu but had spontaneously lost gag gene expression stimulated CD4+ responder T cells but was not recognized by the CD8+ subset. Moreover, a Mab that bound the gp70 envelope protein inhibited the response of the FBL-immune CD4' subset and had no effect on the CD8+ subset. Therefore, the responses to these proteins was more directly examined by employing rat fibroblast lines transfected with either the whole F-MuLV genome or only the gag or enu gene plus the Db Class I molecule to which the FBL-specific CD8+ T cell response is restricted (Gomard et al., 1977; Holt et al., 1986).FBL-specific CD8' T c , generated by in uitro stimulation with FBL of cells from FBL-primed B6 mice, recognized and lysed targets transfected with the entire F-MuLV genome or with the gag gene, but failed to lyse targets expressing only the enu gene. Since all targets were equally lysable by alloreactive CD8' Tc, these results suggested that immunization with FBL generated an immunodominant Tc re-


32 1

sponse to gag antigens but failed to elicit envelope-specific CD8+ Tc (Klarnet et al., 1989a). Since these xenogeneic transfected lines could not be used to directly assess the FBL-specific proliferative response of the CD4+ T cell subset due to stimulation by the xenoantigens, the responses to these lines ofcloned FBL-specific CD4+ T cells were evaluated. In the presence of autologous APC, all CD4+ T cell clones tested responded to the env-transfectant, and none responded to the gag-transfectant (Klarnet et al., 1989a).These results are consistent with another analysis of the response of Class 11-restricted T cells to FBL, in which all CD4+ T cell clones responded to soluble gp70 envelope protein (Matis et al., 1985). Thus, B6 mice exposed to FBL tumor generate a CD8+ T cell response that is primarily restricted to recognition of epitopes encoded by the gag gene, and a CD4+ T cell response that is primarily restricted to recognition of determinants on the envelope protein. This distinction between the F-MuLV proteins recognized by CD4+ and CD8+ T cells occurred despite both env and gag encoding large foreign proteins containing many potentially immunogenic epitopes. The selective response to these tumor antigens could reflect intrinsic qualities of each of these proteins that direct the products predominantly to a single processing pathway, or unique qualities attributable to the FBL tumor that result in both poor entry of the envelope protein into the endogenous pathway for presentation with Class I molecules and limited availability of gag products for uptake and processing by Class 11-expressing APC. Therefore, an alternative method for immunizing B6 mice to these proteins was evaluated. Recombinant vaccinia viruses containing the F-MuLV env and gag gene were constructed (Earl et al., 1986), since immunization with such live recombinant vaccinia viruses has been shown to induce both CD4+ and CD8+ T cell responses to the product of the inserted gene (Moss and Flexner, 1987). B6 mice were primed in vivo with either recombinant vaclenv or vaclgag, and then purified CD4+ and CD8+ T cells were obtained from primed spleen cells for in vitro testing of proliferative responses to FBL (Fig. 8). As predicted from our studies with transfected cell lines, immunization of mice with vaclenv elicited only an FBL-specific response by the CD4+ T cell subset, and immunization with vaclgag elicited only an FBL-specific response by the CD8+ T cell subset. These results suggest that individual tumor-associated proteins may be preferentially excluded from processing via the exogenous Class I1 or endogenous Class I pathway andlor from immunogenic presentation in the context of Class I or Class I1 molecules, and thus will activate predominantly only one T cell subset.

322

PHILIP D. GREENBERG

CD4+ Responders

CD8+ Responders

n

h

E

8 9

Stimulators

6000. 1

al u)

C

0

n

3

4000.

K

-e

.-f

2000.

-i? 2

n 0 .

1 F B L v a c h n v vaclgeg

FBL vacbnv vaclgag

In Vivo Priming FIG.8. Recognition of distinct F-MuLV-encoded antigens by FBL-specific CD4+ and CD8' T cells. Spleen cells from B6 mice primed in oioo with irradiated FBL or vaccinia-FMuLV (BGo~~L live ) , recombinant vaccinia-F-MuLVenvelope (B60vilc,eno), gag (B6,vac,E0K) virus were purified into CD4+and CD8+T cell subsets. Responder cells were cultured with irradiated tumor cells, (FBL),, or irradiated macrophages infected with vaccinia virus, (Mfl),,,, and proliferation measured after 3 days.

The basis for apparent selective immunogenicity of a protein for either Class I- or Class II-restricted T cells are not yet well defined, but some principles of antigen presentation are emerging. Despite a protein having many possible immunogenic epitopes, the T cell response tends to be limited to one or very few immunodominant epitopes (Schwartz, 1985; Manca et al., 1984; Cease et al., 1986; Berzofsky, 1988; Braciale et al., 1989; Bennink and Yewdell, 1988; H. Takahashi et al., 1988).The presence of an immunodominant epitope cannot be reasonably explained by assuming that all the other epitopes in a large complex foreign protein are tolerogenic or recognized as self-proteins, and thus there must be unique characteristics that distinguish immunodominant regions. This would include the abilities to be degraded from the native protein into a peptide of appropriate size, charge, and configuration by the host processing machinery, to bind to a host MHC molecule, and possibly to acquire a secondary structure such as to form an amphipathic a helix that enhances formation of a peptide-MHC complex (Berzofsky et al., 1987, 1988). Moreover, the presented epitope must be recognizable by the host repertoire of T cells. Thus the selective responses observed in our tumor system could


323

reflect the failure to form a recognizable immunodominant epitope from the envelope protein that binds to Class I molecules, and similarly the lack of a recognizable immunodominant epitope derived from gag proteins for presentation with Class I1 molecules. The immunogenic presentation of an antigen is also subject to quantitative restrictions, requiring a threshold number of peptide-MHC complexes on an APC to trigger a response. The peptides derived from a particular antigen must compete successfully with other peptides derived from the same antigen, peptides from other antigens, and self-peptides degraded from host proteins for complexing with the available MHC molecules (Rock and Benacerraf, 1983; Maryanski et al., 1988; Adorini and Nagy, 1990). Thus, the generation of a T cell response to an immunogenic protein will depend on the amount of the relevant peptide available to compete, the avidity of the complex formed between the immunogenic peptide derived from the protein with the MHC molecule as compared to competing peptides, and the affinity of T cells for the peptide-MHC complex (i.e., high affinity would reduce the threshold number of complexes necessary to ensure triggering a response). Therefore, in our tumor model, envelope rather than gag proteins may be good Class I1 immunogens because envelope proteins are membrane antigens that are secreted into the extracellular space and thus are more available for presentation by phagocytic Class II+ APC. Similarly, the relevant gag protein may be a more abundant and persistent cytosolic protein for introduction into the endogenous Class I pathway. Although the molecular mechanisms are presently poorly understood, proteins apparently have intrinsic qualities that promote localization to and/or processing by the Class I or Class I1 pathway. For example, the major CD8+ Tc response to many infectious viruses appears to be to matrix and nucleoproteins, whereas the CD4+ T cell responses to the same viruses frequently recognize envelope antigens. Some features of proteins, such as the expression of sequences that promote translocation into or retention in the endoplasmic reticulum (Bonifacino et al., 1990), or the discoordinate overproduction of an individual chain of a multimeric protein, may result in degradation and enhanced presentation with Class I molecules. Similarly, proteins containing signal sequences that result in transport to the membrane and release into the extracellular space, lacking the retention or intracellular localization sequences necessary to prevent secretion (Wieland e t al., 1987; Rose, 1988),or possessing physicochemical characteristics that promote interactions with the APC surface such as a positive charge (Apple et al., 1988), may exhibit enhanced presentation with Class I1 molecules.

324

PHILIP D. GREENBERC

Regardless of the mechanism, our data evaluating the T cell response to F-MuLV antigens expressed by FBL demonstrate that even large unique foreign proteins expressed by tumor cells may not efficiently induce both CD4' and CD8+ T cell responses. Moreover, even if occasional CD4' T cell clones reactive with gag antigens or CD8' T cell clones reactive with an envelope determinant could be isolated in uitro, such clones might be poorly activated in uiuo by FBL tumor d u e to limited presentation of the relevant epitope, and thus would have minimal efficacy in adoptive therapy of the tumor. Thus, future studies elucidating how candidate tumor antigens are processed and presented may provide valuable insights into the nature of the effector T cell that would be most effective in promoting eradication of a tumor expressing that antigen. V. Accessory Cell, Antigen-Presenting Cell, and Cytokine Requirements for Effective Expression of Antitumor Responses by CD4+ and CD8+ T Cell Subsets

CD4' and CD8' effector T cells, in addition to recognizing potentially distinct tumor antigens and being restricted to different MHC molecules, differ in lymphokine production, lymphokine responsiveness, and requirements for APC and accessory cells. Thus, the capacity of the cells and microenvironment at or near a tumor site to induce and support antitumor responses by each T cell subset influences whether CD4' or CD8' effector T cells will be effective at eliminating tumor. For example, even if a tumor cell expresses a protein containing an immunogenic epitope potentially recognizable by CD4' or CD8' T cells, presentation of that antigen either by an APC lacking accessory functions or in the absence of adequate stimulatory cytokines could result in failure to elicit an endogenous or maintain an adoptively transferred T cell response (Hori et al., 1989; Fearon et al., 1990; Gill et al., 1989). Therefore, predicting the nature of the T effector cell necessary to eradicate a tumor, and determining how to augment the relevant effector functions, requires not only definition of the tumor antigen but also an understanding of the conditions necessary for generating and maintaining the T cell response to the tumor. Under typical in vitro and in uiuo conditions, efficient induction of tumorspecific CD8' T cell responses is dependent on lymphokines such as IL-2 usually provided by a concurrent CD4' THresponse to the tumor (Kern et al., 1986; Fearon et al., 1990). However, since some tumors may express antigens that can be recognized by only Class I- or Class II-restricted T cells and thus can only stimulate responses by one


325

subset, it is essential to define the requirements for inducing and supporting antitumor responses by CD4+ and CD8+ T cells independent of a contribution by the other subset. A. REQUIREMENTSFOR INDUCING PROLIFERATIVE RESPONSESBY CD4+ AND CD8+ T CELLSUBSETS There are many effector functions mediated by CD4+ and CD8+ T cell subsets that can be measured to reflect responses by antigenspecific T cells. However, curative adoptive therapy of established tumors requires not only the expression of differentiated effector functions, but also the in uiuo proliferation of the transferred tumor-specific T cells (Greenberg et al., 1979; Colombo et al., 1985). The biological requirements for inducing such T cell proliferation are more complex and multifactorial than the requirements for only triggering expression of differentiated T cell effector functions, although satisfying the requirements for inducing T cell proliferative responses generally leads to the concurrent expression of such effector functions. Therefore, we have adapted our in uitro culture system to measure the proliferative responses of purified T cell subsets derived from FBL-primed mice, and have used this system to evaluate the immunologic requirements for inducing FBL-specific proliferation of each subset independently (Table 11). The proliferative responses of both purified Class IIrestricted CD4+ T cells and purified Class I-restricted CD8+ T cells to stimulation with FBL were dependent on the presence of macrophages. The CD4+ T cell response required macrophages as APC to process FBL tumor antigen in a chloroquine-sensitive endosomal pathway for presentation with Class I1 molecules (Kern et al., 1986). By contrast, CD8+ T cells required macrophages as accessory cells and not as APC for the proliferative responses to FBL, since the addition of exogenous recombinant IL-1 could substitute for macrophages in this response. This demonstration that 1L-1 was essential for triggering proliferation of the CD8+ subset was not predicted b y previously reported phenotypic studies examining IL-1 receptor (IL-1R) expression on T cells, which had detected IL-1R on CD4' T cells but not on CD8+ T cells (Lowenthal and MacDonald, 1987). However, studies subsequently performed in our laboratory with CD8+ T cell clones rather than CD8+ populations have clarified this apparent discrepancy. Although most cytolytic CD8+ T cell clones fail to make IL-2, a subset of cytolytic CD8+ T cell clones have the capacity to produce IL-2 and support their own proliferation (Widmer and Bach, 1981). We have generated such IL-2 producing CD8+ clones specific for FBL, and

326

PHILIP D. GREENBERG

TABLE I1 ACCESSORY CELLAND CYTOKINE REQUIREMENTS FOR FBL-SPECIFIC PROLIFERATION BY CD4+ AND CD8+ T CELLS Responding population" CD4 + ,FBL

Stiniulator (FBL),

Addition to cultureh, c . d. c . f -

M@ (M@)CHLQ CD~+,FBL

(FBL),

-

M@ ( M@)CHLQ

IL-1

Proliferative response (Acpn+ 2,248 19,180 2,637 1,054 7,465 8,120 9,470 16,548 3,823 14,912 5,748 4,880 120 32,859 10,657 15,271 14,824 4,267 14,388

CD~+,FBL

FBL

+ M@

-

IL-2 I L-4 CD~+,FBL

FBL

+ M@

-

IL-2 IL-4

16,155 28,291 34,008 6,581 27,505 9,742

Purified T cell subsets for each experiment were obtained from FRL-primed B6 spleen cells by selective depletion of macrophages, B cells, and the alternative T cell subset. Macrophages selected from normal spleen cells by plastic adherency, and used untreated or after treatment with 0.1 mM chloroquine (CHLQ), were added to T cell cultures at a ratio of 1 :20. ' Monoclonal aI-A" or uK"/D"was added to selected cultures. Recombinant IL-1 was added to selected cultures at a final titer of 6 U/ml. Monoclonal uIL-2 or aIL-4 was added to selected cultures. IRecombinant IL-2 (25 U/nil) or IL-4 (5.0 ng/ml) were added to selected cultures. Proliferative responses were assessed after 6 days in culture.

