ADVANCES IN
Immunology VOLUME 41
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ADVANCES IN
Immunology VOLUME 41
This Page Intentionally Left Blank
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
COPYRIGHT 0 1987 BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER
ACADEMIC PRESS, INC. Orlando, Florida 32887
United Kingdom Edition published by
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ISBN 0-12-022441-0
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PRINTED IN THE UNITED STATES OF AMERICA
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)
HUMAN T LYMPHOCYTE ACTIVATION
3
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;
4
ARTHUR WEISS A N D JOHN B. IMBODEN
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
H UMAN T LYMPHOCYTE ACTIVATION
5
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.
6
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.
HUMAN T LYMPHOCYTE ACTIVATION
7
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,
8
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-
HUMAN
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9
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
28
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
HUMAN T LYMPHOCYTE ACTIVATION
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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Meuer, S. C., Hussey, R. E., Fabbi, M., Fox, I)., Acuto, O., Fitzgerald, K. A,, Hodgdon, J. C., Protentis, J. P., Schlossman, S. F., and Reinherz, E. L. (1984b). Cell 36, 897. Mizel, S. B. (1982). Zmmunol. Reo. 63, 51. Moldwin, R. L., Lancki, D. W., Herold, K. C., and Fitch, F. W. (1986).J. E x p . Med. 163, 1566. Moretta, A,, Pantaleo, G., Lopez-Botet, M., and Moretta, L. (1985a).J. E x p . Med. 162, 823. Moretta, A , , Pantaleo, G., Lopez-Botet, M., Mingari, M.-C., Carrel, S., and Moretta, L. (1985b). E u r . J. Zmmunol. 15, 841. Mosmann, T. R., Bond, M. W., Coffman, R. L., Ohara, J., and Paul, W. E. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 5654. Nagasawa, R., Gross, J., Kanagawa, O., Townsend, K., Lanier, L., Chiller, J., and Allison, J. (1987).J. Zmmunol., 138, 815. Nidel, J.. Kuhn, L. J., and Vanderbark, G. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 36. Nikaido, T., Shimizu, A,, Ishida, N., Sabe, H., Teshigawara, K., Maeda, M., Uchiyzma, T., Yodoi, J., and Honjo, T. (1984). Nature (London) 311, 631. Nisbet-Brown, E., Cheung, R. K., Lee, J. W. W., and Gelfand, E. W. (1985).Nature (London) 316, 545. Nishizuka, Y. (1986). Science 233, 305. Oettgen, H., Terhorst, C., Cantley, L., and Rosoff, P. (1985). Cell 40, 583.10. Oettgen, H. C., Pettey, C. L., Maloy, W. L., andTerhorst, C. (1986). Nature(London)320, 272. O’Flynn, K., Krensky, A. M., Beverley, P. C. L., Burakoff, S. J., and Linch, D. C. (1986). Nature (London) 313, 686. Ohashi, P., Mak, T. W., Van den Elsen, P., Yanagi, Y., Yoshikai, Y., Calman, A. F., Terhorst, C., Stobo, J. D., and Weiss, A. (1985). Nature (London) 316, 602. Palacios, R., and Martinez-Maza, 0. (1982). J . Zmmunol. 129, 2479. Parker, P., Loussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfeld, M., and Ullrich, A. (1986). Science 233, 853. Persson, H., Henninghausen, L., Taub, R., DuGrode, W., and Leder, P. (1984). Science 225, 687. Prystowsky, M. B., Ely, J. M., Beller, D. I., Eisenberg, L., Goldman, J., Goldman, M., Goldwasser, E., Ihle, J., Quintans, J., Remold, H., Vogel, S. N., and Fitch, F. W. (1982). J. Zmmunol. 129, 2337. Raulet, D. H., Garman, R. D., Saito, H., and Tonegawa, S. (1985). Nature (London) 314, 103. Reed, J. C., Nowell, P. C., and Hoover, R. G. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 4221. Reed, J. C., Alpers, J. D., Nowell, P. C., and Hoover, R. G. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 3982. Reem, G. H., Yeh, N.-H., Urdal, D. L., Kilian, P. L., and Farrar, J. J. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 8663. Reif, A. E., and Allen, J. M. (1966a). Nature (London) 209, 521. Reif, A. E., and Allen, J. M. (1966b). Nature (London) 209, 523. Reif, A. E., and Allen, J. M. (1984).J. E x p . Med. 120, 413. Reinherz, E. L., and Schlossman, S. F. (1980). Cell 19, 821. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. (1979a).J . Zmmunol. 123, 1312. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F. (1979b).Proc. Natl. Acad. Sci. U.S.A. 76, 4061. Reinherz, E. L., Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Acuto, O., and Schlossman, S. F. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 4104. Rosoff, P., and Cantley, L. (1985).J. B i d . Chem. 260, 14053. Rosoff, P., and Teres, 6. (1986). Cell. Biol. 103, 457.
