Advances in Immunology Volume 41

ADVANCES IN

Immunology VOLUME 41

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ADVANCES IN

Immunology EDITED BY

FRANK J. DIXON Scripps Clinic and Research Foundation La Jolla, California

ASSOCIATE EDITORS

K. FRANK AUSTEN LEROYE. HOOD JONATHAN W. UHR

VOLUME 41

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers Orlando San Diego New York Austin Boston London Sydney Tokyo Toronto

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87 88 89 90

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CONTENTS

Cell Surface Molecules and Early Events Involved in Human T Lymphocyte Activation ARTHURWEISSA N D J O H N B. IMBODEN

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

I. Introduction

11. Cell !surface Molecules Involved in T Cell Activation

111. Synergy between Ca2+ Ionophores and Phorbol Esters in T Cell Activation . IV. Receptor-Mediated Signal Transduction during T Cell Activation . . ... V. Role of Intracellular Signals Other Than Ca2+ and pkC . . . . . . . . . . ... VI. Effects of Early T Cell Activation Events upon Gene Regulation . . . . . . . . . . . . VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................. References

1 1 15 19 26 26 30 31

Function and Specificity of T Cell Subsets in the Mouse JONATHAN S P R E N T A N D S U S A N

R.

WEBB

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Cell !surface Molecules Controlling T Cell Specificity and Function . . . . . . . . . 111. H-2-Restricted Recognition of Antigen by Mature T Cells IV. Recognition of H-2 Alloantigens by Mature T Cells . . . . . . . . . . . . . . . . . . . . . . . V. Consequences of T Cell Contact with H-2 Molecules in the Thymus . . . . . . . . VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . ......................................

39 40 51 78 95 110

113

Determinants on Major Histocompatibility Complex Class I Molecules Recognized by Cytotoxic T Lymphocytes JAMES

FORMAN

I. Introduction . . . . . . . . . . . . 11. Exon Shuffling to Produce ................. 111. Recognition of HLA Class IV. Role of Carbohydrate Moieties in Determining CTL Recognition of Class I Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Role of Pz-Microglobulin in T Cell Recognition VI. Use of Monoclonal Antibodies to Block CTL Recognition of Class I Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Class I Heavy Chains Bearing Defined Amino A Polymorphic Determinants Recognized by CTL .................. tic Cell Class I VIII. CTL Recognition of Monoclonal Antibody-Select ................................................ Variants V

135 138 149

156 158 165

vi

CONTENTS

IX . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

167 171

Experimental Models for Understanding B Lymphocyte Formation

PAULW . KINCADE

I . An Introductory Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Organization of Lymphohemopoietic Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... 111. Resolution of B Cell Precursors . . . . . . . . . . . ................. IV . Rearrangement and Utilization of Immunoglo

...................... V . Population Dynamics . . . . . . . . . . . VI . Long-Term Bone Marrow Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . ............................... VII . An Inducible Cell Line ............ VIII . ........................................... IX . Soluble Mediators ................... X . Synthesis and Conclusions . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

181 185 188 203 205 208 220 223 232 235 239

Cellular and Humoral Mechanisms of Cytotoxicity: Structural and Functional Analogies

JOHNDING-EYOUNG

AND

ZANVIL A . COHN

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Nature of Cytotoxicity Mediated by CTL and NK Cells . . . . . . . . . . . . . . . . . . . 111. Cytolytic Mechanisms Proposed in the Past and the Concept of Secretion and Colloid Osmotic Lysis .......................................... IV . Granule Proteins in Cell-Mediated Killing ................... V . Membrane Attack Complex of Complemen ................... VI . Other Cytolytic Pore-Forming Proteins ....................... VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . ................

269 270 273 286 299 311 319 320

Biology and Genetics of Hybrid Resistance

MICHAELB E N N E ~

....................... 1. Introduction . . . . . . . . . . . . I1. Hybrid Resistance to Normal Hemopoietic Cells . . . . . . 111. Hybrid Resistance to LeukemiaLymphoma Cells ........................ IV . Effector Mechanisms of Hybrid Resistance . . . . . . V . Genetics of Antigen Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Proposed Mechanisms of A References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

333 335 358 369 397 401 411

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

447 473

ADVANCES IN IMMUNOLOGY, VOL. 41

Cell Surface Molecules and Early Events Involved in Human T Lymphocyte Activation ARTHUR WEISS* AND JOHN 6. IMBODENt *Deportment of Medicine, Howard Hughes Medical Institute, University of California, San Francisco, California 94 143 and fDepartment of Medicine, Veterans Administration Medical Center, San Francisco, University of California, Son Francisco, California 94 143

1. Introduction

The activation of human thymus-derived (T) cells is the result of ligandreceptor interactions. Under physiologic conditions, such ligand-receptor interactions occur at the interface of the plasma membranes of an antigenspecific T cell and an antigen presenting cell (APC) or target cell. These antigen-specific and non-antigen-specific ligand-receptor binding events result in the transduction of these events into intracellular biochemical signals in the form of “second messengers.” Ultimately, such intracellular biochemical signals influence specific targeted genes receptive to these signals which can become transcriptionally active or inactive. The summation of these events is the expression of the phenotype of an activated T cell. The diverse manifestations of T cell activation include the production of lymphokines, the appearance of new cell surface proteins (which include growth factor receptors), the acquisition of cytolytic effector function, and, as a consequence of the production of growth factors and their receptors, proliferation. In this review, we will focus primarily on the structures and function of the cell surface molecules of the human T cell which appear to initiate activation. Where appropriate, data referring to the murine system will be drawn upon. The events subsequent to initial activation events, i.e., the interaction of interleukin 2 (IL-2) with its receptor and the resultant proliferative response, will not be addressed in this review. 11. Cell Surface Molecules Involved in T Cell Activation

The study of the T cell surface molecules involved in T cell activation has been facilitated through the use of homogeneous cell populations, such as T cell clones, hybridomas and leukemic lines, and the availability of monoclonal antibodies (mAb) which define antigenic epitopes expressed on an 1 Copyright Q 1887 by Academic Press, Inc. All rights of rrproductron in any form reserved.

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ARTHUR WEISS A N D JOHN B. I M B O D E N

array of distinct molecules. By virtue of the agonist or antagonist properties of mAb reactive with these molecules, many of these cell surface molecules are felt to play a role in T cell activation, either in antigen-induced activation or in alternate pathways of activation. Most of these molecules are thought to function as cell surface receptors. The T cell antigen receptor must play a central role in antigen-driven T cell activation and has been most intensively studied, The ligands of these other putative receptors remain to be identified or confirmed. Some of these receptors can play a primary role in activation (the T cell antigen receptor, T11, Thy-1), initiating distinct biochemical events, which alone, following ligand interaction, can lead to T cell activation under appropriate conditions. Triggering of these receptors results in an increase in the concentration of cytoplasmic free calcium ([Ca2+Ii),one of the intracellular events which is generally felt to be required for T cell activation. Other receptors appear to function as accessory molecules [Tp44, T1, interleukin-1 (IL-1) receptor], which when stimulated, are able to synergize with stimuli provided by the T3/Ti complex. These receptors have little effect upon T cell activation when stimulated alone. Still other molecules have been implicated as receptors involved in increasing the overall avidity between the T cell and the APC (or target cell) (LFA-1, T4, T8). The following will attempt to summarize the structure and evidence supporting the role of some of these cell surface molecules in T cell activation. However, we will not attempt to exhaustively review all molecules involved in activation.

A. T CELLANTIGEN RECEPTOR Activation of the T cell induced by an antigen on the surface of an APC must involve an interaction with the T cell antigen receptor. This receptor subserves two functions in antigen-induced activation: (1)a recognitive function in which a specific antigen is recognized in the context of the appropriate major histocompatibility complex (MHC) molecules, and (2) an effector function in which the recognitive event is transmitted across the plasma membrane to the interior of the cell, with the resultant appearance of intracellular second messengers. A fundamental understanding of the structure of the receptor is useful in order to begin to understand the basis for these two functions. 1. Structure of the T Cell Antigen Receptor

The T cell antigen receptor was identified independently in several laboratories by the generation of mAb, which reacted with unique clonally distributed antigenic epitopes on T cell lines, hybridomas, or clones (clonotypic determinants) (Allison et al., 1982; Meuer et al., 1983a; Haskins et al., 1983). These antibodies react with disulfide-linked heterodimer glycoproteins (Ti)

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of 80-90 kDa. These heterodimers are composed of an acidic Ti-a chain of 43-54 kDa and a more basic Ti$ chain of 38-44 kDa (Reinherz et al., 1983; Kappler et' al., 1983a,b). Peptide mapping studies suggested each chain has both constant and variable domains (Reinherz et al., 1983; Acuto et al., 1983; Kappler et al., 198313). Both chains are integral membrane proteins, have two to six N-linked glycosylation sites, and have an intracytoplasmic tail of five amino acids at the carboxy-terminus (McIntyre and Allison, 1984; Yanagi et al., 1984; Sim et al., 1984). The relatively short cytoplasmic tail of these chains suggests that they are by themselves not responsible for transmembrane signaling events. A detailed understanding of the structure of the human Ti has come from study of the complementary DNA (cDNA) clones and genomic clones of the Ti-a and p chains (Yanagi et al., 1984; Sim et al., 1984). The human Ti-a and -p chains have limited homology to immunoglobulin genes, suggesting a common evolutionary origin (Yanagi et al., 1984; Sim et al., 1984; Hood et nl., 1985). Both Ti-a and -p chains are assembled from gene segments which undergo rearrangements and expression during T cell ontogeny (Royer et al., 1984, 1985; Yoshikai et al., 1984; Raulet et al., 1985; Collins et al., 1985). Analogous to immunoglobulin heavy chain genes, Ti-6 chains are assembled from recornbinational events involving variable (V), diversity (D), joining (J), and constant (C) gene segments (Siu et al., 1984). The Ti-a chain genes are similarly assembled from V, J, and C segments, but, to date, no D segments have been identified (Yoshikai et al., 1985). Thus, the diverse antigen-reactive repertoire of T cells can be accounted for, in part, from the joining of different V, J, and D gene segments as well as combinatorial associations between the Ti-a and -6 chains. Transfection studies and cell fusion studies have suggested that the Ti-a and -p chains are sufficient to confer antigen and MHC specificity upon the T cell (Dembic et al., 1986), although primary sequence studies suggest that neither Ti-a nor -p chains are solely responsible for antigen or MHC specificity (Fink et al., 1986). Thus, the evidence strongly implicates Ti heterodimer in the antigen/MHC-specific recognitive events. More preliminary evidence, however, suggests that Ti-a and -p chains may not be the only chains involved in antigen recognition. In the course of attempts to isolate the Ti-a chain, another cDNA, the Ti-y chain, was isolated (Saito et al., 1984). This gene, once thought to be prefentially transcribed in cytolytic cells (Kranz et al., 1985), has now clearly been found to be expressed in helper T cells as well (Zauderer et al., 1986). The Ti-y chain gene, like the a and p chain genes, undergoes rearrangement utilizing V and J region segments linked to constant region segments (Hayday et al., 1985; LeFranc et al., 1986). Interestingly, the Ti-y chain is the first of the Ti chains to rearrange and to be expressed during T cell ontogeny (Raulet et al., 1985;

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Haars et al., 1986) and, thus, has been proposed to be important in thymocyte selection (Raulet et al., 1985; Garman et al, 1986). Until recently, the protein product of the Ti-y chain had not been identified. However, recent studies suggest that it is expressed as a 55-kDa glycoprotein on an small, unusual subpopulation of human peripheral T cells which fail to express T4 (CD4) or T8 (CD8) antigens (Brenner et d., 1986; Weiss et d., 1986c; Lanier and Weiss, 1986). It may exist as a non-disulfide-linked heterodimer or as a single chain in association with T3 (see below). It has also been detected on the surface of T4-/T8- thymocytes felt to represent the most immature population of the thymus (Bank et al., 1986; Lanier and Weiss, 1986). On human T cells, the Ti heterodimer or the protein product of the Ti-y chain gene is associated with three invariant peptides which comprise the T3 (CD3) antigenic complex. T3 consists of at least three distinct integral membrane proteins: The T3-6 chain, a 22-kDa glycoprotein; the T3-Echain, a 21kDa nonglycosylated protein; and the T3-y chain, a 26- to 28-kDa glycoprotein (Borst et al., 1982, 1983a; Kanellopoulos et al., 1983). The cDNAs encoding these three chains have been isolated and sequenced (van den Elsen et al., 1984; Gold et al., 1986; Krissansen et al., 1986). The expression of these T3 genes is limited to T cells. T3-6 and T3-y chains exhibit substantial homology to each other, but not to other known proteins (Krissansen et al., 1986). Homologous chains have been identified in the murine system (Allison and Lanier, 1985; Samelson et al., 1985; Oettgen et al., 1986). However, additional chains have also been identified in the mouse. These include the 5 chain, a disulfide-linked homodimer or heterodimer of 32 kDa, with monomers of 14-17 kDa (Samelson et al., 1985; Oettgen et al., 1986), and a more recently isolated p21, a disulfide-linked dimer of 42 kDa with 21kDa subunits, which is phosphorylated on tyrosine residues with activation by antigen (Samelson et al., 1986). It is likely that homologues to the T3-4 chain and p21 will be identified in the human. Thus, the T3 complex may consist of seven chains. All three of the cloned chains of T3 contain between 40 and 80 cytoplasmic residues (van den Elsen et al., 1984; Gold et al., 1986; Krissansen et al., 1986). This observation, together with the findings discussed below regarding the agonist properties of T3 mAb and the phosphorylation of T3 chains, are consistent with the notion that T3 plays a role in transmembrane signaling events. Several lines of evidence suggest that the antigen receptor exists as a molecular complex composed of Ti and T3, or, in the case of the protein product of the Ti-y chain, Ti-y and T3. The physical association of the Ti-a/P chain heterodimer was demonstrated by comodulation (Meuer et al., 1983a), coimmunoprecipitation (Reinherz et al., 1983; Borst et al., 1983b), and the chemical cross-linking (Allison and Lanier, 1985; Brenner et al., 1985). T3

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has been linked to the protein product of the Ti-y chain by coimmunoprecipitation (Weiss et al., 1986c) and chemical cross-linking (Brenner et al., 1986; Bank et al’., 1986). Evidence suggests that the association between T3 and Ti is obligatory in that mutants of the T cell leukemic line Jurkat, which lack Ti+ chain transcripts, contain T3 proteins trapped intracellularly (Weiss and Stobo, 1984; Ohashi et al., 1985). Reconstitution of the Ti+ chain by transfection into one such mutant resulted in the reexpression of Ti and T3 (Ohashi et al., 1985). Although the close association of T3 and Ti is suggested by such studies, the exact nature of this association is not clear. Under many conditions of immunoprecipitation, T3 and Ti do not coprecipitate (Allison et al., 1982; Haskins et al., 1983; Samelson et al., 1983; Weiss and Stobo, 1984). In ;icross-linking study, the T3-y chain was chemically cross-linked to the Ti+ chain, suggesting a close association between these chains (Brenner et al., 1985). A shortcoming of this study is the observation that neither the Ti-a and -p chains nor the chains of T3 were cross-linked to each other. One striking observation has been made from the sequence analyses of the component chains of Ti and T3. All three of the Ti chains, a, p, and y, of mouse and man contain an unusually placed highly charged basic lysine residue within the putative transmembrane domain (Yanagi et al., 1984; Sim et al., 1984; Saito et al., 1984), whereas the three chains of T3 contain conserved acidic residues of aspartic or glutamic acids within their hydrophobic putative transmembrane domains (van den Elsen et al., 1984; Gold et al., 1986; Krissansen et al., 1986). It has been suggested that these charged amino acids may play a role in the association between T3 and Ti. Collectively, these observations support a model of the T cell antigen receptor as a multisubunit complex composed of five to nine chains consisting of T3 and Ti.

2 . Role of the T3lTi Complex in Activation

The T cell antigen receptor must play a role in antigen-induced T cell activation. However, the direct study of the role of the receptor binding to the antigen is hampered by the inherent difficulty in studying cell-cell interactions and the ill-defined structure of antigen associated with M HC molecules. The use of monoclonal antibodies reactive with Ti or T3, which can function as agonists or antagonists, has facilitated the study of the role of the T3/Ti complex in T cell activation. Thus, such antibodies can serve as probes to elucidate the function of the receptor, mimicking the effects of antigen, without the participation of other cell surface molecules which may interact during T cell-APC interactions. Although this approach has limitations, since the effects of agonist mAb may not fully mimic the effects of antigen-antigen receptor interactions, it provides a first approximation toward the :itudy of the function of the antigen receptor.

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ARTHUR WEISS AND JOHN B . IMBODEN

A large number of studies have revealed that mAb reactive with T3 could function as polyclonal agonists in inducing resting human peripheral blood T cells within peripheral mononuclear cells to secrete the lymphokines IL-2 or interferon-y (IFN-y) (von Wussow et al., 1981; Chang et d., 1982; van Wauwe et al., 1984), to express IL-2 receptors (Meuer et al., 1984a; Schwab et al., 1985; Tsoukas et al., 1985; Ledbetter et al., 1986), or to proliferate (van Wauwe et al., 1980; Chang et al., 1981). These antibodies have also been used to activate T cell clones and tumor lines to produce lymphokines or kill targeted bystander cells (Meuer et al., 1983b; Weiss et al., 1984a; Kranz et al., 1984; Mantzer et al., 1985). Similarly, clonotypic Ti mAb and Ti mAb reactive with nonpolymorphic determinants of Ti, such as mAb WT31, can activate T cells in a manner analogous to T3 mAb (Kappler et al., 1983a; Meuer et al., 1983b; Tax et al., 1983; Kaye and Janeway, 1984; Weiss and Stobo, 1984). It is of interest that under appropriate conditions of antibody immobilization, all anti-T3 or anti-Ti mAb described, with one exception, can function as agonists (Lanier et al., 1986). This implies that, in contrast to the T I 1 molecule (discussed below), perturbation of several distinct sites on the T3/Ti complex can lead to appropriate triggering of the complex. The potency of T3 and Ti mAb suggests that occupancy of relatively few receptors is sufficient to activate T cells (Chang et al., 1982; Kaye and Janeway, 1984). T3 and Ti mAb are also capable of functioning as antagonists, under some circumstances, to block the interactions between T cells and antigen-presenting cells or target cells (Chang et al., 1981; Meuer et al., 1983a; Haskins et al., 1983; Lancki et al., 1983; Samelson et al., 1983). Thus, the use of T3 and Ti mAb has proved to be a powerful tool to examine the role of the T cell antigen receptor in activation. The conditions required for activation of T cells by T3 or Ti mAb are dependent upon the particular manifestation of T cell activation examined. For instance, expression of the IL-2 receptor (IL-2R) has less stringent requirements than T cell proliferation. Hence, IL-2R expression can be induced by T3 or Ti mAb under conditions in which no proliferative response is observed (Schwab et al., 1985; Wakasugi et al., 1985; Tsoukas et al., 1985; Ledbetter et al., 1986). Therefore, production of IL-2 is more stringently regulated than the expression of the IL-2R. Since both the growth factor and its receptor must be produced in order for T cell proliferation to occur, T cell proliferation is primarily limited by the production of IL-2. This view must be qualified by the recent findings that there may be IL-2 independent pathways of T cell proliferation (Moldwin et al., 1986). BSF-1 is produced by T cells and can support the growth of some T cell clones (Smith and Rennick, 1986; Mosman et al., 1986; Yokota et al., 1986; Fernandez-Botran et al., 1986). It is not clear what the requirements are for BSF-1 production or for its role in T cell proliferative responses to antigen.

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Regardless of the growth factors by which T cell proliferation is mediated, the induction of T cell proliferation by anti-T3 or anti-Ti mAb is dependent upon accessory cells (AC) (Chang et al., 1982; Tax et al., 1983; Landegren et al., 1984) In the case of human PBM, these AC are contained within the adherent cell population (Tax et al., 1983). At least two functions of these AC have been demonstrated. One function is dependent upon an interaction of the Fc portion of the T3 mAb and the Fc receptor on these AC (Tax et al., 1983, 1984; Landegren et al., 1984; Smith et al., 1986; Wakasugi et al., 1985; Ceuppens et al., 1985). This function of AC can be bypassed by immobilization of the T3 mAb onto Sepharose beads or onto the surface of culture dishes (Tax et al., 1984; Ceuppens et al., 1985). This suggests that the formation of a cross-linked matrix of antibody and T3 may be critical in activation requirements. Alternatively, as has been suggested, the immobilization of the T3 mAb may be important in preventing receptor internalization which might result in blunting the stimulatory response (Manger et al., 1985; Ledbetter et al., 1986). Indeed, as reviewed below, transmembrane signaling by soluble and immobilized anti-T3 or anti-Ti has been shown to differ. Whereas neither soluble nor immobilized anti-T3 induce IL-2 production or proliferation of highly purified T cells, only immobilized anti-T3 is able to induce IL-2R expression (Wakasugi et al., 1985; Ledbetter et al., 1986). It is likely that immobilized T3 mAb more closely mimics the Ti/T3 interaction with antigen/MHC on the surface of the T cell and APC. The second function of the AC is revealed by the failure of highly purified resting T cells to proliferate to immobilized anti-T3 or mitogenic lectins (Schwab et al., 1985; Williams et al., 1985; Ledbetter et al., 1986; Manger et al., 1986; Weiss et al., 1986a). The requirements for the activation of purified freshly isolated resting T cells and previously stimulated T cell clones or lines appear to differ (Manger et al., 1985; Meuer and zum Buschenfelde, 1986). Immobilized anti-T3 or anti-Ti alone is sufficient to activate T cell clones to produce IL-2 and to proliferate (Meuer et al., 1983b, 1984a; Manger et al. 1985). Similarly, the T cell leukemic line HUT 78, which phenotypically resembles a previously activated T cell, produces IL-2 in response to immobilized but not soluble T3 mAb (Manger et al., 1985). In contrast, the Jurkat cell line, like resting highly purified T cells, fails to respond to immobilized anti-T3 (Manger et al., 1985; Williams et al., 1985; Ledbetter et al., 1986; Weiss et al., 1986a). These findings suggest that resting T cells require an additional stimulus, provided by AC, which is not observed with T cells previously activated. Relatively small numbers of AC can provide this additional stimulus; hence, the notion that a soluble mediator is involved has emerged. Four ligands that bind to the surface of the T cell can mimic the effect of AC in providing this second function: mAb reactive with T1, T11, or Tp44,

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ARTHUR WEISS A N D JOHN B . I M H O D E N

as well as IL-1. These will be discussed separately below. In addition, it should be noted that both functions of the AC can be provided by phorbol myristate acetate (PMA), a potent activator of protein kinase C (pkC) (Hara et al., 1985; Ledbetter et al., 1986; Weiss et al., 1986a). The role of PMA and pkC in T cell activation will be discussed at length later in this review. Thus, mAb reactive with T3 or Ti can function as polyclonal activators of T cells in a manner analogous to that of anti-Ig and B cells. However, simple ligand binding to the T cell antigen receptor does not appear to be sufficient for activation. In view of the fact that T cells do not respond to soluble antigen, but, rather, react with cell-bound antigen, the AC dependence of anti-T3 mAb may be quite consistent with the physiologic situation. In addition to its role in antigen-induced T cell activation, the T3/Ti complex appears to be important in the activation of T cells by the T cellspecific mitogenic lectins phytohemagglutinin (PHA) and concanavalin A (Con A). Both lectins bind to large numbers of T cell surface glycoproteins (Henkart and Fisher, 1975; Sitkovsky et al., 1984); however, the cell surface molecules responsible for the ability of these lectins to stimulate T cells have been undefined. Biochemical analyses of solubilized cell surface proteins have demonstrated that Con A can bind to the T3 chains but not Ti, whereas PHA can interact with the Ti heterodimer but not the isolated T3 chains (Kanellopoulos et al., 1985). Thus, among the many cell surface glycoproteins bound by these lectins are component chains of the T3/Ti complex. Indeed, both Con A and PHA can induce cocapping of T3 (Kanellopoulos et al., 1985). Simple demonstration of binding to components of the T3/Ti complex does not establish that the T3/Ti complex mediates the relevant activation signal induced by these mitogens. Evidence supporting the role of the T3/Ti complex in PHA- and Con A-induced T cell activation is the observation that Jurkat mutants which fail to express the T3/Ti complex lose the capacity to produce IL-2 in response to either PHA or Con A (Weiss et al., 1984b; Weiss and Stobo, 1984). Moreover, reconstitution of the T3/Ti expression in one of these mutants by transfection resulted in the restoration of the PHA and Con A responsiveness of this cell (Ohashi et al., 1985; Weiss et al., 198613). These results are in contrast to those suggesting that the T11 (CD2) molecule may function as the relevant PHA receptor. In these studies, anti-T11 mAb were used as antagonists (O’Flynn et al., 1986). The explanation for this discrepancy is not clear but may reflect differences in experimental approach. The evidence that the T3/Ti complex plays a role in mitogenic lectin-induced T cell activation is compelling. B. T11 (CD2, Leu5, LFA-2) The T11 molecule is a 50-kDa glycoprotein on the surface of all T cells and thymocytes (Howard et al., 1981; Kamoun et al., 1981). This molecule func-

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tions as the sheep erythrocyte receptor on human T cells. As many as six distinct epitopes have been defined by mAb reactive with T11: 9.6/T11,, D66, 35.11, Tll,, T113, and 9.1 (Meuer et al., 1984b; Martin et al., 1983; Brottier ct al., 1985; Yang et al., 1986). Certain epitopes of T11 are not expressed on resting T cells, T11, and 9.1, but can be induced by other mAb reacting with the TS/Ti complex or other epitopes of T11 (Meuer et al., 1984b; Yang et al., 1986). Interest in this molecule has been stimulated by the finding that such mAb can function as agonists or antagonists in inducing T cell activation. Initial studies revealed that an anti-T11 mAb, OKT11, could inhibit lectin and anti-T3-induced lymphokine production T cell proliferation and the lytic activity of cytolytic T cell clones (CTL) (Palacios and Martinez-Maza, 1982; Sanchez-Madrid et al., 1982; Wilkinson and Morris, 1984; Moretta et al., 1985b). This led to the proposal that the T11 molecule might function in immune responses by delivering negative signals (Palacios and MartinezMaza, 1982). Supporting a negative signal role for the T11 molecule is a recent study demonstrating diminished levels of IL-2 transcripts in stimulated T cells preincubated in the presence of mAb 9.6, reactive with T11 (Tadmori et al., 1986). Several studies from independent laboratories have demonstrated that certain combinations of anti-TI1 mAb can activate T cells, as measured by proliferation or IL-2 production (Meuer et al., 1984b; Brottier et al., 1985; Yang et al., 1986). Similarly, non-antigen-specific cytolytic activity of antigen-specific CTL and natural killer (NK) clones can be induced by appropriate anti-T11 mAb (Siliciano et al., 1985).Individual anti-T11 mAb are insuficient in inducing T cell activation. Only certain combinations of appropriate mAb are able to induce the activation of T cells. Whereas mAb reactive with T11, + T l l , or 9.1 + 9.6 can activate T cells in an AC-independent manner (Meuer el al., 1984b; Yang et d., 1986), D66 + 9.6 or D66 + T11, depend upon the presence of Fc receptor-bearing AC (Brottier et al., 1985).Certain T11 mAb can activate resting T cells in the presence of PMA without the addition of a second anti-T11 mAb (Holter et al., 1986). As some combinations of antibodies reactive with different epitopes of T11 do not activate T T l Q , simple cross-linking of molecules does not appear cells (i.e., T11, to account for the ability of certain combinations of mAb to activate. In contrast to the TS/Ti complex, stimulation of T cells via T11 appears to be exquisitely epitope dependent and requires relatively high (probably saturating) amounts of stimulating mAbs (Meuer et al., 198413). The ability to stimulate T cells in the absence of AC with appropriate combinations of antiT11 mAb would appear to exclude the participation of other cell surface molecules in this model of T cell activation. Thus, appropriate triggering of the T11 molecule appears to be able to provide a primary activation signal in