have demonstrated that these clones express a small number of IL-lR, and have a functional requirement for IL-1 to trigger IL-2 production (Klarnet et al., 1989b).By contrast, no IL-1R expression was detected on any IL-2-dependent CD8' clones, which fail to produce IL-2 and require exogenous IL-2 for survival. Thus, the failure to detect IL-1R


327

on unfractionated CD8+ T cells presumably reflected the relatively low frequency of such IL-2 producing CD8+ T cells as well as the small number of IL-1R present on IL-2-producing cells (Klarnet et al., 198913) The importance of IL-1 for activating and inducing IL-2 production by CD8+ T cells in the absence of IL-2 producing CD4+ T cells has also been demonstrated functionally in the allogeneic CD8+ T cell response to mutant Class I molecules (Mizuochi et al., 1988). These results suggest that in uiuo activation of a tumor-specific CD8+ T cell response requires not only recognition of the tumor by antigenspecific CD8+ T cells but also the presence of either CD4+ TH to provide IL-2 or accessory cells to trigger IL-2 production by IL-2producing CD8+ T cells to support proliferation of the CD8+ T cell subset. Expansion of antigen-specific T cells is dependent on the delivery of a proliferative signal at the time of T cell receptor accomodation. Two major T cell growth factors, IL-2 and IL-4, have been identified. Since these lymphokines have potentially distinct effects on the generation and maintenance of responses b y the CD4+ and CD8+ T cells subsets, we have examined the role of each in the proliferative response of CD4+ and CD8+ T cells to FBL (Kern et al., 1988).The addition either of aIL-2 or of aIL-4 neutralizing Mab partially inhibited the FBLstimulated proliferative response of purified CD4+ T cells (Table 11). By contrast, the proliferative response of purified CD8+ T cells was completely inhibited by aIL-2, and aIL-4 had no effect on the response. These results demonstrate that both IL-2 and IL-4 are endogenous growth factors for FBL-specific CD4+ T cells, but that IL-2 is the only endogenous growth factor produced by responding CD8+ T cells. The production of both IL-2 and IL-4 by the CD4+ T cell subset implies that immunization with FBL elicits both T Hand ~ T HCD4+ ~ T cell responses (Mosmann and Coffman, 1989a,b).Although our studies on the effector mechanisms operative in tumor eradication suggest that CD4+ T H cells, ~ which can mediate DTH and activate macrophages (Cher and Mosmann, 1987; Stout and Bottomly, 1989), represent the more important CD4+ effector population, IL-4 has been shown to activate in uiuo non-T effector cells with antitumor activity (Tepper et al., 1989). Therefore, the relative contribution of IL-4-producing T H ~ cells to the generation and expansion of an effector response contributing to tumor eradication requires further evaluation. In contrast to the selective production of IL-4 as an endogenous growth factor only by the CD4+ subset, the FBL-stimulated proliferative response of both CD8+ and CD4+ T cells could be augmented by the addition of either exogenous recombinant IL-4 or recombinant IL-2. This augmenting effect of IL-4 on CD8+ T cell responses is also

328

PHlLIP D. GREENBERG

reflected by increased cytotoxic activity (Kern et al., 1988; Widmer and Grabstein, 1987).Although the CD8+ T cell population produced adequate IL-2 to induce a proliferative response, the substantial increase in proliferation observed with the addition of exogenous IL-2 and the smaller increase with IL-4 suggest that the response of tumor-specific CD8+T cells can be enhanced by the local production of IL-2 and IL-4 by CD4+ TH responding to the same tumor. Moreover, the results imply that the magnitude of the response to a tumor by CD8+T cells in the absence of a concurrent CD4+ TH response will be significantly limited by the amount of endogenously produced IL-2. This conclusion, based on results from the in uitro experiments, is also supported by in uiuo observations. Studies examining the ability of IL-2producing FBL-specific cytolytic CD8+ T cells to eliminate FBL leukemia cells have demonstrated that the amount of IL-2 produced by these T cells limits in uiuo antitumor activity (Fig. 4; and Klarnet et al., 1987). Similarly, the generation and expression of CD8+ T cells to reject allografts disparate only for Class I antigens can be augmented by providing an antigenic stimulus for a concurrent CD4+ TH cell response (A. S. Rosenberg et al., 1988a,b). These studies, in defining the requirements for generating an immune response to tumor cells, have also helped elucidate potential obstacles to the expression of an effective response. CD4' and CD8' T cells require macrophages to function as APC and/or IL-l-producing accessory cells, and thus the presence of a mononuclear infiltrate at sites of tumor growth is likely to be essential for maintenance of the antitumor responses at those sites. Macrophage accumulation at tumor sites can be regulated b y many distinct but potentially interacting mechanisms, such as the production by tumors of inhibitors of monocyte chemotaxis (Snydennan and Pike, 1976; Cianciolo et al., 1981) or alternatively of chemoattractants for mononuclear phagocytes (Bottazzi et al., 1983; Mantovani et al., 1986),the production by neutrophils infiltrating the tumor of factors that recruit T cells and subsequently inflammatory cells (Yamaki et al., 1988),and the production by CD4+ and CD8+ T cells at the tumor site of lymphokines that recruit macrophages and amplify the inflammatory response (Yamaki et al., 1988;Shijubo et al., 1989;Jayaraman et al., 1990; Zangemeister-Wittke et al., 1989; Evans, 1986; Doherty et al., 1990).Tumor masses lacking a significant macrophage infiltrate may inadequately support immune responses, which may explain why some tumor masses appear to be immunologically privileged sites (Spitalny and North, 1977). Therefore, methods to modify the function of the microenvironment at a tumor site need to be explored. One such approach is the administra-


329

tion of recombinant IL-1, which cannot entirely replace the APC function of macrophages but can supplement macrophage accessory function. Recent studies have demonstrated that systemic administration of IL-1 can enhance the immunogenicity of a poorly immunogenic tumor (McCune and Marquis, 1990), and can promote tumor eradication by augmenting ineffective T cell antitumor responses (North et al., 1988). Limited production of T cell growth factors by the T cells participating in an antitumor response can also result in an inadequate effector response. As predicted from the in vitro studies demonstrating enhanced antigen-specific T cell proliferation in response to the addition of IL-2, the administration of exogenous recombinant IL-2 has been shown to augment the in vivo proliferation and therapeutic efficacy of adoptively transferred FBL-specific CD4+ and CD8+ T cells (Cheever et al., 1986; Klarnet et al., 1987). Moreover, insufficient production ofT cell growth factors during induction of an immune response may not only make the T cell response appear inadequate, but alternatively may result in a tumor appearing nonimmunogenic despite expressing recognizable antigenic determinants. Studies have demonstrated that previously undetected host T cell antitumor responses can become evident by the systemic administration of high doses of IL-2 or by the local inoculation of IL-2 directly into a growing tumor mass (Thompson et al., 1986; Forni et al., 1985). More recently, the introduction of the IL-2 gene into an apparently nonimmunogenic tumor has provided a novel approach for enhancing immunogenicity (Fearon et al., 1990). In these experiments, the wild-type tumor expressed an epitope potentially recognizable by Class I-restricted T cells but failed to elicit an antitumor response due to the lack of a Class 11-restricted response by CD4+ TH, whereas the transfected tumor cell expressing the IL-2 gene provided the help necessary to generate a tumor-specific CD8+ T cell response. Although in settings such as this, in which the tumor can only elicit a Class I-restricted response, the host may contain IL-2producing CD8+ T cells that could potentially be immunized in the absence of a CD4+ TH response, the initial low frequency of such cells and the magnitude of IL-2 production may be prohibitive for the generation of an effective primary response. Additionally, analysis of the requirements for priming IL-2 producing CD8+ T cells have suggested that activation may be dependent on expression of the antigen in association with a large number of Class I molecules on the stimulating cell, and thus weakly antigenic tumors may fail to elicit the response (Singer et al., 1987).Thus, in some settings, it may be necessary to provide exogenous lymphokines to induce and/or support T cell responses.

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These studies have emphasized the importance of accessory cells and cytokines in the generation and maintenance of an antitumor response. The availability of large quantities of recombinant cytokines should make it possible to overcome some ofthe obstacles to establishing an effective response to a growing tumor. However, there are additional poorly defined qualities of antigen-presenting cells which may also impact on the expression of T cell responses. Cells can express immunogenic epitopes recognizable by CD4+ and CD8+ T cells but, due to an inability to provide appropriate costimulatory signals, may fail to activate the reactive T cells, and may alternatively tolerize or render the T cells anergic (Azuma et al., 1989; Hori et al., 1989; Schwartz, 1989). Such functional inactivation of responder T cells by the stimulator cell has been the subject of a recent review (Mueller et al., 1989), and may explain the apparent lack of immunogenicity of some tumors. For example, presentation to T cells of an antigen by Class 11-expressing pancreatic islet cells, which like some tumors may be a poor stimulator cell, has been shown to render reactive T cells hyporesponsive to the antigen when subsequently presented by a normally stimulatory APC (Markman et al., 1988).Thus, it is quite possible that many tumors express potentially immunogenic epitopes but the nature of antigen presentation by the tumor leads to clonal inactivation rather than clonal expansion. Further studies defining the principles underlying this phenomenon and elucidating mechanisms to overcome inactivation should increase the frequency with which therapeutic responses can be detected and expressed. OF T CELL B. ROLEOF B CELLSIN THE GENERATION ANTITUMOR RESPONSES APC are a heterogeneous population of cells that include, in addition to the macrophage population discussed, other cells expressing Class I1 molecules, including dendritic cells and B cells (Unanue, 1984; Kakiuchi et al., 1983;Ashwell et al., 1984; Metlay et al., 1989).B cells can process and present antigen similar to other APC (Chesnut and Grey, 1986),but, by virtue of the expression of an antigen-specific surface Ig-receptor, have the potential to be the most efficient APC. Thus, B cells can stimulate T cell responses at much lower antigen concentrations than other APC provided the antigen to be processed and presented contains an epitope that can be bound by the B cell Ig-receptor (Rock et al., 1984; Abbas et al., 1985; Lanzavecchia, 1985). This APC function of B cells has been shown to be important in uiuo during priming to soluble antigens, with B cell-deficient mice demonstrating poor induction of T cell responses (Ron et al., 1981, 1983; Hayglass et al., 1986).

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An efficient APC, such as an antigen-specific B cell, could be important for inducing tumor-specific T cell responses at the critical time when the tumor burden is small or in settings in which the amount of tumor-derived immunogenic protein available for antigen presentation is limited. Therefore, we have evaluated the role of B cells during the induction of T cell responses to the FBL tumor (Schultz et al., 1990). This was examined by depleting mice in uiuo of B cells, priming in uiuo to FBL, and then measuring the in uitro secondary T cell response to FBL. As previously described, mice were rendered B cell deficient by treatment from birth with high doses of rabbit antimouse IgM (anti-p) antibody. Such anti-p-treated mice have no detectable mature B cells or circulating IgM, but have normal T cell function (Hayglass et al., 1986).Lymph node T cells derived from anti-p-treated mice primed in uivo to FBL exhibited no measurable FBL-specific T cell proliferative response, and splenic T cells derived from anti-ytreated mice demonstrated a 50% reduction in the proliferative response to FBL, in comparison to the response of control mice treated with normal rabbit Ig (Fig. 9). These results are consistent with studies of the contribution of B cells to priming with soluble protein, which dso demonstrated that B cells have a more essential role in the genera-

B cell

B cell

Responder

Normal deficient --

Normal deficient --

Source

Lymph Node

Spleen

FIG.9. Role of B cells in priming mice for an FBL-specific proliferative T cell response. Lymph node or spleen cells were obtained after in uiuo priming with FBL of B6 mice previously treated from birth with rabbit immunoglobulin (normal) or anti-p (B cell deficient). Responder cells were stimulated in uitro with FBL or B6 spleen cells for 72 hours and thymidine uptake was determined.