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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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wEnn
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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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 this idea. These workers find that fluorescein-labeled cells isolated from the spleen and lymph nodes within a few hours of intrathymic labeling show essentially normal T cell function and, like mature T cells, consist almost entirely of “single-positive” L3T4+
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and Lyt-2+ cells (556); <1% of the cells have the characteristic doublepositive (L3T4+ Lyt-2+) phenotype of the major component of cortical thymocytes. Recent thymic emigrants appear in thoracic duct lymph within hours of leaving the thymus (554), implying that the cells immediately enter the recirculating lymphocyte pool. Despite their striking similarity to normal mature T cells, recent thymic emigrants do display two subtle differences (555). First, these cells are slightly larger and less dense than mature T cells. Second, nearly all recent thymic emigrants express the B2A2 marker. This marker is present on 98% of thymocytes, but only 10-20% of peripheral T cells. Interestingly, B2A2+ T cells totally disappear from the peripheral lymphoid tissues within 2 weeks of adult thymectomy (555).This finding suggests that all B2A2+ T cells in the periphery are of recent thymic origin. Since the thymus releases only a trickle of cells into the periphery, the vast majority of thymocytes are presumed to die in situ. Although this might seem peculiarly wasteful, there is now good reason to believe that large-scale destruction of cells in the thymus is a reflection of a highly efficient process in which developing thymocytes are screened for H-2-restricted specificity. Before discussing how this process of thymic “education” operates, it is important to consider the salient features of T cell development and ontogeny in the thymus. This subject has been reviewed in detail by several other groups (556-560).
A. T CELLDEVELOPMENT IN THE THYMUS On the basis of expression of the L3T4 and Lyt-2 markers, thymocytes can be divided into four subsets (556-560). Single-positive Lyt-2 and L3T4 +
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cells collectively comprise about 15% of thymocytes. These two populations resemble recent thymic emigrants in function and surface phenotype (including B2A2 expression) and are found largely, though not entirely, in the medulla (the central portion of the thymus). The bulk of thymocytes (8085%)are L3T4 Lyt-2 , and these double-positive cells account for the vast majority of cells in the cortex. L3T4- Lyt-2- cells amount to only about 3% of thymocytes and are concentrated in a thin rim of cells lying in the outer cortex beneath the capsule (561). Various approaches have shown that these double-negative cells act as stem cells for the other thymocyte subpopulations (562-566). In a recent study, it was shown that individual doublenegative thymocytes were able to colonize lymphoid cell-depleted (deoxyguanosine-treated) aggregates of thymic epithelium (565). A key finding was that the thymuses repopulated by individual double-negative stem cells contained double-positive cells as well as single-positive L3T4 and Lyt-2 + cells, suggesting a common origin for these three subpopulations. In fetal life, the thymic anlage descends into the anterior mediastinum at +
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about 10 days of gestation (567); at this stage, the thymus is alymphoid and consists predominantly of sheets of epithelial cells (568,569). Blood-borne stem cells begin to colonize the thymus on days 11-12 (567). In normal marrow, thymic stem cells are concentrated in a very small subset of cells (0.1% of marrow) expressing a low, but detectable density of the Thy-1 marker (570); these cells are L3T4- L ~ t - 2 By ~ . day 14 of gestation, thymocytes consist almost entirely of bright Thy-1 +, L3T4- Lyt-2- blast cells (558,564). Expression of L3T4 and Lyt-2 molecules is first detected at days 16- 17, with Lyt-2 expression slightly preceding L3T4 expression; this applies both in uiuo (571)and in thymic organ cultures in uitro (564). Over the next 2 days, one sees rapid emergence of double-positive and single-positive cells, with a gradual change from blast cells to small lymphocytes. Functional T cells, e.g., CTL precursors, are first demonstrable at about day 18. By day 20--the day of birth-the fetal thymus closely resembles the adult thymus. With regard to TCR expression, fetal thymocytes do not express TCR ol-p heterodimers until about day 17 (572,573). TCR expression corresponds with the initial synthesis of full-length (Y mRNA transcripts (574,575); full-length p transcripts are apparent 1-2 days earlier (573-575). Whereas the level of (Y and p mRNA increases markedly from the time of initial transcription until the day of birth, y transcripts are readily detectable early in ontogeny, e.g., on day 16, but then decline to very low levels thereafter (574,575). This finding has led to the suggestion that early T cells might show sequential expression of y-P heterodimers followed by a-P heterodimers (574,575). Although such a possibility has not been excluded, studies with y-proteinspecific antibodies have yet to demonstrate the existence of y-P heterodimers (see Refs. 70,71). One of the most perplexing questions about T cell development is whether single-positive thymocytes arise from double-negative cells or double-positive cells. Although it was originally thought that double-positive cells [cells able to bind peanut agglutinin (PNA)] could give rise to singlepositive (functional, PNA- ) cells in culture (576-578), these findings probably reflected minor contamination with preexisting single-positive cells (579,580). Most groups now agree that the vast majority of double-positive thymocytes are “end” cells destined for rapid death. Double-positive thymocytes do express TCR a-P heterodimers (572,573), but these cells appear to be functionally inert and survive for only a few days in culture. Similarly, in marked contrast to double-negative thymocytes, double-positive cells disappear rapidly when injected intravenously in vivo (566). The danger in concluding that double-positive cells are functionally inert is that there is always the objection that these cells are excessively “fragile”: Thus, one could argue that even temporary removal of double-positive cells