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resting T cells which is distinct from that induced by the antigen receptor in view of the AC independence of this pathway. The T11 molecule is nonpolymorphic. Therefore, it is not likely to play a major role in antigen binding. The physiologic function of this molecule is not clear. The activation of T cells via stimulation of the T11 molecule has been termed the alternative pathway of human T cell activation to distinguish it from the antigen-dependent T3/Ti mediated pathway (Meuer et al., 1984b). As it is functional in thymocytes, it has also been proposed to play a role in thymocyte ontogeny (Fox et al., 1985). Recent studies have suggested that LFA-3 may represent the physiologic ligand of T11 (Springer et al., 1987). This 55- to 70-kDa glycoprotein is widely expressed on tissues. Antibodies reactive with LFA-3 inhibit a wide variety of T cell-dependent functions. A role for LFA-3 and T11 interactions in the thymus has also been proposed. Binding studies suggest a direct interaction between LFA-3 and T11. The interaction between the T3/Ti complex and the T11 molecule is of some interest. Stimulation of resting T cells via the T11 pathway does not require interaction with the T3/Ti complex. However, prior modulation of the T3/Ti complex inhibits the ability of T11 mAb to activate T cells (Meuer et al., 1984a; Fox et al., 1986). Conversely, modulation of T11 has little effect upon T3/Ti-induced activation. T11 mAb are able to activate NK cells which do not express T3 molecules (Siliciano et al., 1985). As no physical interaction between T11 and T3/Ti has been demonstrated, the explanation of these findings is not clear. However, one possibility is that in addition to their antigen removal effects, the mAb used in such modulation studies may have physiologic effects upon the cell. Of further interest, however, is a recent report suggesting that anti-T3 and anti-T11 mAb can synergize in inducing proliferation in cultures prepared with highly purified T cells (Yang et al., 1986). Thus, in addition to independent pathways of activation, these two pathways may interact under certain conditions. C. Thy-] Thy-1 is included in this review because of its unique structure and the evidence which had accumulated to support its role as a receptor involved in T cell activation in the murine system. The Thy-1 molecule is a 25- to 30-kDa glycoprotein with two allelic forms expressed on mouse thymocytes, peripheral T cells, fibroblasts, epithelial cells, and neurons (Reif and Allen, 1966a,b, 1984). The cDNAs encoding murine and human Thy-1 genes have been cloned and sequenced and exhibit some sequence homology to immunoglobulin genes (Evans et al., 1984; Seki et al., 1985). The murine Thy-1 gene is located on the ninth chromosome and encodes the 112 amino acid polypeptide chain (Blankenhorn and Douglas, 1972; Cohen et al., 1981). A most interesting structural feature of Thy-1 is the finding that the predicted

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membrane anchoring region of the molecule does not span the membrane, but is instead truncated and covalently linked to the membrane lipid phosphatidylirtositol (Tse et al., 1985; Low and Kincade, 1985). This feature is of particular interest as Thy-1 can function as a receptor involved in activation, but has no described associated molecule and cannot communicate with intracellular effector molecules via a transmembrane of cytoplasmic domain. Early work with heterosera demonstrated that antibodies reactive with Thy-1 could be mitogenic for murine T cells (Smith et al., 1982). Subsequently, only certain mAb reactive with Thy-1, used individually, could induce ILA-2R,IL-2, or IFN-y production and be mitogenic for murine T cells, whereas other mAb could not (Gunter et al., 1984; MacDonald et al., 1985). However, most anti-Thy-1 mAb failed to induce T cell activation when used alone (Kroczek et al., 1986a). This difference in the agonist effects of these mAb was interpreted to correlate with the distinct epitopes recognized by iigonist versus nonagonist antibodies (Kroczek et al., 1986a). However, T cell proliferation was observed if cross-linking of Thy-l was induced using a rabbit anti-mouse Ig in combination with nonactivating Thy-1 mAb in the presence of PMA (Kroczek et al., 1986a). This response was independent of AC or the epitope of Thy-1 with which the mAb reacted. The requirement for cross-linking Thy-1 is reinforced by the observation that combinations of two Thy-1 mAb, reactive with distinct noncompeting epitopes, were also effective in inducing T cell activation if used in the presence of PMA (Kroczek et al., 1986a). Why some anti-Thy-1 mAb are able to function as agonists in the absence of additional cross-linking antibodies or PMA is not clear. It is of interest that the antigenic epitope recognized by one of these mAb, which by itself can activate T cells, is lost following transfection of Thy-1 into human T cells, murine B cells, or fibroblasts (Kroczek et al., 198613). Thy-1 has been transfected into the human T cell line Jurkat, and Thy-1 mAb can activate this line in the presence of PMA (Gunter et al., 1986). This important study demonstrates that transfer of the Thy-1 molecule alone is sufficient for the active cell surface receptor. Clarification of the mechanisms of Thy-1-induced activation should be possible in such a transfection system. Although the physiologic ligand of Thy-1 has not been identified, the potent effects of anti-Thy-1 mAb would suggest a potential for involvement in murine T cell activation.

D. RECEPTORSWHICHMAYPROVIDEACCESSORY IN T CELLACTIVATION SIGNALS 1 . Tp44

Several reports have implicated the cell surface molecule Tp44 as potentially playing an important role in T cell activation. The only mAb reactive

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with Tp44, 9.3, identifies an 80- to 90-kDa disulfide-linked homodimer composed of 44-kDa subunits which is expressed on the surface of all T4 and -50% of T8 human T cells (Hansen et al., 1980; Yamada et al., 1985). A murine homologue of this molecule may have recently been identified (Nagasawa et al., 1986). Based on modulation studies of normal T cells and studies of mutants of the Jurkat cell line which fail to express the T3/Ti complex, no physical association between Tp44 and T3 exists (Hara et al., 1985; Moretta et al., 1985a; Weiss et al., 1986a). Initial studies demonstrated that 9.3 could inhibit the cytolytic activity of CTL (Fast et al., 1981). However, more evidence has accumulated demonstrating agonist properties of 9.3. The addition of 9.3 mAb to T cell cultures has demonstrated two distinct effects. In the first, 9.3 can play a primary role in inducing T cell proliferation (Hara et al., 1985; Moretta et al., 1985a). In monocyte-depleted cultures, the addition of PMA was required to observe proliferation (Hara et al., 1985). Addition of 9.3 mAb to monocyte-depleted cultures failed to induce IL-2 production or IL-2R expression, whereas abundant IL-2 production and IL-2R expression was observed in cultures containing monocytes or PMA (Hara et al., 1985). Thus, the effects of 9.3 mAb mimic the effects of anti-T3 or anti-Ti mAb, although in one study the kinetics of the response to 9.3 were delayed compared to anti-T3 (Moretta et al., 1985a). The dependency upon the T3/Ti complex for activation by 9.3 has been addressed by modulation of the T3/Ti complex and the study of a Jurkat cell mutant which failed to express the T3/Ti complex (Hara et al., 1985; Moretta et al., 1985a; Weiss et al., 1986a). The modulation experiments performed in different laboratories led to conflicting results regarding this dependency; however, the finding that the Jurkat mutant could still be activated by 9.3 plus PMA supports the notion that activation via the Tp44 molecule is independent of the participation of the T3/Ti complex. Although the ligand of Tp44 is unknown, it is clear that this molecule can be involved in delivering primary activation stimuli. A second accessory function has been demonstrated for Tp44. Addition of 9.3 mAb can substitute for one of the functions of adherent cells in the response to anti-T3, anti-Ti, or T cell mitogenic lectins. If 9.3 is added to cultures of purified T cells in the presence of cross-linked anti-T3, T cell proliferation is observed (Ledbetter et al., 1985; Martin et al., 1986; Weiss et al., 1986a). Thus, 9.3 substitutes for the second function provided by AC, alluded to above, which may involve a soluble factor. In a similar manner, 9.3 can synergize with anti-T3, anti-Ti, or the lectin PHA in inducing Jurkat to produce IL-2 (Martin et al., 1986; Weiss et al., 1986a). Interestingly, 9.3 cannot reconstitute the response to soluble antibody or calcium ionophore by purified T cells or Jurkat (Weiss et al., 1986a). Thus, it does not fully replace the function of AC.

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The potency of 9.3 in exerting the two effects described is markedly different. Saturating concentrations of 9.3 are required for delivering a primary activation stimulus (Weiss et al., 1986a). The finding that 9.3 can be used at siibsaturating levels of antibody for the accessory function is consistent with the notion that Tp44 is more likely to play an accessory role in T cell activation (Weiss et al., 1986a). The precise role of Tp44 in T cell activation awaits the identification of its relevant physiologic ligand.

2. T1 (CD5, Tp67, L e d ) The T1 antigen is expressed as a 67-kDa protein on all T cells and thymocytes (Reinherz et al., 1979a; Martin et al., 1980). The murine homologue of T1 is Lyt-1 (Ledbetter et al., 1981). The cDNA encoding T1 has recently been isolated (Jones et al., 1986). T1 has a large, 347 amino acid extracellular domain and a 93 amino acid intracytoplasmic domain. Although mAb reactive with T1 have little effect upon T cell activation alone, they appear to have some capacity to deliver accessory stimuli for T cell proliferation. Thus, anti-T1 can augment the proliferative responses and IL-2 production by antiT3-stimulated T cells if immobilized anti-T3 mAb are used (Ledbetter et al., 1985; Ceuppens and Baroja, 1986). The effect of anti-T1 appears to be monocyte independent, since F(ab’)2and Fab are able to provide this accessory function (Ledbetter et al., 1985). Similar effects have been observed in the murine system (Hollander et al., 1981; Logdberg and Shevach, 1985). The accessory function provided by anti-T1 may be distinct from the effect of mAb 9.3 or IL-1, as the effects of these ligands are additive (Ledbetter et al., 1985). The ligand of T1 is unknown.

3. IL-1 Receptor (ZL-1R ) Although the identification of the IL-lR remains somewhat tentative, this receptor 1s included in this discussion because of the numerous studies performed with its ligand, IL-1. Numerous studies in the past have demonstrated that AC function can be, in part, reconstituted by soluble factors in the supernatants of adherent cells (reviewed by Mizel, 1982). One of the most potent of these factors was termed IL-1 (Aarden et al., 1979) and has been purilied to homogeneity (Kronheim et al., 1985). Recently, two cDNAs have been isolated which encode two forms of IL-l,IL-l, and IL-lp(Auron et al., 1984; March et al., 1985). The predicted size of the protein encoded by each of these clones is 31 kDa, though the mature form of the protein is proteolytically processed to 17.5 kDa (Auron et al., 1985). The role of each of these forms of IL-1 remains to be clarified. Interestingly, an activity which would suggest a membrane form of IL-1 has been described (Kurt-Jones et al., 1985). This is of particular interest, since no hydrophobic domain has been identified in the IL-1 sequence (Auron et al., 1984; March et al., 1985).

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Highly purified IL-1, has been used to study the structure of the IL-1R. Radiolabeled IL-lBwas cross-linked to a 75-kDa cell surface protein on the murine T cell leukemic line LBRM-33-1A5 (Dower et al., 1985). This line was used to identify this putative IL-1R because it responds to IL-1 in the presence of suboptimal concentrations of PHA (Gillis and Mizel, 1981). This line was found to express -500 receptors per cell with an affinity of -2 X 10lO/M (Dower et al., 1985). Human T cells were found to express 100 or fewer IL-1R (Dower et al., 1985). Most studies have relied upon the use of purified IL-1, to study the relative function of its receptor. Although IL-1 has little effect upon human T cells by itself, it can substitute for AC if added with appropriate stimuli. IL-1 fails to induce IL-2 production or IL-2R expression when added to cultures of purified T cells or the T cell leukemic line Jurkat. However, it can synergize with PHA or immobilized anti-T3 in the induction of IL-2 production or IL-2R expression (Williams et al., 1985; Manger et al., 1985; Scheurich et al., 1985). Immobilization of the T3 mAb appears to be necessary for the synergistic effects of IL-1 (Williams et al., 1985; Manger et al., 1985). Thus, IL-1 does not completely reconstitute the function of AC, but appears only to substitute for the soluble factors liberated by AC. The phorbol ester PMA can substitute for the role of IL-1 in most systems (deVries et al., 1980; Farrar et al., 1980b). The role of IL-1 in anti-T11 or anti-Thy-l-induced activation remains to be clarified. 4 . Other Accessory Molecules Involved in T Cell Activation Several other T cell surface molecules are thought to play a role in T cell activation. Among these, the most well characterized are LFA-1, expressed on all T cells (Kurzinger et al., 1981), and T4 (CD4, Leu3) and T8 (CD8, Leu2) antigens, which are expressed on the two mutually exclusive major T cell subsets (Reinherz et al., 1979b; Reinherz and Sclossman, 1980). The inhibitory effects of mAb reactive with these antigens upon T cell proliferative responses and cytolytic activity of CTL is the strongest argument for their participation in T cell activation (Davignon et al., 1981; Meuer et al., 1982; Biddison et al., 1982). Whereas the ligand of LFA-1 is unclear, the MHC restriction pattern observed with cells expressing T8 or T4 suggests that these molecules may interact with nonpolymorphic class I or class I1 MHC antigens, respectively. These human cell surface molecules and their murine homologues are generally felt to increase the avidity of the interaction between the T cell and the relevant target cell or APC (Biddison et al., 1982; Swain et al., 1983; MacDonald et al., 1982; Marrack et al., 1983). However, several recent studies have suggested that mAb reactive with the T4 molecule or the murine homologue L3T4 may induce a negative signal apart from diminishing the avidity of the T cell-AC interaction (Bank and

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Chess, 1985; Wassmer et al., 1985). A more detailed discussion of the role of these antigens is beyond the scope of this review and is presented in several recent reviews (Littman, 1987; Springer et al., 1982; Springer et al. ,1987). 111. Synergy between Ca2+ lonophores and Phorbol Esters in T Cell Activation

The cell surface structures that initiate T cell activation must be capable of generating regulatory intracellular signals. One approach to the identification of these signals is to “bypass” the cell surface structures involved in activation by stimulating the cell with pharmacological agents known to activate particular signaling pathways. Application of this approach to T cell activation reveals a remarkable synergy between Ca2 ionophores and phorbol esters. Whereas neither agent alone is mitogenic, the combination of Ca2+ ionophore and phorbol ester activates T cells to produce lymphokines, to express receptors for IL-2, and to proliferate (Weiss et al., 1984a; Truneh et al., 198i3). Of interest, this combination cannot substitute for the growthpromoting effect of IL-2 on T cell lines that are dependent upon exogenous IL-2. In other words, the combination of Ca2+ ionophore and phorbol ester mimics the effect of activation by antigen, but does not bypass the requirement for the IL-%mediated proliferative signal. The implication of these findings is that the early stages of T cell activation involve synergy between at least two discrete intracellular signals, one of which can be supplied by Ca2+ ionolphore and the other by phorbol esters. There is little doubt that the activation signal delivered by Ca2 ionophores is an increase in [Ca2 Ii. There is less certainty as to the identity of the phorbol ester-mediated signal, but because the only known phorbol ester receptor is pkC, the effects of phorbol esters have been attributed to activated pkC. Synergy between increases in [Ca2 Ii and activated pkC appears to regulate cellular activities in a variety of tissues and has been implicated in systems ranging from platelet activation to aldosterone secretion (reviewed by Berridge and Irvine, 1984.; Nishizuka, 1986). +

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A. RECEPTOR-MEDIATED INCREASES IN [Ca2+IiA N D ACTIVATIONOF PROTEIN KINASE c: GENERAL CONSIDERATIONS

It is worthwhile considering the general mechanisms by which receptors regulate increases in [Ca2 Ii and activate pkC. A single receptor-mediated event, the hydrolysis of a membrane phospholipid, phosphatidylinositol bisphosphate (PIP,), can stimulate both intracellular pathways. The turnover of PIP, generates two products with second messenger capabilities: inositol 1,4,5-trisphosphate (1,4,5-1P3), which mobilizes intracellular Ca2 , and diacylglycerol (DG), which activates pkC (Berridge, 1983; Berridge and Irvine, 19134). +

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1 . IP,-Mediated Increases in [Ca2+Ii 1,4,5-IP3, a water-soluble compound, binds to specific receptors within the endoplasmic reticulum, stimulating an efflux of Ca2+ (Streb et al., 1983; Hirata et al., 1985). In intact cells, release of Ca2+ from intracellular stores can increase [Ca2+Iifrom 100 nM to >SO0 nM (Lew et al., 1984). Increases in [Ca2+Ii that are due solely to intracellular mobilization are invariably transient and are usually of only several minutes duration. Certain receptors, such as T3/Ti and the a-1 adrenergic receptors on hepatocytes, can stimulate extracellular Ca2 uptake as well as mobilize intracellular Ca2 (Imboden and Stobo, 1985; Williamson et aZ., 1985). While intracellular Ca2+ mobilization accounts for the initial response, sustained receptor-mediated increases in [Ca2+Ii require extracellular Ca2+ influx. The mechanism by which these receptors regulate extracellular Ca2+ influx is not understood. The metabolism of 1,4,5-IP3 is complex. Sequential phosphatases can remove phosphates from the inositol ring, eventually converting 1,4,5-IP3 to free inositol which can be recycled into phospholipid (Berridge, 1983). Alternatively, a cytoplasmic kinase can phosphorylate 1,4,5-IP3on the 3 position of the inositol ring, yielding inositol 1,3,4,5-tetrakisphosphate (IP,) (Irvine et al., 1986). The existence of alternative pathways of 1,4,5-IP3 metabolism implies that certain metabolites of 1,4,5-IP3 also have regulatory functions, presumably distinct from those of 1,4,5-IP3. Speculation in this regard focuses on the regulation of extracellular Ca2+ influx and, because the cell invests ATP in its formation, on the possible role of IP,. An additional complexity is imparted to the inositol phosphate system by the observations that these compounds can exist in a 1,2 cyclic form and that cyclic inositol phosphates can be demonstrated following receptor stimulation of intact cells (Dawson et al., 1971; Dixon and Hokin, 1985). Both cyclic and noncyclic 1,4,5-IP3can release Ca2 from permeabilized cells, demonstrating that the cyclic configuration is not required for Ca2+ mobilization (Wilson et al., 1985). Whether the cyclic configuration confers some additional regulatory capability on the inositol phosphates remains to be determined. +

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2 . DG- and Phorhol Ester-Mediated Activation of Protein Kinase C The second signaling pathway linked to PIP, hydrolysis is the activation of pkC. The distinctive feature of pkC, which can phosphorylate a wide range of substrates on serine and threonine residues, is that its activation requires the presence of phospholipid (especially phosphatidylserine) and Ca2 (Takai et al., 1979). In vitro, DG greatly increases the affinity of pkC for phospholipid and Ca2+ and allows pkC activation to occur at Ca2+ concentra+

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tions that are within the intracellular range. In an insightful series of experiments, Catagna et al. observed that biologically active phorbol esters have effects on pkC similar to those of DG, activating pkC at Ca2+ concentrations in the nanomolar range (Castagna et al., 1982). Subsequent studies demonstrated that pkC activity and phorbol ester binding activity copurify (Kikkawa et al., 1983; Kraft and Anderson, 1983; Nidel et al., 1983). In view of the diverse biological effects of phorbol esters, the identification of pkC as the high-affinity phorbol ester receptor underscores the potential importanoe of pkC as a regulator of cellular activities. Recent cloning studies, however, clearly demonstrate that pkC activity and phorbol ester binding are mediated by a family of closely related, but distinct polypeptides (Knopf et al., 1986; Parker et al., 1986; Coussins et d.,1986). This finding, of course, implies that the biology of pkC is considerably more complex than previously appreciated. It is possible, for example, that there is differential expression of pkC subtypes, that the different species of pkC have distinct substrate specificities, and that there are constraints on the interactions between particular receptors and the different forms of pkC. Since all functional studies to date have treated pkC activity as a single enzyme, we will, of necessity, continue to discuss pkC as if it were a single entity. It is likely that pkC, which has an apparent M , of 80,000, is composed of a regulatory region which binds Ca2+, phospholipid, and DG (or phorbol ester), and a catalytic region (Nishizuka, 1986). The putative catalytic region of pkC has extensive sequence homology with the catalytic subunit of CAMPdependent kinase (Knopf et al., 1986; Parker et al., 1986). In most unstimulated cells, including T lymphocytes, pkC activity is recovered from the cytosol. Activation of pkC, whether mediated by receptor-ligand interaction or phorbol esters, is temporarily associated with a loss of pkC activity from the cytosol and a proportionate recovery of pkC activity in the membrane fraction (Farrar and Anderson, 1985). This translocation of pkC to the membrane is thought to be a critical event in its physiologic activation (Bell, 1986). The binding of pkC to membranes has been studied in a reconstitution system using purified enzyme and inside-out erythrocyte vesicles. As expected, phorbol esters promote the binding of pkC to membranes in this system (Wolf et al., 1985a). Increasing the Ca2+ concentration from 100 nM to 500 nM also promotes the association of pkC with membranes (Wolf et al., 1985a). It is noteworthy that the Ca2 -mediated binding is reversible and occurs at (;a2 concentrations that are within the range of receptor-mediated increases in [Caz+lI(but well below the 5 to 50 F M required for Ca2+ to activate pkC in the absence of DG). Of interest, the effects of Ca2+ and phorbol esters on pkC binding are synergistic. An effect of pkC binding, therefore, may in part explain the widely observed synergy between Ca2+ +

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ionophores and phorbol esters. Taken together, these binding studies suggest a model for the physiologic activation of pkC in which receptor-mediated increases in [Ca2+Ii, while probably not of sufficient magnitude to activate pkC directly, “prime” pkC by promoting its binding to plasma membranes. This binding facilitates contact between pkC and DG, leading to the formation of a stable, active complex composed of pkC, phospholipid, Ca2+, and DG. The termination of receptor-mediated pkC activation may be quite complex. Intuitively, a critical factor in the maintenance of an active pkC complex must be continued receptor-mediated generation of DG. Indeed, in reconstitution experiments, a stable membrane-pkC complex requires the continuing presence of phorbol ester. Even in the presence of phorbol ester, however, this complex dissociates when ATP is added (Wolf et d., 1985b). The ATP-induced dissociation is nucleotide specific and requires Mg2+, implying that the release of pkC from membranes is due to phosphorylation of a membrane protein (possibly the autophosphorylation of pkC). Whether a similar ATP-mediated mechanism releases pkC from membranes in vivo is not known. A finding that argues against such an event in vivo is the sustained (>1hour) translocation of pkC that invariably follows the addition of phorbol esters to intact cells. An interesting observation of potential importance for the physiologic regulation of pkC is that pkC can serve as a substrate for a cytosolic proteinase, termed calpain (Inoue et d., 1977). The calpain-mediated proteolysis of pkC releases a 50-kDa fragment which is fully catalytically active in the absence of Ca2 and phospholipid and which is thought to represent the catalytic subunit of pkC freed of its regulatory region (Inoue et al., 1977). Activation of calpain was initially thought to require supraphysiologic concentrations of Ca2+, raising doubts as to whether proteolyltic cleavage of pkC occurs in viuo. Recent studies, however, demonstrate that micromolar concentrations of Ca2+ promote the binding of both pkC and calpain to partially purified plasma membranes (Melloni et al., 1985). Under these conditions, binding is followed by time-dependent proteolysis of pkC. In the presence of membranes, therefore, the in vitro conversion of pkC to its Ca2 and phospholipid-independent form occurs at Ca2 concentrations that approach the intracellular range. These studies suggest an alternative mechanism for the regulation of pkC in which receptor-mediated pkC translocation and increases in [Ca2+Iiresult in the generation of an irreversibly activated fragment of pkC. Implicit in the identification of pkC as a signaling pathway is the assumption that pkC-mediated phosphorylation of particular proteins in some way influences the functions of those proteins. In vitro, a wide range of proteins can serve as substrates for pkC, and a number of cell surface receptors, +

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cytoskeletal proteins, and enzymes have been proposed as in vivo substrates for pkC (Nishizuka, 1986). The addition to PMA to intact T lymphocytes leads to the phosphorylation of the IL-2 receptor, T200, the transferrin receptor, and T3 chains of the T3/Ti complex as well as to the hyperphosphorylation of HLA class I antigens (Shackelford and Trowbridge, 1986; Cantrell et al., 1985; Samelson et al., 1985). Compelling data indicate that, at least in the case of the IL-2 receptor, this phosphorylation is directly mediated by pkC. Purified pkC phosphorylates the cytoplasmic domain of immunoprecipitated IL-2 receptor (Shackelford and Trowbridge, 1986). By tryptic peptide analysis, these in uitro phosphorylation sites are identical to those induced in vivo by PMA. The functional consequences of this and other pkC-mediated phosphorylations, however, are far from certain. Studies of the consequences of pkC activation on T lymphocytes and other cells have relied heavily on phorbol esters to stimulate pkC and, to varying degrees, have equated the effects of phorbol esters with pkC activation. The use of phorbol esters for this purpose has several limitations that deserve emphasis. First, phorbol esters bind to more than one species of pkC, but it is not yet known whether there are constraints on receptor interactions with pkC subtypes. Second, although pkC remains the only convincingly demonstrated phorbol ester receptor, it is possible that phorbol esters, in addition to activating pkC, may directly stimulate other signaling pathways. A recent report of the isolation of a phospholipid-dependent, Ca2 -independent kinase that is activated by phorbol esters serves to emphasize this point (Malviya vt al., 1986). Finally, there are marked differences in the metabolism of phorbol esters and DG. Phorbol esters are metabolized slowly, if at all, whereas DG turns over readily. As a result, there can be dramatic differences in the duration and magnitude of pkC activation following the addition of phorbol esters and following receptor stimulation. Phorbol estermediated activation, as reflected by the translocation of pkC to the membrane, is virtually irreversible, whereas receptor-mediated activation of pkC can be transient. +

IV. Receptor-Mediated Signal Transduction during T Cell Activation

The ability of Ca2+ ionophores and phorbol esters to deliver activation signals to T cells implies that T cells express cell surface receptors which regulate increases in [Ca2+Iiand activate pkC. Studies of receptor signaling during physiologic T cell activation are limited to a certain extent by the ambiguities inherent in studying a cell-cell interaction (a situation in which several types of receptors might signal simultaneously). As a result, studies of signal transduction in T cells have relied heavily on the use of mAbs as agonists to stimulate specific receptors. This approach has identified three

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separate T cell surface structures which appear to signal by increasing [Ca2+Ii:T3/Ti, T11, and Thy-1. At least one of these, T3/Ti, also activates pkC. Three additional cell surface structures, Tp44, Tp67, and the IL-1 receptor, can deliver signals during the early stages of T cell activation which are not readily explained by a direct effect on either [Ca2+Iior pkC. We will review recent studies of the mechanisms of signal transduction by these six cell surface structures.