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tion of lymph node than splenic T cell responses ( Janeway et al., 1987; Ron and Sprent, 1987). Analysis of CD8+ T cell responses demonstrated that mice depleted of B cells generated very weak cytolytic responses to FBL. This poor priming of CD8+ T cells presumably resulted in large part from inefficient priming of FBL-specific CD4+ TH, since we have previously observed that the generation of optimal CD8+ T cell responses to FBL require CD4+ TH (Kern et al., 1986). These results demonstrate that B cells can make an important contribution to the priming of T cells with tumors. Thus, in contrast to our results showing that B cell responses to a tumor are not important during the effector phase of tumor rejection mediated by T cells (Fig. 6), efficient induction of an antitumor response may be greatly facilitated by B cell recognition, processing, and presentation of tumor antigens in the context of Class I1 molecules. These studies suggest that the generation of an effective T cell response to a tumor by the host will frequently be dependent upon recognition by the immune system of multiple tumor-defined determinants. For example, induction of a cytolytic CD8+ T cell response to tumor may not only require the presence of a tumor antigen that provides a unique Class I-restricted epitope, but also a determinant that can be recognized by Class IIrestricted CD4+ TH to provide adequate IL-2 for the CD8+ T cell response, as well as an epitope recognized by B cells for efficient antigen presentation and triggering of the CD4+ TEI. Although many tumors may express novel or mutated proteins, the derived antigens are not likely to frequently satisfy all these criteria, which could result in poor or absent T cell responses to a potentially immunogenic tumor. Thus, developing approaches for priming that employ and enhance the potential contributions of B cells and CD4+ T H , such as by constructing fusion proteins for immunization containing a wellcharacterized B cell determinant linked to a potential tumor antigen or by modifying a tumor cell to add helper determinants (Yoshioka et al., 1986), or that partially substitute for the roles of B cells and CD4+ TH, such as b y the administration of cytokines, will likely permit detection of specific T cell reactivity in a greater frequency of tumors. Potentially, such T cell responses could then be expanded and augmented to promote tumor eradication. VI. Concluding Statements

Studies in animal models, as discussed in this review, have defined many of the underlying principles, and provided many insights for the development of adoptive T cell therapy as a modality for the treatment


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of human malignancy. The growing numbers of laboratories examining T cell responses to human tumors, and the increasing frequency with which investigators using in nitro culture technologies are detecting T cells specifically reactive to autochthonous human tumors, suggest that the clinical application of adoptive T cell therapy will have significant therapeutic impact during this decade. However, despite the enormous potential and likely benefits that many individual patients will receive from such therapy, it is clear that many obstacles still exist before this approach can have widespread applicability. With the current methodologies being employed, tumor-specific T cells can only be generated in a fraction of cancer patients, and complete tumor eradication is only achieved in a subset of the patients treated with such T cells. Thus, it is essential that the existing results from animal models b e critically examined in an effort to elucidate the impediments to therapeutic success, and future studies be designed to develop methods to overcome these hurdles. As presented in this review, studies from our laboratory initially focused on determining the effector mechanisms essential for T cellmediated tumor rejection, based on an assumption at that time that a unique effector mechanism might be responsible or obligatory for tumor eradication. These studies, and supporting results from other laboratories, have demonstrated that: (1)both CD4+ and CD8+ T cells can independently promote complete tumor elimination in the absence of a contribution b y T cells of the other phenotype; (2) CD4+ T cells can directly lyse selected Class 11+cells, but the major antitumor effect of this subset results from the recruitment and activation of other effector cells, particularly macrophages; (3)CD8+ T cells can mediate tumor eradication not only by direct lysis, but similar to CD4+ T cells, also by the recruitment and activation of other effector cells such as macrophages; (4) the induction of tumor-specific antibody responses following T cell transfer is not an essential component of tumor eradication; and (5)NK cells do not make a significant contribution to tumor elimination by T cells, except potentially under special circumstances in which a subpopulation of Class I- tumor variants may be present. Thus, the data demonstrated that CD4+ and CD8+ T cells mediate tumor eradication by overlapping effector mechanisms, and failed to identify a unique effector role in tumor eradication for either subset. Although these results could be interpreted as suggesting that either T cell subset should be effective in tumor therapy, studies in a large number of experimental tumor therapy models have demonstrated that for many tumors only CD4+ or only CD8' T cells can mediate tumor eradication. These observations have prompted an evaluation of alter-

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native explanations for the settings in which there is preferential activity of a single subset. Studies examining the requirements for T cell activation in response to a tumor have subsequently demonstrated that the ability of a T cell subset to specifically recognize and proliferate in response to a tumor may be the major determinant of therapeutic efficacy. Many factors could contribute to the preferential activation by a tumor of CD4' or of CD8' T cells, including the expression of MHC antigens, the presence of a mononuclear infiltrate at the tumor site containing APC and accessory cells, and the production or availability of adequate amounts of cytokines, including IL-1, IL-2, and IL-4. Additionally, presentation of antigens in the context of Class I or Class I1 molecules requires biochemical processing via distinct intracellular pathways, and individual proteins may be preferentially directed to or excluded from one pathway, or may be efficiently processed to relevant peptides by only one pathway. Thus, tumors containing unique or mutated and potentially immunogenic cellular proteins may selectively or predominantly elicit only CD4' or CD8' T cell responses because the antigen is adequately presented in the context of only one MHC molecule, and in this setting only T cells from the subset restricted to that MHC molecule will appear effective in tumor therapy. There are several goals for the next decade with regard to T cell therapy that need to be realized. These include not only improved methods to isolate tumor antigens, but the development of approaches to determine if such antigens will be efficiently processed and presented with Class I and/or Class I1 molecules. This should make it possible to predict the nature of the effector T cell and the cellular and cytokine requirements necessary for an effective antitumor response. Moreover, the relation of tumor antigens to normal host proteins needs to be more extensively explored. For example, it must be determined if immunogenic tumor antigens have to be distinct from host proteins, such as resulting from a mutation or the expression of a gene from a transforming virus, or could result from overexpression of normal proteins. T cell tolerance to many self-proteins does not result from clonal deletion but rather reflects peripheral inactivation of reactive T cells following recognition of the antigenic epitope presented by normal resting cells, and significant changes in the amount of a protein expressed by a tumor cell, as well as biological changes in the transformed tumor cell expressing and presenting the protein, could permit induction of an immune response that preferentially recognizes the tumor cell. Although the consequences to normal host tissues of breaking tolerance to such proteins will need to be determined, and


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may require methods to terminate the response, rendering such proteins immunogenic would significantly broaden the potential range of antigens expressed by tumors that might serve as targets for immunologic attack. One additional goal, which is already being pursued, is to modify the function or activity of effector T cells by the introduction of genes coding for relevant proteins. For example, the introduction of genes into CD4+ T cells resulting in the production of chemotactic factors that recruit macrophages to a tumor site, or of genes into CD8+ T cells resulting in the production of growth factors that promote cell proliferation, could substantially modify the therapeutic efficacy of a potential effector T cell. Several approaches, including the use of retroviral shuttle vectors, appear useful for promoting the introduction and expression of such genes into T cells. The continued application of advances in cellular and molecular biology to the field of T cell therapy of tumors should enhance the prospect that such therapy will significantly impact on our ability to control malignant disease during the next decade.

ACKNOWLEDGMENTS I wish to thank M. Jensen, D. Kern, J. Klarnet, C. 6hl&n,K. Okuno, S. Riddell, K. Schultz, and H. Sugawara for their efforts in designing, performing, and evaluating many of our laboratory’s experiments discussed in this review; M. Cheever for his contributions as a collaborator to many of the studies; S. Emery, J. Smith, and K. Slaven for their expert technical assistance; and Joanne Factor and Anita Rogers for preparation of this manuscript. The work was supported in part by U.S. Public Health Service Grant CA 33084 and American Cancer Society Grant IM-304.

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Takahashi, H., Cohen, J., Hosmalin, A., Cease, K. B., Houghten, R., Cornete, J. L., DeLisi, C., Moss, B., Germain, R. N., and Berzofsky, J. A. (1988).An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes. Proc. Natl. Acad. U . S. A. 85,3105-3109. Takahashi, K., Ono, K., Hirabayashi, Y., and Taniguchi, M. (1988). Escape mechanisms of melanoma from immune system by soluble melanoma antigen. J . Immunol. 140, 3244-3248. Tanaka, K., Yoshioka, T., Bieberich, C., and Jay, G. (1988). Role of the major histocompability complex class I antigens in tumor growth and metastasis. Annu. Reu. Zmmunol. 6,359-380. Tepper, R. I., Pattengale, P. K. and Leder, P. (1989). Murine interleukin-4 displays potent anti-tumor activity in uioo. Cell (Cambridge, Mass.) 57,503-512. Thompson, J. A., Peace, D. J., Klarnet, J. P., Kern, D. E., Greenberg, P. D., and Cheever, M. A. (1986). Eradication of disseminated murine leukemia by treatment with highdose interleukin-2. J . Immunol. 137,3675-3680. Thorley-Lawson, D. A. (1980). The suppression of Epstein-Barr virus infection in uitro occurs after infection hut before transformation of the cell. 1.Immunol. 124,745-751. Thorley-Lawson, D. A., Chess, L., and Strominger, J. L. (1977). Suppression of in uitro Epstein-Barr virus infection. A new role for adult human T 1ymphocytes.J. E x p . Med. 146,495-508. Ting, C. C., and Rodrigues, D. (1980). Switching on the macrophage-mediated suppressor mechanism by tumor cells to evade host immune surveillance. Proc. Natl. Acad. Sci. U. S . A. 77,4265-4269. Tite, J., and Janeway, C. A., Jr. (1984). Cloned helper T cells can kill B lymphoma cells in the presence of specific antigen: Ia restriction and cognate vs. noncognate interactions in cytolysis. Eur.]. Immunol. 14,878-886. Tomita, S., Fujiwara, H., Yamane, Y., Sano, S., Nakajima, H., Izumi, Y., Arai, H., Kawanishi, y., Tsuchida, T., and Hamaoka, T. (1986). Demonstration of intratumoral infiltration of tumor-specific Lyt-l+2- T cells mediating delayed-type hypersensitivity response and in uiuo protective immunity.1pn.J. Cancer Res. 77, 182-189. Topalian, S. L., Muul, L. M., Solomon, D., and Rosenberg, S. A. (1987). Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials. J . Immunol. Methods 102,127-141. Topalian, S. L., Solomon, D., and Rosenberg, S. A. (1989). Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J . Immunol. 142,3714-3725. Townsend, A. R. M., and Bodmer, H. (1989). Antigen recognition by class I-restricted T lymphocytes. Annu. Rev. Immunol. 7,601-624. Townsend, A. R. M., McMichael, A. J., Carter, N. P., Huddleston, J. A., and Brownlee, G . G. (1984). Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell (Cambridge, Mass.) 39, 13-25. Townsend, A. R. M., Rothbard, J. M., Frances, M., Gotch, G., Bahadur, J., Wrath, D., and McMichael, A. J. (1986). The epitopes of influenza nucleoproteins recognized by cytotoxic lymphocytes can be defined with short synthetic peptides. Cell (Cambridge, MUSS.)44,959-968. Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Muller, P., Falk, W., Debatin, K. M., and Krammer, P. H. (1989).Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245,301-305. Treves, A. J., Cohen, I. R., and Feldman, M. (1975). Immunotherapy of lethal metastases by lymphocytes sensitized against tumor cells in uitr0.J.Natl. Cancer Znst. (U.S.)54, 777-780.

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ADVANCES IN IMMUNOLOGY,VOL. 49

The Development of Rational Strategies for Selective lmmunotherapy against Autoimmune Demyelinating Disease LAWRENCE STEINMAN Deparfments of Neurology and Neurological Sciences, Pediatrics, and Genetics, Stonford Univemity School of Medicine, Stonford, Colifomio 94305

1. Introduction

The pace of research on the pathogenesis and treatment of multiple sclerosis (MS), the principal human demyelinating disease of the central nervous system (CNS), has intensified in the past 3 years. In part, this is due to the application of advances in molecular biology, such as the polymerase chain reaction (PCR), and to developments in cellular immunology, such as technology for the growth of T cell clones. Many lessons that have been learned in an animal model of CNS demyelinating disease, experimental allergic encephalomyelitis (EAE), have been verified in the human disease MS. Indeed, certain successful approaches for treatment of EAE are being attempted in MS at the present time. Recent work on EAE in my laboratory has been the subject of extensive reviews in each of the last 2 years, 1989 and 1990, in Annual Reviews of Immunology (1,2) and elsewhere (3-6). The reader is kindly referred to these texts for background. This review describes the strong parallels that exist between T cell receptor (TCR) usage in the pathogenesis of EAE, and TCR usage in myelin basic protein (MBP)-specific T cells in the peripheral blood of MS patients (7-9) and in T cells in demyelinative plaques in MS brain (10). Based on these similarities, selective immunotherapy that targets either class I1 molecules of the major histocompatibility complex (MHC) or TCR-variable (TCR-V) regions will be described in EAE, with consideration given to application of these principles in MS. These new therapeutic approaches involve monoclonal antibodies (Mabs) directed to either HLA class I1 molecules or TCR-V region molecules, or peptides that compete with HLA class I1 molecules or vaccination against TCR-V regions. II. Multiple Epitopes of Myelin Basic Protein in Mice and Humans

Chronic EAE with relapses and remissions and with pathologic evidence of demyelination can be induced in mice with peptide frag357 Copyright 6 1991 by Academic Press, Inc. All rights of reproduction in any forni reserved.