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from their natural microenvironment in the thymus nullifies further differentiation. Hence, although it is incontestable that nearly all double-positive cells do die rapidly in situ, the possibility that a minor subset of these cells could give rise to single-positive cells has certainly not been excluded. In this respect, L. Smith and N. A. Mitchison (personal communication) have preliminary evidence that repeated injection of mice with anti-Lyt-2 antibody impairs the generation of the single-positive L3T4+ subset. If confirmed, this finding would provide prima facie evidence that double-positive cells can give rise to single-positive cells, or at least to the L3T4+ subset. (Since Lyt-2 + cells are evident slightly before double-positive cells, the possibility that L3T4+ cells arise from a subset of Lyt-2+ cells cannot be excluded. ) Although double-negative thymocytes show rapid differentiation to double-positive and single-positive cells in thymic organ cultures (562,564), dispersed cell suspensions of double-negative cells show only incomplete differentiation in uitro (581-587). A large proportion of double-negative thymocytes express IL-2R (583-588), and it is now possible to grow these cells with a cocktail of soluble stimuli, e.g., PMA ionomycin IL-2 (587). Some double-negative thymocytes show rapid transition to double-positive cells in culture (581,582), and recent work suggests that under certain conditions, double-negative cells can differentiate to an unusual population of Lyt-2 single-positive cells (H. von Boehmer, personal communication). As yet, however, there is no clear-cut evidence that suspensions of doublenegative thymocytes can be coerced to generate typical single-positive L3T4+ and Lyt-2+ cells expressing TCR a-P heterodimers. As discussed later, the production of typical single-positive cells probably depends crucially on direct cell contact with thymic epithelial cells. It should be emphasized that the above description of thymic differentiation is a very brief overview of a highly complex subject. For simplicity, we have subdivided thymocytes into only four groups on the basis of L3T4 and Lyt-2 expression. However, if one also considers expression of other markers such as Lyt-1, B2A2, and IL-2R, thymocytes can be divided into at least a dozen different subsets (557,558,560,589); this heterogeneity also applies at the level of L3T4- Lyt-2- cells. The important issue is whether all of these various subpopulations belong to a single lineage of cells or whether there are two lineages, one for the single-positive (medullary) cells and the other for the double-positive cells (cortical cells). Although there is one report that stem cells can selectively repopulate the medullary region (590), the conservative view is that cortical and medullary thymocytes both have a common origin from the rim of double-negative cells in the outer cortex (see Refs. 556-560, 589 for detailed discussion). The notion that the microenvironment of the thymus is vital for T cell formation is challenged by the fact that congenitally athymic nude mice
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(591-593) end rats (594) contain low, but significant numbers of cells expressing T cell markers. These cells are rat-e in young nudes, but can account for >20% of lymph node cells in older animals (595). Though apparently incapable of mediating alloaggression in uiuo (596), nude T cells do express CTL activity for H-2 alloantigens in vitro provided that the cells are supplemented with IL-2 (597). Nude T cells can respond to haptenated stimulators (598), but responses to minor HA (598) and typical protein antigens (599) are low or absent, which suggests that the repertoire of nude T cells is fairly limited. Nude T cells are also unusual in their surface markers, singlepositive Lyt-2+ cells being much more common that L3T4+ cells (600); double-positive cells are very rare (
Since the discovery in the early 1970s that T cells preferentially recognize antigen in the context of self H-2 determinants (154-160), there has been intense interest in the issue of how T cells are imprinted with H-2-restricted specificity. After the initial report of Katz et al. (155) that primed T cells failed to interact with H-2-different B cells, Bechtol et al. (603) reported that allophenic mice prepared by embryonic fusion of strains giving high versus low responses to the synthetic antigen, TGAL, produced TGAL-specific antibody of low responder allotype. The authors suggested that this finding [which was subsequently retracted (604)] might reflect interaction between H-2-incompatible T and B cells. Direct support for this idea came from