A. T3/Ti COMPLEX Several lines of evidence indicate that an increase in [Ca2+],serves as an intracellular signal for T3/Ti-mediated activation. Soluble T3/Ti mAb and Ca2+ ionophores, such as A23187 and ionomycin, display similar requirements in their abilities to activate T cells. Neither soluble T3/Ti mAb nor Ca2+ ionophores are effective alone, but both can synergize with PMA to elicit lymphokine production (Weiss et al., 198413; Truneh et al., 1985). If PMA is present, Ca2 ionophores activate T3/Ti-negative mutants of Jurkat, demonstrating that ionophore-mediated activation does not require cell surface expression of T3/Ti (Weiss et d . , 198413). The recent development of techniques to monitor [Ca2+Ii in intact, small cells has provided direct evidence that perturbation of T3/Ti increases [Ca2+],.The addition of T3/Ti mAb to T lymphocytes loaded with the Ca2+-sensitive fluor, quin2, induces substantial sustained increases in [Caz+li (Weiss et al., 1984b; Imboden et al., 1985; M. Weiss et al., 1984; Oettgen et a l . , 1985). T3/Ti-mediated increases in [Ca2+], have been demonstrated in peripheral T cells, T cell lines, and T cell clones and are not simply a consequence of the interaction of mAb with the cell surface. Flow cytometric analysis of [Ca2+Ii using the second-generation Ca2 indicator, Indo-1, demonstrates that T3-mediated increases in [Ca2+Ii are not restricted to any T cell subset, but occur in essentially all peripheral T cells exposed to a T3/Ti mAb (June et al., 1986). The nature of the perturbation required for triggering the T3/Ti complex has received limited attention. It is clear that mAb reactive with distinct sites of this complex can induce comparable increases in [Caz+li. Hence, anti-T3 or distinct anti-Ti mAb can all induce greater than $fold increases of [Ca2+Ii (Weiss et al., 1984b; Imboden et al., 1985; Oettgen et al., 1985; O’Flynn et al., 1985; Lanier et al., 1986). These mAb do not appear to trigger T3/Ti-mediated signal transduction by cross-linking receptors as univalent Fab fragments of T3 mAb also induce [Ca2+Iiincreases (Oettgen et al., 1985). This would imply that conformational changes of T3/Ti may be induced by relevant ligand binding events, and this results in receptormediated signal transduction. In support of this notion, one pentameric IgM anti-Ti mAb binds to an epitope of the HPB-ALL Ti effectively, but fails to induce substantial increases in [Ca2+Ii(Lanier et al., 1986). +

+

H U M A N T LYMPHOCYTE ACTIVATION

21

While there is little doubt that perturbation of TS/Ti by mAbs leads to increases in [Ca2+Ii, there are legitimate concerns as to how accurately T3/Ti mAbs mimic physiologic activation of TS/Ti. Antigen-primed AC, however, stimulate increases in [Ca2+Ii when added to quin%loaded, antigen-specific T cell clones (Nisbet-Brown et al., 1985; Shapiro et ul., 1985). When considered together with the studies of T3/Ti mAb, the latter observation strongly supports the notion that physiologic activation of T3/Ti induces an increase in [Ca2+Ii. As is the case with many other receptors that signal via increases in [Ca2 Ii, perturbation of TS/Ti stimulates the turnover of polyphosphoinositides and the generation of inositol phosphates (Imboden and Stobo, 1985). The addition of TS/Ti mAb to Jurkat cells leads to a prompt (detectable in <20 seconds) increase in IP, that is sustained for >30 minutes. Similary, antigen recognition by a human T cell clone is associated with a substantial, prolonged increase in IP, (Imboden et ul., 1987). When inositol phosphates are resolved by high-performance liquid chromatography (HPLC), it is clear that the T3/Ti-mediated change in IP, is due in large part to increases in the Ca2+-mobilizing isomer 1,4,5-IP3 (Stewart et al., 1986). A substantial proportion of the 1,4,5-IP, generated by T3/Ti stimulation is converted to IP,, an observation that underscores the possibility of a regulatory role for IP, in T cell activation (Stewart et d., 1986). In all cellular systems studied, receptor-mediated increases in I,4,5-IP3 are associated with the release of Ca2+ from intracellular stores (Berridge and Irvine, 1984). In the presence of extracellular Ca2+, the addition of T3/Ti mAb to Jurkat cells leads to an increase in [Ca2+Ii from 80 nM to a peak of >400 nM within 60 seconds (Imboden et al., 1985). [Ca2+Iithen falls to a plateau of 200-250 nM and remains elevated above basal levels for >30 minutes. When care is taken to minimize the Ca2 -buffering effect of intracellular quin2, depletion of extracellular Ca2 has little effect on the initial peak TS/?’i-mediated increase in [Ca2 Ii, but completely prevents the sustained increase (Imboden and Stobo, 1985). This finding indicates that the initial peak increase in [Ca2+Iifollowing perturbation of T3/Ti Jurkat cells is due to intracellular Ca2 mobilization, while the sustained T3/Ti-mediated increases in [Ca2+Ii require extracellular Ca2+. In addition to mobilizing intracellular Ca2 , therefore, perturbation of T3/Ti must either open a Ca2+ channel in the plasma membrane or regulate the transport of Ca2+ across the plasma membrane. Distinction between these two general mechanisms cannot be made using intact quin2-loaded cells and requires either study of (=a2+ transport in a cell-free system or the application of patch clamping techniques to directly study channel conductance. Initial patch clamping of T lymphocytes failed to demonstrate Ca2+ channels under conditions in which voltage-gated Ca2+ channels, if present, should have been +

+

+

+

+

+

22

ARTHUR WEISS A N D J O H N B . I M B O D E N

identified (Decoursey et al., 1984; Matteson and Deutsch, 1984). Recently, however, Kuno and colleagues have identified a Ca2+ channel in T lymphocytes that is not voltage gated, and its frequency of opening increases following the addition of PHA (Kuno et al., 1986). This channel, whose tissue distribution and gating mechanisms are of considerable interest, is an attractive candidate to explain T3/Ti-mediated extracellular Ca2 influx. One immediate consequence of the T3/Ti-mediated increase in [Ca2 Ii is enhanced activity of the plasma membrane Na+ / H antiporter, leading to a sustained increase in intracellular pH (Rosoff and Cantley, 1985). Activation of the Na+ / H antiporter has been implicated as an important signaling mechanism in the stimulation of quiescent cells by growth factors and in B lymphocyte differentiation. Dimethylamiloride, an inhibitor of the Na+ / H antiporter, inhibits IL-2 production by Jurkat cells stimulated with a T3 mAb and PMA. While the specificity of dimethylamiloride has been questioned, it is of interest that cyclosporine A also blocks the T3/Ti-mediated increase in intracellular pH (Rosoff and Teres, 1986). The link between T3/Ti and polyphosphoinositide turnover suggests that perturbation of T3/Ti, in addition to increasing [Ca2+Ii,also activates pkC. Direct support for this notion stems from the demonstration that T3 mAbs induce the translocation of pkC activity from the cytosol to the membrane fraction. In unstimulated peripheral T cells, pkC activity is recovered almost entirely from the cytosolic fraction. Following the addition of a T3 mAb, there is a >90% decrease in cytosolic pkC activity and a proportionate, concomitant increase in membrane-associated pkC (Farrar and Ruiscetti, 1986). Under these conditions, the TS/Ti-mediated translocation of pkC is maximal at 10 minutes and of <40 minutes’ duration. The observation that T3/Ti mAb activate pkC leads to an apparent paradox, because in order to activate T cells, T3/Ti mAb require an additional stimulus that can be supplied by PMA. The potential pitfalls in interpreting the effects of PMA, which are discussed in the preceding section, are applicable here. The duration of pkC activation appears to be critical for T cell activation. While the translocation of pkC induced by soluble T3 mAb is transient, PMA-induced translocation appears to be irreversible (Farrar and Ruiscetti, 1986). When T cells are stimulated with immobilized T3 mAb, however, pkC is translocated for >2 hours, demonstrating that the duration of the TS/Ti-mediated translocation can be considerably influenced by the form of the ligand (Manger et al., 1987). Immobilized, but not soluble, T3 mAb can activate certain T cell lines and clones in the absence of additional stimuli. On the other hand, immobilized T3 mAb do not activate resting peripheral T cells and Jurkat cells, suggesting that in these cells PMA may activate an as yet unidentified signaling pathway in addition to pkC. In support of this notion, either IL-1 or 9.3 (an mAb reactive with Tp44) can +

+

+

+

+

H UMAN T LYMPHOCYTE ACTIVATION

23

synergize with immobilized T3/Ti to activate resting peripheral T cells and Jurkat cells, yet neither IL-1 nor 9.3 induce detectable translocations of pkC (Weiss et al., 1986; Manger et al., 1987). At least one function of T3/Ti during signal transduction is to activate the phosphodiesterase that hydrolyzes PIP,. In membranes prepared from several nonlymphoid tissues, the addition of guanosine triphosphate (GTP) increases the activity of the PIP, phosphodiesterase, suggesting that Ca2+mobilizing receptors may be coupled to this enzyme by GTP binding proteins (G proteins) (Cokcroft and Gomperts, 1985). One approach to the identification of these putative G proteins has been to take advantage of the ability of certain bacterial toxins, such as cholera toxin and pertussis toxin, to covalently modify and functionally alter G proteins (Cassel and Pfeuffer, 1978; Katada and Ui, 1982). Exposure of Jurkat cells to cholera toxin completely inhibits TS/Ti-mediated polyphosphoinositide turnover and increases in [Ca2+Ii (Imboden et al., 1986). This effect of cholera toxin on T3/Ti is temporally dissociated from its well-recognized ability to ADPribosylate the stimulating G protein (Gs) of adenylate cyclase and is not mimicked by directly activating adenylate cyclase with forskolin. These observations suggest that a cholera toxin substrate, presumably a G protein other than Gs, regulates signal transduction by T3/Ti. An attractive candidate for this substrate is Go, a G protein that can functionally interact with muscarinic receptors in reconstitution experiments, but whose physiologic role is not known (Florio and Sternweis, 1985). Although originally thought not to be a substrate for cholera toxin, the cloning of Go revealed that it contains a cholera toxin ADP-ribosylation site (Angus et al., 1986). The multimeric complexity of T3/Ti appears to be unique among receptors that are linked to polyphosphoinositide turnover. Even those receptors such as the hepatic a,-adrenergic receptor, which stimulates extracellular Ca2+ influx as well as mobilizes intracellular Ca2+, have no known associated T3-like chains. If we assume that Ti is sufficient for antigen recognition, then the complexity of T3 suggests either that the regulation of signal transduction by T3/Ti is exceptionally complicated or that T3/Ti has a signaling role in addition to activating the hydrolysis of PIP,. Of interest in this regard is the demonstration that T3 polypeptides can be phosphorylated in response to activation by antigen or by treatment with PMA. Antigen recognition by a murine T cell hybridoma results in serine phosphorylation of the T3-6 chain and tyrosine phosphorylation of p21 of the antigen-receptor complex (Samelson et al., 1985, 1986). The kinase(s) involved is (are) not known, but treatment of this T cell hybridoma with PMA also induces serine phosphorylation of the T3-6 chain, raising the possibility that activated pkC phosphorylates this chain during physiologic T cell activation. However, PMA, but not antigen, also stimulates phosphorylation of the

24

ARTHUR WEISS AND J O H N B. I MB O D E N

nonglycosylated 26-kDa E chain of the antigen-receptor complex. Phorbol esters stimulate the phosphorylation of T3 components of the human antigen-receptor complex (Cantrell et al., 1985). Exposure of either human T lymphoblasts or the T cell line, HPB-ALL, to phorbol dibutyrate leads to a rapid (detectable in <5 minutes) phosphorylation of the T3-y chain and, to a lesser degree, phosphorylation of T3-6 (Cantrell et al., 1985). While the functional significance, if any, of these phosphorylations is not known, phorbol esters (and synthetic DGs) induce a selective >50% decrease in the cell surface expression of T3/Ti in human T cells and can render T cell clones unresponsive to antigen (Cantrell et al., 1985; Ando et al., 1985). One possible consequence of phorbol ester-induced phosphorylation of T3, then, is the desensitization and down-regulation of T3/Ti. B. T11 As discussed in Section II,B, perturbation of T11 by the appropriate combination of mAb activates human T lymphocytes. These same combinations of T11 mAb also trigger increases in [Ca2+Iiin T cells, suggesting that an increase in [Ca2+Ii constitutes an intracellular signal for T11-mediated activation (M. Weiss et al., 1984). Perturbation o f T l l also increases [Ca2+Iiin human thymocytes and NK cells that do not express T3, clearly demonstrating that the T11-mediated increase in [Ca2+Ii does not require the cell surface expression of T3/Ti (June et al., 1986; Alcover et al., 1986). It is not known whether perturbation of T11 stimulates the generation of IP,. An indirect argument against a link between T11 and polyphosphoinositide turnover stems from the observation that the T11-mediated increase in [Ca2+], in quin2-loaded T cells is due exclusively to extracellular Ca2+ influx (Alcover et al., 1986). In studies of Indo-1-loaded T cells, however, T11-induced intracellular Ca2+ mobilization has been reported (June et al., 1986).The discrepancy between these two observations may reflect the need for higher intracellular indicator concentrations when using q u i d (all intracellular Ca2+ indicators chelate Ca2+ and, in sufficient concentrations, can prevent increases in [Ca2+Iidue solely to intracellular Ca2+ release) (Lew et al., 1984). Clearly, direct measurements of inositol phosphates following stimulation of T11 are needed. In the absence of other stimuli, perturbation of T11 can activate T lymphocytes, including resting peripheral T cells (Meuer et al., 1984b). Because an increase in [Ca2+Iialone is not sufficient to activate T cells (Weiss et al., 1984a; Truneh et al., 1985), it is very likely that signal generation by T I 1 involves more than an increase in [Ca2+Ii.Moreover, in its ability to provide all the requirements for the activation of resting peripheral T cells, T11 appears to have a signaling capability that is not observed with T3/Ti.

HUMAN T LYMPHOCYTE ACTIVATION

25

Whether these additional signaling effects of T11 are mediated through pkC and/or some other intracellular pathway remains to be determined. C. Thy-1 In the presence of PMA, most Thy-1 mAb activate murine peripheral T lymphocytes when a second-layer anti-Ig antibody is added (Kroczek et al., 1986a). This synergy between the Thy-l-mediated signal and PMA suggests that perturbation of Thy-1 increases [Ca2 Ii. Thy-l-induced increases in [Ca2+Ii have been clearly demonstrated in several cellular systems. Quin2loaded murine T cells and thymocytes that have been pretreated with Thy-1 mAb exhibit an increase in [Ca2+Iiin response to the addition of an anti-Ig antibody (Kroczek et al., 1986a). While most Thy-1 mAb require “crosslinking” with anti-Ig, one Thy-1 mAb, G7, elicits a prompt, sustained increase in [ Ca2+Ii when added directly to quin2-loaded T cell hybridoma cells. G7 activates these hybridomas in the absence of other stimuli. The Thy-l-mediated increase in [Ca2+Ii has been studied further by introducing the Thy-1 gene into cells that do not normally express Thy-1. Following transfection with the Thy-1.2 gene, Jurkat cells and several B cell lines express abundant cell surface Thy-1. The addition of Thy-1 mAb to transfected Jurkat cells, but not to the parental cells, leads to a sustained increase in [Ca2+Ii and, in the presence of PMA, elicits the production of IL-2 (Gunter et al., 1986). Of considerable interest, Thy-l-mediated increases in [Ca2+Iican be demonstrated in two of three B cell transfectants (Kroczek et al., 1986b). These observations establish that Thy-1 can deliver an activation signal to human T cells and that the Thy-l-mediated signal is independent of any other T cell-specific molecule. Thy-1 is anchored to the plasma membrane by a covalent attachment to phosphatidylinositol (or its phosphorylated derivatives) and not by a hydrophobic domain (Low and Kincade, 1985; but see also Seki et al., 1985). This conclusion is particularly intriguing in view of the ability to Thy-1 to transmit an activation signal. The known association between PIP, hydrolysis and Ca2 mobilization suggests that the covalent attachment of Thy-1 to phosphatidylinositol is in some way causally linked to the Thy-l-mediated increase in [Ca2+Ii. It is difficult, however, to develop a mechanistic model along these lines because the inositol ring attached to Thy-1 is extracellular (inositol phosphates are hydrophilic and have only been implicated as intracellular messengers). Alternatively, Thy-l-mediated signaling may require interaction with another plasma membrane protein which in turn triggers an increase in [Ca2+Ii.The transfection studies demonstrate that this putative effector molecule cannot be T cell specific and must be sufficiently well conserved that murine Thy-1 functionally interacts with the human effector. +

+

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ARTHUR WEISS A N D JOHN B . IMBODEN

D. Tp67, Tp44,

AND THE

IL-1 RECEITOR

There are few studies of signal transduction by Tp67, Tp44, and the IL-1 receptor during T cell activation. These cell surface structures have been variably described as delivering signals that mimic the effects either of Ca2 ionophores or of phorbol esters. Indeed, high concentrations of a Tp44 mAb, 9.3, stimulate IP, generation and increases in [Ca2+Iiwhen added to Jurkat cells (Weiss et al., 1986a). The 9.3-mediated increases in IP,, however, are only a fraction of those observed following stimulation with T3/Ti mAb and are not associated with detectable pkC translocation. A direct effect of Tp44 on either [Ca2+Iior pkC, therefore, does not provide a satisfactory explanation for the ability of low concentrations of 9.3 to synergize with immobilized T3/Ti mAb in the activation of Jurkat and resting peripheral T cells. By a similar argument a direct effect on polyphosphoinositide turnover is not likely to be the only signal transducing mechanism for Tp67 and IL-1 receptor, given the abilities if these structures to synergize only with immobilized T3/Ti mAb in activation. +

V. Role of Intracellular Signals Other Than Ca2+ and pkC

There is indirect evidence for a mitogen-derived intracellular signal other than Ca2 and pkC during T cell activation (Kaibuchi et al., 1985; Gelfand et al., 1985). PHA, for example, can augment the proliferative response of peripheral T cells to the combination of ionomycin and synthetic DG (Kaibuchi et al., 1985). The mechanism of this PHA-mediated effect and the cell surface structure(s) involved is not known. Signaling pathways that have been implicated in T cell activation include changes in cyclic nucleotides (Wedner and Parker, 1976) and the opening of voltage-gated K+ channels (Decoursey et al., 1984). Voltage-gated K + channels, which have been recently reviewed, are the predominant ion channel expressed by T lymphocytes (Decoursey et al., 1984; Matteson and Deutsch, 1984). The gating characteristics of these channels are altered by PHA such that the channels open more frequently and at more negative membrane potentials following the addition of PHA to patch-clamped T cells. K + channel blockers inhibit PHA-mediated mitogenesis (Chandy et al., 1984), but the specificity of these inhibitory effects has been questioned (Gelfand et al., 1986). Specific cell surface structures that regulate the opening of K + channels have not yet been identified. +

VI. Effects of Early T Cell Activation Events upon Gene Regulation

As detailed above, activation of resting T cells is initiated by cell surface ligand-receptor interactions which result in second-messenger generation.

H U M A N T LYMPHOCYTE ACTIVATION

27

Subsequently, poorly understood events lead to the transcriptional activation of a certain set of genes which are responsible for the early manifestations of T cell activation. It is not clear whether the activation of the relevant genes is in response to the second messengers described above or, more likely, through an as yet undefined cascade of events which leads to the appearance of elements that can bind to targeted sequences and regulate this set of responsive genes. It is likely that following the initial activation of a certain set of targeted genes, a cascade of later gene activation events is initiated by the secondary effects of the products of these primary target genes. Indeed, the activation of IL-2 and its receptor can result in a second wave of gene activation, leading to mitogenesis (Stern and Smith, 1986). The regulation of T cell activation would appear to be most tightly regulated at the level of the activation of this initial set of responsive genes and will thus be the focus of further discussion. Individual T cells appear to respond to activating stimuli with the expression of different overlapping menus of responsive gene products. Hence, heterogeneity among the different lymphokines secreted by individual T cell clones is well documented (Kelso et al., 1982; Prystowsky et al., 1982). The basis for this response heterogeneity of target genes is not clear. Among the many genes that are activated during the initial phase of T cell activation are the oncogenes c-myc and c-fos, the IL-2 receptor, and a variety of lymphokines, including IL-2 and IFN-y.

A. c-myc AND c-fos The protooncogenes c-myc and c-fos encode nucleoproteins (Curran et al., 1984; Eisenman et al., 1985). They have attracted particular interest because they are among the earliest genes to become transcriptionally active in stimulated T cells (Persson et al., 1984; Kaczmarek et al., 1985; Reed et al., 1985). Thus, c-fos and c-myc transcripts can be observed within 10 minutes following the stimulation of resting PBL with PHA and persist for 2 or 48 hours, respectively (Reed et al., 1986). Based on nuclear runoff technology in which the synthesis of nascent RNA chains is examined, this appears to represent true transcriptional activation of c-myc (Kronke et al., 1985). The requirements for the expression of c-myc and c-fos have not been intensively studied. Whereas it is clear that the lectin PHA, a calcium ionophore, or PMA alone can induce c-myc expression (Reed et al., 1985), a synergistic effect is observed if PMA is used in combination with either PHA or Ca2+ ionophore (Yamamoto et al., 1985; Granelli-Piperno et al., 1986). The presence of adherent cells can also increase the inductive effects of PHA upon c-myc expression (Kern et al., 1986). These results support the notion

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ARTHUR WEISS A N D JOHN B . IMRODEN

that activation of c-myc and c-fos involves the synergistic effects of an increase in [Ca2+],and the activation of pkC, as discussed above. As the appearance of c-fos and c-myc transcripts precedes the accumulation of IL-2 and IL-SR, it was tempting to speculate that these nucleoproteins might influence the expression of IL-2 and IL-R (Kronke et al., 1985; Reed et al., 1985). However, the addition of a protein synthesis inhibitor, cycloheximide, failed to affect the expression of c-myc, c-fos, IL-2, or IL-2R transcripts (Kronke et al., 1985; Reed et al., 1985). This result has recently been challenged (Weiss et al., 1987). Thus, the role of c-myc and c-fos expression in T cell activation is not clear.

B. INDUCTION OF IL-2

AND

IFN-y

The cloning of the IL-2 and IFN-y genes has greatly facilitated the study of their regulation (Taniguchi et al., 1983; Holbrook et al., 1984; Gray and Goeddel, 1982). The appearance of IL-2 and IFN transcripts correlates well with the production of the protein products of these genes (Efrat et al., 1982). This involves the transcriptional activation of both these genes (Efrat and Kaempfer, 1984; Kronke et al., 1984; Weiss et al., 1986b). Posttranscriptional control is suggested by the ability of cycloheximide to superinduce IL-2 mRNA (Efrat and Kaempfer, 1984). The appearance of both transcripts requires simultaneous stimulation of purified resting T cells or Jurkat cells with PMA together with ligands which increase [Ca2+Ii,such as lectins PHA or Con A, mAb reactive with the T3/Ti complex, or calcium ionophores, although small amounts of IL-2 and IFN-y transcripts may be seen with the lectins alone (Weiss et al., 1984a; Wiskocil et al., 1985; Granelli-Piperno et al., 1986; Kern et al., 1986; Yamamoto et al., 1985). Exceptions to this general model have been well documented, as in the case of the murine EL-4 and the human HUT-78 lines in which PMA alone may be sufficient for IL-2 activation (Farrar et al., 1980a). The basis for this discrepancy is not clear, but, at least in the case of HUT-78, may reflect the more activated phenotype of the cell (Manger et al., 1985). Hence, the synergistic effects of the activation of pKC and increases in [Ca2+Iiappear to be involved in the activation of IL-2 and IFN-y genes in resting T cells. It takes 2-6 hours for IL-2 or IFN-y transcripts to accumulate following stimulation (Wiskocil et al., 1985; Kronke et al., 1985; Reed et al., 1986). It is not clear what events are required to occur during this time period. The failure of cycloheximide to inhibit the appearance of IL-2 or IFN-y transcripts would argue against the participation of another protein whose synthesis is induced earlier (Kronke et al., 1985; Reed et al., 1986). However, conflicting findings have recently been obtained (Weiss et al., 1987). The ability of cyclosporin A to inhibit the induction of IL-2 and IFN-y transcrip-

HUMAN T LYMPHOCYTE ACTIVATION

29

tion may provide some clues regarding these earlier events (Wiskocil et al., 1985; Kronke et al., 1984; Reed et al., 1985). Since cyclosporin A does not interfere with the increase in [Ca2+Ii or activation of pkC in stimulated Jurkat cells, it must interfere with an event more closely linked to the transcriptional activating event (Wiskocil et al., 1985; Manger et al., 1986). Recent reports suggest that cyclosporin A binds to and inhibits calmodulin (Colombani et al., 1985). Another set of studies has identified yet another cyclosporin binding protein, cyclophyllin (Handschumacher et al., 1984). The role of these two proteins in the activation of the lymphokine genes is not yet clear. Apparent coordinate regulation of IL-2 and IFN-y has been observed. Thus, the stimuli required for their activation, their kinetics of appearance, and their inhibition by cyclosporin are sirnilar (Wiskocil et al., 1985). Whether other lymphokines are similarly coordinately regulated is not clear. In contrast, as discussed below, IL-2R is not coordinately regulated with its ligand, IL-2 (Kronke et al., 1984, 1985). The unique conformation of regulatory regions of genes in intact nuclei can render these regions unusually sensitive to digestion by DNase I. The mapping of DNase I hypersensitive sites is one approach to the identification of DNA sequences which are potential sites of transcriptional regulation. The application of this approach to the human IFN-y gene reveals a prominent hypersensitive site within the first intron in nuclei obtained from Jurkat cells (Hardy et al., 1985). This site is far less sensitive to DNase I in a spontaneous variant of Jurkat that has lost the ability to produce IFN-y and is not present in B cells and nonlymphoid cells. The presence of this hypersensitivity, therefore, correlates with the capability of a cell to produce IFN-y. Computer scanning of this intronic hypersensitive region reveals a 25 bp sequence, with 83% homology to a sequence located 300 bp upstream from the promoter of the human IL-2 gene and may be relevant for their coordinate regulation. A similar analysis has been applied to the IL-2 gene (Siebenlist et d.,1986). In order to identify functional DNA sequences of the human IL-2 gene, Fujita et al. (1986) fused potential regulatory regions of the IL-2 gene to the chloramphenicol acetyltransferase (CAT) structural gene. The expression of these recombinants was then studied in resting and activated T cell 1'ines as well as in several non-T cell lines. In an elegant series of experiments, Fujita et al. identified a 200 bp segment in the 5' flanking region of the IL-2 gene that can mediate inducible T cell-specific gene expression. This sequence functioned in an orientation-independent fashion, suggesting this region of the IL-2 gene is a regulatory enhancer whose function is restricted to activated T lymphocytes. Similar results have recently been obtained by Durand et al. (1986).

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ARTHUR WEISS A N D JOHN B. IMBODEN

C. REGULATIONOF

THE

IL-2 RECEPTOR

The IL-2 receptor (IL-2R) is expressed on activated T cells, but more recently also has been identified on other cells (reviewed in Waldman, 1986). From the partial protein sequence, the cDNA encoding the IL-2R has been isolated (Leonard et al., 1984; Cosman et al., 1984; Nikaido et al., 1984). Its expression is transcriptionally regulated (Leonard et al., 1985b; Kronke et al., 1985). At least three distinct initiation sites can be used and appear to be differentially utilized, depending on how the gene is induced (Leonard et al., 1985a). Its regulation in T cell activation is distinct from the regulation of its ligand, IL-2. As discussed above, its expression can be induced by PMA only or by other ligands which effectively activate pkC (Leonard et al., 1985b). Moreover, unlike IL-2 or IFN-7, its activation is not inhibited by cyclosporin A (Kronke et al., 1984). In addition to the effects of the primary activation stimuli, it is up-regulated by its ligand, IL-2 (Leonard et al., 1985b; Hemler et al., 1984; Smith and Cantrell, 1985; Reem et al., 1985). This may explain some of the synergistic effects observed with stimuli used together with PMA, which induce IL-2. Thus, the effect may not be upon the transcription of the IL-2R directly, but may be an indirect effect mediated by the IL-2 induced. A more detailed review of the regulation of the expression of the IL-2R has recently been published (Waldman, 1986). VII. Summary

The physiologic activation of human T cells by antigen involves events that occur between ligands and receptors at the interface of the T cell and antigen-presenting cell (or target cell). These events have been examined by identifying the cell surface receptors involved in such interactions using mAb. Whereas the T3/T cell antigen receptor plays a central role in such interactions, other T cell receptors have been identified which may also contribute to T cell activation in providing primary activation signals or by functioning as accessory molecules. Although the ligands of these other receptors are currently unknown or ill defined, it is likely that this will provide a fruitful area of investigation. The use of mAb as probes to mimic these putative ligands has facilitated the study of the requirements for activation and the biochemical events initiated by the receptors involved. The T cell receptor, a multisubunit complex, has been most intensively studied. Ligands that bind to T3/Ti cannot initiate activation by themselves and require the participation of accessory molecules. Stimulation of T3/Ti results in the formation of at least two potent intracellular second messengers, IP3 and DG, through the hydrolysis of PIP,. These second messengers, in turn, induce an increase in

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31

[Ca2+Iiand the activation of pkC. These two events appear to be essential in the transcriptional activation of certain targeted genes through ill-defined pathways leading to the manifestations of T cell activation.

ACKNOWLEDGMENTS We would like to thank Mr. Michael Armanini and Ms. Denise Go for their excellent secretarial assistance in the preparation of the manuscript. This work was supported in part by a grant from the Arthritis Foundation to A.W. J.I. is a Pfizer Scholar.