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LAWRENCE STEINMAN

ments of MBP (11-16). Other peptide fragments of MBP are also immunogenic, but instead of inducing disease these fragments can protect mice from the induction of EAE caused by a pathogenic fragment. Thus, not all immunogenic epitopes of MBP are pathogenic. In fact, some epitopes that are protective are actually immunodominant in comparison to pathogenic epitopes (17). The isolation of MBP-reactive T cell clones that mediate EAE facilitated the identification of individual encephalitogenic epitopes. The encephalitogenic T cell epitope within MBP 1-37 in H-2" mice was the first to be identified and has been characterized in greatest detail (11,12,18).In fact, within this epitope, the amino acids that contact the MHC and TCR have been deduced (19,20). We had initially observed that separate forms of native (intact) MBP, varying in their N-terminal sequences, differed in their ability to stimulate individual MBP clones that were encephalitogenic and responded to MBP fragment 1-37, restricted by I-A". In fact, bovine MBP, which is less encephalitogenic in PL/J mice, was less stimulatory than rat or mouse MBP. Because bovine MBP 1-37 differs from the mouse MBP 1-37 sequence at residues 2 and 17 only (Fig. l), we predicted that the epitope recognized by these clones would include one of these two residues (12). Using overlapping synthetic peptides containing these two residues, we determined that the encephalitogenic epitope was located within the first 11 residues. Peptides 1-11 and 1-16 were equipotent with intact rat or mouse MBP. Shorter peptides, pl-7 and pl-9, were less stimulatory (12). A few features of this T cell epitope were intriguing. First we noted that Acl-l1[4A], with an alanine-for-lysine substitution at position 4, produced a heteroclitic proliferative response compared to Acl-11 in encephalitogenic T cell clones reactive to the N terminus (18-20). Using a photoaffinity probe to measure direct binding to I-A", Wraith and co-workers showed that Acl-l1[4A] binds to I-A" with at least a 10-fold higher relative affinity when compared to Acl-11 (19,20). In contrast, peptides Acl-l1[3A] and Acl-ll[GA] did not stimulate T cell clones or T cell hybridomas reactive to Acl-11 (20). However, peptides Acl-l1[3A] and Acl-l1[6A] both significantly inhibited binding of the photoprobe to I-A" at 1000-fold molar excess. This implies that their inability to activate Acl-ll-reactive T cells reflects a defect in TCR interactions rather than in I-A" binding. To determine whether (PL/J X SJL)F1 mice possess T cells capable of responding to Acl-l1[3A] or Acl-l1[6A], they were immunized with these two substituted peptides as well as with the original Acl-11 peptide. Ten days later, their lymph node cells were stimulated in

359

AUTOIMMUNE DEMYELINATING DISEASE 10

20

RaVGuinea Pig MBP

Ac-AS O K R P S O R

H G SK Y L A T A S T M D H A R

Mouse MBP

Ac-

0

Bovine MBP

AC--A

(

-1

30 HG FL P R HR DTG I

S

FIG. 1. Amino acid sequences of the N-terminal portion of various myelin basic proteins from different species.

uitro with these same peptides. Peptide 89-101 was used as a negative control. When mice were immunized with Acl-11 they generated a good proliferative response when stimulated in vitro with Acl-11, but generated apoor response to either Acl-l1[3A] or Acl-ll[GA]. In the reciprocal experiment, mice immunized with Acl-l1[3A] generated a good proliferative response to Acl-l1[3A], but a very poor response to Acl-11. Similarly, mice immunized with peptide Acl-ll[GA] generated a good response to Acl-ll[GA], but a relatively poor response to Ac 1- 11. These results show that (PL/J x SJL)F1 mice are able to generate T cell responses to both Acl-l1[3A] and Acl-ll[GA], but that the majority of these responses are mutually non-cross-reactive with the response to Acl-11. They also explain why these substituted peptides failed to stimulate hybridoma 1934.4 and the panel of encephalitogenic T cell clones tested previously, even though they were able to bind to I-A". Taken together with the peptide binding analysis, these lymph node proliferation experiments show clearly that residues 3 and 6 of Acl-11 determine TCR interactions rather than I-A" interactions. MBP occurs naturally acetylated at its N terminus. Acetylation of residue 1, NAc-Ala, was essential for stimulation of all encephalitogenic clones that recognize the N terminus of MBP. Using a photoaffinity probe to measure direct binding of peptides to I-A", Wraith and co-workers showed that unacetylated 1-11[4A] (peptide 1-11, with alanine substituted at residue 4 for lysine) bound to I-A" weakly compared with Acl-l1[4A]. However, despite its decreased binding to I-A", unacetylated 1- 11[4A] effectively activated encephalitogenic T cell hybridoma 1934.4,which was produced by fusion of an encephalitogenic T cell clone reactive to Acl-11 and restricted by I-A" (20). Peptide 1-11 does not bind to I-A" or stimulate encephalitogenic T cell clones reactive to the N terminus of MBP (12). This implicates the N-acetyl group as a determinant important in interactions with I-A", but not absolutely necessary for effective TCR interactions. Taken

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LAWRENCE STEINMAN

together, these data confirm the assignment of function to particular residues of Acl-11. Thus, Ac-Ala residue 1contacts the MHC, as does residue 4,Lys. Residues 3 and 6, Glu and Pro, interact with the TCR (Fig. 2). We have described other epitopes in the N-terminal region of MBP that are protective rather than pathogenic. Thus, PLSJ F1 mice immunized with MBP give better proliferative responses to AcN9-20 than to Acl-11. AcN9-20 can protect mice from EAE induced with Acl-11 ( vide infru ). AcN9-20 also elicited a stronger proliferative response in PLSJ mice than did pN35-47, which is encephalitogenic (21). A third epitope within the N terminus was identified. Nonencephalitogenic T cell clones isolated from homozygous PL/J or (PLSJ)F1 mice, restricted by Aa"Afi" or Aa"AF, respectively, recognize MBP 1-37 of rat or guinea pig MBP, but not mouse (self) MBP. Rat and guinea pig MBP contain His-10 and Gly-11, which are deleted in the mouse MBP sequence (Fig. 1). The epitope recognized by these clones is located within residues 9-16. Thus, in I-A" and in I-A"'s mice, certain peptides within the N terminus were encephalitogenic and others were immunogenic and protective (17). It is not clear what features of certain peptides render them encephalitogenic. Even encephalitogenic peptides do not always trigger EAE-inducing T cell clones that bear receptors for these peptides and that proliferate in response to them. We hypothesized that perhaps lymphokine activity in T cell clones could correlate with their pathogenicity. Thus, lymphokine activity in seven MBP-specific T cell clones was examined (22). All of these clones recognize MBP peptide AcN1-9. Five of these clones have the same VP and V a gene usage and exhibit similar Va-Ja and VP-DP-JP rearrangements (22). All of these clones were stimulated at similar concentrations of MBP, except one clone that proliferated at a 5- to 10-fold lower dose of NAcl-9. A strong positive correlation was found between levels of lymphotoxin (LT)and tumor necrosis factor-a! mRNA and the capacity of these clones to induce paralysis (22). No correlation was found

-

+

-

+

AC A - S - 0 - K - R P - S - 0 - R - H - G

' MHC interaction

+

TCR interaction

FIG.2. Putative interactions of N-terminal amino acids of myelin basic protein with TCR or MHC.

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AUTOIMMUNE DEMYELINATING DISEASE

between interleukin-2 or interferon-y production and encephalitogenicity (22). Encephalitogenic fragments within p89-169 of MBP, the fragment that causes EAE in H-2s strains, have been identified. Epitopes were predicted using Rothbard's template for TCR recognition of peptide (23). The encephalitogenic epitope p89-101 contains the tetramer (HFFK) that Rothbard's guidelines predicted to be immunogenic (13,14). In contrast with the encephalitogenic response to the N terminus, there is more than one discrete population of encephalitogenic I-A" restricted T cells for SJL/J mice. Two distinct, overlapping encephalitogenic peptides were identified ( 13,14).One group of encephalitogenic clones recognizes both p89-101 and p89-100 ( 1 3 ~ 4 )The . clones recognizing p89-101 utilize the TCR gene Vpl7, whereas the p89-100 clones do not (13,14) (Table I). Both peptides p89-100 and p89-101 are encephalitogenic for SJL/J mice. Another encephalitogenic peptide in SJL/J mice, p96-109, was identified by Kono and colleagues (15)(Table I). Another I-AS-restrictedencephalitogenic epitope was discovered by Fritz and co-workers; an SJL/J MBP-specific T cell line that recognizes p17-27 causes EAE in recipient mice (16). Other cryptic encephalitogenic epitopes have been identified in H-2" mice. An epitope recognized by an encephalitogenic T cell clone, restricted to a hybrid I-E molecule (Table I), was identified. This T cell epitope, p35-47, contains sequence 42-45 RFFS, as predicted from Rothbard's template (23);p35-47 causes an EAE in H-2" mice that is as severe as that caused by MBP pNAcl-11. Thus, in Table I we enumerate seven immunologic epitopes of MBP in just two inbred mouse strains. The implications of this diversity of TABLE I MULTIPLEDISCRETE T CELLEPITOPES OF MYELINBASICPROTEIN Peptide

Encephalitogenic potential

Class I1 restriction

pl-11

+

Aa"Ai3"

p5-16 p17-27 p35-47 p89-100 p89-101 p96- 109

-

AaUAPL1, AaSAP Aa"Ap Ea"EP", Ea"EW AaSAp Aa'Ap AdAp

ND. Not determined.

?

+ + + +

VlX

VP

Va4.2 Va2.3 ND" ND ND ND ND ND

Vp8.2 Vp13 Vp8 ND Vp8VP17Vp17 ND +

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LAWRENCE STEINMAN

encephalitogenic and nonencephalitogenic epitopes for outbred human populations will be discussed below. It should be noted that other myelin antigens, such as proteolipid protein (PLP),also cause EAE. A T cell line directed against PLP can adoptively transfer EAE. An encephalitogenic region, p139-151, has been identified for SJLIJ mice (24). Several groups have now identified immunogenic epitopes of MBP in MS patients and in healthy human controls. Hafler and colleagues defined the T cell specificity toward MBP in patients with MS, other neurologic diseases, and normal controls (8).Both MS patients and controls who were DR2,DQwl had T cell lines that proliferated to MBP 84-102, which included the encephalitogenic epitope for H-2s mice. The frequency of these lines was somewhat higher in MS patients (7.2 & 2.4%) compared to patients with other neurologic disease (4.1 1.0%)or normals (4.7 1.6%). A second region between MBP residues p143-168 elicited proliferation in T cell lines whose occurrence was equal among MS patients and controls. This epitope was associated with the DRwll phenotype. Significant but less striking increases in the frequency of reactivity to MBP residues 61-82 and 124-142 were also observed in MS patients. It is difficult to know whether an immunodominant epitope, determined by reference to its ability to proliferate and incorporate [3H]thymidine in response to antigen, would be pathogenic. Pathogenic epitopes are not always immunodominant, and immunodominant epitopes can be protective (17). Hafler and colleagues are acutely aware of these limitations and provide the following suggestions: _+

_+

To show that MS is a cell-mediated autoimmune disease analogous to EAE, certain criteria can be proposed. First, an association should exist between an immunodominant region of the presumed autoantigen and disease-associated MHC haplotypes (like HLA-DR2, DQwl]. Second, there should be an increase in frequency of T cells that react with this immunodominant epitope. Finally, the course of the disease must be altered by elimination of autoreactive T cells or by inducing immune tolerance to the autoantigen identified in the first two criteria. This final condition implies that in oitro experiments on their own cannot prove the association of an autoantigen with a disease, and instead clinical trials are necessary (8).

Martin and colleagues have obtained similar results in studies of cytotoxic T lymphocyte lines that recognize MBP and its fragments in association with HLA class I1 molecules. Both MS patients and healthy controls responded to MBP p87-106 in association with HLA-DR2 and DR4. Of note is that HLA-DR2,Dw2,DQwl and HLA-DR4(DQw7) share amino acid sequences between residues 71 and 83 of the third hypervariable region of the HLA-DQP chain. Martin and colleagues

AUTOIMMUNE DEMYELINATINC DISEASE

363

also described cytotoxic T cell lines recognizing MBP p154-172 in DR2 controls, in DR4 MS patients, and in DR6 individuals with MS and in controls. HLA-DR6,DQwG shares amino acid sequences with HLA-DR2 in the third hypervariable region of the HLA-DQP chain. The sequence p154-172 is encephalitogenic in monkeys (25), and p87-106 includes an encephalitogenic fragment seen in SJL/ J mice (13,14,16)and in Lewis rats (26).

111. Human and Rodent TCR Usage Restriction in T Cells Responding to Specific Epitopes of Myelin Basic Protein

The identification of multiple encephalitogenic epitopes of MBP indicated that the potential repertoire of MBP-specific T cells includes more than one population of T cells. However, the T cell response to each epitope appears limited to discrete populations of T cells. For example, encephalitogenic N-terminal MBP-specific T cell clones could not be distinguished from one another on the basis of their reactivity to peptides of MBP or class I restriction. Furthermore, there was a concordance between in vitro T cell recognition and encephalitogenic potential after active immunization. Both of these results suggested that the TCR repertoire of encephalitogenic N-terminal MBPspecific T cells in H-2” was limited. Recent advances in molecular biology have made it possible to examine the T cell receptor of individual T cells. With this technology it is possible to examine TCR gene expression of T cells mediating EAE, and to address whether T cells that appear phenotypically similar in their Ag/MHC recognition express common TCR genes. TCR gene expression has been examined for the encephalitogenic response to MBP 1-9 and MBP 89-101. A. THEENCEPHALITOGENIC N TERMINUS

TCR gene expression of MBP pl-9-specific T cells has been examined by three approaches: (1) cell surface staining with TCR VPspecific monoclonal antibodies; (2) Southern blot analysis, and (3)TCR gene sequencing. T cell clones from PL/J mice were initially stained with monoclonal antibodies specific for TCR VP8 (27-29). TCR VP8 is a three-member family of TCR genes encoding TCR (28,29) expressed b y several strains, including PL/J. This TCR gene family is deleted in certain strains, including SJL/J and SW/R. When a panel of 18 pl-9specific T cell clones isolated from 14 separate PL/J mice were stained with these antibodies, 14 (78%) expressed TCR VPS (30). This high percentage was a minimum estimate, as potential “sister” clones from