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studies of von Boehmer et al. (605,606) on double (“tetraparental”) bone marrow chimeras, i.e., heavily irradiated F, mice reconstituted with Tdepleted bone marrow cells taken from both parental strains. The significant finding here was that each population of parental strain T cells which developed in the F, hybrid recipients was able to collaborate with B cells of either parental strain; both populations were fully tolerant in terms of alloreactivity to the host (606). To account for these findings, von Boehmer and Sprent (605) offered two different explanations. First, it was suggested that T cells differentiating from stem cells in an H-2-different environment might undergo a process of “adaptation” which enabled the cells to interact with H-2-different B cells (see also Ref. 434). The alternative suggestion was that normal unprimed T cells might comprise a mixture of self H-2-restricted and all0 H-2-restricted cells. Only self H-2-restricted T cells would undergo priming in normal mice, whereas in double chimeras the presence of a mixture of syngeneic and allogeneic APC would lead to priming of all0 H-2-restricted as well as self H-%restricted T cells. Despite intensive investigation by many different groups during the past 10 years, the pros and cons of these two possibilities continue to be debated. With regard to the first possibility, Bevan (607) made the crucial discovery that transfer of T-depleted (a x b)F, marrow cells to heavily irradiated parental strain a mice (F, + a chimeras) generated a population of F, T cells with skewed H-2-restricted specificity. Using minor HA as antigen, Bevan observed high CTL activity against strain a target cells, but only weak lysis of strain b targets. Since normal F, T cells lysed both targets, the data suggested that raising F, T cells in a strain a environment generated a marked preponderance of a-restricted T cells with very few b-restricted T cells. Since the lymphohematopoietic system in F, + parent chimeras is almost entirely of donor origin, the skewed T cell repertoire seen in these chimeras suggested that a radioresistant component of the host, probably the thymus, was responsible for imprinting T cells with H-2-restricted specificity. In support of this idea, Fink and Bevan (608,609) and Zinkernagel et al. (610612) demonstrated that thymectomized F, mice reconstituted with F, marrow cells and then grafted with a strain a thymus developed large numbers of a-restricted CTL, but very few b-restricted CTL; this applied both for minor HA (608,609) and antiviral (610-612) responses. These findings strongly suggested that the thymus itself imposes H-&restricted T cell specificity. Although the initial data on thymic selection were highly convincing, problems arose when investigators began to study other types of chimeras and thymus-grafted mice. As first reported by Matzinger and Mirkwood (613), it rapidly became clear that strong restriction to thymic H-2 determinants was not the rule in totally H-2-different a + b chimeras, i.e., in
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irradiated :itrain b mice reconstituted with T-depleted strain a marrow cells (613-619). Indeed, in the case of virus-specific CTL, the pattern of responsiveness seen in totally H-2-different chimeras varied from complete restriction to host (thymic) H-2 determinants, to complete restriction to donor H-2, to total unresponsiveness, depending on the virus and the particular strain combination used (617). It was also reported by some workers (620), though not others (621), that thymectomized irradiated F, mice given strain a marrow cells and a strain a thymus graft contained considerable numbers of b-restricted CTL in addition to a-restricted CTL. The data on nude mice grafted with H-2-different thymuses were even more disturbing. Thus, the T cells developing in these mice often showed complete restriction to host (nonthymic) H-2 determinants (348,622-624), even when the T cells were primed to antigen (virus) in the context of thymic H-2 (622). Finally, it was found that normal unprimed strain a T cells (624,625) or strain a T cells acutely depleted of b alloreactivity (see below) (626-629) contained substantial numbers of b-restricted CTL; similar results applied to T cells from neonatally tolerized mice (630), although this was not an invariable finding (631). Although there is considerable opposition-at least from some quartersto the view that the thymus imprints class I restriction, the data on class I1 restriction are rather more convincing. The most impressive data have come from Singer, Kruisbeek, and co-workers. From studies on a wide variety of experimental models, including totally H-2-different chimeras and nude mice given H-2-different thymus grafts, Singer, Kruisbeek et al. (344,632-637) have concluded that, unlike Lyt-2 CTL, L3T4 Ia-restricted T helper cells show complete restriction to thymic H-2 determinants. A considerable amount of evidence from other workers is consistent with this viewpoint (314,348,638-643). But there is also evidence to the contrary (201,615,61.8,644-647). The main area of controversy is whether normal strain a T cells can interact with strain h B cells and/or APC. This is a difficult question to address because, to prevent alloaggression (which can stimulate B cells nonspecifically), one has to deplete the T helper cells ofalloreactive cells. Three main approaches have been used to deplete T cells of alloreactivity: (1) acute blood-to-lymph recirculation of T cells through MHC-different rats (644) or mice (648); (2) exposing T cells to H-2-different stimulators in the presence of bromodeoxyuridine (BUdR) plus light (which destroys dividing cells) (201,646,647,649), and (3)inducing tolerance in parental strain mice by injection of F, hybrid lymphoid cells at birth (640,650). With each of these three techniques, some groups observe effective interaction with H-2-different B cells/APC (644-647,650), whereas others see virtually no such interaction (640,648,649). Although space constraints prevent a thorough discussion of these conflicting results, it is our view that the various examples of T cells +