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ADVANCES IN IMMUNOLOGY, VOL. 41

Function and Specificity of T Cell Subsets in the Mouse JONATHAN SPRENT AND SUSAN R. WEBB Department of Immunology, IMM4A, Research Institute of Scripps Clinic, La Jollo, California 92037

1. Introduction

Specific antibody molecules play a vital role in counteracting infectious organisms in the extracellular milieu, but antibodies and antibody progenitors, B cells, are poorly equipped to react against organisms harbored inside cells. To deal with intracellular organisms, the immune system has evolved a quite different set of immunocompetent cells, T cells. These cells are imbued with a number of interesting properties (1).First, in contrast to B cells and antibody molecules, T cells generally do not manifest specificity for free antigen, despite the fact that T cells are highly antigen specific. Second, unlike B cells, T cells do not secrete their antigen-specific receptors. Third, under physiological conditions, T cells only respond to antigen displayed on the surface of living cells. Fourth, T cells show the intriguing requirement that, to be immunogenic, antigen has to be aligned on the cell surface in association with gene products of the major histocompatibility complex (MHC),’ the H-2 complex in mice. With their disregard for free antigen, T cells are thus programmed to concentrate their attentions on parasitized cells, these cells being flagged by breakdown products of the pathogen linked to surface H-2 molecules. Especially in the case of viral infections., parasitized cells recognized by T cells are destroyed by cytotoxic T lymphocytes (CTL), a subset of T cells with cytolytic properties. 1 Abbreviations: Ab, antibody; AC, accessory cells; APC, antigen-presenting cells; ATS, antithymocyte serum; “B” mice, thymectomized, irradiated mice reconstituted with T-depleted marrow cell:;; BUdR, bromodeoxyuridine; CAS, supernatant from concanavalin A-stimulated lymphoid cells; CFA, complete Freund’s adjuvant; CML, cell-mediated lympholysis; Con A, concanavalin A; cOVA, chicken ovalbumin; CTL, cytotoxic T lymphocytes; DC, dendritic cells; dCuo, deoxy guanosine; DTH, delayed-type hypersensitivity; GVHD, graft-versus-host disease; HA, histocompatibility antigens; HAN, hemagglutinin; HEV, high endothelial venules; HRC, horse red blood cells; Ig, immunoglobulin; IL, interleukin; KLH, heyhole limpet hemocyanin; mAb, monoclonal antibody; MHC, major histocompatibility complex; MLR, mixed-lymphocyte reaction; M+, macrophage; PHA, phytohemagglutinin; PMA, phorbol myristate acetate; self + X, foreign non-MHC antigen seen in association with self MHC determinants; SRC, sheep red blood cells; TCR, T cell receptor; TNP, trinitrophenol.

39 Copyright 0 1987 by Academic Press, Inc. All rights of‘reproduction in any form reserved.

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In addition to destroying cells harboring pathogenic organisms, T cells also play a major role in controlling the quantity and quality of specific antibody made by B cells. This function of T cells is controlled by a different subset of T cells, T helper cells. Whereas T killer cells provide a negative (cytotoxic) signal to antigen-bearing cells, T helper cells provide a positive signal. Thus, when a T helper cell encounters a foreign antigen linked to H-2 molecules on the surface of a B cell, the T cell delivers a helper signal which enables the B cell to differentiate into an antibody-forming cell. Another interesting property o f T cells is that, although both T killer and T helper cells generally ignore self components, including self H-2 molecules per se, T cells show a marked propensity to respond to foreign H-2 molecules-the phenomenon of alloreactivity. The issue of how T cells discriminate between self and nonself H-2 determinants while remaining reactive to antigen is one of the main themes of this review. The principal aim of this article is to outline how the specificity, function, and induction of T cells and T cell subsets are under the strict control of H-2 molecules. The scope of the subject matter covered in this article is quite large: It should be emphasized that our underlying intention is not to discuss each topic in exhaustive detail, but rather to give an overview of a highly complex field. II. Cell Surface Molecules Controlling T Cell Specificity and Function

Formulating concepts of how and why T cells display H-2-restricted specificity inevitably hinges on understanding the various receptor-ligand interactions which take place during T cell recognition. Three types of molecules are of obvious importance: antigen-specific T cell receptors (TCR), H-2 molecules, and so-called accessory molecules on T cells. A brief description of these molecules and their genes is given below.

A. THE T CELLRECEPTOR Although the basic structure of antibody molecules has been known for many years, the nature of the TCR remained a near-total mystery until only a short time ago. During the 197Os, a considerable body of evidence from a number of influential investigators led to the view that Ig genes encoded the TCR (reviewed in Ref. 2). Although some of this evidence looked highly convincing on paper, a sizable cross section of immunologists remained skeptical as typified by the extensive review on T cells by Jensenius and Williams (2) in 1982. The first direct information on TCR molecules came from studies in 19821983 with monoclonal antibodies (mAb) reacting with clonotype-specific structures on a T lymphoma (3) and on antigen-specific T cell clones and

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hybridomas (4-7). These studies revealed that the TCR consists of a disulfide-linked, glycosylated heterodimer consisting of two chains, a and p. Each chain is a transmembrane protein and, in mice, has an MW of 40,00043,000, the MW of the heterodimer being about 82,000. Like certain other surface proteins, both chains of the TCR show considerable homology with immunoglobulin (Ig) and are thus viewed as forming part of the “Ig supergene family” (reviewed in Ref. 8). By isoelectric focusing (9,10), peptide mapping (10-12), polypeptide sequencing (13- 15), and gene sequencing (see below), TCR a and f3 chains show extensive polymorphism. As for Ig molecules, polymorphism is largely limited to the variable (V) NH,-terminal domain of each chain, the constant (C) COOH-terminal domain anchored to the cell membrane showing little variability. In a brief %year period from 1984 to 1986, recombinant DNA technology has provided a detailed picture of the genetic organization of the genes encoding the TCR. Using the technique of subtractive cDNA-mRNA hybridization, initial studies on mouse (16) and human (17) T cells led to the isolation of cDNA clones for segments of DNA which rearranged exclusively in T cells. The rearranged genes were shown to encode the V and C regions of the TCR p chain. Subsequent work has revealed the following information on TCR p genes (18-34). The p gene complex in mice is situated on chromosome 6 (19,20) and contains a cluster of about 30 V gene segments (27,29);in contrast to Ig genes, the family of V genes is divided into a large number of subfamilies (19,27,29),each containing relatively few members, usually 1-3 (19,27,29). Downstream from the V genes, there are two consecutive clusters of D (diversity), J (joining), and C region segments, the two C regions being almost identical (21-25); each cluster has 1 D region and 6 J regions. Recently, a third C region, CPO, has been mapped between Jp1 and C p l (30). A single Vp gene has been mapped 3’ to Cp2 (28); curiously, this gene is in inverted transcriptional polarity relative to all the other known genes in the p complex. Soon after the initial description of p chain genes, subtractive cDNA hybridization led to the isolation of cDNA clones for both mouse (35,36) and human (3‘7) TCR a chain genes. The a complex is situated on chromosome 14 in mice (38) and shows close similarities with the p complex, although there are some distinct differences (38-43). Thus, in contrast to the p complex, there are large numbers of V a genes, perhaps 100 (33). Only one C a gene segment has been identified and, to date, no D a regions have been found. A unique aspect of the a complex is that there are probably of the order of 50 Jasegments which are dispersed over at least 60 kb of DNA 5’ to

Ca (39,40). TCR genes show the same type of ordered rearrangement observed for Ig genes (8,34). Thus, through flanking sequences analogous to those for Ig

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genes, D-J joining of p genes is followed by V-D-J rearrangement to form a single exon. VQ gene segments can rearrange to either of the two Cp segments, since each C p segment has its own set of upstream D and J segments. At present, there is no evidence that individual T cells express more than one type of a-p heterodimer on their surface. Although both chromosomes can show rearrangement of (Y and Q genes, productive rearrangements seem to be restricted to one chromosome, rearrangements on the other chromosome being incomplete or abortive (26). A number of different mechanisms shape the diversity of the TCR (22,27,31,33):(1) combinatorial rearrangement of different V, D, and J gene segments, (2) junctional diversity, i. e., imprecise V-D- J joining plus V-D joining in any of three translational reading frames, and (3) N-region diversity, i.e., addition of extra nucleotides at the V-D-J joint. It has been calculated (33) that these mechanisms in toto can generate in the order of 8 X lo6 combinatorial associations of expressed a,p genes. This is quite close to the estimate of 2 X lo7 different associations for Ig (V,-V,) molecules. An interesting difference between T and B cell receptors is that somatic hypermutation seems to be very rare in T cells (22,26,27,44). Thus, whereas primed B cells and myeloma cells show considerable hypermutation (which leads to increased affinity for antigen) (reviewed in Ref. 45), there is currently only one example of such mutation occurring in T cells, and these changes were observed in a T hybridoma (46). T cell clones do occasionally change their specificity over prolonged periods in vitro (47-49), but so far these alterations in specificity have not been shown to involve point mutations. In a recent report, the change in specificity found in a long-term cytotoxic clone was attributed to secondary rearrangement and expression of Q chain gene segments (48). Although the available evidence is clearly consistent with TCR being intrinsically resistant to somatic hypermutation (perhaps to guard against the emergence of self-reactive cells), the evidence is still too fragmentary to make firm statements on this important issue. In addition to TCR (Y and p chains, considerable attention is now being focused on a third chain, y (49-61). Originally mistaken for the a gene, the y complex of genes is situated on chromosome 13 in mice (50) and contains a fairly small number of V gene segments, of which at least four are functional (59). There are currently four known Cy segments (one silent), but each Cy gene is flanked 5’ by only a single J region. The potential diversity of y gene products thus appears to be fairly limited. Although both T cell subsets can express y mRNA (53,59,60), many of these transcripts might be nonfunctional. Indeed, until very recently (see below), the protein product of the y complex has been a topic of mere speculation. Although models have been proposed in which the y chain forms heterodimers with (Y or p chains (62), the function of at least some typical T cells clearly does not depend on

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functional y transcripts (52,55,56,63,64). The possible role of y in thymic differentiation will be considered in Section V,A. The discovery of y gene products hinged on the fact that classic a-P heterodimers are linked to T3, a heterogeneous complex of polypeptides containing, in mice, at least 3 monomeric glycoproteins and a family of homo- and heterodimers (65-69). Under defined conditions, mAb to human T3 molecules precipitate the a-f3 dimer from nearly all T cells. However, a small proportion of T cells (1-3%) in the thymus and blood are T 3 + , but lack a-p TCR heterodimers. Studies on cell lines of this type isolated from blood showed that anti-T3 mAb coprecipitated two non-disulfide-linked chains of MW 55,000 and 40,000 (70). Significantly, the 55,000 MW protein was precipitated by antisera raised against synthetic peptides corresponding to nucleotide sequences of Cy and Vy genes. Since the cell lines expressed potentially functional y mRNA transcripts, the data strongly suggest that the 55,000 MW is the y product. The 40,000 MW protein, provisionally termed 6, was not precipitated by anti-y antisera, and its relationship to the 55,000 MW protein is unknown. In parallel studies by another group (71), an a-pT3+ cell line was isolated from human thymus. Anti-T3 antibodies co-precipitated two chains from the cell line, with MWs of 62,000 and 44,000. Only the 44,000 MW protein was precipitated with anti-y antibodies. More recently, 7-6 heterodimers have been found on mouse T cells (D. Pardoll, personal communication); y-6 heterodimers are disulfide linked in mice and, as in man, appear to be expressed largely and perhaps solely on the minority population of a- p- T3+ cells. At this time, the functions of a-6 heterodimers on T cells is totally obscure, although it is intriguing that ybearing cells triggered by anti-T3 antibodies develop cytolytic activity (71).

B. H-2 MOLECULES Early studies on histocompatibility loci controlling skin allograft rejection in mice showed that histocompatibility differences encoded by one particular locus, the Histocompatibility-2 (H-2)locus (now known to be a complex of genes), led to a particularly rapid rate of graft rejection (72-74). It soon became clear that all mammals examined and many lower species have a major histocompatibility complex (MHC) and that the MHC controls all forms of “strong” histocompatibility reactions, including allograft rejection and graft-versus-host disease (GVHD), as well as the in vitro counterparts of these reactions, such as the mixed-lymphocyte reaction (MLR) and assays for cell-mediated lympholysis (CML) (reviewed in Ref. 74). At a time when “cellular” immunology is rapidly becoming subcellular, it is instructive to reflect that contemporary T cell immunobiology had its humble beginnings in studies on transplantation reactions. Evidence from these early studies led to a number of key conclusions. First, the studies of

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Owen (75) on tolerance in chimeric cattle twins led Burnet and Fenner (76) to propose that self tolerance is an immunological process and is acquired in neonatal life; proof for this theory was obtained by Billingham et al. (77) and HaSek (78). Second, Simonsen (79,80),working with chickens, demonstrated that a very high proportion of normal lymphoid cells express MHC alloreactivity. Third, Gowans (reviewed in Ref. 81) proved that alloreactivity is mediated by a population of long-lived small lymphocytes which recirculate continuously from one lymphoid organ to another; this was a decisive breakthrough because small lymphocytes at that time were regarded as ignominious cells with a limited life span whose main function was to act as trephocytes, i.e., to die quietly and make their DNA available for reutilization by other more “important” cells (82). Fourth, the studies of Miller (83) on the effects of neonatal thymectomy in mice demonstrated that alloreactivity is controlled by thymus-derived (T) lymphocytes. Fifth, groups headed by Benacerraf (84) and McDevitt (85) showed that the inability of certain members of a species to respond to particular foreign antigens was controlled by “immune response” (Ir) genes; these genes mapped within the MHC. Finally, a number of workers in the early 1970s (reviewed in Refs. 1,74) demonstrated that, in addition to serving as alloantigens, self MHC molecules guide T cell reactivity to typical foreign (non-MHC) antigens. Following these pioneering studies, the genetic makeup of the MHC and the gene products of this complex have come under close scrutiny. Although the MHC encodes a variety of different cell surface molecules, two types of MHC molecules, termed class I and class 11, are of particular relevance to T cell function. These molecules differ from TCR and Ig molecules in always being expressed codominantly. An overview of the H - 2 complex is given below; for detailed information, see Refs. 74, 86-92. Class I molecules are expressed on nearly all cells in the body and consist of two glycoprotein chains: (1) a heavy 45,000 highly polymorphic a chain encoded by the H - 2 complex on chromosome 17, and (2) a light (12,000) nonpolymorphic p chain, &-microglobulin, encoded on chromosome 2. The two chains are noncovalently associated and only the heavy chain is anchored to the cell membrane. Class I a chains consist of three external “domains” (yet to be confirmed by X-ray crystallography), termed a1 (NH,-terminal), a2,and a3; intrachain disulfide bonds exist in the a2 and a3 domains, but not in al. The a3 domain shows relatively little polymorphism and associates with &-microglobulin. The a1 and a 2 domains, by contrast, show extensive polymorphism and probably express all of the sites (epitopes) recognized by T cells. Polymorphic class I a chains are the products of two regions of the H - 2 complex, K and D. The K region encodes at least two different types of class I molecules (K, K’), although it is not yet clear whether these molecules are products of different loci or, conversely, reflect

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differential processing of a single species of mRNA (93). The D region encodes a varying number of molecules, ranging from a single molecule, in H-2‘’ mice to as many as 5 molecules in H-2“ mice (Dd, L“, R“, M”, and L2d) (9495). Precisely how many loci encode these molecules is still unclear, although protein sequencing analysis has shown that H-24 mice have at least three structurally distinct molecules, Dq, Lq, and Rq (96). As mentioned above, typical K and D molecules show extensive polymorphism, with possibly as many as 100 alleles for each locus (97). K and D molecules are not separable structurally, i. e., the alleles do not display obvious “K-ness” or “D-ness.” Thus, although it seems highly likely that the K and D regions developed by gene duplication from an ancestral gene, this process must have occurred quite early in evolution and been followed by rapid diversification (89). Class I gene segments consist of 8 exons (88).The first exon encodes the hydrophobic leader peptide which guides insertion of the class I molecule through the cell membrane. The second, third, and fourth exons encode the three external domains of the molecule, while the remaining exons encode the transmembrane and cytoplasmic regions; as expected from protein sequencing, extensive polymorphism is apparent only in the second and third exons. Although there are only a limited number of known class I gene products (mice of the H-2b haplotype express only two molecules, K” and D’)), there are many class I genes--26 in the B10 (H-2”) mouse (98) and 36 in the BALBIc (Ei-2”)mouse (99,100). Most of these genes, however, are situated in the Qa and Tla regions, which are telomeric to the K and D regions. The function of’these genes is still unclear, although it has long been known that the QaITZa region encodes a series of class I-like molecules, collectively termed Qa molecules. These molecules differ from typical class I molecules in several respects (87). First, the expression of Qa molecules appears to be restricted to lymphohematopoietic cells; it was initially thought that Qa molecules were expressed only on subsets of T cells, but more recent evidence suggests that all T cells and at least some B cells are Qa+ (87).Second, Qa molecules display only limited polymorphism, with only 2-6 alleles for each molecule (87). Third, although Qa molecules can serve as targets for allo (foreign) CML responses and can be detected serologically, there is currently 110 evidence that these molecules can serve as “restriction elements”: in other words, unlike typical class I (and class 11) molecules, T cells do not appear to respond to antigen in the context of self Qa molecules. Fourth, in contrast to typical class I molecules, certain Qa molecules, e.g., Qa-2 and (210, are secreted in a soluble form (101,102). Recently, it has been suggested that QaITla genes might act as a reservoir of diversity for class I KID genes (103-105). This evidence stems from ex-

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periments on spontaneous class I mutant strains of mice, i.e., mice whose class I H - 2 K molecules differ at 1-3 amino acids relative to the “wild-type” strain of origin (106). Examination of one of these mutants, Kbml, at the DNA level indicated that a 13 nucleotide region of the H-2K region had 7 nucleotide changes relative to the wild-type Kb gene, these changes leading to 3 amino acid substitutions at the protein level (104,105). This finding was difficult to explain by simple, random point mutations. Stimulated by recent studies on globin genes (107), two groups (104,105) took the view that the changes at the DNA level might reflect a gene conversion-like event in which a segment of the K b gene had been replaced by a segment from some other gene. In support of this idea, it was found that one of the QalTla genes, QlO, had a stretch of DNA that was identical to the “new” segment of DNA in the KbnL1gene (106).On the basis of this finding, the authors suggest that gene conversion might be the main mechanism responsible for generating H-2 polymorphism. Although proof for gene conversion, as defined in lower eukaryotes, is not yet available, it is striking that for many of the “bg” series of mutants (of which Kbml is the prototype), potential donor genes have now been found in the QalTla region (106). Unlike class I molecules, the expression of class I1 molecules is limited to certain cell types, especially B cells and dendritic cells (a class of cells involved in presenting antigen to T cells) (see Section II1,C) (74). Various other cell types, such as macrophages, endothelial cells, epithelial cells, and fibroblasts, express class I1 molecules, but only when induced by interferon-? (IFN-7) (108,109). Whereas there appears to be a wide variety of different class I1 molecules in man (11O), mice have only two sets of molecules, termed I-A and I-E (88,97,111).Each of these molecules (often collectively termed Ia molecules) is composed of an a and p chain. These chains, which are not covalently linked, are glycoproteins of similar size (-35 kDa for the a chain and 29 kDa for the p chain). Both chains are transmembrane proteins and show sequence homology with TCR and Ig molecules, and are thus members of the Ig supergene family (88). The p chain has two intrachain disulfide bonds and is presumed to form two domains, p l and p2; the a chain also probably has two domains, a1 and 012, although only a2 (closest to the cell membrane) has an intrachain disulfide bond. Before being expressed on the cell surface, the a and p chains are noncovalently associated with a nonpolymorphic chain, termed the invariant chain (111,112). This chain is encoded on a different chromosome and has little or no homology with the a and p chains (or with the other members of the Ig supergene family) (113). The main function of the invariant chain is presumably to regulate the intracellular transport andlor association of the a and p chains (113-115). Curiously, the invariant chain is reported to appear on the cell

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surface after dissociating from the a dnd P chains (116). Since the invariant chain lacks a signal sequence, it has been suggested that it lies “upside down” in the cell membrane, the carboxy-terminus being extracellular (114,115). The biological significance of cell-surface invariant chain is obscure. At the DNA level there are at least 8 class I1 gene segments in mice, three AP, three E P , one A a , and one Ea gene (117-120). These genes reside in the 1 region of the H - 2 complex between the K and D loci, the order of the genes (and gene clusters) being K , AP, A a , E P , Ea, D (except for one EP gene which maps between Ea and D). Interestingly, the a and P genes are transcribed off opposite strands of DNA, i.e., the genes are in tail-to-tail orientation. As yet, it is not clear how many of the P genes are expressed, although at least one of these genes (AP3) is known to be a pseudogene (120). Strain variation in the expression of Ea and EP genes will be considered later. The arrangement of the exons and introns of Ia a and P chain genes is slightly different. For a typical a chain, a signal sequence exon encodes the leader peptide and the first few amino acids of the mature protein. The second and third exons encode the a 1 and a 2 domains, and the fourth exon encodes the transmembrane region and the cytoplasmic tail as well as part of the 3’ untranslated sequence; the remainder of the untranslated sequence is encoded by a fifth exon. The arrangement of the genes for the P chain is essentially the same, except that the transmembrane and cytoplasmic regions are encoded by two separate exons. Based on nucleotide sequencing, the P chains for both I-A and I-E molecules show extensive polymorphism in their N-terminal domains. Polymorphism is less marked in the I-A a chain and is virtually absent in the I-E a chain. Although the a chains (especially Ea) play a comparatively minor role in contributing to polymorphism of class I1 molecules, a chains are essential for the cell surface expression of the P chains. In this respect, it should be mentioned that a considerable number of mouse strains, e.g., mice of the s, b, f a n d y H - 2 haplotypes, do not express an I-E molecule. In these mice, the E a chain is not expressed. Defective E a expression in these mice involves at least three different mechanisms (121): (1) a deletion in the Ea gene for the b and s haplotypes; (2) an E a mRNA of aberrant size for thef haplotype; and (3) a defect in mRNA processing and/or mRNA instability for the y haplotype. In the absence of the E a chain, the EP chain can be synthesized in the cytoplasm, but fails to be expressed on the cell surface. This situation occurs in mice of b and s haplotypes. If these mice are crossed with mice that do express an E a chain, transchain association can occur. Thus, the E a chain of one parent associates with the EP chain of the other

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parent to form a functional heterodimer which is then expressed on the cell surface. These same heterodimers can be expressed by cis-chain association in appropriate H - 2 recombinant mice, e.g., in recombinants carrying the E; allele and a functional Ea allele. In contrast to the b and s haplotypes, mice of thefand q haplotypes do not express an EP product. These mice are thus incapable of synthesizing an I-E molecule even when crossed with an E,+ strain. Because of transchain association, H-2 heterozygous mice can express unique “F, hybrid” Ia molecules lacking in the two parental strains (122). Thus, in the case of F, hybrids between I-Aa (A;-A$ and I-A“ (At-Abp), one finds four I-A heterodimers: the A:-A; and At-A; heterodimers of the two parental strains plus 2 sets of F1-unique heterodimers, Ai-A; and At-A;. Because of the nonpolymorphism of the E a chain, trans-chain association can create only one F,-unique I-E molecule (with the proviso that one of the parents is EL). H-2 heterozygous mice can therefore express a total of 6 Ia molecules, 4 I-A molecules and 2 I-E molecules. It should be pointed out that, in theory, two additional mechanisms might generate further sets of Ia heterodimers. First, mixed heterodimers might exist between I-A and I-E a and (3 chains, thus creating Ea-AP and/or Aa-EP molecules. Although heterodimers of this type have yet to be observed under normal physiological conditions, gene transfection experiments suggest that Aa-EP heterodimers do form in certain situations (123). Second, it is by no means clear that the clusters of three EP and three AP genes each contain only one functional gene. If several of these genes were functional, the potential diversity of Ia molecules would obviously be considerable. By classical genetic methods involving studies on a variety of H - 2 recornbinant mice, it was originally concluded that the I region of the H - 2 complex was divided into 5 subregions: A, B , J , E , C (reviewed in Ref. 87). It is now generally accepted that the A subregion (a term no longer used) encodes the A@,A a , and EP genes and the E subregion encodes the E a chain. The B, 1, and C subregions, however, appear to be nonexistent at the DNA level (117-120). In view of this disturbing discrepancy, the A, B , J , E , C terminology has fallen into rapid disfavor. The phenomenology that led to the postulated existence of the B , J , and C subregions is outside the scope of this review (see Ref. 87). In addition to the genes for class I and class I1 molecules, the H - 2 complex also contains a number of “passenger” genes, e.g., genes for neuraminidase-1 and the C4 component of complement (87);these genes map between Ea and the D locus. Why these genes reside in the H - 2 complex is obscure. As suggested by Klein et al. (87), the simplest explanation is that these genes were entrapped accidentally during evolution.

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C. ACCESSORYMOLECULES ON T CELLS Like other cells, T cells display a multiplicity of different cell surface molecules, and antibodies to some of these molecules provide invaluable tools for isolating T cells and separating these cells into functionally distinct subsets (124). Defining the physiological role of the various molecules expressed on T cells is still in its infancy, but two sets of molecules have aroused particular interest, Lyt-2/3 (125, 126) and L3T4 (127). These molecules are of importance for two reasons, one practical and one theoretical. First, the expression of L3T4 and Lyt-2/3 molecules on extrathymic T cells is niutually exclusive. Thus, with the exception of rare “doublepositive” ‘r cell clones maintained in uitro (128,129), peripheral T cells are either L3r4 , Lyt-2/3 -, or L3T4 -, Lyt-213 (127). Antibodies to these markers are therefore extremely useful for isolating phenotypically distinct T cell subsets. As discussed in more detail in a later section, the second reason for the high interest in L3T4 and Lyt-213 molecules is that the expression of these molecules correlates quite closely with the class of H-2 molecules recognized by T cells. Moreover, there is reason to believe that L3T4 and Lyt-2/3 molecules might actually bind to H-2 molecules. Before discussing these topical issues, it is usefd to consider recent information on the structure and genetic organization of L3T4 and Lyt-2/3 molecules and genes and their homologues in other species, e.g., CD4 and CD8 molecules in man (130,131) and W3/25 and OX8 molecules in the rat (132). Except for a subset of natural killer (NK) cells (133), the expression of Lyt-2/3 appears to be restricted to T cells (124). The Lyt-2/3 molecule is a 70-kDa heterodimer of two covalently linked chains, an a chain (35-38 kDaj expressing epitopes detected by anti-Lyt-2 antibodies, and a p chain (30-34 kDa) expressing Lyt-3 epitopes (124, 134-137). The Lyt-2 chain is the homologue of the CD8 chain in man, the CD8 molecule consisting of homodimers and multimers of a single chain; there is no apparent counterpart of the Lyt-3 chain in man. The Lyt-2 and Lyt-3 chains are both transmembrane glycoproteins and are encoded by two closely linked genes on chromosome 6. The Lyt-2 gene segment contains 5 exons (138, 139). The first exon encodes the signal peptide and an amino terminal domain showing close homology with Ig V, light-chain regions, including cysteines for an intrachain disulfide bond. Exons 2-5 encode, in order, the spacer region, the transmembrane region, and two cytoplasmic regions, C 1 and C2. Interestingly, although there is only a single Lyt-2 gene segment, there are two types of Lyt-2 chains, a and a’,of slightly different size (124,134,140143). The two chains are identical except that the a‘ chain lacks the C1 and +

+

+

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C2 intracytoplasmic regions of the a chain (138,139). The chains arise from separate species of mRNA and are presumed to reflect alternative modes of mRNA splicing (138,139). The tissue distribution of a and a’chains is somewhat different (124,134). Thymocytes express both chains, whereas lymph node cells express only the a chain (despite the presence of a’mRNA in the cytoplasm). Interest in this differential expression of the a‘ chain is muted by reports that there is no counterpart of the a’chain in human T cells (see Ref. 139). Likewise, the absence of the Lyt-3 chain in human T cells suggests that the biological function of the Lyt-213 heterodimer is largely determined by the Lyt-2 chain. For simplicity, we shall henceforth refer to “Lyt-2 molecules” rather than Lyt-213 molecules. The structure and genetic organization of L3T4 molecules have only recently come under close scrutiny. Whereas Lyt-2 molecules are heterodimers, L3T4 and their homologues in other species are monomeric glycoproteins and are somewhat smaller (55K)than Lyt-2 (127,143-147). Genes for the human (CD4) molecule have now been cloned and sequenced (144) and reveal a single polypeptide chain with three extracellular domains: an amino-terminal Ig V-region-like domain, a joining (J)-like region, and a third extracellular domain. There is also a membrane-spanning domain analogous to class I1 MHC Q chains and a highly charged cytoplasmic domain; the intron-exon organization of the genes is still unclear. Sequencing at both the cDNA (144) and protein (147) levels suggests that the three external domains each have intrachain disulfide bonds. The V-like domain shows significant homology with v k and also with the amino-terminal domain of the CD8 (Lyt-2) molecule. However, the degree of homology between the amino-terminal domains of CD4 and CD8 is fairly weak [28% for CD8 vs CD4 (144)l; in fact, the domains of these two molecules are less homologous to each other than each is to v k (144). Like CD8 molecules, CD4 (L3T4) molecules show considerable divergence from their homologues in other species, suggesting that these molecules have undergone rapid evolution to maintain complementarity with their respective “ligands” (? MHC molecules-see below) (147). In addition to Lyt-2 and L3T4 molecules, T cells also express several other types of “accessory” molecules that might play a role in T cell recognition of antigen, e.g., LFA-1, LFA-2, and LFA-3 (148). Moreover, in the case of human T cells, a variety of mAb are being used to separate T cells into a bewildering complexity of subsets displaying different functions (149,150). We have elected not to discuss this phenomenology, largely because, as yet, there is little or no evidence that murine T cells-the main subject of this review-are divided into more than two phenotypically and functionally distinct subsets.