364

LAWRENCE STEINMAN

a single T cell line were excluded in this calculation. When all clones were included, 85% expressed TCR Vp8 (31). In many strains, inlcuding PL/J and BlO.PL, Vp8 is the predominant TCR Vp family expressed, accounting for 16-25% of peripheral T cells. Therefore, we asked whether the high frequency of usage ofTCR Vp8 was an in vitro cloning artifact, or whether it represented Vp8 usage in uiuo. Second, we examined whether the use of TCR Vp8 was specific for MBP 1-11. Lymph node cells from MBP 1-11-primed PLIJ mice were sorted by fluorescein-activated cell sorting (FACS) into CD4+lVP8+and CD4+/VP8- subpopulations. When stimulated i n uitro, >90% of the proliferative response to MBP 1-1 1occurred in the VpS' subpopulation. Thus, the high frequency of Vp8 usage was not a cloning artifact. Furthermore, I-E"-restricted encephalitogenic T cell clones specific for p35-47 are Vp8- (31).When p35-47-specific CD4+/ VpS' and CD4+/VP8- subpopulations were examined in primary cultures, the proliferative response occurred within the TCR Vp8- subpopulation (18).However, TCR Vp8 usage in the response to MBP is not specific for MBP 1-9. When independent I-A"-restricted T cell clones specific for the nonencephalitogenic epitope p5- 16 were examined with monoclonal antibodies specific for TCR Vp8, six of seven (84%) utilized TCR Vp8 (32).These results are also intriguing in that earlier studies by Morel (33)demonstrated that most Vp8' T cell clones specific for myoglobin were I-Ed restricted in DBAI2 (H-2d) mice. From their results, they suggested that V/38 usage correlated with I-E restriction. IfVp usage does correlate with class I1 restriction, based on our results, Vp8 expression may correlate with I-A restriction in PL/J mice. Heterogeneity in the T cell response to MBP 1-9 was further evaluated by molecular genetic techniques. By Southern blot analysis, Vp8.2 was identified as the TCR Vp gene used by Vp8' T cell clones (31,32). This was confirmed by the sequencing of TCR genes of eight MBP 1-9-specific T cell clones. Of these eight clones, seven utilized Vp8.2; one encephalitogenic clone expressed Vp4 (Table 11).There was less restrictive use of D p and Jp, with four clones utilizing Jp2.7, two using Jp2.3, and two clones expressing Jp2.5. Thus, the predominant Vp-Jp, expressed by four (50%) of these clones, was V8.2-Jp2.7. Even less heterogeneity was observed in a-chain gene usage. All eight clones used the same Va, Va4.3, a new member of the Va4 family [also referred to as VaPJR-25; see Acha-Orbea et al. (18)l.Six of these clones utilized JaTA31, one used JaTT11, and one used JaF1-12. The predominant Va-Ja, expressed by six (75%) clones, was Va4.3-JaTA31 (Table 11).Thus there was a striking degree of restriction in the a-and


365

TABLE I1 SUMMARYOF TCR SEQUENCES Clone

VP

JP

Va

Jff

8.2 8.2 8.2 8.2

2.7 2.7 2.7 2.7

PJR-25 PJR-25 PJR-25 PJR-25

TA31 TA31 TA31 TA3 1

8.2 8.2

2.3 2.3

PJR-25 PJR-25

TA31 TA3 1

8.2

2.5

PJR-25

T T ll

4

2.5

PJR-25

F1-12

Group 1

PJB-20 PJpR-2.2 PJpR-6.2 F1-21 Group 2 PJR-25 PJB-18 Group 3 PJpR-7.5 Group 4 F1-12

p-chain TCR gene usage in response to the encephalitogenic N terminus. TCR gene expression for MBP 1-9-specific T cells was examined in another H-2" strain, B1O.PL (34,35). This strain contains the same MHC, the H-2" haplotype, on a BlO background. As in PL/J mice, MBP 1-9 is encephalitogenic in BlO.PL, and pl-9-specific T cells are restricted by I-A" (12).Of 33 MBP 1-9-specific hybridomas, 79% utilized Vp8.2 with Jp2.7 (referred to as Jp2.6 or Jp2.7, depending upon whether or not the sixth J gene of the Jp2 cluster, a pseudogene, is considered in the numerical order) (32,36), and 21% used Vp13 with Jp2.2. Although p-chain gene usage was very similar to that seen for PL/J pl-9-specific T cell clones, a-chain gene expression was somewhat different. In contrast with the PL/J clones analyzed, all having used Va4.3, of the B1O.PL clones examined, 58% used Va2.3 and 42% expressed Va4.2. Both Va2.3- and Va4.2-bearing T cell hybridomas utilized the same J gene,ja39 (34,35). Within PL/J and B1O.PL mice, the expression of TCR genes in the MBP pl-9-specific response is quite strikingly limited. However, when comparing TCR gene expression in these two strains, certain differences are apparent. Even though Vp8.2 is used to the same extent by both strains, it is unclear why Va2.3, which was not expressed by any of the PL/J clones, was used more frequently than Va4 in B1O.PL mice. Polymorphic differences in TCR gene expression may exist among these strains. Clones expressing different V a genes may differ in their pl-Y/I-A" affinity. If so, they may differ in their proliferative capability and/or in uivo function.

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LAWRENCE STEINMAN

Despite these differences in TCR V gene usage in PL/J and B1O.PL mice, in T cell clones recognizing MBP pl-9 in association with I-A”, there is a striking conservation of amino acid sequences in the Va-Ja junction and in the V@-D@-J@junction (18,35). C TERMINUS B. THEENCEPHALITOGENIC TCR gene usage in the encephalitogenic T cell response of SJL/J mice to the C terminus has been examined, although not as extensively as for MBP pl-9. The T cell response appears more complex. Three encephalitogenic peptides have been identified, p89-101, p89-100, and p96-109 (13-15). TCR V@ expression has been examined for T cells that respond to p89-101. Approximately 50% of T cells that proliferate in response to p89-101 also respond to p89-100. The other 50% require Pro-101 for stimulation. TCR V@gene expression for these two populations has been examined with a monoclonal antibody that recognizes Vp17, a single gene family expressed by several I-A+/I-Estrains, including SJL/J (37,38). Interestingly, all clones that recognize p89-101, but not p89-100, use TCRVP17. All clones that proliferate in response to p89-100 are V@17- (13). The TCR V@(s)expressed by Vp17- clones is not known at this time. Examination of TCR a-chain genes and further analysis ofthe @-chaingenes is currently in progress. Examination of susceptibility to EAE in different strains indicates that the MHC genotype, and not the TCR repertoire, controls susceptibility induced with MBP p89-101. H-2’ (1-As) strains SJL/J and A.SW, and H-2“ (I-A“) strains SW/R and BlO.T(GR), strains that differ in non-MHC genes, are all susceptible to EAE induced with MBP p89101.Sequence analysis of A a (39)and A@(40) suggest that I-As and I-Arl are very similar. Interestingly, SJL/J and SW/R have deleted approximately 50% of their VP genes, including V@S. However, these two strains express [email protected] contrast, ASW and BlO.T(GR) express V@8but not Vp17. Thus, susceptibility in this case does not correlate with the absence ofV@8or the expression ofVp17. By examination oftransgenic mice expressing various “susceptible” class I1 genes, it may be possible to assess the relative contribution of the MHC and the TCR repertoire in individual encephalitogenic responses.

C. EAE IN THE RAT The Lewis strain is the most extensively studied rat in EAE. In this strain, MBP p68-88 contains an encephalitogenic determinant, although another encephalitogenic epitope(s) probably exists (41). Encephalitogenic T cells specific for p68-88 are CD4+ and class I1 restricted, although there is some ambiguity in the identification of the


367

exact class 11-restricting element (42).Approximately 50% of CD4+ T cell clones raised against rat MBP are specific for p68-88 (42). The TCR composition of rat T cells specific for p68-88 has been examined by TCR sequencing of one clone and by subsequent probing of other clones by Northern and Southern analysis. The TCR data from the rat clearly support previous studies of MBP pl-9-specific T cells in H-2" mice, demonstrating a marked restriction in usage of Va and Vp in the T cell response to an individual encephalitogenic determinant (43).Of the p68-88-specific T cell hybridomas, 70% utilize the same Va gene and 100% express the same Vp gene. Interestingly, the V a gene is 77% homologous to Va2, one of two V a genes utilized by MBP pl-9specific mouse 'r cell clones. The Vp gene used by these clones is most closely related to mouse Vp8.2, sharing 80% homology. Although there is considerable homology between the V a and Vp genes used in rat and mouse T cell clones, rat T cells do not recognize MBP pl-9 on H-2" antigen-presenting cells (APCs), and conversely, mouse T cells do not respond to p68-88 cultured with rat APCs (43). Offner et al. (44) described an I-E-restricted sequence (p87-99) of rat MBP that is encephalitogenic in the Lewis rat. The TCR recognizing p87-99 in the context of I-E also appears to express Va2 and Vp8. Because of the similarity in Va and Vp gene usage among Lewis rats and H-2" mice, Heber-Katz (43) has suggested that TCR V-region usage is the critical determinant in EAE, independent of Ag/MHC. Although this hypothesis is intriguing, it does not account for all encephalitogenic T cell epitopes. In mice, multiple distinct encephalitogenic T cell epitopes include pl-9, p35-47, and p89-101. Thus, within one species, it is known that at least three separate Vp genes are used in encephalitogenic T cell responses to MBP (Table 11).Nevertheless, the TCR data from the rat clearly support previous studies of recognition of MBP pl-9 in H-2" mice in at least one aspect: there is a marked restriction in usage of V a and Vp in the T cell response to an individual encephalitogenic determinant. TCR usage has been studied by Hafler and colleagues in human peripheral blood-derived T cell clones that respond to epitopes on MBP (7). TCR usage in MS brain plaques has been evaluated by Oksenberg and colleagues (10).The Vp gene usage in 83 peripheral blood-derived T cell lines from MS patients and from healthy subjects reactive with MBP residues 84-102 was studied (7). One MS patient, Hy, who was HLA-DR2,DR7, had 24 out of 31 clones that used Vp17. Other p84-102-reactive clones from this patient used Vpl, Vp2, Vpll, Vp4, Vp7, and Vp14. A second MS patient who was also DR2,DR7 had one out of four clones utilizing Vp17, with Vp3, Vp4, Vp5, VPS,

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LAWRENCE STEINMAN

and Vp8 being expressed in other clones from this patient (Fn). A DR2, DRwll MS patient responding to p84-102 used Vpl2, Vp17, Vpl, Vp2, and Vp7. A DR2,DR4 patient used Vpl2 and Vp17, as well as Vpl, Vp3, Vp5, Vp7, and Vp14. One control (DR2) responding to p84-102 used Vp17 in all five clones, and two other DR2 controls used Vpl2 and Vp5, and Vp6, Vp8, and Vp18, respectively. Comparison of the VDJ sequences of the six Vp17' T cell lines from subject Hy demonstrated only two different Dp sequences and only one Jp sequence, Jp2.l. T cell lines reactive to MBP p143-168 were studied in patients and controls. The DR2,Dwll patient who had other T cell lines responding to p84-102 used Vp14 in six of nine lines responding to p143-168. However, three other patients and two controls who had pl43-168-reactive T cell lines used Vp sequences other than Vp14 in the response to p143-168. These Vp genes included Vpl, Vp2, Vp3, Vp4, Vp5, Vp7, Vp8, Vpl2, and Vp17 (7). The identification of activated T cells in the brain of individuals with MS indicates that these cells are critical in the pathogenesis of this disease. In an attempt to elucidate the nature of the lymphocytic infiltration, we used the polymerase chain reaction (PCR) to amplify T cell antigen receptor V a sequences from transcripts derived from MS brain lesions. In each of three MS brains, only two to four rearranged TCR V a transcripts were detected. Va transcripts encoded by the Va12.1 region showed rearrangements to a limited number of Ja region segments. These results imply that TCR V a gene expression in MS brain lesions is restricted. Vp usage has also been studied in plaques from these patients. Vp usage included TCR Vp genes 5.1,5.2,7, and 18.Three Vp genes were expressed in each of these brains.

IV. Possibilities for Future Immune Intervention in Multiple Sclerosis

A. ANTIBODIESTO HLA CLASSI1 MOLECULES Nearly 60% of MS patients of Northern European Caucasoid background are HLA-DR2,DQwG (45). Comparison of nucleotide sequences in the membrane distal domain of DQp chains of haplotypes associated with susceptibility to MS reveals that the major subtypes [DR2,DQw6; DR4,DQw7; DR4,DQw8; DRw6,DQwG (18); and DRw6,DQwG (IS)]are identical or nearly identical for long stretches, including the highly polymorphic region encoding amino acids 71-83 (46).


369

1 . Prevention and Treatment of EAE with Anti-Class 11 Antibodies In 1981 it was demonstrated that EAE could be prevented by injection of anti-I-A prior to immunization with spinal cord homogenate (47). Anti-I-A treatment reduced the influx of radiolabeled lymphocytes that home to the CNS in EAE (48). When anti-I-A treatment is given after the first appearance of paralysis in EAE, mice return to normal within 48 hours. Anti-I-A treatment also reduced the number of relapses and mortality in chronic relapsing EAE (49). In rhesus monkeys, treatment of paralytic disease was successful with polymorphic mouse anti-HLA-DQ or HLA-DR antibodies that react with Rh-LAD (50).Therapy with monoclonal anti-class I1 antibodies is partially specific, blocking only responses restricted by a given class I1 isotype. Thus, although anti-I-A blocks EAE, experimental autoimmune myasthenia gravis (EAMG), and thyroiditis, in each of these diseases responses to purified protein derivative (PPD) were left intact (51,52).