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interacting with H-2-different B cellslAPC probably all reflect nonphysiological interactions. For example, the capacity of T cells to provide help for allogeneic B cells in the response to SRC (644)is in line with evidence that help for anti-SRC responses in uitro is not necessarily antigen specific (651). Likewise, incomplete depletion of alloreactive cells could explain the capacity of BUdR plus light-treated T cells to respond to antigens presented by H-2different APC (201).The one report that T cells from neonatally tolerized mice can interact with H-2-different B cells (650)is not so easily dismissed. But as the authors of this study point out, the data can be explained in terms of self X mimicry. In other words, a T cell with primary specificity for H-2”(thymic H-2)plus antigen X1might have cross-reactive specificity for H-2b (nonthymic H-2) plus antigen X2.Studies of Hunig and Bevan (350)provide a precedent for this idea. Self X mimicry could also explain the finding of some groups (615,618)that a + b chimeras contain significant numbers of a-restricted T helper cells (in addition to b-restricted T helpers); as mentioned earlier, other groups (635) report that T helper cells from a b chimeras are totally restricted to thymic H-2 determinants. Although one can make a reasonably strong case that the thymus imprints class I1 restriction, the issue of how T cells acquire class I-restricted specificity is still unsettled. There are three main possibilities. First, one might argue that the thymus has nothing to do with imposing class I restriction. To sustain this argument, one has to dismiss the highly convincing evidence of Fink, Bevan, Zinkernagel, and others on F, + parent chimeras and F, “B” mice given parental strain thymus grafts (607-612,652,653). To account for the strong restiction to thymic H-2 determinants seen in these studies, one either has to invoke the existence of haplotype-specific suppressor T cells (654) or argue that CTL induction depends on a form of haplotype-specific help in which Ia-restricted T cells interact with thymic H-2-restricted CTL, but not with CTL restricted by nonthymic H-2 (346). Zinkernagel and Althage (655) and Fink and Bevan (621,656)could find little or no evidence in favor of either of these possibilities. Second, one could argue that the thymus does imprint class I-restricted specificity and that all of the examples of T cells restricted by nonthymic H-2 determinants reflect self X mimicry. In our view, this idea is still tenable. Third, as suggested by Singer, Kruisbeek et al. (634,636), it is conceivable that CTL differentiation involves two quite separate pathways: (1) a conventional pathway involving T cell maturation in the thymus, and (2) an extrathymic pathway, i.e., the pathway that generates T cells in nude mice (see Section V,A). Singer, Kruisbeek et al. favor this idea because, in their hands, class I-restricted Lyt-2+ CTL precursors show strong restriction to thymic H-2 determinants when harvested from the thymus itself, but not when taken from the spleen [interestingly, in marked contrast to Ia-restricted T helper cells, the failure of CTL in spleen
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to show restriction to thymic H-2 determinants applies not only to typical class I-restricted Lyt-2+ CTL, but also to the minor subset of Ia-restricted IA3T4+CTL (344)l. CTL precursors in the peripheral lymphoid organs thus might comprise a mixture of (1) thymus-derived T cells strongly restricted by thymic H-2 determinants, and (2) thymus-independent T cells displaying a heterogeneous pattern of H-2 restriction. Without markers for these two putative subsets of CTL, this intriguing possibility is difficult to assess. Taken as a whole, the evidence that the thymus imposes the H-2-restricted specificity of typical thymus-derived T cells is, in our view, quite strong, both for T helper cells and CTL. If one accepts a central role for the thymus in imprinting H-2 restriction, one is faced with the question of how restriction is imposed and which cell types control this process. To explain the pattern of restriction seen in F, + parent chimeras, one has to postulate that T cells are imprinted with H-2-restricted specificity as the result of encountering H-2 determinants on a radioresistant component of the thymus. Initially, it was tacitly assumed that restriction was controlled by thymic epithelial cells. Subsequently, however, a series of papers by Longo and co-workers (657-659) provided impressive evidence that la restriction of T helper cells is imprinted not by thymic epithelial cells, but by immigrant bone marrow-derived cells, presumably M 4 and/or DC. To explain the restriction to thymic H-2 determinants seen in F, + parent chimeras, this group argues that donor-derived M+/DC have considerable difficulty in gaining entry to the host thymus. With chimeras prepared with -900 rad of irradiation--the dose used by most investigators-Longo and Schwartz (657) found that within 1 month of irradiation and marrow reconstitution, nearly all of the M+/DC recovered from the host thymus were of host origin; donor-derived F, M+/DC were not evident until 2 months postreconstitution. By contrast, with chimeras prepared with a very high dose of irradiation, i.e., 1200 rad, donor-derived F, M+/DC were easily demonstrable in the thymus by 3 weeks postreconstitution (658). Three lines of evidence support the view that intrathymic M+/DC impose H-2-restricted specificity. First, Longo and Schwartz (657) prepared F, + parent chimeras with a dose of 900 rad and then left the chimeras for several months so ;is to allow effective entry of donor M+/DC into the host thymus. When the chimeras were treated with anti-thymocyte serum (ATS) and cortisone to remove mature T cells, the new wave of T cells generated in the chimeras showed restriction to both parental strains. [It may be noted that two other groups have failed to confirm this finding for class I-restricted CTL (660,661).]Second, Longo and Davis (658) found that, in contrast to F, + parent chimeras prepared with 900 rad, T cells from chimeras given 1200 rad did not show preferential restriction to thymic H-2 determinants, even without ATS plus cortisone treatment. Third, Longo et al. (659) observed that