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111. H-2-Restricted Recognition of Antigen by Mature T Cells

After the discovery of H-2-linked Ir genes (85), the first evidence that T cell function is controlled by H-2 gene products came from studies on T-B collaboration. In the late 1960s, experiments of Claman et al. (151) and Davies et al. (152) followed by the definitive experiments of Mitchell and Miller (153)showed that antibody responses to “T-dependent” antigens involve cooperation between thymus-derived (T) helper cells and bone marrow-derived antibody-forming cell precursors (B cells). In 1972-1973, Kindred and Shreffler (154) and Katz et al. (155) provided convincing evidence that this interaction between T and B cells exhibits H-2 restriction, i.e., T-B collaboration fails to occur unless the two cell types share H-2 determinants; the restricting determinants were shown to map to the 1 region of the H-2 complex. Similar restriction was observed by Rosenthal and Shevach (156) in an in uitro system involving proliferative responses of T cells to antigen presented by syngeneic versus allogeneic macrophages. In 1974, the studies of Zinkernagel and Doherty (157) on antiviral responses and Shearer (158)on responses to the hapten TNP indicated that CTL lyse H-2-compatible but not H-2-incompatible target cells; lysis requires a sharing of class I (K or D) H-2 determinants. Similar restriction to self H-2 determinants was found for CML to minor histocompatibility antigens (HA) by Bevan (159) and Gordon et al. (160). Although Zinkernagel and Doherty were not the first to discover the phenomenon of H-2 restriction, the speculations of these two workers on the physiological significance of H-2 restriction (161,162) made the phenomenon “accessible” to the immunological community at large. Doherty and Zinkernagel put forward two models to account for H-2 restriction: altered self and dual recognition. Both models assert that T cells have joint specificity for self H-2 plus foreign antigen. According to the altered-self model, T cells express a single recognition unit (receptor) with specificity directed neither to self H-2 determinants per se nor to antigen, but to new antigenic determinants (NADs) created by an association of the two ligands. The opposing model, dual recognition, argues that T cells express two linked recognition units, one specific for self H-2 and the other for antigen, the two recognition units either being expressed on two different (though linked) receptors or on a single polypeptide chain. The exposition of these two models was enormously influential, and even now there is no direct proof for either model, although the “two receptor” variant of the dual recognition model would seem to be ruled out. Although the three-dimensional structure of the TCR is still uncertain, the conservative view is that the TCR binding site will show close similarities

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with the binding site of Ig molecules. Thus, despite the accumulating evidence that T cells have specificity for small peptides (see below) whereas antibody (Ab) molecules recognize three-dimensional configurations on native molecules, it is not unreasonable to suppose that the combining sites of TCR and Ab molecules both simply bind complementary “shapes” of best fit. Although it is relatively easy to study antibody-antigen interactions, attempting to establish how T cells recognize antigen is fraught with two imposing difficulties. First, the fact that TCR are not secreted makes it very difficult to prepare these molecules in pure form. Second, we still have only a vague idea of precisely “what” T cells recognize. Before dealing with the complex issue of how T cells recognize antigen, one apparent difference between the V regions of TCR and Ab molecules should be mentioned. In the case of Ab, it has long been known from protein sequencing studies that the V region displays three discrete areas of hypervariability and that these complementarity-determining regions (CDRs) converge in the three-dimensional structure of the combining site to form the contact residues for antigen (163). Whether this also applies for TCR molecules is uncertain, since hypervariability is seen throughout the TCR V region (18, 164). Therefore, it is possible that a wide area of the TCR V region can act as a combining site (or sites). The physiological response of a virgin T cell to antigen can be envisaged as having three components: (1)an induction phase in which the T cell recognizes an immunogenic form of the antigen and is induced to blast transformation, (2) a proliferative phase where the induced T cell undergoes clonal expansion, and (3) a stage of differentiation where the proliferating T cells acquire some type of effector function, e.g., the capacity to lyse appropriate target cells or provide help for B cells. These three components of the immune response need to be addressed separately. Examining the induction phase of the T cell response is complicated by the fact that activation of unprimed T cells depends not only on recognition of antigen plus H-2, but also on additional signals from other cells. Primed T cells are less dependent on these other signals, and most of the information on antigen recognition by T cells has come from studies with preactivated T cells, especially class 11-restricted antigen-specific T helper clones and hybridomas.

A. TRIGGERING OF ACTIVATEDT CELLSAND HYBRIDOMAS In trying to establish how T cells are triggered under physiological conditions, one has to work backward from the simplest system currently available: the capacity of T hybridomas to synthesize the lymphokine interleukin-2 (IL-2)after contact with specific antigen or other ligands (165). With this system one can address a very basic question: Is TCR-ligand interaction

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alone sufficient to deliver a signal to the T cell? Studies with anti-TCR antibodies-a very simple type of ligand-suggest that this is indeed the case. Thus, aggregated anti-TCR mAb, e.g., mAb attached to beads, is highly efficient at inducing IL-2 production by T cell hybridomas and clones (6,9,166,167). This finding suggests that cross-linking of TCR is all that is required to deliver at least one type of positive signal to a T cell. As discussed earlier, the physiological ligand for T cells is presumed to be an association or juxtaposition of antigen and self H-2 molecules. Until recently it has generally been assumed that T cell triggering requires that antigen plus self H-2 be displayed on the surface of viable “antigen-presenting cells” i(APC)(168). In the case of Ia-restricted T helper cells, the APCs have to express class I1 molecules, i.e., be I a + . For complex antigens, it has long been argued that native antigens have to be broken down (“processed”) by the APC into small immunogenic peptide fragments which then align themselves with surface Ia molecules (168-171). Indirect support for this notion stemmed from findings that (1)peptides cleaved from native antigens are strongly immunogenic (168,170-175) and (2) that, after feeding native antigens to APCs, effective presentation of antigen to T cells requires a lag period of about 1 hour at 37°C (176-178). The first direct evidence for antigen processing by APCs came from the experiments of Shimonkevitz et ul. (179,180) on the capacity of T hybridomas to respond to chicken ovalbumin (cOVA) presented by glutaraldehyde-fixed APCs. The key finding was that although fixed APCs were not able to present native cOVA to the T hybridomas, fixed APCs were fully capable of presenting enzymatically degraded or chemically disrupted fragments of cOVA. This observation, since confirmed by other groups (181-183), strongly suggests that antigen processing simply involves partial proteolysis or unfolding of native antigen. The issue of how the TCR recognizes peptide fragments aligned with Ia molecules was first addressed in depth by the group of Heber-Katz, Hansburg, Schwartz et uZ. (170,184-188). Using T cell lines and hybridomas specific for fragments of cytochrome c and two sets of APCs expressing slightly different Ia molecules (E);-Ef: vs E$-E$), these workers have assembled impressive evidence that T cell recognition of antigen involves the formation of a trimolecular complex between the TCR, the immunogenic peptide, arid Ia molecules on the APC. This group envisages that the immunogenic peptide has two distinct contact points, an “epitope” recognized by the TCR and an “agretope” that binds to the Ia molecule. Likewise, the polymorphic part of the Ia molecule also has two contact points, a “histotope” recognized by the TCH and a “desotope” that binds the peptide. As proposed earlier by other workers (189; see below), it is argued that association of the peptide with the Ia molecule results in “determinant selection.” In other words, the desotope of the la molecule orients the peptide in an

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“immunogenic” position such that the epitope can be seen by the T cell, the type of orientation being unique for each Ia allele. An accumulating body of evidence (190-192) supports this model. The trimolecular model for T cell recognition of antigen raises two questions. First, is there any physicochemical difference between the peptide epitope recognized by the TCR and the Ia-binding agretope? An interesting suggestion here is that immunogenic peptides are amphipathic and have an a-helical structure with hydrophobic and hydrophilic polarities (170,193195); the hydrophobic aspect of the peptide makes contact with the Ia molecule, whereas the hydrophilic portion is recognized by the TCR. [Note that if immunogenic peptides do indeed have an a-helical structure, one would be able to discard the popular view that, unlike B cells, T cells see linear sequences of amino acids (169); T cell recognition of peptides in the context of Ia molecules would then be closely analogous to recognition of conformational (“discontinuous”) determinants by antibody molecules (see also Ref. 196).] The second, and crucial, question is whether the immunogenic peptide and the Ia molecule do actually enter into a physical association. This question-which is obviously central to the altered-self versus dual-recognition controversy-has been a topic of speculation €or several years. Although a number of groups have reported the existence of complexes of Ia and antigen released from APCs (197-199), most investigators were not able to reproduce these findings. Recently, several groups have reinvestigated the question of antigen-Ia association by examining whether complexes occur when immunogenic peptides are incubated with purified Ia molecules in a cell-free system. Before considering the results of these experiments, the concept of Ir genes needs to be discussed (for a comprehensive review, see Ref. 170). It has long been known that many antigens, especially simple synthetic antigens and viruses, are under H-2-linked Ir gene control (85,170). Such antigens are strongly immunogenic in some strains (high responders), but not in others (low or nonresponder strains). The nature of Ir genes gradually became apparent when it was discovered that T cells from a high responder strain recognized an Ir gene-controlled antigen in the context of one self H-2 molecule, but not another. For example, T killer cells from a high responder strain, e.g., H-2k, were found to mount CTL responses to a particular virus only in the context of the Kk molecule, but not the Dk molecule (1).Likewise, T helper cells recognized a particular protein only in the context of the I-E molecule and not the I-A molecule (170,200). These and other findings led to the now well-established view that H-2 molecules and Ir gene products are one and the same, and that only certain class I or class II alleles are “permissive” for a particular antigen. Two main theories have been advanced to explain T cell unresponsiveness to antigens under Ir gene control:

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(1)creation of “holes” in the cell repertoire, and (2) a defect at the level of antigen presentation by APC (156,169-171,188,189,201,202). The essence of this second possibility-the determinant selection theory-is that unresponsiveness reflects a failure of antigen to enter into an immunogenic association or alignment with H-2 molecules. Thus, in the words of Rosenthal et al. (189), the original proponents of determinant selection, Ir gene products “focus or orient distinct regions of the antigen for presentation to the T cell.” In assessing this theory, one is forced back to the central issue of whether antigen and Ia can form stable associations in the absence of T cells. If such associations do occur normally, a peptide under Zr gene control might be expected to show significant binding affinity for high responder Ia molecules, but not for low responder molecules. Babbitt et al. (203,206) recently addressed this issue with the aid of an equilibrium dialysis system in which 16 amino acid fragments of hen egg lysozyme, HEL (46-61), were incubated with purified I-Ak (high responder) versus I-A“ (low responder) molecules. There were two important findings. First, the HEL (46-61) peptide did form quite a strong association with high responder I-Ak molecules, with an apparent equilibrium constant of 2 x 10-6 M . Second, the HEL (46-61) peptide did not show measurable binding to low responder I-Ad molecules. These findings thus demonstrate that antigen and Ia can indeed physically associate with one another (see also Refs. 204-206). The data also suggest that a failure of antigen-Ia association accounts for (or at least correlates with) Zr gene-controlled unresponsiveness. Striking confirmation of the data of Babbitt et al. (203) has since been reported by Buus et al. (207). Using an essentially similar approach, these workers repeated the observation of Babbitt et al. that H E L (46-61) binds to I-Ak, but not to I-Ad molecules; in addition, binding failed to occur with a second low responder molecule, I-Ek. With another antigen, a peptide of ovalbumin, Buus et al. (207) found a quite different binding pattern, i.e., binding to I-A“ (high responder), but not to I-Ak or I-Ek (both low responders). Collectively, these two studies provide powerful support for the view that the immunogenicity of peptides depends upon physical association with Ia molecules. The data also strongly support the view that l r genes act at the level of antigen presentation. Although the above studies are highly convincing, Watts et al. (192) have recently reported somewhat different findings. On the basis of examining resonance-energy transfer from donor peptides to acceptor I-A molecules on a solid matrix, these workers concluded that significant peptide-Ia association only occurs when specific T cells are present. It is not clear how this finding can be reconciled with the above evidence that antigen and Ia do associate in the absence of T cells. An obvious possibility is that the system used by Watts et al. is less sensitive than equilibrium dialysis. Whatever the

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explanation for this discrepancy, the important finding of Watts et al. is that the interaction between antigen and Ia molecules is stabilized by the TCR. The data thus provide further strong support for a trimolecular complex of antigen, Ia, and TCR molecules. It was mentioned earlier that cross-linking of TCRs with anti-TCR antibodies is sufficient to trigger IL-2 production by T hybridomas in the absence of APCs. Similar findings might therefore be expected for antigenspecific T cells exposed to a cross-linked association of antigen plus self Ia molecules. Watts et al. (195,208) have verified this prediction with the APCfree system described above, i.e., purified Ia molecules plus peptide antigens supported on glass coverslips. Antigen-specific hybridomas placed on the coverslips responded with vigorous production of IL-2. These data provide formal support for the view that, after processing of antigen, the role of APCs in presenting antigen plus self H-2 to activated T cells is simply to display these two ligands in cross-linked form. Until recently, the evidence that antigen processing is a prerequisite for T cell recognition of native antigens has come almost entirely from studies with Ia-restricted T cells. In the case of class I-restricted T killer cells, it has usually been assumed that CTL recognize membrane proteins, such as viral envelope glycoproteins or minor HA, which associate in some way with cellsurface class I molecules. As pointed out by Germain (209), however, the evidence that the particular antigenic epitopes recognized by CTL do exist as intact transmembrane proteins is fairly sparse. Interest in this issue has been kindled by the finding that a sizable proportion of influenza virusspecific CTL are reactive to viral nucleoprotein, i. e., to a component that does not have a recognizable leader sequence and is therefore incapable of being inserted in the cell membrane (210-213). The explanation of Townsend et al. (214) for this paradox is that nonmembrane viral proteins are processed in the cytoplasm: The proteins are degraded to small peptides which then somehow reach the surface and associate with class I molecules. In support of this idea, Townsend et al. (214) demonstrated that nucleoprotein-specific CTL were able to lyse target cells incubated with short (14 amino acid) synthetic peptides derived from the nucleoprotein sequence; lysis was antigen specific as well as H-2 (Db)specific. Since a wide variety of virus-infected cells can act as targets for CTL under physiological conditions, the authors suggest that processing of viral antigens might be a property of a multiplicity of different cell types. An interesting feature of processing of viral nucleoprotein is that treatment of virus-infected cells with the lysosomotropic agent, chloroquine, does not interfere with the display of the surface epitopes recognized by nucleoprotein-specific CTL (214). This is surprising because chloroquine is highly effective at inhibiting processing of antigens recognized by Ia-re-

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stricted T cells, including Ia-restricted CTL (178,215-219). Further information on this issue has come from studies by Morrison et al. (219). Using T cell clones specific for influenza hemagglutinin (HAN), these workers have assembled impressive evidence that the intracellular handling of HAN for class I- and class 11-restricted CTL is quite different. In the case of HAN detected by class I-restricted CTL, the expression of HAN was chloroquine resistant and depended on endogenous synthesis of the new HAN antigens, either by infectious virus or by transfected HAN genes; as found by Townsend et al. (214) with whole (nondegraded) nucleoprotein, exposing the target cells to exogenous HAN failed to elicit lysis. In marked contrast, expression of HAN detected by class 11-restricted CTL was chloroquine sensitive ancl depended solely on exogenous uptake of HAN. To accommodate the findings of the above two groups, Germain (209) proposes that the differential processing of viral antigens for class I- versus class 11-restricted T cells involves two quite separate mechanisms. For class I-restricted T cells, he suggests that endogenous viral proteins are degraded to simple peptides in a portion of the Golgi apparatus; these peptides then associate with class I molecules, either locally or after migration to the cell surface. For class 11-restricted CTL, the viral proteins have to make their way to enclosomes before being processed into small peptides; these peptides then associate with class I1 molecules, either after export to the cell surface, or possibly within the endosomes (see Ref. 220). A key aspect of this scheme is that, except for artificially degraded proteins or synthetic peptides, proteins introduced into cells exogenously associate only with class I1 and not class I molecules. A critical assessment of this attractive theory will obviously require a great deal of additional information on the intracellular handling of viral antigens. It will also be essential to obtain comparable information on other antigens seen by class I-restricted CTL, especially minor HA. Though excellent targets for CML (159,160), minor HA are very poorly characterized, largely because these antigens are extremely difficult to detect serologically (221). By analogy with CTL responses to viruses, one can now toy with the possibility that minor HA are not integral membrane proteins, but instead represent breakdown (processed) products of certain endogenous intracellular proteins, the display of these products on the cell surface being dependent on their capacity to associate with class I molecules. Although the results discussed above are still fragmentary, it seems quite possible that all typical protein antigens seen by H-2-restricted T cells, whether class I or class 11 restricted, are handled in much the same way: Native antigens are degraded into simple peptides, which then associate immunogeriically with permissive H-2 molecules. This pathway is unnecessary if the antigens are already in the form of “pre-processed” simple pep-

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tides (or if H-2 molecules are directly modified with a hapten). Of course, we are still left with the fundamental enigma of how foreign antigens make contact with H-2 molecules in the presence of a sea of selfproteins (222,223). This paradox is unlikely to be resolved until we have detailed information on the capacity of autologous proteins and peptides to associate with H-2 molecules (see Ref. 224). Despite the increasing evidence that class I and class I1 molecules have binding sites for processed antigens, the relationship of these sites (desotopes) to the histotopes recognized by the TCR is still unclear. Since polymorphism in H-2 molecules is largely limited to the membrane-distal domains, i.e., the a1 and 012 domains of class I molecules and the a1 and p l domains of class I1 molecules, one would obviously expect both recognition sites to be displayed on these domains. Studies with recombinant H-2 molecules prepared by transfection with “exon-shuffled’ genes imply that this is indeed the case (reviewed in Ref. 225). H-2 gene transfection experiments and studies with mutant H-2 molecules also suggest that the membranedistal domains interact closely with one another to form a single quaternary structure, both for class I and class I1 molecules (106,123,226-228). It seems likely, therefore, that desotopes and histotopes are both combinatorial in nature. But, at present, this seems to be the limit of our knowledge. Although the basic concept of the trimolecular complex is now gaining general acceptance, it should be borne in mind that so far there is no compelling evidence which sheds light on the altered-self versus dual-recognition controversy. Despite widespread evidence to the contrary, there are now several reports that under certain circumstances H-2-restricted T cells can bind and respond to free antigen, e.g., haptens conjugated to polymers on protein carriers (229-234) or (undenatured) proteins held in liposomes (235). Although these data are easier to interpret in terms of dual recognition than altered self, the possibility that the data reflect covert association of antigen with H-2 molecules, e.g., from the responding T cells (and/or contaminating APCs), has not been totally ruled out; alternatively, T cells might indeed be able to bind free antigen, but with much lower affinity than antigen plus H-2.2 2 Given the enormous diversity of TCRs, it is quite possible that some unprimed T cells do have the capacity to bind free antigen, e.g., native ovalbumin, with high affinity. But how could one isolate these particular T cells? As discussed later (Section III,C), it appears that inductive signals provided by APCs are essential for the activation of resting T cells. Hence, the induction of a resting T cell specific for native ovalbumin would require that this antigen be displayed on a living APC without being degraded. Such presentation would be most unlikely under physiological conditions. Under normal conditions, the antigen would be broken down and presented in association with H-2 molecules. This complex of processed antigen plus self H-2 would then be immunogenic for a different set of T cells. These cells, at best, would have only low binding

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It would be interesting to repeat the above experiments on T cell binding of free antigen with a cell-free system using soluble TCRs prepared by exonshuffling techniques (236). Another important issue that could be addressed with a cell-free system is whether the TCR has any specific binding affinity for self H-2 molecules in the absence of antigen. This question is central to the problem of how T cells “learn” restriction to self H-2 (see Section V). Finally, cell-free systems will undoubtedly be the key to establishing the nature of the binding site(s) of the TCR and how the TCR a and p chains contribute to this site. It should be mentioned that gene transfection experiments have shown that, at least in one situation, the TCR binding site for class I-restricted CTL is assembled solely from the products of the TCR a and p genes (64). It therefore seems unlikely (though not proved) that other chains, e.g., y chains, contribute to the binding site of typical H-2-restricted T cells. It also seems clear that the pattern of TCR V a and Vp genes used by class I- and class 11-restricted T cells is very similar (237-239). What is not clear is how the various polymorphic a and p gene elements assemble a binding site that has dual specificity, i. e., specificity for antigen and self H-2. The obvious question here is whether the H-2-restricted specificity of the TCR correlates with the usage of particular gene segments (e.g., certain Ja genes), whereas specificity for antigen is controlled by other genes. The available data suggest that this is unlikely (see Ref. 239), although some groups do report provocative correlations in some instances (e.g., Ref. 240).

B. FUNCTION OF T ACCESSORYMOLECULES Although the specificity of T cells is probably controlled solely by the TCR, there is increasing evidence that interaction with antigen also involves other molecules on T cells, especially L3T4 and Lyt-2 molecules. As discussed earlier, extrathymic T cells generally express either L3T4 or Lyt-2 molecules, but not both. It was mentioned that occasional clones express both markers (128,129), but this phenotype is very rare outside the thymus. Double-positive T cells are virtually undetectable in the peripheral lym-

&nity for the native antigen. The point to emphasize is that T cells with specificity for native antigen would not be triggered under physiological circumstances and so would go undetected. In theory, T cells specific for a native antigen could be isolated by exposing rinprimed T cells to cross-linked native antigen plus a soluble source of the inductive signals provided by APCs. The stumbling blcick here is that the nature of the signals provided by APCs is still unclear (see Section III,C), so the experiment is unfeasible at present. It may be noted that the capacity of primed T cells/hybridomas to bind and respond to anti-TCR mAb could be viewed as an example of T cells binding a native antigen. It is also worth pointing out that T cells generally recognize H-2 alloantigens in native form (Section IV) and that the strong immunogenicity of these particular antigens for unprimed T cells is in part a reflection of the fact that H-2 molecules reside on the cell surface of APCs in intact (nondenatured) form.

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phoid organs of mice, although 3% of human peripheral blood T cells are reported to be C D 4 + , CD8+ (241). Interestingly, quite a high proportion of mitogen-activated human T cells can show transient expression of both markers (241);for obscure reasons, double-positive cells are also common in rats treated with cyclosporine (242). There are also reports of a phenotype switch for both human (243) and mouse (244) T cell subsets. The general finding, however, is that the expression of accessory molecules by mature T cells is mutually exclusive and remarkably stable (245, 246). Studies on a wide variety of T cell clones and hybridomas have shown a close correlation between (1) Lyt-2 expression and restriction by class I molecules and (2) L3T4 expression and class I1 restriction (247-254). (Exceptions to this correlation will be discussed later in Section IV.) To explain this finding, the prevailing view is that accessory molecules function by binding to mononiorphic sites on H-2 molecules and thus strengthen the avidity of binding via the TCR (247-254). A wide body of evidence supports this idea. First, antibodies to T accessory molecules are generally highly efficient at inhibiting T cell responses to antigen plus H-2, T cell clones with apparently low binding avidity being more easily inhibited than high avidity clones (247,251,255-257). Second, one group has reported that T cell triggering via anti-TCK antibodies is not inhibited by antibodies to accessory molecules (258). Third, T hybridomas which show spontaneous loss of accessory molecules in vitro often show a reduction in their capacity to respond to antigen, consistent with a lowering of binding avidity (S. Webb, unpublished data). Fourth, an unusual L3T4+ class I-restricted T hybridoma is inhibited by anti-L3T4 antibody if the APCs express both class I and class I1 molecules, but is not inhibited if the APCs are Ia- (253). Collectively, these data would seem to make a strong case that L3T4 and Lyt-2 molecules do bind selectively to H-2 determinants and that the blocking effects observed with anti-L3T4 and anti-Lyt-2 antibodies cannot be attributed simply to down-regulation of T cells. Surprisingly, however, there is accumulating evidence that, under certain conditions, antibodies to accessory molecules can down-regulate T cells. First, the capacity of L3T4+ T cells to respond to mitogens such as phytohemagglutinin (PHA) can be inhibited by anti-L3T4 antibody in the absence of Ia+ cells (259-262); this effect is most prominent if limiting doses of mitogen are used. Second, it has recently been reported that the proliferative response of resting T cells to anti-TCR antibodies can be inhibited by anti-L3T4 antibodies in the absence of Ia+ APC (263); likewise, stimulation of human T cells with anti-T3 antibody can be inhibited with anti-CD4 antibody (264). Third, the capacity of Lyt-2+ CTL to induce lectin-mediated lysis of class I-negative target cells (the lectin “glues” the CTL and target cells together) can be inhibited by anti-Lyt-2 antibody (265). To account for these discrepant findings, Tite et

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al. (262) have suggested that interaction between T cells and APCs involves two phase:;: (1)an antigen-independent phase of binding between T accessory m o l e d e s and monomorphic H-2 determinants on APCs, followed by (2) formation of a trimolecular complex between the TCR and Ia plus antigen on APCs. Tite et al. argue that if the phase of binding between T accessory molecules and H-2 is not followed by TCR interaction with antigen, e.g., if the APC 1.acks antigen, the T cell receives a down-regulation signal, which enables th'e T cell to break free from the APC and wander away. TCR contact with antigen counteracts this negative signal and the T cell is triggered. Though fitting most of the available facts, this theory raises certain questions. In particular, one has to explain how interaction of T accessory molecules with H-2 determinants (or with antibodies to T accessory molecules) transmits a negative signal to the T cell whereas TCR contact with antigen plus H-2 molecules results in a positive signal. Fazekas de St. Groth et al. (129)offer a somewhat different explanation for the blocking effects of antibodies to accessory molecules. These workers isolated two rare class 11-restricted T cell clones that express both L3T4 and Lyt-2 molecules. The interesting finding was that only anti-L3T4 and not anti-Lyt-2 antibodies blocked the response of the clone to antigen (hapten) coupled to Ia+ APCs. Since the APCs expressed both class I and class I1 determinants, the selective inhibition observed with anti-L3T4 antibody is clearly difficult to explain in terms of simple competitive inhibition. The provocative suggestion of the authors is that the L3T4 molecules on the clones form some type of inultiinolecular complex with TCRs during interaction with antigen, Lyt-2 molecules being excluded from this complex; antiL3T4 antibodies inhibit the formation of this complex and thereby impair the capacity of the TCR to recognize (or respond to) antigen." None of the above evidence is inconsistent with the notion that T accessory molecules do bind to H-2 molecules, but it should be emphasized that this hypothesis is based almost entirely on circumstantial evidence. There are two main problems with this theory. First, one has to argue that all class I and class 11 molecules express class-unique monomorphic regions accessible to one set of accessory molecules, but not the other. This has yet to be proved. Second, an ingenious experiment by Golding et al. (266) suggests that if H-2 molecules do have unique binding sites for accessory molecules, In the case of Lyt-2+ cells, recent studies of N . Crispe and M . Bevan (personal coinmunication) have shown that attachment of anti-Lyt-2 inAb to a solid matrix next to an artificial antigen (anti-TCR rn.4b) can lead to enhanced responses of Lyt-2 + cells to the antigen; conipara1)le findings have been observed for L3T4 cells with iniiinobilized anti-LX1'4 mAl) (K. Eichlnann, unpublished data). Although these findings are open to various interpretations, the data are in line with the suggestion of Fazekas de St. Grot11 rt ul. (129) that T cell triggering involves the formation of a complex between the TCR and appropriate T accessory molecules. +