2. Peptides that Block lnteraction of T Cells with the M H C EAE is a prototypic T cell-mediated autoimmune disease. MBP, a major component of myelin in the central nervous system, is one of the autoantigens capable of sensitizing encephalitogenic T cells. Several immunogenic determinants in MBP have been identified by using pepsin-digested peptides and synthetic oligopeptides. Although these determinants induce strong T cell immune responses in the context of a certain MHC class I1 molecule, not all the determinants are encephalitogenic. The N-terminal peptide AcN1-20 contains an 1-A"restricted, dominant encephalitogenic epitope. When the N-terminus of AcN1-20 is deacetylated, this peptide loses its encephalitogenicity. However, the immunogenicity of this deacetylated peptide, N1-20, still remains. Detailed mapping of T cell epitopes revealed the existence of another I-AU-restricted epitope, N9-16, within the deacetylated peptide, and this epitope was found to nonpathogenic. The known epitopes of MBP, their capacity to induce EAE, their MHC class I1 restriction, and the nature of the variable region gene of the T cell receptor /3 chain (Vp) are given in Table 111. It has been demonstrated that peptide fragments of antigenic proteins directly bind to the MHC class I1 molecule to be recognized by T cells. It has been suggested that a given MHC molecule has a single functional antigen-binding site. Peptides from unrelated antigens can compete with one another for T cell activation. Extending the observations, the possibility has been proposed that the relative immunodominance of an epitope might be determined in part by its affinity for the MHC class I1 molecule.

370

LAWRENCE STEINMAN

TABLE I11 DISCRETE T CELLEPITOPES OF MBP IN MICE Peptide AcN 1-20 N1-20 AcN9-16

N35-47 N89- 101 N89-100

Encephalitogenicity

+ c

-

+ t

+

Class I1 restriction I-AEA); I-AEA); I-AEA); I-AkAI; I-EEE); I-EEEj I-AzAj I-A;AsP

TCR" Vp usage

Vp8 predominantly Vp8 predominantly Vp8 predominantly Not known Not Vp8 Not Vp8 Vp17 predominantly Not Vp17

TCR, T cell antigen receptor.

Based on these findings we have attempted to see whether in uiuo competition between pathogenic and nonpathogenic self-peptides can be applied to the prevention of autoimmune disease. We first predicted which competitor peptides might be efficacious in viuo by screening their ability to block in uitro the stimulation of an encephalitogenic T cell clone that recognizes AcN1-20 with I-A". Peptides N 1-20 and AcN9-20 were shown to inhibit proliferative responses to the encephalitogenic peptide AcN1-11 both in uitro and in uiuo (17). AcN1-11 is a strong pathogenic peptide for PL/J and (PLSJ)F' mice, and T cells that can recognize this self-antigen mediate autoimmune encephalomyelitis in these strains of mice. The demonstration that peptide N1-20 can compete in the in uiuo induction of AcN 1-1 1-primed T cells suggested that this nonpathogenic peptide might be able to reduce the induction of autoaggressive T cells and thereby prevent EAE. Thus, the preventive effect of the competitor peptide N1-20 on induction of EAE with AcN1-11 was tested. As shown in Table IV, neither N1-11 nor AcN2-11 could prevent disease, even at a 6 : 1 ratio relative to AcN1-11, whereas injection of N1-20 significantly ( P < 0.001; Fisher's exact test) prevented the clinical development of EAE at a 3 : 1 ratio ( P < 0.001). In addition, AcN9-20 had a preventive effect on EAE at a 3 : 1 ( P < 0.001) or 6 : 1 ( P < 0.001) ratio. Injection of N1-20 at a 3 : 1 or 5 : 1 ratio did not prevent EAE induced with the I-A'-restricted peptide N89-101 in SJL/J mice. In reviewing representative sections of 20 mice treated with competitors (Nl-20 and AcN9-20), which did not show any clinical signs of EAE, no perivascular cuffs or submeningeal cell infiltrates were evident.

37 1


Further experiments with peptide inhibition have been performed. Peptide Acl-l1[4A] binds with greater affinity than does Acl-11 to I-A". Mice coimmunized with Acl-l1[4A] and Acl-11 were protected from EAE (20).Similar results were reported by Urban et al. (53). In a first experiment to test the protective effect of Acl-l1[4A] on EAE induction with Acl-11, Acl-l1[4A] completely inhibited disease induction, with 0 of 14 mice paralyzed compared with 8 of 13 control mice that were paralyzed ( P < 0.001) (20).In a second experiment, the protective effect of coimmunization with peptide Acl11[4A] was confirmed. The overall incidence of disease was substantially reduced ( P < 0.001), with 3 paralyzed out of 15 coimmunized mice, versus 14 of 15paralyzed control mice. The onset of disease was significantly delayed in the coimmunized group. Early disease began at day 8 in the control Acl-11 group. By day 14,9 of 15 control mice were paralyzed versus 0 paralyzed of 15 coimmunized mice ( P < 0.001). Late disease did not begin until day 16 in the coimmunized mice (20). TABLE IV PREVENTION OF EAE IN (PLSJ)F,AND SJL/J MICE WITH THE COMPETITOR PEFTIDES" Encephalitogen (nmol)

Mice

Competitor (nrnol)

Incidence of E A E ~

Day of onset

Severity

13.5 2 0.5 13.6 f 1.0

3.1 ? 1.1 3.0 f 1.2

15.1 f 1.7 11.0 17.4 t 0.5 17.0 15.0 2 1.5 12.0 2 0 12.6 5 0.6 15.2 5 2.1 15.8 2 1.6

2.9 t 1.2 2.0 3.8 ? 1.6 4.0 3.1 5 0.6 2.0 f 1.7 3.6 t 0.6 2.2 t 1.2 3.0 2 1.0

~

AcNl-ll(100)

PLSJFl

AcN1-ll(lO0)

PLSJFl

AcN1-ll(lO0)

PLSJFI

AcNl-ll(100)

PLSJFl

N89-101 (200)

SJL/J

N89-101(200)

SJLlJ

None AcN2-11 (600) N1-20 (600) None N1-20 (300) None AcN9-20 (600) None AcN9-20 (300) None N1-20 (1000) None N1-20 (600)

6/11 8/12 0/10* 10119 1/16* 5/11 1/12* 7/12 0/10* 3/14 319 4/11 518

Incidence of EAE is expressed as number of mice with clinical EAEhumber of mice immunized; day of onset as mean day of onset ? SD; severity as mean severity of sick mice SD. For the induction of EAE, mice were immunized with MBP peptide AcN1-I1 (100 nmol) or N89-101 (200 nmol) that had been dissolved in phosphate-buffered saline (PBS)and emulsified with complete Freund's adjuvant (CFA) in a 1 : 1mixture of PBS and CFA containing H37Ra. For prevention of EAE, the mixture included the competitor peptide (300,600,or loo0 nmol). On the same day and 48 hours later, pertussis toxin (List Chemicals) was injected intravenously. Mice were examined daily for signs of EAE and assessed for clinical severity, graded from 1to 5 as described (17). Some animals were killed 23-27 days after immunization for histological examination. *, Significant at P < 0.01 (x2 or Fisher's exact test).

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LAWRENCE STEINMAN

B. ANTIBODIESTO TCR V REGIONMOLECULES The monoclonal antibody F23.1 depletes VP8' T cells from the peripheral blood (29). T cells reactive with Mab F23.1 constitute 25% of the T cells in lymph nodes of normal PL/J mice. In the (PLSJ)Fl mouse this percentage is 14%. The depletion of T cells reactive with Mab F23.1 is 98% complete 3 days after intraperitoneal (ip)administration of a dose of 0.5 mg (32). EAE was first induced with T cell clone PJR-25. This clone is fully encephalitogenic, capable of inducing paralysis and demyelination (11,54). PJR-25 expresses the epitope recognized by Mab F23.1 (31). Therapy was begun 24 hours after the mice first developed paralysis. In two experiments (PLSJ)F1 mice were randomly divided into two groups, with 16 mice each receiving two 100-pg injections of F23.1 ip at 72-hour intervals and 16 mice each receiving Mab L e u 9 b (S5.2),an isotype-matched control reactive with the CD2 antigen (a pan T cell marker on human but not on mouse T cells). Within 2-4 days, mice receiving F23.1 showed a marked reversal in their paralysis, and 13out of 16 were completely free of disease 10 days after therapy started. Only one relapse with tail weakness was seen, on day 35, in the animals given Mab F23.1. Next we tested whether EAE induced with pl-11 in complete Freund's adjuvant (CFA) in (PLSJ)Fl mice could be prevented with Mab F23.1. Immunization with MBP peptide pl-11 in CFA can induce clones that are both F23.1-positive and -negative and that are fully encephalitogenic. Successful prevention of disease with F23.1 would indicate that the F23.l-positive T cell clones predominate in the development of disease and that the depletion of these T cell clones in viuo would not simply result in an escape to F23.l-negative T cell clones that would cause disease. Results shown in Table V indicate that whereas 1 of 19 mice receiving Mab F23.1 developed EAE, 9 out of 20 mice given Mab S5.2 became paralyzed ( P < 0.001). These results serve to indicate that the Vp8-expressing clones function in the induction of EAE. (PLSJ)F1mice were immunized with guinea pig MBP. In (PLSJ)Fl mice there are at least two distinct encephalitogenic epitopes for MBP, pl-11 and p35-47. The response to p35-47 is restricted to I-EL'and involves mostly Vp8- T cells. After paralysis was present, mice were given 0.2 mg ip of the Mab F23.1 or KJ23,, a monoclonal antibody specific for the product of the TCR VP17, gene product (38,39).KJ23, prevents EAE induced with T cell lines responsive to MBP p89-101 in the SJL mouse. Of 19 (PLSJ)F1 mice given F23.1, 12 returned to

373


TABLE V PREVENTION OF MBP PEETIDE P1-I I-INDUCED EAE WITH Mab F23.1 ~~~

Monoclonal antibodya

Incidenceb

Clinical disease mean onset (day)

F23.1 S5.2

1/19 9/20

20 15'

a MabF23.1orS5.2 wasgivenip(500wg)ondays -1, 1, and 9; immunization with pl-11 was on day 0. The ratio of number of paralyzed mice to the total number of mice. All mice were examined through day 40. The standard deviation was 1.7days.

'

normal within 72 hours and 21 of 22 mice given KJ23, had moderate to severe paraplegia after 72 hours (Table VI). Relapses were seen in 5 of 19 F23.1-treated mice in the next 14 days. Thus, treatment with F23.1 reversed EAE in a situation wherein multiple encephalitogenic epitopes were present. VP8- T cells capable of responding to MBP pl-11 or P35-47 may have accounted for the relapses seen in the F23.1treated mice. In contrast, the SJL (I-As) mouse strain recognizes a peptide from MBP (p89-101) with at least three overlapping epitopes. There is evidence for limited TCR gene usage in recognition of one of these epitopes (15,16). However, depletion of this subset of T cells did not prevent antigen-induced EAE; elimination of a single V/3 subset, in a TABLE VI REVERSAL OF GUINEA PIGMBP-INDUCED EAE WITH Mab F23.1

Number of mice with clinical symptoms 72 hours after treatmentb Treatment" F23.1 KJ23a

Number of mice with clinical symptoms 14 days after treatment

None

Mild

Severe

None

Mild

Severe

Deaths

12 1

5 12

2 9

14 9

3 2

1

7

1 4

Treatment was begun 24 hours after mice exhibited EAE. At this time the mice were separated randomly into two groups. Mice in each group received one 2OO-pg ip injection of F23.1 or S5.2. Nineteen mice received F23.1 and 22 mice received KJ23a. 'Clinical status was graded as follows: none, no neurologic symptoms; mild, flaccid tail andlor mild paraparesis; severe, severe paraparesis or complete paraplegia.

374

LAWRENCE STEINMAN

polyclonal autoimmune disease such as this, may not be sufficient to prevent or reverse disease. C. VACCINATIONTO TCR V REGIONS Cohen and associates have shown that it is possible to use autoimmune T cell clones or lines as vaccines to prevent or reverse autoimmune disease. An inoculum of T cell clones below the threshold for triggering disease, or irradiated, fixed, or pressure-treated T cells, can serve as vaccines (55-58). The inoculated animals remained free of disease for prolonged times, and EAE could not be induced with T cell lines, T cell clones, or MBP in adjuvant. T cell clones specific for EAE-inducing T cells have been isolated from rats that recovered from EAE, suggesting an antiidiotypic mechanism for protection. These T cell clones are either CD4+ or CD8+. The CD8+ T cells lyse their targets specifically, and this cytotoxicity is not blockable with antiCD4, anti-CD8, anti-class I, or anti-class I1 antibodies. Recently, EAE in the Lewis rat was prevented by immunization with a nonapeptide spanning the V-D-J region of Vp8, expressed on about three-fourths of T cell clones recognizing encephalitogenic MBP p72-86 (59).Vandenbark and associates protected against EAE with a peptide from the CDR2 region of Vp8 (60). Highly selective therapies with antibodies or peptide directed against TCR or HLA class I1 molecules thus appear feasible for treatment of MS, especially because the elucidation of target TCR and HLA molecules is proceeding rapidly. It is worth noting that Teitelbaum and co-workers have treated EAE with a random copolymer (termed COPI) of tyrosine, alanine, lysine, and glutamate. This peptide was successfully employed in therapy of relapsing-remitting MS (61). COPI blocks MHC binding of MBP (62). Highly selective approaches with either monoclonal antibodies or peptides directed against TCR or HLA will be likely to involve several reagents per patient. Zaller and coworkers (63), and Sakai and coworkers (13) have noted that cocktails of monoclonal antibodies directed against different TCR V regions might be necessary to optimize antibody treatment. Thus, even the pathogenic response to a single epitope involves, in most cases, multiple TCRs, though one particular TCR may have a dominant influence on pathogenesis. In human diseases such as MS, the immune response, especially after disease is established, may involve multiple epitopes on several myelin antigens, including, but not limited to, MBP or PLP. In any case, it is likely that, given an individual patient's particular TCR repertoire and HLA type, therapy will be customized to a certain extent. Given the ease


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with which it may now be possible to produce Mabs to human TCR V regions (64), and given current technologies to humanize and chimerize Mabs, it should not be difficult to envision being able to select from a set of humanized Mabs for any combination of TCR V a or V/3 chains that the physician wishes to target. A pharmaceutical company with such an armamentarium might be in an enviable position. Similarly, the formulation of peptide-based TCR vaccines should be feasible, again allowing for a physician to customize therapy from an available set of peptides from all human TCR V a or Vp regions. Finally, the design of pharmaceuticals that interfere with TCR-MHC interactions should be pursued vigorously in light of the success attained thus far with peptides that block TCR-MHC interactions in

EAE. ACKNOWLEDGMENTS The editorial assistance of T. Montgomery is appreciated. This work was supported by the National Institutes of Health, the National Multiple Sclerosis Society, and the Phil N. Allen Trust.