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when (a x b)F, nude mice were grafted with thymuses taken from long-term b + a chimeras, i. e., thymuses containing strain a epithelial cells and strain b M+/DC, the T cells differentiating in these grafts showed preferential restriction to b rather than to a. In assessing these data of Longo et al., a central question is whether the strong restriction to thymic H-2 determinants seen by many groups in typical F, + parent chimeras prepared with =900 rad does indeed reflect retarded intrathymic entry of donor M+/DC. In fact, with the exception of Longo et al., most groups find that donor-derived M+/DC enter the thymus quite quickly after exposure to 900-1000 rad (517,662-666). Indeed, Ron et al. (666) observed that the M+/DC recovered from thymuses of F, + parent and a + b chimeras prepared with 1000 rad were almost entirely of donor origin at 3 weeks postreconstitution. With lower doses of irradiation, i. e., 800-900 rad, a considerable proportion of the chimeras showed a failure of marrow engraftment. This problem was not encountered in parent + F, chimeras: In these chimeras, even low doses of irradiation, e.g., 800 rad, led to effective engraftment with rapid appearance of donor M+/DC in the thymus. In view of these findings, Ron et al. suggested that a host-versusgraft reaction might have explained the slow intrathymic entry of donor M+/DC observed in the F, + 900 rad parent chimeras studied by Longo and Schwartz (657). Although Ron et al. (666) found that the thymuses of F, + 1000 rad parent chimeras were almost completely repopulated with donor M+/ DC within 3 weeks of irradiation, the T cells developing in these chimeras showed marked restriction to thymic (host) H-2 determinants. Some of the chimeras were left for a period of several months and then depleted of T cells by exposure to 800 rad. In striking disagreement with the findings of Longo and Schwartz (657)on chimeras treated with ATS plus cortisone, the T cells from twice-irradiated chimeras showed stringent restriction to thymic H-2 determinants (666). These data are clearly in conflict with the view that M+/DC imprint restriction. Further conflicting evidence has come from recent studies of Lo and Sprent (667). These workers examined the effects of grafting mice with thymuses depleted of M+/DC. With use of the technique of Jenkinson et al. (668), 14-day fetal thymuses were cultured in vitro with deoxyguanosine (dGuo); after 5 days, the thymuses were almost entirely devoid of lymphoid cells and M+/DC, and consisted largely of epithelial cells. Parental-strain dGuo-treated thymuses were grafted to thymectomized F, mice; 2 weeks later, the recipients were exposed to heavy irradiation (1100 rad) and reconstituted with F, marrow cells. The significant finding was that the T cells differentiating in these recipients of M+/DC-depleted thymuses showed strong restriction to thymic H-2 determinants.
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The above results are unquestionably confusing! On the one hand, Longo et al. (657-659) provide elegant and comprehensive support for the view that H-2 restriction is imprinted solely by M+/DC. On the other hand, the data of Ron et al. (666)and Lo and Sprent (667)provide equally firm support for the notion that thymic epithelial cells control restriction. At present, it is impossible to reconcile these diametrically opposite results. Until this conflict is resolved, one is forced to choose between two fundamentally different models for thymic selection. The essence of the scheme proposed by Kruisbeek and Longo (669)is that “some unknown interaction between medullary dendritic cells and medullary thymocytes determines the specificity of the class-II-specific T-cell repertoire.” Strong support for this hypothesis is provided by the striking observation of Kruisbeek et al. (670-672) that mice treated from birth with anti-Ia antibody develop only Lyt-2+ T cells and not L3T4+ cells. Since thymuses of anti-Ia-injected mice are grossly depleted of Ia+ M+/DC (cells with APC function), the authors suggest that the absence of these cells removes the “instructive” stimulus for generation of L3T4+ cells. The notion that Ia+ M+/DC imprint H-2 restriction is also consistent with the findings of Rock et al. (673,674). These workers observe that thymocytes can proliferate in the presence of autologous M+/DC in vitro, and that culturing ( a x b)F, thymocytes with strain a M+/DC (without antigen) selects for a subset of T helper cells able to help only strain a and not strain b B cells in collaborative responses to exogenous antigens. The authors suggest, therefore, that T cell acquisition of self + X specificity is a consequence of initial contact with self H-2 on thymic M+/DC. The model for thymic selection favored by Lo and Sprent (667,675) (enlarged upon here by J. Sprent) is as follows: After undergoing initial differentiation in the specialized microenvironment of the outer cortex, early T cells-probably a subset of double-positive cells-make contact with cortical epithelial cells. Through random expression of TCR c.-p heterodimers, a very small fraction of cells display TCR with significant binding affinity for the particular H-2 molecules expressed on epithelial cells. For selection of Ia-restricted cells, the double-positive precursors bind to Ia molecules on epithelial cells through the combined action of their TCR and L3T4 molecules6; the cells then down-regulate the expression of Lyt-2 molecules. Conversely. binding to class I molecules leads to down-regulation of L3T4 G As discussed in Section III,B, there is still very little direct evidence on the function of L3T4 and Lyt-2 accessory molecules, and the evidence that these molecules physically bind to H-2 molecules is still incomplete. It is quite possible that L3T4 and Lyt-2 molecules play a crucial role in thymocyte maturation, but at present there is no experimental evidence in favor of this possibility.