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these H-2 sites must be represented on the polymorphic domains. By exon shuffling and gene transfection techniques, these workers fashioned a recombinant H-2 molecule consisting of the NH,-terminal (polymorphic) domain of a class I1 p chain (A:,) covalently attached to the a3, transmembrane, and intracytoplasmic portions of a class I molecule. An L3T4+ T cell line raised against allogeneic I-Ak (A~,,,--A~~,,) determinants proved capable of lysing target cells expressing the hybrid molecule, implying that at least some of the T cells were specific for Abl epitopes. The key finding was that lysis directed to these epitopes was totally inhibited by anti-L3T4 antibody. Although the possibility that T accessory molecules bind to polymorphic epitopes on H-2 molecules still remains open, the results of Golding et al. are clearly much easier to explain in terms of the model of Fazekas de St. Groth et al. (see above). What is clearly needed is direct evidence on whether T accessory molecules bind to H-2 molecules. Before leaving this issue, one should mention that other accessory molecules on mouse T cells, such as LFA-1, also might help to stabilize T-APC interactions (259,267270) (see also Section IV,C). As for L3T4 and Lyt-2 molecules, the function of these other accessory molecules is still poorly understood. C. TRIGGERING OF UNPRIMEDA N D RESTINGT CELLS As discussed earlier, simple cross-linking of TCRs through contact with anti-TCR antibodies or antigen plus H-2 is sufficient to cause T hybridoma cells to synthesize IL-2. Triggering of normal T cells, especially resting T cells, is much more complex. Addressing this issue necessitates defining what stimuli are required to induce a small resting T cell in Go to enter cell cycle and initiate synthesis of growth-promoting lymphokines such as IL-2. The ideal system for approaching this question would be to prepare purified populations of unprimed antigen-specific T cells and then define what particular signals are required to induce these cells to respond to purified antigen plus H-2 on a solid matrix. There are two insuperable problems with this type of approach. First, there are no known techniques for isolating antigen-specific unprimed T cells from other T cells. Second, the precursor frequency of unprimed T cells for antigen plus self H-2 is extremely low. Indeed, with one exception (201), no one to our knowledge has been able to reproducibly demonstrate proliferative responses to protein antigens in vitro with unprimed T cells as responders. In view of these problems, investigators have had to resort to artificial systems for studying the induction of unprimed T cells. Most of the information on this issue has come from studying the response of normal T cells to three sets of stimuli which trigger a high proportion of unprimed cells:

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(1) H-2 alloantigens (dealt with in Section IV), (2) T cell mitogens such as concanavalin A (Con A) or PHA, and (3)antibodies directed to the TCR/T3 complex of molecules. Recent information gained from the latter two systems can be summarized as follows. Purified small resting T cells can proliferate and synthesize IL-2 when cultured with anti-T3 antibodies (tested only for human T cells) or mitogens in the presence of accessory cells (macrophage/monocytes or dendritic cells) (271-275). 'There is general agreement that, in the abs'ence of added factors, T cell stimiilation by these ligands fails to occur if the T cells are rigorously depleted of accessory cells (AC). Precisely how AC control T cell responses to mitogens, however, is far from clear. One possibility is that AC merely provide a source of H-2 molecules, stimulation of T cells by mitogens being dependent on corecognition of H-2 molecules, especially Ia molecules. This idea seems unlikely in view of reports that Ia- cells can act as AC, even for L3T4+ cells (261,274,275). A more plausible possibility is that AC function simply by cross-linking the ligand. Although AC undoubtedly do play an important role in cross-linking ligand, there is increasing evidence that a predominant function of AC is to display or release certain activation signals required by resting T cells. The nature of these signals is a source of continuing controversy. For both mouse and human cells, it is well accepted that activation of resting T cells leads initially to synthesis and surface expression of receptors for IL-2 (IL-2R) (276-278). In order for the T cells to proceed to the stage of cell division and proliferation, the cells have to make contact with IL-2, either exogenous IL-2 or IL-2 made by the cells themselves. Contact with IL-2 through IL-2R then activates the cells to enter cell cycle. With regard to the initial events in T cell induction, the bulk of the evidence from studies with mitogens and anti-TCR/T3 antibodies suggests that cross-linking of the TCR/T3 complex, e.g., by using anti-T3 antibodies on Sepharose beads or mitogens, is sufficient to trigger at least a proportion of T cells to produce IL-2R (271.274). The subsequent signals required to induce the cell to secrete IL-2 (and thus enter cell cycle), however, are still poorly understood. For human T cells, a convincing case has been made that IL-2 synthesis is under the control of IL-1, a lymphokine synthesized by typical AC, but not T cells (279). The evidence implicating IL-1 in the activation of unprimed T cells is as follows (271,280,281): When AC-depleted resting T cells are exposed to anti-T3 antibodies cross-linked on Sepharose beads or attached to glass dishes, the cells express IL-2R, but do not proliferate. If the cells are supplemented with IL-1 (or IL-2), however, vigorous proliferation occurs; addition of IL-1 to normal (unsensitized) T cells causes no proliferation, resting T cells being insensitive to both IL-1 and IL-2 (and a mixture of IL-1

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and IL-2). Although the precise mechanism of action of IL-1 is not known (271,280-283), the prevailing view is that IL-1 somehow induces the cells to begin endogenous synthesis of IL-2. The main problem with this scheme is that although IL-1 is highly effective in the hands of some groups, other workers, especially those working with murine T cells, have had considerable difficulty in finding a convincing role for IL-1 in T cell induction (263,274,275,284,285) [although there is at least one murine T cell clone that responds dramatically to anti-TCR antibodies supplemented with IL-1 (283)l. Most of the skepticism concerning IL-1 has come from studies on the differential triggering requirements of purified populations of unprimed Lyt-2 and L3T4 cells responding to mitogens or anti-TCR antibodies. In the case of mitogens, most groups find that purified resting Lyt-2+ cells respond well to mitogens such as Con A providing AC are present (274,275,284,285-287). If AC are removed, responses to mitogens are abolished or considerably reduced, but can be restored to high levels by addition of crude supernatants of Con A-activated spleen cells (CAS). Since CAS contains a wide variety of different lymphokines, including IL-2, much effort has been devoted to defining which particular lymphokines can overcome the unresponsiveness of AC-depleted Lyt-2+ cells to Con A. Several groups find that IL-2 alone is sufficient to restore the response provided that high doses of IL-2 are used; one group (275) has the extra proviso that responsiveness of Lyt-2+ cells to Con A plus IL-2 in the absence of AC depends on reducing the net negative charge on the T cells, e.g., by pretreating the cells with neuraminidase. If Lyt-2+ cells are cultured with Con A plus low doses of IL-2, some groups argue that additional factors, e.g., certain factors present in CAS, are required to cause optimal proliferation and differentiation into CTL (285,287); these factors, some of which are still not well characterized, include IL-2R-inducing factor (RIF)4(285), CTL differentiation factor (CTDF) (285,288), and interferon-y (287). The important point to emphasize is that IL-1 has virtually no effect in this system. Thus, even in high doses, IL-1 added to AC-depleted Lyt-2+ cells fails to induce these cells to proliferate in response to Con A (274,284,285). In marked contrast to Lyt-2+ cells, the response of L3T4+ cells to mitogens depends heavily on the presence of AC (274,284,285). Thus, addition of even very high doses of IL-2 fails to allow purifed AC-depleted +

+

Recent studies of Wagner’s group (H. Wagner, personal communication) suggest that RIF is synthesized by dendritic cells and that AC-depleted, small, resting, high-density Lyt-2+ cells cannot respond to Con A plus high concentrations of IL-2 unless the cells (used in small numbers) are supplemented with RIF. This finding suggests that at least two signals, mitogen and RIF, are required for IL-2R expression by Lyt-2+ cells.

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L3T4 cells to respond to mitogens such as Con A; adding a mixture of IL-2 and IL-1 is similarly ineffective. Interestingly, however, AC-depleted L3T4+ cells can be induced to respond to mitogens by supplementing the cells with a mixture of IL-2 and the synthetic compound, phorbol myristic acetate (PMA), an activator of protein kinase C (274). Even better stimulation occurs with a mixture of IL-2, PMA, and the calcium ionophore, ionomycin (284). The above data suggest that in response to mitogens such as Con A the requirement for AC is weak for Lyt-2+ cells, but strong for L3T4+ cells. Very similar findings have been reported for activation of resting T cells by an anti-TCR antibody, F23.1; this antibody reacts with about 25% of Lyt-2+ and L3T4+ T cells from normal mice and is specific for TCR P-chain determinants (VP8) (289). When the F23.1 antibody is coupled to Sepharose beads, bulk cultures of AC-depleted purified T cells (a mixture of Lyt-2+ and L3T4+ cells) give high proliferative and CTL responses provided that the cells are supplemented with CAS (290); IL-2 (used only at low doses) is less effective than CAS, and IL-1 is totally ineffective. In the presence of CAS, the vast majority of the T cells stimulated by F23. l-beads are Lyt-2+ cells,
+

+

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Although a clear picture is now emerging on the intracellular events which follow T cell activation (reviewed in Ref. 293), it is apparent from the above discussion that the extracellular signals required for the induction of resting T cells are still largely mysterious. To compound the confusion on this topic, three additional points should be made. First, in addition to triggering via the TCR/T3 complex, there is now increasing evidence that other molecules on T cells can convey activation signals; these molecules include Thy-1 (294) and Ly-6 (295) in the mouse and T11 (296) in man. The physiological significance of T cell triggering via these molecules is obscure. The second point to be stressed is that recent studies on cloned lines of T cells indicate that T cells themselves can produce a multiplicity of different factors, many of these being capable of acting on T cells at an early stage of differentiation; these factors include an intracellular form of IL-1 (297), B cell-stimulating factor 1 (BSF-1) (298; W. E. Paul, personal communication), a T cell-activating factor provisionally termed I L 4 A (299), and IFN-y (287,300). Establishing precisely how these and AC-derived factors interact under physiological conditions will be an imposing task. The final point to be made is that the longstanding notion that IL-2 is a mandatory growth signal for all T cells has now been called into question. In the case of Lyt-2+ cells, there is at least one well-documented example of a clone that fails to synthesize IL-2 and yet is capable of being triggered to proliferate in the absence of exogenous IL-2 (301). Likewise, there are now many examples of L3T4+ clones that make a variety of lymphokines, but do not synthesize IL-2 (302; see Section 111,D). The growth-promoting factors required by these clones is unknown, although BSF-1 is a likely candidate (298). Although studies on in vitro responses to mitogens, etc., will ultimately provide us with a clear picture of what particular signals are needed to trigger resting T cells, there will always be concerns whether this information is relevant to immune responses occurring in vivo. Thus, one hopes that triggering via mitogens and anti-TCR antibodies is a good model for “normal” antigens, but this is by no means certain. Studying the activation requirements for antigen-specific T cell clones might seem a more relevant approach, but a major problem here is that even when “rested” for prolonged periods in vitro, most T cell clones do not totally revert to resting cells, e.g., to a state where the cells lose their IL-2R and reenter G,, (S. Webb, unpublished observations) or begin to reexpress the homing receptors characteristic of resting T cells (303,304). More sophisticated tissue culture systems might solve this problem, but there will still be the concern that any procedure used to manipulate or culture T cells in vitro has the potential to introduce unphysiological stimuli. In uivo systems have the advantage of being at least semiphysiological, but these systems have their own inherent problems: They are costly, cumber-

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some, and, unlike in vitro systems, can only give information on T cell behavior at a population level. Nevertheless, in vivo approaches do provide unique information on such issues as how T cells are primed under physiological conditions, which tissue compartments are involved in priming, and where T cells migrate after activation. Before considering how T cells respond to antigen in vivo, it is important to emphasize that normal T cells are not resident in any one lymphoid tissue, but move constantly from one organ to another via the bloodstream and the lymphatic system (81,305,306); this applies both to unprimed T cells and to most memory T cells, i.e., T cells that have responded to antigen and then reverted to resting cells. The highest concentration of T cells is found in lymph nodes and thoracic duct lymph. T cells enter lymph nodes from the bloodstream by penetrating the walls of high endothelial venules (HEV) with the aid of homing receptors, which are now well characterized biochemically (307,308). After passing through the paracortex (the T-dependent area), T cells exit from lymph nodes via efferent lymphatic vessels. These vessels terminate in the thoracic duct, which in turn empties into the bloodstream in the neck. Large numbers of T cells are also found in the spleen. T cells enter the spleen via the splenic artery and localize in the periarteriolar sheaths of the white pulp. Most T cells exit from the spleen via the splenic vein, the mean transit time through the spleen being about 6 hours. Recirculation of T cells through the lymphoid tissues is presumably a device for enabling specifically reactive cells to congregate at sites where pathogenic organisms enter the body (306). To illustrate this point, one can consider an artificial situation in which a mouse is injected intravenously with a large dose of sheep red blood cells (SRC). Being particulate antigens, SRC localize mostly in the liver and spleen after intravenous injection and do not reach the lymph nodes. The spleen has a large blood supply and nearly all recirculating T cells randomly pass through this organ at least once a day. Thus, when SRC localize in the spleen, percolation of T cells through the spleen over a 24-hour period will screen virtually the entire recirculating pool for SRC-reactive cells. If the dose of antigen reaching the spleen is high enough, the result is that all SRC-specific T cells become temporarily trapped in this organ. In support of this notion, one finds that within 1 day of injecting mice intravenously with a large dose of loy SRC, SRC-specific T cells (but not other T cells) totally disappear from thoracic duct lymph and lymph nodes (309-312). During this stage of “negative selection”-which lasts for 1-2 days-the specifically reactive T cells accumulate in the spleen and undergo blast transformation and division in response to SRC. After extensive proliferation, the progeny of the responding T cells then reenter the circulation in expanded numbers as activated T helper cells. This stage of “positive selection’’ is maximal at 3-6 days after contact with antigen. A similar sequence of events

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occurs in lymph nodes if antigen is given subcutaneously rather than intravenously (313). Negative and positive selection of resting T cells has proved a useful tool for examining the nature of T-APC interactions occurring in viuo, especially the influence of H-2 determinants on these interactions. Evidence that in uivo responses display H-2 restriction in the induction phase came from studies with a system in which T cells were recirculated from blood to thoracic duct lymph through irradiated mice (314). [Note that exposure of mice to high doses of irradiation, e.g., 1000 rad, destroys nearly all T and B cells, but leaves APC relatively intact (315).] Transfer of T cells plus SRC intravenously into irradiated mice causes the same sequence of negative and positive selection seen in intact mice. This is best demonstrated with a helper assay in which the donor T cells entering thoracic duct lymph are transferred to further irradiated mice together with B cells and a mixture of SRC and a different antigen, horse red blood cells (HRC). As a manifestation of negative selection, T cells recovered from the lymph of irradiated mice at 1 day after injection of T cells + SRC are selectively depleted of SRCspecific T helper cells: When transferred with B cells, the T cells elicit high numbers of antibody-forming cells for HRC, but not for SRC. By contrast, at 5 days post-transfer, the lymph contains very large numbers of SRC-specific T helper cells (indicating positive selection), whereas help for HRC remains unchanged. The significant finding is that T cell selection to antigen fails to occur across H-2 barriers (316). Thus, if purified strain a T cells are transferred with SRC to irradiated H-2-different strain b mice, the T cells entering the lymph 1 day later show no depletion of SRC-reactive T cells, i.e., no negative selection; this applies even when massive doses of antigen are injected. Likewise, there is no evidence of positive selection to SRC when the cells are harvested at 5 days posttransfer (note that here it is essential to deplete the injected T cells of alloreactive T cells; otherwise, the lymph is full of alloreactive blast cells). These findings only apply if there is no sharing of H-2 determinants between the donor and host; with semiallogeneic (a x b)F, irradiated hosts, selection of strain a T cells to SRC is as efficient as in syngeneic hosts. The observation that T cells fail to undergo selection to antigen in totally H-2-different hosts indicates that, as in vitro, T cells have to see antigen in the context of self H-2 determinants. Thus, when strain a T cells are exposed to SRC in a strain b environment, the T cells are unable to recognize antigen presented by the strain b APC. The obvious prediction here is that selection of strain a T cells in strain b mice could be induced by supplementing the T cells with a source of strain a APC. This is indeed the case (314; J. Sprent, unpublished data). The selection system described above can be used to separate T cells into

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distinct subsets of H-2-restricted T cells. For example, by using various H-2recombinant mice as selection hosts, one can show that T cells from homozygous mice expressing both I-A and I-E molecules consist of two discrete groups of I-A- and I-E-restricted T cells (317). Likewise, heterozygous mice are found to contain multiple subsets of T cells restricted by the I-A and I-E molecules of the two parental strains (318,319). As for in vitro T cell responses, it seems highly likely that presentation of antigen in vivo is controlled largely by dendritic cells (DC) and macrophages (M+) (168,320). The tissue distribution of these two cell types is somewhat different (320-327). M+ are distributed throughout the body, but reach their highest concentration in the marginal zone of the spleen, the subcapsular sinus of lymph nodes and the liver (Kupffer cells). DC tend to reside in microenvironments frequented by T cells, especially the periarteriolar sheaths oft he spleen, the paracortex of lymph nodes, and the corticomedullary junction of the thymus; in these sites, DC are termed interdigitating cells (321). DC in lymph nodes probably arise from immature precursors in the skin, termed Langerhans cells (328,329). These cells enter lymph nodes via afferent lymphatics, where the cells are called veiled cells (330). In addition to the T-dependent areas of the lymphoid tissues, DC are also found scattered in other tissues, e.g., the lung (331) and intestines (332). Phenotypically, M+ and DC display two important differences (320). First, the expression of Ia molecules seems to be constitutive for DC, but not for M+, Ia expression by M+ depending heavily on induction by IFN-y. Second, in contrast to M+, typical DC isolated in vitro are not phagocytic, although this point now needs to be reexamined in view of recent reports that DC (interdigitating cells) are highly phagocytic in v i m , especially for dead nucleated cells (333). Whether M+ and DC are both capable of presenting antigen to unprimed T cells has excited considerable controversy. Inaba and Steinman (320,334,335) have made a strong case that, in contrast to activated T cells, antigen presentation to unprimed and memory T cells in vitro is controlled solely by DC. For unprimed T cells, these workers observed that the failure of AC-depleted populations of T and B cells to mount primary antibody responses to hapten-protein conjugates or SRC in vitro could be restored by adding very small numbers of DC, i.e., lo4 DC for 4 x lo6 T + B cells (335). By contrast, even 10-fold higher doses of M+ or B cells failed to restore the response. However, when primed (activated) T helper cells were used, all three types of AC were able to restore the response. These data imply that DC play an obligatory role in stimulating unprimed T cells (and also resting memory T cells), whereas a variety of cell types are able to stimulate activated T cells. Although the data of Inaba and Steinman are highly convincing, the notion

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that responses of resting T cells are controlled solely by DC has yet to reach universal acceptance. For example, Ramila et al. (336), working with memor y T cells, contend that although antigen presentation by DC leads to high T proli$erative responses, generation of T helper function is induced only by M 4 and not by DC. Since DC are held to be nonphagocytic, there is also the problem of accounting for how DC are able to present large particulate antigens, such as SRC or undenatured proteins. On this point, Kapsenberg et al. (337)have reported that, although DC are highly efficient at presenting soluble antigens, these cells are unable to present insoluble antigens such as ovalbumin coupled to latex beads. Interestingly, the failure of DC to present such antigens could be overcome by adding very small numbers of M 4 . The authors suggest therefore that M+DC interaction is required for antigens that need to be processed before presentation to T cells. Further evidence on the APC function of DC and other AC is discussed in Section IV,C on T cell responses to alloantigens. Direct evidence on the nature of APC functioning in vivo is sparse. With the aid of the T cell selection system described above, it was found that cells able to present SRC to unprimed T cells in vivo were at their highest concentration in the spleen and in peritoneal exudates (338), the latter being rich in M4, but containing relatively few DC; suspensions of lymph node cells and thoracic duct lymphocytes (poor sources of both M 4 and DC) had little or no APC function. These studies need to be repeated using highly purified populations of M 4 and DC. In the case of primed T cells or hybridomas, it is now well accepted that B cells can act as APC (339).There is little if any evidence, however, that small B lymphocytes can present exogenous antigen to resting T cells. The finding that lymph node cells and thoracic duct lymphocytes, i.e., populations containing 20-50% B cells, have very poor APC function for SRC-specific T cells (338) is consistent with this view. Paradoxically, however, recent evidence suggests that in certain in vivo situations, B cells play a decisive role in antigen presentation. The strongest evidence for APC function by B cells in vivo has come from studies on priming of T cells in the draining lymph nodes of mice given antigen subcutaneously (340). Provided that the antigen, e.g., keyhole limpet hemocyanin (KLH), is injected together with an adjuvant such as complete Freund’s adjuvant (CFA), T cells taken from the draining lymph nodes a week later give strong secondary proliferative responses to the antigen when the cells are cultured in vitro. The intriguing finding, made initially by Ron et al. (341), is that mice depleted of B cells from birth by repeated injection of p chain-specific antibody fail to undergo T cell priming in lymph nodes after injection of antigen in CFA. Similar findings occur when T cells are exposed to antigen in lymph nodes of irradiated mice (340). Thus, if

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irradiated mice are injected with purified T cells intravenously and then given antigen in CFA subcutaneously, the T cells harvested from the draining LN fail to show evidence of priming when tested in uitro. In this situation, as in anti-k antibody-suppressed mice ( p m ) , the lymph nodes contain M+/DC but not B cells (B cells being highly radiosensitive). In both situations, priming of T cells in lymph nodes can be restored by injecting purified B cells subcutaneously just before injection of antigen; injection of M+containing populations, e.g., peritoneal exudate cells, is ineffective. Why B cells are required for presentation of antigen to T cells in lymph nodes is unclear. Perhaps the most likely possibility is that the initial induction of T cells in lymph nodes is controlled by M+/DC, the role of B cells being to induce the clonal expansion of activated T cells after M+/DC become limiting. According to this idea, one would expect that T cells exposed to antigen in B-depleted lymph nodes would show at least a low degree of activation as the result of contact with M+/DC. This indeed appears to be the case. Thus, T cells primed in B-depleted LN do display a low but significant degree of sensitization if the T cells are tested in a highly sensitive in vivo T helper assay (340). Antigens such as SRC and KLH appear to be recognized solely by L3T4 cells and not by Lyt-2+ cells. To examine antigen recognition by Lyt-2+ cells, one has to resort to very different types of antigens, e.g., minor HA or viruses. Minor HA are a particularly useful model for studying how unprimed T cells recognize antigen in uiuo, and a brief overview of this subject is given below. T cell induction to viruses has been reviewed in detail by other workers (1,342). Though less immunogenic than MHC differences, minor HA can evoke quite strong primary responses in uiuo in terms of skin graft rejection (74) and GVHD (343). Minor HA also elicit strong CTL responses, but only after priming in viuo followed by boosting in vitro (159,160). Evidence that minor HA are recognized in association with self H-2 determinants was first demonstrated for cytotoxic cells (159,160). Most minor HA-specific CTL are restricted by class I K and D molecules, although a small proportion of CTL show Ia restriction (344). In the case of CTL, a broad body of evidence suggests that generation of minor HA-specific Lyt-2 effector cells is under the control of Ia-restricted L3T4+ T helper cells (1,345-348). It is important to point out, however, that most of this evidence is based on rather indirect assay systems. A key issue is whether Ia-restricted T helper cells are essential for the induction of Lyt-2+ cells or whether Ia-restricted T cells merely amplify the response. At face value, the most direct approach to this question would be to examine whether purified populations of unprimed Lyt-2+ cells can elicit CTL responses to minor HA in uitro. The major problem here is that, even in the +

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presence of Ia-restricted T helper cells or their products, primary CTL responses to minor HA are virtually undetectable in vitro. Another problem is that anti-L3T4 antibodies-essential for the purification of Lyt-2 cellsonly became available quite recently. As an alternative to examining primary CTL responses, the approach currently being used by Bevan and co-workers (M. J. Bevan, personal communication) is to transfer purified unprimed Lyt-2+ cells to syngeneic irradiated mice and then inject the recipients with a priming inoculum of minor HA-different spleen cells (depleted of T cells). The Lyt-2 cells are then harvested from the recipients and tested for their capacity to mount secondary CTL responses to the priming antigen in vitro in the presence of optimal amounts of “help” (IL-2 in the form of CAS). Preliminary data from this approach indicate that Lyt-2+ cells can be primed to minor HA in vivo in the absence of L3T4+ cells, although the degree of priming is quite low. Thus, L3T4+ cells do not seem to be obligatory for priming, at least in this particular situation. With the advent of T cell cloning techniques, several groups have succeeded in isolating minor HA-specific T cell clones in vitro from bulk populations of in vivo-primed cells (349-353). These clones include L3T4+ Iarestricted T helper clones as well as typical Lyt-2+ K/D-restricted CTL clones. Most of the Lyt-2+ clones show a marked requirement for exogenous help. In other words, these clones fail to proliferate in response to antigen in the absence of exogenous IL-2 (CAS). In addition to these helperdependent clones, however, there are several examples of minor HA-reactive Lyt-2+ clones (and CD8+ cells in man) which do not require exogenous help and are capable of producing their own source of IL-2 (286,349,353). Indeed, early cloning of minor HA-specific CTL has shown that as many as 30% of CTL show a helper-independent phenotype (286). Further evidence on Lyt-2+ cell induction and the role of help has come from studies on GVHD to minor HA (343). When T cells are transferred to heavily irradiated H-2-compatible mice expressing multiple minor HA differences, a large proportion of the recipients succumb to lethal GVHD (354356); in some strain combinations, quite small doses of cells (<105) cause 100% incidence of lethal GVHD (354). A key feature of this assay system is that lethal GVHD is induced by unprirned T cells. One can thus use induction of lethal GVHD as a means for studying whether primary responses to minor HA require L3T4+ cells. Although L3T4+ T helper cells and their products are clearly important for amplifying minor HA-specific CTL, L3T4 cells in isolation generally fail to induce lethal GVHD (357;unpublished data, R. Korngold and J. Sprent). Depleting T cell populations of Lyt-2 cells virtually abolishes the incidence +

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of lethal GVHD in several different strain combinations, although symptoms of chronic GVHD have been seen by other workers (B. Hamilton, personal c o m m ~ n i c a t i o n )Lyt-2 .~ cells, by contrast, cause heavy mortality. The significant finding is that lethal GVHD mediated by unprimed Lyt-2+ cells does not seem to depend on the presence of L3T4+ cells. Thus, depleting Lyt-2+ cells of all detectable L3T4+ cells has little or no effect on either the incidence of lethal GVHD or the mean survival time of the recipients (357; R. K. and J . S . , unpublished data). These data imply that primary minor HA responseshf Lyt-2+ cells occur independently of donor L3T4+ cells. Additional evidence on the role of L3T4 cells on Lyt-2 cell induction has come from studies with the negative selection system considered earlier. Negative selection to minor HA can be induced by transferring unprimed T cells to minor HA-different irradiated mice and then harvesting the donor T cells from thoracic duct lymph of the recipients 1 day later (358).These T cells show a complete lack of reactivity to the minor HA of the host. Thus, if the T cells are transferred to further irradiated mice syngeneic with the first host, no lethal GVHD is observed. This finding only applies when T cells are filtered through H-Qcompatible or H-2-semiallogeneic hosts. With totally H-2-different hosts, the donor T cells totally ignore the minor HA of the host. In this situation, the donor T cells recirculate into thoracic duct lymph and retain full reactivity to minor HA on further transfer. The requirement for H-2 Compatibility between donor and selection host applies only to K and D molecules, Ia compatibility being unnecessary. From these and other studies, it was concluded that T cells causing lethal GVHD to minor HA consist solely of self class I (K/D)-restricted T cells; donor-derived Ia-restricted cells played no detectable role, either as effector cells for GVHD (but see footnote 5 ) or as helper cells for the KID-restricted cells. Although there is no formal demonstration that GVHD reflects CTL activity, collectively the above data indicate that, in unprimed situations, lethal GVHD to minor HA is caused largely and perhaps entirely by Lyt-2+ K/D-restricted T cells, a requirement for help from Ia-restricted L3T4+ cells being surprisingly inconspicuous. It does not necessarily follow, however, that all Lyt-2+ cells participating in GVHD reactions to minor HA are helper independent. Thus, it is quite possible that many Lyt-2+ cells are helper dependent, the growth and function of these cells being controlled by IL-2 released by a minority population of helper-independent Lyt-2 cells. It is also conceivable, though unlikely, that radioresistant T cells of the host +