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10. Oksenberg, J. R., Stuart, S., Begovich, A. B., Bell, R., Erlich, H., Steinman, L., and Bernard, C. C. A. (1990).Limited heterogeneity of rearranged T cell receptor transcripts in brains of multiple sclerosis patients. Nature (London)345,344-346. 11. Zamvil, S., Nelson, P., Mitchell, D., Knobler, R., Fritz, R., and Steinman, L. (1985). Encephalitogenic T cell clones specific for myelin basic proterin: An unusual bias in antigen presentation. J. Exp. Med. 162,2107-2124. 12. Zamvil, S. S., Mitchell, D. J., Moore, A. C., Kitamura, K., Steinman, L., andRothbard, J. B. (1986). T cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis. Nature (London)324,258-260. 13. Sakai, K., Sinha, A., Mitchell, D. J., Zamvil, S. S., McDevitt, H. O., Rothbard, J. B., and Steinman, L. (1988).Involvement of distinct T cell receptors in the autoimmune encephalitogenic response to nested epitopes of myelin basic protein. Proc. Natl. Acad. Sci. U.S.A.85,8608-8612. 14. Sakai, K., Zamvil, S. S., Mitchell, D. J., Lim, M., Rothbard, J. B., and Steinman, L. (1988).Characterization of a major encephalitogenic T cell epitope in SJL/J mice with synthetic oligopeptides of myelin basic protein. J. Neuroimmunol. 19,21-32. 15. Kono, D. H., Urban, J. L., Horvath, S. J., Ando, D. G., Saavedra, H. A., and Hood, L. (1988).Two minor determinants of myelin basic protein induce experimental allergic encephalomyelitis in SJL/J mice.J. E x p . Med. 168,213-227. 16. Fritz, R. B., Skeen, M. J., Chou, C.-H. J., and Zamvil, S . S. (1991).Localization ofan encephalitogenic epitope for the SJL mouse in the N-terminal region of myelin basic protein. J . Neuroimmunol. (in press). 17. Sakai, K., Mitchell, D. J., Hodgkinson, S. I., Zamvil, S. S., Rothbard, J. B., and Steinman, L. (1989). Prevention of experimental encephalomyelitis with peptides that block interaction of T cells with major histocompatibility complex proteins. Proc. Natl. Acud. Sci. U.S.A.86,9470-9474. 18. Acha-Orbea, H., Mitchell, D. J., Timmerman, L., Wraith, D. C., Waldor, M. K., Tausch, G . S., Zamvil, S. S., McDevitt, H. O., and Steinman, L. (1988). Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell (Cambridge,Muss.)54,263273. 19. Wraith, D. C., McDevitt, H. O., Steinman, L., and Acha-orhea, H. (1989). T cell recognition as the target for immune intervention in autoimmune disease. Cell (Cambridge,Mass.) 57,709-715. 20. Wraith, D. C., Smilek, D. E., Mitchell, D. J., Steinman, L., and McDevitt, H. 0. (1989). Antigen recognition in autoimmune encephalomyelitis and the potential for peptide mediated immunotherapy. Cell (Cambridge, Mass.)59,247-255. 21. Zamvil, S., Mitchell, D., Moore, A,, Schwarz, A., Stiefel, W., Rothbard, J. B., and Steinman, L. (1987). T cell specificity for class I1 (I-A) and the encephalitogenic N-terminal epitope of the autoantigen myelin basic pr0tein.J. Immunol. 139, 10751079. 22. Powell, M. B., Mitchell, D., Lederman, J., Buckmeier, J., Zamvil, S. S., Graham, M., Ruddle, N. H., and Steinman, L. (1990). Lymphotoxin and tumor necrosis factoralpha production by myelin basic protein specific T cell clones correlates with eucephalitogenicity. l n t . lmmunol. 2,539-544. 23. Rothbard, J. B., and Taylor, W. R. (1988).A new sequence pattern common to T cell epitopes. E M B O J . 7,93-100. 24. Sato, J., Sakai, K., Endoh, M., Koike, F., Kunishita, T., Namikawa, T., Yamamura, T., and Tabira, T. (1987). Experimental allergic encephalomyelitis mediated by murine encephalitogenic T cell lines specific for myelin proteolipid apopr0tein.J. It?1mU710l. 138,179-184.


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25. Karkhanis, Y. D., Carlo, D. J., Brostoff, S. W., and Eylar, E. H. (1975). Allergic encephalomyelitis. Isolation of an encephalitogenic peptide active in rhesus monkey.J. Biol. Chem. 250, 1718. 26. Vandenbark, A. A., Hashim, G. A., Celnik, B., Galang, X., Li, E., Heber-Katz, E., and Offner, H. (1989). Determinants ofhuman myelin basic protein that induce encephalitogenic T cells in Lewis rats.+/. Immunol. 143,3512. 27. Haskins, K., Hannum, C., White, J., Roehm, N., Kubo, R., Kappler, J., and Marrack, P. (1984).The antigen-specific major histocompatibility complex-restricted receptors on T cells. VI. An antibody to a receptor a1lotype.J. E x p . Med. 160,542-571. 28. Staerz, U. D., Rammensee, H.-G., Benedetto, J. D., and Bevan, M. J. (1985).Characterization of a murine monoclonal antibody specific for an allotype determinant on T cell antigen receptor. J . Immunol. 134,3994-4000. 29. Behlke, M. A., Henkel, T. J . , Anderson, S. J., Lan, N. C., Hood, L., Braciale, V. L., Braciale, T. J., and Loh, D. (1987).Expression of a murine polyclonal T cell receptor marker correlates with the use of specific members of the Vp8 gene segment subfamily. J . E x p . Med. 165,257-262. 30. Zamvil, S. S., Mitchell, D. J., Lee, N. E., Moore, A. C., Waldor, M. K., Sakai, K., Rothbard, J. B., McDevitt, H. O., Steinman, L., and Acha-Orhea, H. (1988). Predominant expression of a T cell receptor Vp gene subfamily in autoimmune encephalomyelitis. J . E x p . Med. 167, 1586. 31. Zamvil, S. S., Nelson, P. A., Steinman, L., and Mitchell, D. J. (1989). Treatment of autoimmune encephalomyelitis with an antibody to T cell receptor /3 chain. In “Cellular Basis o f Immune Modulation” (J. G. Kaplan, D. G. Green, and R. C. Bleackley, eds., pp. 461-464. Alan R. Liss, New York. 32. Burt, D. S., Mills, K. H. G., Skehel, J. J., and Thomas, D. B. (1989). Diversity of the class I1 (I-I”/I-Ek)-restrictedT cell repertoire and influenza hemagglutinin and antigenic drift. Six nonoverlapping epitopes on HA1 subunit are defined by synthetic peptides. I . E x p . Med. 170,383-397. 33. Morel, P. A., Livingstone, A. M., and Fathman, C. G. (1987). Correlation of T cell receptor Vp gene family with MHC restricti0n.J. E x p . Med. 166,583-589. 34. Urban, J. L., Kumar, V., Kono, D. H., Gomez, C., Horvath, S. J., Clayton, J., Ando, D. G., Sercarz, E. E., and Hood, L. (1988).Restricted use ofT cell receptor Vgenes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy. Cell (Cambridge, Mass.) 54,577-592. 35. Kumar, V., Kono, D. H., Urban, J. L., and Hood, L. E. (1989). The T cell receptor repertoire and autoimmune diseases. Annu. Rev. Immunol. 7,657-682. 36. Fink, P. J., Matis, L. A., McEllingott, D. L., Bookman, M., and Hedrick, S. M. (1986). Correlation between T cell specificity and the structure of the antigen receptor. Nature (London)32,219-226. 37. Kappler, J. W., Wade, T., White, J., Kushnir, E., Blackman, M., Bill, J., Roehm, N., and Marrack, P. (1987).AT cell receptor Vp segment that imparts reactivity to a class I1 major histocompatibility complex product. Cell (Cambridge, Mass.) 49, 263271. 38. Kappler, J. W., Roehm, N., and Marrack, P. (1987).T cell tolerance by clonal elimination in the thymus. Cell (Cambridge, Mass.) 49,273-280. 39. Benoist, C. O., Mathis, D. J., Kanter, H. R., Williams, V. E., and McDevitt, H. 0. (1983). Regions of allelic hypervariability in the murine A a immune response gene. Cell (Cambridge, Mass.) 34, 169-177. 40. Estess, P., Begovich, A. B., Koo, M., Jones, P. P., and McDevitt, H. 0. (1986). Sequence analysis and structure-function correlations of murine q,k,u,s, and f haplotype I-AP cDNA clones. Proc. Natl. Acad. Sci. U.S.A. 83,3594-3598.

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41. Happ, M. P., and Heber-Katz, E. (1987).Differences in repertoire of the Lewis rat T cell response to self and non-self myelin basic proteins. J . E x p . Med. 167,502-513. 42. Heber-Katz, E . , and Acha-Orbea, H. (1989). The V-region disease hypothesis: Evidence from autoimmune encephalomyelitis. Immunol. Today 10, 164-169. 43. Burns, F. R., Li, X., Shen, N., Ofher, H., Chou, Y. K., Vandenbark, A. A., and Heber-Katz, E. (1989).Both rat and mouse T cell receptors specific for the encephalitogenic determinants of myelin basic protein use similar V a and Vp chain genes. J . E x p . Med. 169,27-39. 44. Offner, H., Hashim, G. A., Celnik, B., Galang, A., Li, X., Burns, F. R., Shen, N., Heber-Katz, E., and Vandenbark, A. A. (1989).T cell determinants of myelin basic protein include a unique encephalitogenic I-E-restricted epitope for Lewis rats. J. E x p . Med. 170,355-367. 45. Tiwari, J. L., and Terasaki, P. I. (1985).“HLA and Disease Associations.” SpringerVerlag, New York. 46. Todd, J. A., Acha-Orbea, H., Bell, J. I., Chao, N., Fronek, Z., Jacob, C. O., McDermott, M., Sinha, A. A., Timmerman, L., Steinman, L., and McDevitt, H. 0.(1988). A molecule basis for MHC class 11-associated autoimmunity. Science 240, 1003-1009. 47. Steinman, L., Rosenbaum, J. T., Sriram, S., and McDevitt, H. 0. (1981). I n uiuo effects of antibodies to immune response gene products: Prevention of experimental allergic encephalitis. Proc. Natl. Acud. Sci. U.S.A.78,7111-71 14. 48. Steinman, L., Solomon, D., Zamvil, S., Lim, M., and Sriram, S. (1983).Prevention of EAE with anti-I-A antibody: Decreased accumulation of radiolabeled lymphocytes in the central nervous system. J . Neuroimmunol. 5 , 9 1 4 7 . 49. Sriram, S., and Steinman, L. (1983).Anti-I-A antibody suppresses active encephalomyelitis: Treatment model for IR gene linked diseases.J . E x p . Mcd. 158,1362-1367. 50. Jonkers, M., van Lambalgen, R., Mitchell, D., Durham, S. K., and Steinman, L. (1988). Successful treatment of EAE in rhesus monkeys with major histocompati1)ility complex class I1 specific monoclonal antibodies. J . Autoimmun. 1,399-414. and Steinman, L. (1983).In oiuo therapy 51. Waldor, M., Sriram, S., McDevitt, H. 0.. with monoclonal anti-I-A antibody suppresses immune response to acetylcholine receptor. Proc. Natl. Acad. Sci. U.S.A.80,2713-2717. 52. Vladutiu, A., and Steinman, L. (1987).Inhibition of experimental autoininiune thyroiditis in mice by anti-I-A antibodies. Cell. Zmmunol. 109, 169-180. 53. Urban, J., Horvath, S., and Hood, L. (1989). Autoimmune T cells: Immune recognition of normal and variant peptide epitopes and peptide-based therapy. Cell (Cambridge, Moss.)59,257-271. 54. Zamvil, S., Nelson, P., Trotter, J., Mitchell, D., Knobler, R., Fritz, R., and Steinman, L. (1985).T cell clones specific for myelin basic protein induce chronic relapsing EAE and demyelination. Nature (London)317,355. 55. Ben-Nun, A., Wekerle, H., and Cohen, I. R. (1981).Vaccination against autoimmune encephalomyelitis with T lymphocyte line reactive against myelin basic protein. Nature (London)292,60-61. 56. Holoshitz, J., Naparstek, Y., Ben-Nun, A., and Cohen, I. R. (1983). Lines of T lymphocytes induce or vaccinate against autoimmune arthritis. Science 219,56-58. 57. Maron, R., Zerubavel, R., Friedmann, R., and Cohen, I. R. (1983).T lymphocyte line specific for thryoglobulin produces or vaccinates against autoimmune thyroiditis in mice. J . Zmmunol. 131,2316-2322. 58. Lider, O., Reshef, T., Beraud, E., Ben-Nun, A., and Cohen, I. R. (1988). Antiidiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis. Science 239, 181-183.