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expression with retention of Lyt-2 expression. In view of the paucity of evidence for somatic hypermutation of TCR, it is not necessary to postulate that T cell binding to epithelial cells leads to “education,” i.e., to a progressive change in TCR specificity: The T cells might simply receive some form of positive (protective) signal from the epithelial cells which allows the T cells to survive and make their way out of the thymus without further division. All other T cells, i.e., T cells failing to express TCR with appropriate binding affinity for epithelial H-2 molecules, do not receive a positive signal: These cells undergo rapid destruction, perhaps as a consequence of programmed cell death (“apoptosis”) (676). The notion that thymic selection involves binding of T cells to H-2 molecules on epithelial cells is consistent with electron microscopic studies of Farr et al. (677). These workers observed that thymocytes come into close contact with cortical epithelial cells and that TCR molecules undergo clustering at the point of cell contact. The interesting question of whether T accessory molecules undergo comparable clustering has yet to be studied. Lo and Sprent (667,675) envisage that, after positive selection in the cortex, single-positive T cells migrate to the corticomedullary region where the cells come into close contact with M+/DC. Interaction with H-2 molecules on M+/DC leads to deletion (negative selection) of cells with dangerously high affinity for self H-2 (see Section V,C); cells with low-to-intermediate affinity (cells with typical self X specificity) pass through the filter of M+/DC and exit to the periphery. The essence of this scheme is that T cell contact with M+/DC in the thymus leads only to negative selection (deletion of auto-H-2-reactive cells) and not to positive selection (selection of T cells with self X specificity). This contrasts with the view of Kruisbeek and Longo (669), who argue that M+/DC control both positive and negative selection. Lo and Sprent (678) also suggest that T cell contact with M+/DC might lead to IFN-y production, diffusion of IFN-y into the cortex being important for maintaining Ia expression by cortical epithelial cells (678). The corollary to this idea is that depleting the thymus of M+/DC would diminish epithelial Ia expression and thereby prevent positive selection of Ia-restricted L3T4+ cells. This line of reasoning is consistent with (1)the evidence of Kruisbeek et al. (671) that L3T4+ cells fail to develop in anti-Ia-treated mice, and (2) the finding of Kingston et al. (565)that transfer of stem cells to dGuo-treated thymuses, i.e., to M+/DC-depleted thymuses, generates Lyt-2+ and L3T4+ Lyt-2+ T cells, but very few L3T4+ cells. (These two sets of observations are, of course, equally compatible with the view that positive selection of L3T4+ cells is controlled by M+/DC per se.) Clearly a great deal of additional evidence will be needed to assess the relative merits of the above two models for thymic selection. It should be
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stressed that our knowledge of T cell differentiation in the thymus is still rudimentary. It is quite probable that thymocyte maturation depends on highly complex interactions involving “thymic hormones” (679) and lymphokines such as 11,-2 (583-588) and IL-1 (680), but the study of these mediators is still in its infancy. The thymus is known to contain at least two types of epithelium, one in the cortex and the other in the medulla (681), but there is currently almost no concrete information on how these epithelial cells function. In the future, the primary goal will be to dissect the thymic stroma into individual components and then study the function of each component separately. But we are a long way from this goal.
C. ROLE:OF THE THYMUS I N INDUCINGTOLERANCE TO SELF H-2 DETERMINANTS Although the enigma of the “self MLR” is still unexplained (682-684), it is generally agreed that most mature T cells do not react with autologous APC in the absence of exogenous antigen. There are reports that T cells can be tolerized pi-ethymically (685,686), but the lack of any definite evidence that pre-T cells express TCR suggests that tolerance to self H-2 is either induced within the thymus or after export to the periphery. The finding that stem cells differentiating in allogeneic thymuses in uitro develop full tolerance to thymic H-2 alloantigens (687-689) indicates that the thymus itself is capable of tolerance induction. Although it is quite possible that self H-2 tolerance is controlled in part by T suppressor cells (690,691), the more popular view is that tolerance involves T cell deletion: Young T cells confronting high concentrations of H-2 determinants in the thymus are destroyed as a reflection of their immaturity (77,606,692,693). The basic question here is whether induction of tolerance in the thymus is controlled by epithelial cells or M+/DC. A consensus of opinion is now emerging that thymic epithelial cells play little or no role in tolerance induction. The first direct evidence on this topic came from studies on T cell tolerance in nude mice given allogeneic thymus grafts. If nude mice are grafted with H-2-different thymuses from neonatal mice, the T cells developing in the recipients generally show full tolerance to graft-type €1-2 determinants (694). Zinkernagel et al. (622) observed that, in marked contrast to untreated neonatal thymuses, nude mice grafted with irradiated H-2-different adult thymuses failed to induce tolerance to the graft alloantigens; in terms of both MLR and CTL activity, the T cells from the grafted mice showed high reactivity to donor strain H-2 determinants. To explain this finding, the authors suggested that “lymphohemopoietic” cells in the thymus are required for tolerance induction and that these cells gradually disappear after irradiation. Zinkernagel et al. pointed out that the