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are capable of significant IL-2 release. These issues will be discussed later in the section dealing with T cell responses to H-2 alloantigens (Section IV). The nature of the APC required for primary minor HA responses is poorly understood, although studies on T cell selection in various types of bone marrow chimeras (357)and priming for rejection of minor HA-different tumor cells (359) suggest that presentation of minor HA by Ia+ bone marrowderived cells is important. Whether APC function is controlled solely by Ia+ M+/DC, however, is unknown. It is of interest that minor HA presented by certain Ia- cells, especially activated T cells, cause marked inhibition of priming-the “veto” effect (128,360). Studies on the phenomenon of “cross-priming” suggest that under certain conditions, T cells can recognize processed minor HA. As originally observed by Bevan (361,362), injection of mice with minor HA presented on H-2-incompatible cells primes T cells for recognition of minor HA self H-2 determinants (360-366). The explanation for this finding of cross-priming is that minor HA elute from the H-2-incompatible cells used for sensitization and somehow become displayed on host cells. T cells can then be primed to minor HA in the context of self H-2. Recently, it was reported that crosspriming is less effective in neonatal mice than in adult mice (366); since neonatal mice show a selective deficiency of Ia+ APC (367), the authors (366) suggested that cross-priming is controlled, at least in part, by M+/DC. Although cross-priming occurs very rapidly for Ia-restricted T cells, crosspriming of K/D-restricted T cells is relatively inefficient in vivo and is impossible to demonstrate in vitro (343,357,361,363,365). These findings imply that eluted minor HA associate more readily with class I1 molecules than class I molecules. There is an interesting parallel here with T cell responses to viruses. It was mentioned in an earlier section (Section II1,A) that although exogenous native viral proteins can be processed by cells, i.e., broken down into immunogenic peptides, the processed antigens seem to associate only with class I1 molecules: For the peptides to associate with class I molecules, the native proteins either have to be synthesized endogenously by the cell or be degraded to peptides artificially. This idea is supported by the finding that viruses rendered noninfectious by exposure to ultraviolet (UV) light are immunogenic for Ia-restricted cells, but not for K/ D-restricted cells (219). Thus, if influenza virus-specific CTL are exposed in vitro to target cells pulsed with UV-inactivated virus, only Ia-restricted and not K/D-restricted CTL clones are able to recognize antigen. In terms of their susceptibility to processing, UV-inactivated virus and minor HA thus show close similarities: both easily become associated with class I1 molecules, but not with class I molecules. Since the structure and function of minor HA are totally obscure, trying to understand cross-priming at a molecular level is somewhat difficult! For Ia-

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restricted 1’ helper cells, it seems probable that minor HA, whatever their nature, are processed by M+/DC: and then displayed in association with surface Ia molecules. But what about processing of minor HA seen by class Irestricted CTL? By analogy, with viruses, one possibility is that some minor HA are displayed on the cell surface in preprocessed form, i.e., as small peptides associated with class I molecules. Cross-priming would then simply involve slow movement of these peptides in intact form from one cell to another. On this point, it is of interest that allelic forms of p,-microglobulin (&M) show certain features in common with minor HA. Thus, P,M-specific CTL are class I (K) restricted and are demonstrable only in secondary responses in vitro after priming in vizjo (368);although it is not clear whether P2M differences act as transplantation antigens, it is of interest that P,M is encoded by a gene tightly linked to the H-3 minor HA locus (369).The point to emphasize is that P,M per se is not a transmembrane protein, but rather a small polypeptide which associates noncovalently with class I molecules (see Section 11,B). Moreover, target cells readily absorb P,M from serum in vitro (368), implying that P,M might be able to move freely from one cell to another in oivo, presumably without any form of processing. Although it is too early to classify P,M as a minor HA, it would be of obvious interest to examine whether the type of in oivo cross-priming found for minor HA responses also applies to @,MI To sum up, the following statements can be made concerning primary T cell responses to minor HA in vivo. First, T cell induction to minor HA exhibits stringent H-2 restriction, both for K/D-restricted CTL and Ia-restricted T helper cells. Second, Ia-restricted T helper cells seem to be important for controlling the clonal expansion of K/D-restricted T cells (at least for CTL), but are probably not essential for initial induction of these cells. Third, the question of which particular APC control induction of minor HA-specific T cells, especially K/D-restricted cells, has yet to be resolved. Fourth, the issue of how minor HA are processed and presented to T cells is still a provocative mystery.

D. THE EFFECTORPHASE As in the induction phase, the effector phase of T cell responses depends critically upon recognition of antigen plus H-2 molecules. Thus, expression of lytic function by CTL requires recognition of the same association of antigen plus H-2 encountered during induction (1). The important difference between the induction and effector phases, however, is that the strict requirement for a specialized class of APC in the induction phase is lifted for the effector phase: Thus, after induction, Lyt-2+ CTL are able to lyse virtually any cell that expresses the requisite association of antigen plus class I molecules. Likewise, L3T4+ cells are probably able to interact with

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any cell expressing antigen and Ia molecules. The consequences of secondary recognition of antigen plus H-2 molecules by activated effector T cells are highly complex, and only the barest essentials of effector function are discussed below. The effector function of T cells can be viewed as having two components: (1)direct cell-to-cell contact with antigen-bearing target cells, and (2) bystander effects caused by T cells releasing various lymphokines. In the case of CTL, the consequence of contact with target cells is obviously quite simple: The target cells die. Precisely how CTL mediate lysis is outside the scope of this review (370). [It is worth pointing out that although target cell lysis probably always involves H-2 recognition under physiological conditions, this requirement can be bypassed in vitro, e.g., by using a lectin to glue CTL and irrelevant target cells together (371) or by coating target cells with anti-TCR antibodies (372,373).] The type of bystander effects mediated by CTL is presumably a reflection of the types of lymphokines released by the cells. CTL can synthesize a variety of lymphokines, including IL-2 (374,375), IFN-y (376-378), and lymphotoxin (378-380). These lymphokines can exhibit pleiomorphic effects. For example, IFN-y can induce Ia expression by various cell types (108,log), facilitate CTL differentiation (287,381), and inhibit viral replication (382); likewise, local release of IL-2 has the potential to stimulate the growth of neighboring CTL (375) and possibly also to facilitate B cell differentiation (383). Lymphokine release presumably accounts for the capacity of some Lyt-2+ cells, especially helper-independent CTL, to mediate delayed-type hypersensitivity (286,384). Effector functions of CTL and Lyt-2+ cells to transplantation antigens are discussed in a later section (Section IV,D). The most well-characterized effector function of L3T4+ T helper cells is T-B collaboration. This phenomenon has been the subject of numerous reviews (e.g., 385,386), and only the essential elements of T-B interaction need be mentioned. It is now generally agreed that stimulation of antigenspecific B cells by T helper cells can proceed either through direct cell contact or via local release of lymphokines. B cell stimulation through lymphokines (387-389) is often termed bystander help. Activated L3T4 T cells are known to synthesize several different lymphokines (302), and some of these lymphokines, such as BSF-1 (390-393), and B cell-growth factor I1 (BCGF 11) (394,395) act as growth and differentiation factors for B cells; BSF-1 also stimulates resting B cells to express an increased density of Ia molecules (392,396,397). In the presence of a high local concentration of these factors, contact of B cells with specific antigen leads to differentiation into antibody-forming cells. Bystander help is most easily demonstrated with large or activated B cells and with a subset of B cells expressing the Lyb-5 marker (386,389); small B cells and Lyb-5- B cells are relatively resistant to +

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bystander help. In general terms, bystander help is much more readily induced in vitro than in vivo. Bystander help is seen in vivo under certain circumstances (398), but this might be a reflection of nonspecific B cell activation induced by adjuvant (CFA). With antigens that do not require adjuvant, e.g., SRC, bystander help in vivo is low or absent (314,399). Although it has generally been assumed that all L3T4+ cells are capable of mediating bystander help, this issue needs to be reexamined in view of evidence that L3T4+ T cells fall into two discrete groups in terms of their pattern of lymphokine release (302). Type 1T cells produce IL-2, IFN-y and other lymphokines, but not BSF-1; conversely, type 2 cells produce BSF-1, but not IL-2 or IFN-y. Although both types of T cells are reported to express T helper fknction for B cells, whether there is a clear-cut distinction between these L3T4+ T cell subsets in terms of producing bystander help versus direct contact (cognate) help has yet to be resolved. In contrast to bystander help, cognate help exhibits marked H-2 restriction (155,314,386,399-402). This form of help is usually seen with small resting or Lyb-5- B cells, although cognate help can also be important for large, low-density B cells (403). To account for H-2-restricted interactions between T and B cells, it was suggested several years ago that B cells must express the same association of antigen plus Ia molecules that the T helper cells recognized on M+/DC during initial induction (314). Given that T cells generally recognize antigen in processed form, i.e., as small peptides (see Section III.A), how do B cells come to express antigen in peptide form on their cell surface? The prevailing view is that, like M+/DC, B cells have the capacity to process native antigens (404). B cells bind native antigen via their Ig receptors and degrade the antigen to peptide form after internalization; the peptides then move to the cell surface and associate with Ia molecules. T cells recognize this complex and deliver an activatiorildifferentiation signal to the B cells. Under the combined influence of the signals provided by (1) triggering of the B cell Ig receptors, (2) direct T-B contact, and/or (3)local release of lymphokines by the T helper cells, the B cells then differentiate into antibody-forming cells. Whether there is an essential difference between cognate and bystander help is not clear. Thus, it is quite likely that a common pathway is involved, the involvement of one or other type of help simply being a reflection of the local concentration of lymphokines and the physical state of the B cells (see Ref. 386). Local release of lymphokines probably accounts for the other effector functions of T helper cells, e.g., the expression of delayed type hypersensitivity and the activation of M+ leading to destruction of facultative intracellular microorganisms. This topic has been covered in other reviews (e.g., 168).

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IV. Recognition of H-2 Alloantigens by Mature T Cells

Bearing in mind that T cells probably never encounter all0 H-2 molecules under physiological circumstances, the conspicuous attention that has been focused on T cell alloreactivity might seem misplaced. In fact, there are at least three pressing reasons for studying T cell responses to alloantigens. First, the increasing use of allogeneic tissue transplants to cure certain human diseases creates an obvious pressure to determine howlwhy transplants are rejected/accepted. Second, with their striking capacity to stimulate unprimed T cells, H-2 alloantigens are a particularly useful tool for examining the triggering requirements of resting T cells. Third, understanding the mechanism(s) responsible for generating alloreactivity has the potential to provide important insights into how the T cell repertoire for conventional antigens is created.

A. ACCOUNTING FOR ALLOREACTIVITY From studies in several different species, including chickens (405,406), rats (407-409), humans (410), and mice (411-413), it has long been known that a very high proportion of unprimed lymphoid cells responds to allogeneic MHC determinants. In both rats and mice, up to 3% of peripheral T cells from spleen or thoracic duct lymph undergo blast transformation or differentiate into CTL in vitro in response to stimulator cells from an MHCdifferent strain. In rats, an even higher proportion (2.12%)of lymphoid cells undergoes specific negative selection after transfer to irradiated MHC-different recipients (408). In this situation, only about one-half of the alloreactive cells enter DNA synthesis (409); the remainder of the cells become temporarily sequestered in the spleen and lymph nodes, but do not undergo blast transformation, presumably indicating that these particular cells are of low affinity. On this point, it is of interest that a very high proportion of mouse T cells displays specific H-2 alloreactivity when T cells are activated nonspecifically in vitro in a limiting dilution assay, i.e., under conditions that activate CTL irrespective of their specificity for antigen (414,415). In this situation, Beretta et al. (415) find that 1 in 9 T cells display lectindependent CTL activity. Of these CTL, about 10%of the cells show specificity for a given all0 H-2 haplotype. As suggested initially by Wilson et al. (416), the fact that up to 12% of T cells express alloreactivity for single MHC haplotype differences coupled with the existence of dozens of different MHC haplotypes leads to the inescapable conclusion that the vast majority of T cells are alloreactive. Accordingly, most T cells would be expected to express joint reactivity for MHC alloantigens and self X (foreign non-MHC antigens seen in association with self MHC determinants). In support of this idea, it became apparent in

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the late 1970s that murine T cells stimulated against self X in vitro manifested cross-reactivity to particular allo H-2 determinants, and vice versa (417--419).With the advent of techniques for T cell cloning, it was soon established that many T cell clones selected for specificity for self X express concomitant reactivity for H-2 alloantigens (420-423). Estimates of the proportion of self X-reactive clones displaying detectable H-2 alloreactivity range from 16 to 61% (424-427). Since relatively small panels of allogeneic haplotypes were used in these studies, the data are clearly consistent with the notion that all self X-reactive T cells show joint specificity for H-2 alloantigens. The one reservation here is that Bux et al. (428) reported that only 5 of 13 self + X-reactive T cell clones and hybridomas revealed H-2 alloreactivity when tested on a very large panel of 33 different H-2 haplotypes. Although this group concludes that “most T cell clones may not be alloreactive,” the estimate that there could be as many as 100 different MHC haplotypes (97) suggests that it is more than likely that all self Xreactive clones do have the potential to exhibit some form of alloreactivity. In accounting for the existence of T cells displaying joint specificity for self + X and allo H-2 determinants, the obvious issue is whether one or more than one set of TCR is involved. If two distinct TCK molecules were used, a series of T cell clones reactive to particular self + X determinants would be expected to display a random pattern of H-2 alloreactivity. This question has been addressed by a number of groups (424-429), and there now seems to be a consensus of opinion that the type of H-2 alloreactivity displayed by self + X-reactive clones is nonrandom. In the most extensive study on this issue, Ashwell et d.(427) prepared large numbers of T cell clones specific for three different sets of self + X epitopes and found that a strikingly high proportion of the clones, 61%, reacted with one or more of a panel of 9 different allogeneic H-2 haplotypes. Each of the three groups of self X-reactive clones showed considerable heterogeneity in their pattern of alloreactivity: Repeat patterns were quite rare and most (67-81%) of the alloreactive clones displayed unique patterns of alloreactivity. Despite this heterogeneity, each group of clones showed a statistically significant skewing of reactivity to particular H-2 alloantigens (or groups of alloantigens). In light of the evidence that self + X-reactive clones display nonrandom H-2 alloreactivity, it seems improbable that T cell recognition of alloantigens is mediated through a separate set of receptors. Moreover, it has been reported that monoclonal antibodies to idiotypic (clonotypic) determinants on dual-reactive T cell clones are capable of inhibiting responses to self X as well as to allo H-2 (430,431). Although these and the above data do not exclude “one and a half receptor” models, e.g., the involvement of putative a-y or P--y heterodimers in recognition of alloantigens (62), the conservative view is that H-2 alloantigens and self + X epitopes are both recog-

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nized through a single set of a-(3 heterodimers. Proof for this idea will inevitably hinge on gene transfection experiments with a and (3 genes taken from cloned lines of dual-reactive T cells. If T cells do use one receptor to recognize both self + X and all0 H-2, one is faced with the question of whether one or two binding sites are involved. Since there is still no direct evidence on the number of binding sites needed for recognition of self X, it is obviously impossible to discuss allorecognition at the TCR level in more than speculative terms. On a priori grounds, there are two broad possibilities: (1)the TCR has a single binding site which reacts with common epitopes shared between self X and particular all0 H-2 determinants (419); (2) the TCR has two binding sites, one for self H-2 and the other for antigen X: One or other of these sites binds to all0 H-2 determinants (see 432). Without direct information on the nature of the binding site(s) of the TCR, there seems little point in discussing the pros and cons of these two theories. Irrespective of how H-2 alloantigens are recognized at a molecular level, the central issue is why the precursor frequency of T cells for H-2 alloantigens is so much higher than for self X. The suggestion of Bevan (433) is that this difference might be simply a reflection of determinant density. Arguing that the association between self H-2 and antigen X is usually fairly weak, Bevan proposes that the density of self X complexes formed at any particular moment on APCs is quite low. Recognition of self + X is therefore limited to a very small proportion of T cells expressing high-affinity binding for self + X complexes. Alloantigenic determinants, by contrast, are expressed at a high density on APC and are thus immunogenic for a wide spectrum of T cells, including low affinity cells. Although Bevan’s hypothesis accounts for the difference in precursor frequency for all0 H-2 versus self X determinants, one still has to explain why alloreactivity seems to be directed selectively to H-2 molecules rather than to other (non-H-2) cell surface molecules. The answer to this paradox is presumably connected in some way with the preoccupation of T cells with recognition of self H-2 determinants. Here it should be pointed out that B cells, which by and large do not exhibit self H-2 restriction (but see Refs. 434-436), display only a low level of H-2 alloreactivity. If T cells recognize all0 H-2 and self X through a single combining site, one could envisage that T cell specificity is directed to slight perturbations of self H-2 molecules (altered self), these perturbations being expressed naturally on a proportion of all0 H-2 molecules, i.e., molecules closely related to self H-2 molecules, but only very rarely on other (non-H-2) molecules (419). Alternatively, for a two-binding site model, one might argue that a TCR binding site with low affinity for self H-2 would randomly show significantly higher affinity for a proportion of closely related molecules (allo H-2 molecules) than for more

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distantly related molecules (non-H-2 molecules). Until techniques are devised for direct measurement of TCR affinities, such models for allorecognition are impossible to assess objectively. The notion that alloreactivity reflects the close resemblance of allo H-2 molecules to self H-2 molecules-an idea originally proposed by Klein (437)-is strongly supported by the studies of Wilson and Fox (438) on MLR directed to allo- versus xenoantigens. These workers found that although rat lymphocytes gave high MLR to a variety of different rat strains, rat lymphocytes gave only very low primary MLR to stimulator cells from mice, guinea pigs, hamsters, and humans; in fact, when the responder cells were taken from germ-free rats, MLR to xenoantigens were virtually undetectable unless presensitized cells were used. In contrast to these dramatic findings for cells responding in M LR, Fischer-Lindahl and Wilson (439) subsequently reported that the precursor frequency of mouse CTL for rat alloantigens was only marginally lower than for mouse alloantigens. The notion that MHC xenoantigens are poorly immunogenic for T cells therefore may not be a general rule. The converse of examining T cell responses to MHC xenoantigens is to ask whether T cells show conspicuously high alloreactivity to mutant H-2 molecules, i . e . , to alloantigens which are very closely related to self H-2 molecules. It is well accepted that T cells do show high alloreactivity for mouse strains expressing mutant class 1(437,440-443) or class I1 (444-446) molecules; as discussed earlier (see Section 11,B), the mutant molecules differ from the corresponding molecules of the wild-type strain of origin by only 23 amino acids, and probably arose by gene conversion. If high alloreactivity to mutant H-2 differences does reflect the similarity of these molecules to self H-2, ?' cells restricted by more distantly related H-2 molecules would be expected to show decreased alloreactivity to the mutant molecules. To assess this idea, Bevan and Hunig (447) used appropriate bone marrow chimeras (see Section V,B) to prepare T cells exhibiting self-restriction to H-2" versus H-2" determinants. When these T cells were tested for their capacity to mount allo CTL responses to Kbml (B6.C-H-2b1n1)(bml) and bm4 mutant strains, i.e., mice expressing mutations in class I K" molecules, higher responses were found with the H-2"-restricted T cells than with H-2d-restricted cells. The authors concluded therefore that "T cells preferentially respond to antigens similar to self H-2." Very similar findings were observed by Gress and Hodes (448). Again using T cells from bone marrow chimeras, these workers found that, in marked contrast to H-2"-restricted T cells, H-2d-restricted cells responded only very poorly to the b m l mutant and gave almost no response to another Kb mutant, bm6. Although these two studies seem highly convincing, contrary findings were reported in a subsequent study by von Boehmer et al. (449) in which alloreactive T cells were

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quantitated by limiting dilution analysis. This group found only borderline differences in the capacity of H-2k-restricted versus H-2"restricted chimera T cells to respond to b m l alloantigens. Similar findings applied to the response of chimera T cells to the Kk mutant, M523. Interestingly, von Boehmer et al. (449) did find decreased alloreactivity for the b m l (Kb) mutant when H-2d T cells were depleted of H-2b alloantigens by acute negative selection in vivo. In the case of the M523 (Kk) mutant, however, alloreactivity to this mutant was no lower for H-2b T cells depleted of H-2k alloreactivity than for normal H-2k T cells. Drawing firm conclusions from the above studies is clearly difficult. However, the notion that alloreactivity reflects the propensity of T cells to recognize slight perturbations of self H-2 molecules still seems the most attractive and logical explanation for alloreactivity being directed to H-2 molecules rather than to other cell surface molecules, but considerably more data will be needed to validate this hypothesis. For simplicity, we have tacitly assumed in the above discussion that T cell alloreactivity is directed to unmodified H-2 alloantigens. This assumption requires qualification. First, largely on theoretical grounds, some workers have argued that alloreactivity is not directed to foreign H-2 molecules per se, but to multiple complexes created by the association of all0 H-2 molecules with various other cell surface molecules, especially minor HA (450). The obvious corollary to this theory is that alloreactivity would be appreciably lower with H-2-congenic mice, i. e., mice differing only at the H-2 complex, than with H-2-different mice expressing multiple background (minor HA) differences. With the exception of one report (451), there is little evidence to support this idea. Thus, most groups find that very few H-2alloreactive CTL are able to discriminate between H-2 congenic and nonH-2-congenic stimulators (452,453). Although this finding makes it highly unlikely that alloreactivity is directed to all0 H-2 molecules complexed with polymorphic minor HA, one could still argue that T cells respond to complexes involving monomorphic minor HA. The most direct test of this idea is to examine whether alloreactive T cells are able to respond to purified H-2 molecules. In the case of class I molecules, it is well established that activated T cells are able to recognize all0 class I molecules on liposomes (454)or planar membranes (455). Likewise, Coeshott et al. (456) reported that 7 of 9 all0 Ia-reactive T hybridomas were able to recognize purified Ia molecules attached to glass beads. Unless one invokes large-scale association of the purified H-2 molecules with T cell-derived minor HA, these data provide a strong case that alloreactivity is indeed directed to H-2 molecules per se rather than to H-2 complexed to other molecules. Another assumption that needs discussion is whether all0 H-2 molecules have to be processed and recognized in association with self H-2 molecules.

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The above data of Coeshott et al. (456) would clearly seem to rule out this possibility. Curiously, however, there are reports that T cell binding to all0 H-2 deterrninants requires the presence of syngeneic H-2 molecules (457), and that syngeneic APC are required for MLR to a110 H-2 determinants (458). Other groups, however, have found that T cells rigorously depleted of APC show no impairment in their responsiveness to a110 H-2 determinants (459; D. Raulet, personal communication). Moreover, the capacity of anti-Ia antibodies to inhibit MLR depends only on antibody recognition of stimulator, not responder Ia (460). For these reasons, most groups have concluded that typical responses to H-2 alloantigens do not require antigen processing Nevertheless, it has recently become apparent that a small proportion of ‘r cells do respond to alloantigens seen in association with self H-2 molecules. Thus, there are now well-documented examples of T cells which recognize (1) all0 class I in the context of self class I1 (461), (2) all0 class I1 in the context of self class I (462), and (3) all0 class I1 in the context of self class I1 (463). It is important to emphasize, however, that these particular types of alloreactive T cells are probably quite rare in unprimed populations. Before turning to the question of how T cells respond to H-2 alloantigens at the cellular level, the nature of the particular antigenic epitopes recognized by alloreactive T cells warrants brief comment. As a broad generalization, it appears that all0 H-2 epitopes are much the same as the epitopes on self H-2 molecules which act as restriction elements for self X responses. Thus, it is clear from transfection experiments with exon-shuffled genes that, like self H-2 epitopes, all0 H-2 epitopes are situated on the polymorphic domains of class I and class I1 molecules (106,225,464-466). Dating back to the experiments of Fathman et al. (467) on F, hybrid Ia molecules, it also seems clear from many different approaches that all0 H-2 epitopes represent conformational determinants rather than linear sequences of amino acids (106,225427,465-471). The one apparent difference between all0 H-2 and self H-2 epitopes is that T cells frequently react with allo-epitopes shared between a number of different foreign strains (427,428,443). The restricting elements for self X responses, by contrast, are usually “private” (unique to one strain), although “public” restricting elements are encountered occasionally (472,473). If the binding affinity of T cells for allo-H-2 epitopes is lower than for self X, as suggested by Bevan (433), the fact that alloreactivity is skewed to recognition of public H-2 determinants is not surprising.