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This article was accepted for publication in September 1990.


ADVANCES IN IMMUNOLOGY, VOL 49

The Biology of Bone Marrow Transplantation for Severe Combined Immune Deficiency ROBERTSON PARKMAN Division of Reswrch Immunolagy/BoneMarrow Transplantation, Children%Hospital of Lor Angeles, and Pediatrics ond Microbiology, Univsrrity of Southem California School of Medicine, 10s Angeles, Colifomia wo54

1. Introduction

Allogeneic bone marrow transplantation for the treatment of infants with severe combined immune deficiency (SCID) has been a model system for many of the developments in clinical bone marrow transplantation during the last 20 years. The first successful human allogeneic bone marrow transplant was performed in a child with SCID in 1967; the first successful fetal liver transplant was reported in 1975; the first successful transplant with a histocompatible unrelated donor in 1977, and the first successful T lymphocyte-depleted histoincompatible bone marrow transplant in 1982 (1-5). Thus, transplantation in SCID patients has represented the first clinical application of many of the advances that have occurred in clinical bone marrow transplantation during the last 20 years. The biological problems present in the bone marrow transplantation of children with SCID, especially when histoincompatible bone marrow is used (i.e., lack of stem cell engraftment, graft-versus-host disease, posttransplant immune dysfunction) continue to present challenges in clinical bone marrow transplantation. II. Severe Combined Immune Deficiency

SCID was initially described as “the Swiss form of agammaglobulinemia” in a group of lymphopenic infants who had developed disseminated infections after vaccination with Bacille Calmette-Gukrin (BCC) (6).It is now clear, however, that SCID is not a single primary defect but is a clinical phenotype characterized by an absence of antigen-specific T and B lymphocyte immunity. At least a dozen different primary defects have been identified which can produce the SCID phenotype. As modern molecular genetic techniques improve, additional primary defects will be defined since at present the primary defect can be identified in less than 50% of SCID patients. The primary 381 Copyright 0 1991 by Academic Press, Inc.

All rights of reproduction in any form resewed.

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defects in SCID represent a continuum starting with the absence ofthe pluripotent stem cell (reticular dysgenesis) and ending with the presence of phenotypically normal T lymphocytes capable of responding to mitogenic stimulation, but incapable of responding to specific antigenic stimulation. Abnormalities that occur at either the pluripotent or lymphoid stem cell level will result in an absence of both circulating T and B lymphocytes. Defects that occur later in T lymphocyte differentiation result in an absence of mature T lymphocytes with the presence of normal B lymphocytes, or the presence of dysfunctional T lymphocytes and normal B lymphocytes. The majority of SCID patients, therefore, have selective defects in T lymphocyte differentiation/function and have phenotypically and functionally normal B lymphocytes.

A. RETICULAR DYSCENESIS The most primitive defect that can produce the clinical phenotype of SCID is reticular dysgenesis, an autosomal recessive disorder, in which an absence of the pluripotent stem cell results in a lack of circulating lymphoid, erythroid, myeloid, and megakaryocytic elements (7).Affected infants suffer from infections due to the absence of both myeloid and lymphoid immunity. In addition to the usual infections from which SCID patients suffer (respiratory bacteria, viruses, fungi, and protozoa), patients with reticular dysgenesis suffer from infections with enteric bacteria as patients with neutropenia do.

B. ABSENCEOF LYMPHOID STEMCELL The initial patients described with severe combined immune deficiency (Swiss form of agammaglobulinemia) were lymphopenic and represented a selective absence of the lymphoid stem cell (6,8). The patients’ hematopoietic stem cells were normal, and, therefore, the patients had normal erythrocyte, granulocyte, and platelet counts. A primary defect at the lymphoid stem cell level results in the absence of both T and B lymphocytes, although natural killer (NK) cells can be present. The presence of NK cells in these patients, plus the determination that NK cells in adenosine deaminase (ADA)-deficient patients following the transplantation of ADA positive bone marrow are still ADA negative, indicates that NK cells are derived from the hematopoietic stem cell and not from the lymphoid stem cell.

C. ADENOSINEDEAMINASE DEFICIENCY The functional absence of the enzyme ADA is the most common definable cause of SCID (9). Twenty-five percent of all autosomal

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recessive cases of SCID are due to ADA deficiency. The patient’s leukocyte ADA levels vary (0.1-lo%),producing clinical heterogeneity. Most cases of ADA deficiency are due to point mutations leading to the presence of a dysfunctional enzyme (10,ll).Some point mutations have resulted in RNA that is rapidly degraded, resulting in the lack of detectable enzyme. A few cases ofADA deficiency are due to deletions of the ADA gene (12).The biological effects of ADA deficiency can be mimicked by the addition of the ADA inhibitor deoxycoformycin to cultures of pre-T lymphocytes (13).The absence of functional ADA results in the toxic accumulation of phosphorylated adenosine metabolites, particularly deoxyadenosine triphosphate (dATP) (14). The elevated levels of dATP and other phosphorylated nucleotides result in the inhibition of both ribonucleotide reductase and S-adenosylhomocystine hydrolase. The in uitro toxic effects of the deoxymetabolites are more pronounced in T, as compared to B, lymphocytes. Similar differences are presumed to exist in uiuo explaining why patients with ADA deficiency may have normal numbers of B lymphocytes but reduced numbers of T lymphocytes. In vitro improvement in nonspecific immunological function (mitogen responsiveness) has been observed following the incubation of peripheral blood leukocytes of some ADAdeficient patients with exogenous ADA (15).The in uitro improvement has been mirrored by in uiuo improvement in some ADA-deficient patients with the exogenous administration of ADA, including ADAcontaining human erythrocytes and polyethylene glycol-coupled bovine ADA (PEG-ADA) (16,17).Patients treated with exogenous ADA may have an increase in phenotypic T lymphocytes and the acquisition of mitogen responsiveness. However, few patients have developed adequate antigen-specific T and B lymphocyte function to protect them from exogenous infectious organisms. The patients who have responded have been older patients with higher endogenous levels of ADA, further demonstrating the clinical heterogeneity of the ADAdeficient form of SCID. Final conclusions concerning the potential role of exogenous ADA in the treatment of ADA-deficient SCID patients await further clinical investigation.

D. INTRATHYMIC DEFECTS DiGeorge syndrome is a congenital abnormality involving the third and fourth pharyngeal pouches, that can result in an absence of the parathyroid and thymus glands, midline cardiac defects, and facial abnormalities (18). The primary immunological defect in DiGeorge syndrome was felt to b e a quantitative decrease in the amount of thymic tissue resulting in a decrease in both the amount of thymic

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stroma and the production of thymic hormones. In uitro incubation of peripheral blood leukocytes from DiGeorge patients with thymic hormones (thymosin, TP5, etc.) can result in an increase in the number of phenotypic T lymphocytes and of their proliferative responses to mitogenic stimulation (19).The in uitro improvements have been the basis for the clinical administration of thymic hormones to DiGeorge patients (20). In the majority of DiGeorge syndrome patients, there is no intrinsic abnormality of the lymphoid stem cells since the transplantation of fetal thymic stroma or the administration of thymic hormones results in the peripheral immunological reconstitution of patients with cells of recipient origin (21,22). The experiments of Gelfand and Pike, however, suggest that some forms of SCID are due to intrinsic defects of the thymus (23). The incubation of a patient’s bone marrow mononuclear cells with normal thymic stroma resulted in the appearance of phenotypically normal T lymphocytes, whereas incubations of the patient’s bone marrow with his or her own thymic stroma resulted in no improvement. These in uitro experiments, along with the clinical improvement seen in some SCID patients following the transplantation of thymic epithelium, suggest that the primary defect in some cases of SCID is due to an intrinsic defect of the thymic epithelium. The evaluation of the thymic stroma from such patients may demonstrate abnormalities in adhesion molecules necessary for normal positive and negative thymic selection (24,25). The presence ofTCR alp’, CD3+, CD4-, and CD8- autoreactive T lymphocytes in some SCID patients suggests that negative selection of autoreactive T lymphocytes may be defective (26).

E. INTERLEUKIN-I DEFICIENCY Interleukin-1 (IL-1) is produced by a large range ofcell types including monocytes and macrophages (27). In normal immunological function, processed antigen bound to the major histocompatibility complex is presented b y antigen-presenting cells (APC) to T lymphocytes through their antigen-specific T lymphocyte receptor (TCR). Costimulation with IL-1 produced by the APC is necessary for the initial activation of the antigen-specific CD4+ T lymphocytes, resulting in the production of IL-2. SCID patients with defects in IL-1 production have been identified. Monocytes from five SCID patients were incapable of IL-1 production after in uitro stimulation with lipopolysaccharide (28). In two cases, the defective IL-1 production was due to excessive prostaglandin production, since IL-1 production was normal after the addition of indomethacin. In four cases, the defect in IL-1 production appeared to be primary. Thus, the clinical phenotype of

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SCID can be produced by aprimary defect in macrophages/monocytes derived from the hematopoietic stem cell.

F. INTERLEUKIN-I RECEPTORDEFICIENCY The activation of T lymphocytes by IL-1 requires the specific interaction between IL-1 and its ligand receptor, the IL-1 receptor. A patient with SCID was identified whose T lymphocytes had defective or absent IL-1 receptors as determined by the inability of the patient’s T lymphocytes to absorb IL-1(29).The patient’s T lymphocytes could be normally activated when phorbol myristate acetate (PMA) was used as a costimulant. The inability of the patient’s T lymphocytes to be activated by IL-1 resulted in a patient with the SCID phenotype.

G . INTERLEUKIN-2 DEFICIENCY Interleukin-2 (IL-2) is the central lymphokine regulating the proliferation and differentiation of T lymphocytes. IL-2 is produced by a minority of mature T lymphocytes (5-15%). The IL-2 produced by the activated subpopulation is then capable of stimulating the proliferation and terminal differentiation of antigen-specific IL-2-dependent T lymphocytes expressing high-affinity IL-2 receptors. Patients have now been identified who have a selective inability to produce IL-2 (30).The phenotype of the patient’s circulating T lymphocytes is relatively normal, although a decrease in CD4+ T lymphocytes was seen in one case. A significant percentage (40%) of the peripheral blood T lymphocytes expressed IL-2 receptors in uiuo. Patients with a selective defect in IL-2 production displayed no in vitro proliferative responses to either mitogenic or antigenic stimulation; however, normal proliferative responses can be detected in the presence of exogenous IL-2. Analysis of the peripheral blood lymphocytes of the patients following in uitro stimulation with PMA and calcium ionophore showed the presence of RNA for y-interferon, but no transcripts for IL-2. Southern blot analysis showed the presence of an intact IL-2 gene. Clinical studies are presently under way to determine the role of recombinant exogenous IL-2 as therapy for the IL-2-deficient form of SCID.

H. INTERLEUKIN-2 RECEPTORDEFICIENCY Following activation, the majority of antigen-specific T lymphocytes express a series of new cell surface proteins including Class I1 histocompatibility antigens, the transferrin receptor, and the high-affinity IL-2 receptor. The presence of the high-affinity IL-2 receptor is necessary for the further proliferation and differentiation of the activated T

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lymphocytes. The congenital inability to express the IL-2 receptor would, therefore, result in a lack of proliferation and terminal differentiation by T lymphocytes. A patient has been described whose cells, following in vitro activation with PHA, produced IL-2 and y-interferon normally, expressed normal levels of Class I1 histocompatibility antigens and transferrin receptors, but expressed no high-affinity IL-2 receptors (31).Southern blot analysis revealed the gene encoding the @chain of the IL-2 receptor to be present. The in vitro proliferative defect could not be corrected by the addition of IL-2 or other recombinant cytokines (IL-1, IL-4).

I. GENERALIZED ACTIVATION DEFECTS A heterogenous group of SCID patients has been described over the last 15 years in whom a generalized defect in lymphocyte activation has been demonstrated (32-35). When the patients’ T lymphocytes were stimulated with PHA or anti-CD3 antibody, lymphocyte activation as determined by an increase in intracellular calcium or the production of messenger RNA for cytokines such as IL-2, IL-4, GM-CSF, or y-interferon was not detected. Defects in activation can be due to abnormalities of the CD3-TCR complex, the transducing G proteins, or phospholipase C. Patients can be evaluated for defects at each step of activation. Many of the patients initially described as having “membrane abnormalities” probably represent defects of activation. In some cases, the defect can be bypassed by the addition of calcium ionophore (A23187 or ionomycin), indicating that the defect lies in the signal transduction mechanism, but that the cytoplasmic and nuclear mechanisms are intact. Ultimately, a series of primary defects in the signaling mechanism can be expected to be identified, all of which will result in defective activation. The patients with general activation defects differ from the patients in whom a selective defect in IL-2 production is present (35).

J. DEFECTSOF THE TCR-CD3 COMPLEX The antigen-specific activation of T lymphocytes requires triggering through the TCR. Effective signaling requires both a functional TCR molecule and a fully mature CD3 molecule. A SCID patient has been identified in whom abnormalities of the CD3 (-chain resulted in decreased surface TCR and CD3 expression(36). The intracellular assembly of the TCR alp-CD3 complex was normal; however, the defective CD3

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