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B. ALLORECOGNITIONBY RESTINGT CELLSUBSETS Of the various assay systems used to quantitate alloreactivity, it is generally held that MLR and GVH reactions detect class I1 differences, whereas CTL assays usually define class I differences (1,86). Until quite recently, determining which particular subsets of T cells respond to all0 class I versus

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class 11 determinants has been approached largely by separating T cells into subsets on the basis of expression of Lyt-1 and Lyt-2 molecules. The early studies of Cantor and Boyse (474,475) and others (476-481) demonstrated that Lyt-1+2- T cells responded well in MLR and GVHD assays, whereas Lyt-l-2+ cells accounted for all0 CTL responses. Although isolated Lyt-1 2- cells reacted to alloantigens autonomously, the response of Lyt-1- 2 cells usually depended critically on the presence of exogenous help (IL-2) from the Lyt-1+2- subset. In contrast to Lyt-1+2- cells, unprimed Lyt-l-2+ CTL precursor cells were thus viewed as crippled cells incapable of functioning on their own. A nagging problem with this scheme was that although some CTL precursors were Lyt-l-2+, other precursors were typed as Lyt-l+2+ (reviewed in Ref. 482). When stimulated with alloantigen, some of the Lyt-1 2 cells switched to Lyt-1-2' cells, whereas others retained their Lyt-1+2+ phenotype. To add to the confusion, a switch in CTL phenotype was observed for responses to some alloantigens, but not to others (483). This confusion can now be attributed largely to the inadequacy of the Lyt-1 marker for subdividing T cells into subsets. Thus, the use of monoclonal antibodies and high titered alloantisera has shown that Lyt-1 molecules are expressed on virtually all T cells (484), including CTL precursors (482,485). Hence, the procedure of isolating CTL precursors by treating T cells with anti-Lyt-1 antibody plus C generates only a very small proportion of Lyt-2+ cells, i.e., a subset of cells expressing only a low density of Lyt-1. The size of this fraction of cells obviously depends on the titer of the antibody used for cell separation. On this point it should also be mentioned that the proportion of T cells lysed by anti-Lyt-1 antibody plus C can vary greatly depending on the Lyt-1 allele involved. Thus, antibodies to the Lyt-1.1 allele generally destroy nearly all T cells, whereas anti-Lyt-1.2 antibodies, including monoclonal antibodies, can spare 20-30% of T cells (446,486). Although some workers tried to avoid the problems intrinsic in the use of anti-Lyt-1 antibodies by using positive selection procedures (panning) to select for Lyt-2+ cells (487), it was not until the availability of monoclonal antibodies to the L3T4 marker (127) that concerted attempts were made to compare the properties of Lyt-2+ and Lyt-2- T cells. With the aid of cell separation on a fluorescence-activated cell sorter (FACS) (284,286) or a combination of antibody plus C treatment followed by positive selection on antibody-coated plates (488), it is now possible to prepare very highly purified populations of L3T4+ Lyt-2- and L3T4- Lyt-2+ cells. With the aid of these well-characterized nonoverlapping T cell subsets, the issue of which particular T cells respond to class I versus class I1 differences can be addressed anew. Mice expressing mutations of class I or class I1 molecules are the obvious +

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choice for examining T cell responses to defined alloantigenic differences. In the case of class I1 differences, unseparated T cells from the C57BL/6 (B6) (H-2”) strain give high MLR to the bm12 strain (444), i.e., to mice expressing three amino acid differences in the @ chain of the I-Ab molecule as the result of gene conversion from the E P gene (489-491). Recently, it has been found that, both for MLR (488) and IL-2 production (374,375), the response of unseparated B6 T cells to bm12 is abolished by adding anti-L3T4 antibody to the cultures; adding anti-Lyt-2 antibody, by contrast, fails to inhibit the response. By the same token, purified B6 L3T4+ cells give strong MLR and IL-2 production to bm12, whereas B6 Lyt-2+ cells show almost no response. Since B6 L3T4 cells fail to give MLR to class I differences, e.g., to the Kb mutant b m l , these data corroborate the view that L3T4 is a selective marker for T cells reactive with class I1 molecules. It should be mentioned, however, that although primary MLR by L3T4+ cells against class I differences are weak or absent as manifested by T cell proliferation, low but significant responses are detectable in terms of IL-2 production (375). These responses appear to be mediated by a minor proportion of L3T4+ cells which recognize a110 class I molecules presented in association with self class I1 molecules. Turning to responses to class I differences, it has long been recognized that MLR of unseparated T cells to allelic class I differences tend to be quite low, whereas responses to certain mutant class I differences can be spectacularly high. Thus, the MLR of B6 T cells to the class I-different b m l mutant can be as intense as to a whole H-2 difference (437,440). Although one might explain this finding in terms of T-T interaction, i.e., proliferation of class I-restricted Lyt-2+ cells aided by help from Ia-restricted L3T4+ cells, in practice, purified B6 Lyt-2+ cells give excellent MLR (286,488) and IL-2 responses (375) to bml, even when T-depleted stimulators are used. In the case of MLR, the response of B6 Lyt-2+ cells to b m l peaks quite early, i. e., on day 3 or day 4, and then falls precipitously to background levels by day 6 (488). This contrasts with the response of L3T4+ cells where peak responses occur on day 5 or 6. The unusual kinetics observed with Lyt-2+ cells probably reflect destruction of the stimulator cells by CTL, high levels of CTL being readily apparent by day 4 of culture (488). Although purified B6 Lyt-2+ cells respond to a variety of class I-different mutant strains, as mentioned earlier these cells give little or no response to the class II-different mutant, bm12 (488). In view of the dogma that responses involving Lyt-2+ cells are heavily dependent on exogenous help, one might argue that minor contamination with L3T4+ cells accounts for the responsiveness of Lyt-2+ cells. This possibility seems most unlikely. Thus, irrespective of whether unseparated T cells or purified Lyt-2+ cells are used as responders, B6 anti-bm1 MLR +

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are completely resistant to inhibition by anti-L3T4 antibody (488); anti-Lyt-2 antibody, by contrast, ablates the response. It should be noted that B6 anti-bm1 MLR are appreciably higher with purified Lyt-2+ cells than with whole T cells, presumably reflecting the higher precursor frequency in isolated Lyt-2+ cells. The failure of anti-L3T4 antibody to inhibit anti-bml MLR also applies to CTL production and IL-2 release (374,375). As in uitro, purified B6 Lyt-2+ cells give strong responses to b m l in various in vivo assays (492). Transfer of B6 Lyt-2’ cells to heavily irradiated b m l or (B6 x bml)F, mice leads to intense proliferation in the spleen followed by emergence of large numbers of blast cells into thoracic duct lymph (positive se!ection). Lyt-2+ cells are also highly effective at causing GVHD. Thus, B6 Lyt-2+ cells cause pronounced splenomegaly when transferred to neonatal b m l mice and elicit 100% mortality in heavily irradiated adult b m l mice. When transferred to nude mice (493) or thymectomized, irradiated marrow-protected B6 mice (492), syngeneic Lyt-2 cells cause rapid rejection of b m l skin allografts. The effector functions of Lyt-2+ (and L3T4+) cells in uiuo will be discussed later (see Section IV,D). If purified Lyt-2+ cells can give high MLR to class I differences, how does one account for the long-standing evidence that MLR with unseparated T cells as responders are directed predominantly to class I1 rather than class I differences (494,495)? Several points can be made here. First, it is important to emphasize that the capacity of Lyt-2+ cells to respond in MLR varies considerably from strain to strain (492,496). B6 mice and mice of the C57BL/10 (B10) background tend to be high responders, Lyt-2+ cells fro,m these strains generally giving strong MLR to whole H-2 differences and to certain allelic class I differences. By contrast, other strains, e. g., CBA/Ca and DBA/2, give much lower responses. It appears, therefore, that “background’ genes somehow affect the responsivenss of Lyt-2+ cells. On this point, it is of interest that in rats, MLR by purified OX-8+ (Lyt-2 equivalent) cells vary from very high (497) to intermediate (498) to very low (499), relative to W3/25+ (L3T4+ equivalent) cells; in humans, purified CD-8+ cells generally seem to give only low MLR (500). Second, it should be stressed that certain class I differences are only poorly immunogenic for Lyt-2+ cells, even for high responder strains. Thus, whereas B6 Lyt-2+ cells give very high MLR to several class I-different mutants (e.g., bml, bm4, and b m l l ) , other mutants (e.g., bm9 or the identical mutant bm6) elicit only very low MLR (488,496). Similarly, certain allelic class I differences are much less immunogenic than others, especially for Lyt-2 cells from low responder strains. The third point to emphasize is that significant responses to class I differences, particularly to “weak”allelic differences, are difficult to detect with unseparated T cells because of the problem of “autoMLR,” i. e., the response observed with syngeneic stimulators. Unlike whole T cells and L3T4+ cells, Lyt-2+ cells give almost no auto-MLR (488). +

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For this reason, MLR to class I differences are much easier to detect with purified L4yt-2 cells than with unseparated T cells. The reason for the marked variability in the response of Lyt-2+ cells to class I diflerences is unclear. An obvious possibility is that such variation is simply a reflection of differences in precursor frequency. This explanation cannot account for the difference in the response of B6 Lyt-2 cells to brnl versus bm6/9. Thus, whereas MLR to bm6/9 can be 10-fold lower than to b m l , the generation of CTL to b m l versus bm6/9 under limiting dilution conditions in the presence of exogenous help leads to very similar precursor frequencies (442). It is also noteworthy that purified Lyt-2+ cells give quite high primary CTL responses to haptenated (TNP-coupled) syngeneic stimulators when supplemented with IL-2, yet give virtually no response in the absence of added help (374,375,496). It was mentioned in an earlier section that some Lyt-8+ clones specific for self X, e.g., for minor HA, are helper independent, whereas others rely heavily on the availability of help (IL-2) from other cells, either from L3T4+ cells or from helper-independent Lyt-2 cells. Singer and co-workers (375,493) argue that the distinction between helper-independent (HI) and helper-dependent (HD) Lyt-2+ cells applies at the level of unprimed cells. These workers suggest that the variability in the responsiveness of Lyt-2 cells simply reflects the ratio of HI to H D precursor cells. In other words, HI cells are prominent in the response of B6 Lyt-2+ cells to b m l , but are very rare in the response to bm6/9 or to haptenated stimulators. In support of this idea, Singer et al. (375,493) have observed that the number of B6 Lyt-2+ cells able to produce IL-2 in a limiting dilution system is high with brnl stimulators, low with bm6/9, and virtually undetectable with haptenated stimulators. These data thus correlate closely with the relative capacity of purified Lyt-2 cells to respond to these three stimuli in terms of MLR and CTL production (see above). What is the essential difference between HI and H D Lyt-2+ cells? Perhaps the simplest possibility is that HI and H D cells comprise distinct lineages. If this were the case, the phenoype of HI and H D cells should be stable, HI cells being incapable of changing to H D cells, and vice versa. This prediction is inconsistent with the finding of von Boehmer et al. (286) that under certain conditions, Lyt-2+ cells can readily be induced to switch from HI to HE) cells. As mentioned in an earlier section, these workers observe that when Lyt-2 cells are cloned soon after initial stimulation, many of the clones show an HI phenotype. With repeated stimulation, however, these clones gradually change to H D cells. The authors suggest, therefore, that most Lyt-2+ cells are initially HI, but then switch to an H D state. A problem with this idea is that one has to account for why purified Lyt-2+ cells give virtually no response to some antigens, e.g., haptenated stimulators, without added IL-2, and yet give quite high responses in the presence of +

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help. One possibility is that the precursors for haptenated stimulators have already switched from HI to HD cells, e.g., through contact with crossreactive antigens encountered in viuo. Can the difference between HI and H D Lyt-2+ cells be explained in terms of differences in affinity for antigen? There are two problems with this idea. First, to account for HI + HD phenotype switches, one must postulate that the TCRs of mature T cells are susceptible to somatic hypermutation. As discussed in an earlier section, most of the available evidence is against this possibility. Second, if HI cells were of higher affinity than H D cells, one would have to argue that responses to certain antigens, e.g., to haptenated stimulators, are induced almost exclusively by low-affinity cells. This seems rather unlikely (though not impossible). In summary, it is quite clear that Lyt-2 cells do give conspicuously high primary anti-class I responses in some situations, but rely heavily on exogenous help in other situations. At present, the reason for this conspicuous variability is still unclear. The striking role of T accessory molecules in controlling responses to alloantigens deserves special comment. As mentioned parenthetically earlier in this section, studies on the response of unprimed B6 T cell subsets to class I- versus class II-different mutants in vitro suggest that L3T4+ cells respond only to class I1 and not class I differences, whereas Lyt-2+ cells show reciprocal specificity. This specificity also applies in various in vivo assays, such as T cell proliferation in irradiated mice, induction of GVHD, and skin allograft rejection (492). Thus, for skin graft rejection, B6 Lyt-2+ cells cause rapid rejection of b m l grafts, but do not reject bm12 grafts; B6 M T 4 + cells, by contrast, reject only bm12 and not b m l grafts. The failure of Lyt-2+ cells to respond to bm12 is surprising in view of reports that appreciable numbers of all0 class II-reactive Lyt-2 CTL can be isolated after secondary stimulation to class I1 differences in vitro (445,501,502). As suggested by Haas and von Boehmer (501), manifestation of class I1 alloreactivity by Lyt-2+ cells might reflect cross-reactivity, the “primary” allospecificity (and self specificity) of these cells being directed to class I determinants. Lyt-2+ cell recognition of class I1 alloantigens might thus be of comparatively low affinity. The failure of purified Lyt-2+ cells to give primary responses to class I1 differences would then be easily explained. In this respect, we are not aware of any examples of class II-reactive Lyt-2 cells displaying an HI phenotype. +

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C. ANTIGEN-PRESENTING CELLSFOR RESPONSES TO H-2 ALLOANTICENS Although it is generally accepted that Ia+ cells provide the main stimulus for primary MLR (494), the issue of which particular types of Ia+ cells stimulate MLR has aroused considerable controversy. In line with their data

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on responses to self + X, Steinman and his co-workers (503)have concluded that primary responses of T cells to H-2 alloantigens are controlled almost entirely by DC. This group finds that DC, M+ and B cells are all effective stimulators for secondary MLR, but only DC stimulate M L R by unprimed T cells. Strong support for this scheme has come from studies on the effects of treating stimulator cells with a monoclonal antibody, 33D1, which reacts with DC, but not other Ia+ cells such as M+ or B cells (503,504). Using unseparated T cells as responders, Steinman et al. (503) reported that primary MLR were virtually abolished when DC-enriched populations of stimulator cells were pretreated with 33D1 + C. A particularly impressive finding was that 33D1 + C treatment caused a 70-90% reduction in the stimulatory function of normal unseparated spleen cells, despite the fact that the antibody lysed < I % of these cells; the residual stimulatory function of 33D1 C-treated spleen cells was attributed to incomplete removal of DC. A decisive role for DC as stimulators of primary MLR has also been reported for rat T cells (499,505). Thus, Green and Jotte (505) observed very high primary MLR against DC, but only negligible responses against M+ or B cells (although only low doses of these cells were tested). DC were also the most effective stimulators in secondary MLR, although significant responses were also elicited by B cells; curiously, in contrast to the above findings of Steinman et al. in mice, Ia+ M+ were found to be totally nonstimulatory for secondary MLR of rat T cells (505). The above findings provide a strong case that DC are the only cell type capable of presenting H-2 alloantigens to unprimed T cells. Since secondary anti-H-2 responses can be elicited by a spectrum of Ia+ cells, including IFN-y-induced fibroblasts (506,507) in addition to DC, M+, and B cells, one is led to the assumption that DC express a special form of “second signal” which is mandatory for stimulation of unprimed T cells. The possibility that the efficacy of DC is simply a reflection of high endogenous Ia expression is unlikely because the Ia density of IFN-y-induced M+ can be almost as high as on DC (320,503). Although the essential difference between DC and other forms of APC is still far from clear, Inaba and Steinman (508)made the interesting observation that DC have the unique capacity to induce antigenindependent clustering of activated T cells. Such clustering was observed only at 37°C and not at 4°C; by contrast, antigen-dependent clustering-a manifestation of direct binding to all0 H-2 molecules-occurred at either temperature and was seen with M+ and B cells in addition to DC. A major difference between the two forms of clustering was that antigen-independent clustering was as prominent with syngeneic DC as with allogeneic DC. Green and Jotte (505) reported similar findings for rat DC, but this group made two additional observations. First, DC formed antigen-independent clusters not only with activated T cells, but also with activated B cells.

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Second, in contrast to antigen-dependent clustering, formation of antigenindependent clusters with DC and L3T4+ (W3/25+)T cells was not inhibited by anti-L3T4 or anti-Ia antibody. It is apparent, therefore, that antigenindependent clustering is not specific for T cells and does not involve L3T4Ia interaction. The most likely explanation for this puzzling phenomenon is that clustering is mediated through LFA-1 molecules. Thus, Hamann et al. (509) recently reported that cluster formation with mouse lymphoid cells was effectively inhibited by anti-LFA-1 antibodies, but not by antibodies to other cell surface molecules. Is membrane-bound IL-1 (mIL-1) a contender for the second signal of DC? At first glance, this idea might seem unlikely because mIL-1 was first described on M+ (292) and has since been detected on B cells (510). It is worth emphasizing, however, that neither of these cell types seems to express mIL-1 constitutively. Thus, Kurt-Jones et al. (510) found mIL-1 on B cells only when these cells were stimulated in vitro with a combination of anti-Ig antibody and “T cell-conditioned medium.” Likewise, freshly isolated M+ failed to express mIL-1 unless these cells had been stimulated in uivo with lipopolysaccharide or Listeria organisms (292); other stimuli such as protein antigens or SRC were ineffective. Culturing normal M+ in vitro led to mIL-1 expression, but only for a brief period (2 days). In the case of DC, Nagelkerken and van Breda Vriesman (511) recently reported that, unlike M+, rat DC released only negligible amounts of soluble IL-1 when stimulated with LPS/silica. Significantly, however, unstimulated DC expressed quite high levels of mIL-1. Collectively, these data suggest that constitutive expression of mIL-1 might be a property unique to DC. Without detailed additional information, however, the notion that mIL-1 is the “real” second signal of DC has to be regarded as merely an interesting possibility. A useful test of this idea would be to determine whether unprimed T cells can respond in MLR to Listeriainduced allogeneic M+, i.e., cells known to express high levels of mIL-1 for at least 4 days in vitro (292). Although the powerful accessory function of D C is not contested (see also Refs. 512-517), some workers are unwilling to accept that DC are the only cell type able to stimulate primary MLR. Thus, several groups report high MLR with purified M+ as stimulators (512,513,516,518). In view of the extreme potency of DC, however, the possibility of minor contamination with DC is difficult to exclude. It would be interesting to repeat these experiments with purifed M+ pretreated with 33D1 C. Unlike IFN-)Iinduced fibroblasts, it has been reported that IFN-y-induced endothelial cells are excellent stimulators of primary MLR (519,520). Since endothelial cells remain immunogenic after serial passage in vitro, contamination of these cells with D C seems rather unlikely. In the case of B cells, there are

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well-documented examples of certain B lymphoma or hybridoma cells being able to stimulate primary MLR (521,522). Although most groups find very poor MLR with resting small dense B cells as stimulators, Webb et al. (459) observed appreciable MLR with a highly purified population of low-density B cells; since these cells were rigorously depleted of adherent cells by culture on plastic dishes followed by double passage through G10 columns prior to density separation, it seems unlikely that the stimulatory function of the B cells reflected minor contamination with DC. Since relatively large doses of B cells (5 x 105/culture) were needed for optimal responses in this study, the potency of the low-density B cells was probably weak relative to DC. Cowing and Chapdelaine (523) have put forward the provocative suggestion that the poor stimulatory function of typical high-density B cells does not reflect the paucity of a putative second signal, but simply that Ia molecules on B cells are heavily sialated. Arguing that T cells need to recognize desialated Ia molecules, these workers report that pretreatment of small B cells with rieuraminidase leads to a dramatic increase in stimulatory function. Although the data of Cowing and Chapdelaine are most impressive, one would like to see confirmation of their findings by other groups. The bulk of the evidence on the nature of the stimulators for primary MLR has come from studies with unseparated T cells. This raises the question of whether isolated L3T4+ and Lyt-2+ cells both respond to the same type of APC or whether different APC are involved. Since unprimed Lyt-2+ cells respond selectively to allo class I differences and are not inhibited by antiL3T4 antibody, it is difficult to envisage why Ia+ APC would be needed for anti-class I MLR. Yet, a number of groups have found that pretreatment of spleen stimulator cells with anti-Ia antibody C ablates anti-class I MLR (286,488,524). Bearing in mind that most Ia- cells in spleen are typical Thy-l+ T cells (Thy-1 is a pan T cell marker), the possibility arises that a variety of Ia- cell types might be stimulatory for Lyt-2+ cells, T cells being the notable exception. In this respect, Sprent and Schaefer (525; unpublished data) have observed that purified Lyt-2+ cells give quite high primary MLR to the minor component of Thy-l- Ia- cells isolated from spleen and also to the major component of these cells in bone marrow. Unseparated T cells (526) and purified Lyt-2+ cells (525) also give high MLR to certain Thy-1- Ia- tumor cells, e.g., to P815 mastocytoma cells and L929 transformed fibroblasts. Except for one particular T tumor line (EL4), purified Lyt-2+ cells are totally unresponsive to H-2 alloantigens presented by T cells, including several T tumors and purified populations of Lyt-2+ or L3T4+ T blasts (Sprent and Schaefer, unpublished data). Since the unresponsiveness of Lyt-2+ cells to T stimulator cells can be partly overcome by adding rIL-2, the defective APC function of T cells presumably reflects the lack of some form of second signal. On this point, it is of interest that, like

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rIL-2, addition of rIL-1 partly restores MLR with T stimulators (Sprent and Schaefer, unpublished data). However, the reconstitution observed with rIL-1 applies only when relatively high doses of responders (2 x 105) are used. The possibility arises, therefore, that rIL-1 does not act on Lyt-2+ cells directly, but on some rare contaminant in the Lyt-2+ cell suspension. The finding to be emphasized in the above studies is that the response of Lyt-2+ cells in MLR does not depend on Ia+ cells. Unlike L3T4+ cellswhich probably respond largely to Ia+ DC-Lyt-2 + cells can be stimulated by a variety of Ia- cell types, although it is quite likely that Ia+ cells (e.g., DC) are the main stimulators for Lyt-2+ cells under normal physiological conditions.

D. THEEFFECTORPHASE Certain effector functions of allo-H-2-activated T cells, e.g., CTL activity, can be measured in vitro, but to understand typical “transplantation reactions” one has to resort to in uiuo systems. It is not our intention to review the vast literature on transplantation immunity, but rather to give a brief outline of how T cell confrontation with H-2 alloantigens in uiuo leads to allograft rejection and how the cell interactions involved in this type of reaction correlate with T cell alloreactivity measured in vitro. By analogy with in uitro responses, the effector phase of graft rejection and other transplantation reactions is probably the consequence of initial T cell induction by DC (527). It has been known for many years that T cell sensitization leading to allograft rejection depends on “passenger leukocytes” in the graft (526-528). Removing these cells, e.g., by culturing the grafts in uitro at high oxygen concentration (526,529) or by pretreating the grafts with anti-Ia antibody plus C (530) or deoxyguanosine (531), leads to long-term graft survival. Whether the stimulatory function of passenger leukocytes is controlled solely by DC rather than by a mixture of cell types is unclear. Especially for skin allografts, the induction phase of graft rejection probably reflects T cell contact with graft-derived DC (Langerhans cells) in the paracortex of the draining lymph nodes (527); since interruption of donorhost lymphatic connections prevents graft rejection (532), DC from the graft presumably reach the lymph nodes via afferent lymphatic vessels. For GVHD, the widespread distribution of host DC leads to sensitization of donor T cells throughout the body, although the spleen is probably the main site of induction (305,306). In each of these situations (and also in other types of transplantation reactions), the sensitized T cells leave the lymphoid tissues after extensive proliferation and enter the circulation as effector cells. In the context of this article, two questions concerning T effector function in uiuo need to be discussed. First, can Lyt-2+ and L3T4+ cells both

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function autonomously in vivo or is T-T interaction essential? Second, do the two T cell subsets display separate effector functions or is a common mechanism involved? With regard to the first question, it is highly likely that L3T4+ and Lyt-2+ cells do function autonomously in vivo in certain situations. It was mentioned earlier (see Section IV, B) that transfer of purified B6 Lyt-2 cells to “B” mice, i. e., thymectomized, irradiated, marrow-protected B6 mice, leads to rapid rejection of class I-different b m l grafts, but long-term acceptance of class 11-different bm12 grafts (492); likewise, with transfer of L3T4+ cells, only bm12 and not b m l grafts are rejected. Since the recipients were exposed to heavy irradiation (1100 rad) and conditioned with a large dose of opsonizing anti-Thy-1 mAb, it is unlikely that graft rejection reflected T-T interaction with host T cells. For L3T4 cells, and also W3/25 cells in rats, there is now firm evidence from a number of groups that these cells are highly competent at causing rapid rejection of skin allografts (492,533-535); although “B” recipients of L3T4+ cells can show de novo formation of Lyt-2+ cells (536), it is doubtful that these cells contribute significantly to the acute graft rejection seen after L3T4+ cell transfer. Although the notion that L3T4+ cells function autonomously in vivo is now gaining general acceptance, the situation with Lyt-2+ cells is more complex. It should be stressed that the evidence that Lyt-2+ cells function in vivo in the absence of U T 4 + cells is based largely on studies with the highly immunogenic b m l mutant (492,493). It is quite possible that future studies will reveal many situations in which Lyt-2+ cells function only very poorly in rdvo without help from L3T4+ cells. In this respect, in rats, purified OX-8 cells seem to have only a limited capacity to reject allografts (534);likewise, in mice, in vivo administration of anti-L3T4 antibody is much more effective in preventing allograft rejection than anti-Lyt-2 antibody (535).However, it is difficult to generalize from these two findings because the data are based on only a limited number of strain combinations. On this point, it was mentioned earlier (Section IV,B) that in vitro responses of Lyt-2+ cells to class I differences can vary considerably depending on (1)the particular class I difference involved and (2) the genetic background of the responder strain. It will be of obvious interest to see whether this variability in the responsiveness of Lyt-2+ cells also applies in vivo. The data on the response of B6 mice to the bm6/9 mutant illustrate that extrapolating from in vitro to in vivo responses can be a precarious exercise. Rosenberg et al. (493) recently reported that B6 mice rejected only about 30% of bmB/S skin grafts and that this relative unresponsiveness correlated with a near absence of bm6/9-specific IL-%producing Lyt-2 cells in vitro; with the bnil mutant, by contrast, there was rapid graft rejection and a high frequency of IL-%producing Lyt-2 cells. Although this is an interesting +

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correlation, it should be pointed out that some groups find that B6 mice reject 100% of bm6/9 skin grafts in <20 days, the mean survival time for bm6/9 grafts being only fractionally longer than for brnl grafts (537,538). Moreover, in the case of heart grafts, B6 mice reject bm6/9 grafts more readily than brnl grafts (537)!Although these data are not easily interpreted, it is of interest that classic second-set graft rejection applies only to brnl and not to bm6/9 differences. Thus, the relative inability of B6 mice to reject b m l heart grafts, and also thyroid grafts, can be easily overcome by presensitizing the mice with a b m l skin graft (537,538). In marked contrast, prior rejection of a bm6/9 skin graft by B6 mice leads to enhanced survival of bm6/9 heart grafts and permanent acceptance of thyroid grafts. The precursor frequency of IL-2-producing Lyt-2 cells therefore seems to correlate better with second-set graft rejection than first-set rejection. Alternatively, the differences in the response to bm6/9 versus brnl grafts might be controlled solely by L3T4+ T helper cells, i.e., cells responsive to all0 class I molecules in the context of self Ia molecules. Clearly, the above experiments on heart and thyroid grafts need to be repeated using mice depleted of L3T4+ cells. The issue of whether L3T4+ and Lyt-2+ cells express separate effector functions in uivo merits close scrutiny. As for responses to self + X, the effector function of all0 H-2-activated T cells is probably a reflection of the combined effects of lymphokine release and direct cell contact with H-2bearing target cells (527,539). The most popular scenario is that L3T4+ T helper cells mediate lymphokine release (DTH-like reactions), whereas Lyt-2+ cells account for CTL activity. This idea, however, is probably a considerable oversimplification. Thus, as mentioned earlier, L3T4 and Lyt-2+ cells are each able to release a wide range of lymphokines, mediate DTH reactions, and express CTL activity; this applies at both a clonal and population level. Although the potentid effector functions of L3T4+ and Lyt-2+ cells seem to be almost indistinguishable, at least at a population level, the fact that these cells display marked differences in their H-2 specificity might suggest that the two T cell subsets interact with a different range of target cells. Thus, for skin allograft rejection, one might argue that activated Lyt-2+ cells interact with a spectrum of different cell types, whereas L3T4+ cells interact selectively with Ia+ Langerhans cells. Again, this is probably an oversimplification because many different cell types can become Ia+ in the presence of IFN-7, including keratinocytes (540), dermal fibroblasts (541), and vascular endothelium (541-543). The final effector mechanisms of Lyt-2+ and L3T4+ cells in graft rejection might thus be very similar: One could envisage that each subset (1)releases lymphokines which attract nonspecific mediators, such as monocytes and M+, (2) induces Ia expression +

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through local release of IFN-y, and (3) exerts CTL activity against virtually any target cell in the graft. Likewise, it is equally conceivable that L3T4+ and Lyt-2+ cells use a common mechanism for eliciting lethal GVHD. The notion that L3T4 and Lyt-2 cells might show close similarities in their effector functions in viuo is obviously only tenable at a population level. Thus, typical H D Lyt-2+ CTL and noncytotoxic L3T4+ T helper cells probably manifest quite different effector functions in uiuo. Likewise, there are presumably distinct differences in the effector functions of HI versus H D Lyt-2 cells and BSF-l-producing versus IL-%producing L3T4 cells (302). Obviously there is a clear need for detailed histological evidence on the pathogenesis of graft rejection and GVHD mediated by purified Lyt-2 and L3T4+ cells and, ultimately, by the further subsets within these lineages. As a footnote, brief mention should be made of the role of IL-2 in uiuo. Evidence that IL-2 influences allograft rejection has come from the finding that injection of mice with anti-IL-2R antibody significantly delays graft rejection (544,545). Likewise, injection of mice with anti-IL-2R antibody retards the incidence of GVHD (546). Conversely, GVHD is exacerbated when mice are given repeated injections of IL-2 (J. Sprent, unpublished data). As in uitro, the function of IL-2 in vivo is presumably to enhance the clonal expansion of effector cells. Whether IL-2 acts selectively on Lyt-2 cells or on both T cell subsets in vivo is unclear. +

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V. Consequences of T Cell Contact with H-2 Molecules in the Thymus

The decisive studies of Miller (83) on the effects of neonatal thymectomy in mice eslablished that the formation of T cells depends heavily on the presence of an intact thymus. Although initial reports suggested that the thymus might function largely as an endocrine organ, i.e., as a source of thymic “hormones” (reviewed in Ref. 547), most immunologists rapidly became disillusioned with this idea. It is now well accepted that although thymic horinones probably do play an important (if still obscure) role within the thymus, the main function of the thymus is to produce mature functional T cells and release these cells into the peripheral lymphoid tissues. Despite f he enormous turnover of cells in the thymus (548), intrathymic labeling studies indicate that only very small numbers of T cells are exported to the periphery, i.e., about 2 x 106/day in young mice (549). Although it was originally thought that T cells left the thymus in an immature state and required a period of maturation in the spleen (550-552), recent studies of Scollay et al. (553-556) do not support