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apparent failure of the epithelial (radioresistant) component of the graft to induce tolerance correlated with the evidence that organ grafts depleted of passenger leukocytes are usually not rejected, i.e., are not immunogenic for mature T cells (see Section IV,D). In agreement with this reasoning, it was noted that although grafting irradiated allogeneic thymuses to nude mice restored T cell function, the grafts themselves were not rejected. Essentially identical findings have since been reported for fetal thymuses depleted of M+/DC by in vitro culture with dGuo. Thus, dGuo-treated thymus grafts survive for prolonged periods in H-2-different normal mice (531,667) and fail to induce tolerance when used to reconstitute T cell function in nude mice (695,696). Likewise, stem cells differentiating in dGuotreated thymuses in vitro do not develop tolerance to the H-2 alloantigens of the graft (697). It is also reported that nude rats grafted with MHC-different thymus fragments cultured in vitro for 6-8 days develop near-normal T cell function, but fail to show tolerance to graft-type MHC determinants (698); the grafted thymus fragments were largely depleted of “lymphoid elements.” Three other lines of evidence suggest that thymic epithelium is poorly tolerogenic for immature T cells. Firs!, if parent -+ F, chimeras prepared with heavy irradiation are left for prolonged periods, host-type lymphohematopoietic cells, including M+/DC, are eventually almost totally replaced by donor cells (699). The T cells developing in these chimeras show a form of split tolerance to the host alloantigens, i. e., tolerance for CML, but not for MLR; by contrast, T cells developing in double (a b + F,) chimeras show full tolerance to host-type alloantigens (606). Second, split tolerance is also observed in surgically fused frog embryos, i. e., when the anterior region of l-day-old frog embryos containing an alymphoid thymic anlage but no stem cells is fused to the posterior (stem cell-containing) region of MHC-different embryos (700). These chimeric frogs accept skin grafts of the thymus donor (anterior portion) strain, but are nontolerant to the thymus donor in terms of MLR. Third, studies with “nurse” cells, i.e., aggregates of cortical thymocytes contained within thymic epithelial cells (701), have shown that the thymocytes in nurse cells of chickens are not selftolerant (702);thus, the T cells in the nurse cells are able to induce syngeneic pock formation in the chorioallantoic membrane assay. Collectively, the above data would seem to provide a strong case that thymic epithelial cells do not play a conspicuous role in tolerizing early T cells. Nevertheless, there are at least two studies suggesting that thymic epithelial cells are capable of tolerance induction. First, Good et al. (689) observed that CTL differentiating from stem cells in 10-day allogeneic fetal thymuses were almost completely tolerant to the graft H-2 alloantigens despite the fact that the thymuses were “. . . entirely devoid of infiltrating hemocytoblasts. ” Second, Jordan et al. (703) reported that fetal thymuses
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depleted of lymphohematopoietic cells by low-temperature culture in uitro induced full tolerance when grafted to H-2-different nude mice. Although one might attribute the tolerance seen in these two studies to minor contamination of the thymuses with M+/DC, the possibility that thymic epithelial cells do play at least some role in tolerance induction certainly cannot be ignored Three lines of evidence suggest that intrathymic marrow-derived cells, probably M+/DC, play a decisive role in tolerance induction. First, in the various situations described above in which lymphoid cell-depleted allogeneic thymuses failed to induce tolerance, T cells developing in normal untreated thymuses were invariably tolerant to the graft alloantigens (695698); likewise, supplementing lymphoid cell-depleted thymuses with lymphohematopoietic cells restored tolerogenicity (697). Second, T cells differentiating in H-2-different thymuses always show tolerance to autologous H-2 determinants (687-689,695-699). Third, the degree of T cell tolerance seen in parental -strain mice given tolerizing injections of semiallogeneic cells at birth correlates well with the entry of donor lymphohematopoietic cells into the host thymus (704). The finding that T cells differentiating in an H-2-different thymus develop tolerance to self H-2 determinants leads to two important conclusions: (1) Thymic epithelial cells do not play a mandatory role in tolerance induction, and (2) intrathymic contact with lymphohematopoietic cells alone is sufficient to induce tolerance. Although there is no direct evidence on which particular lymphohematopoietic cells control tolerance induction, M+/DC (especially DC) are the most likely candidates, particularly for class I1 tolerance. [Note that in the case of class I1 tolerance, depleting thymic stem cells of Ia+ cells-or inhibiting Ia expression-would be expected to prevent tolerance induction. Three lines of evidence are consistent with this prediction (705-‘iO8).] The mechanism by which lymphohematopoietic cells, presumably M+/DC, induce tolerance is obscure. Given that allogeneic M+/DC are highly immunogenic for mature T cells, one has to assume that intrathymic T cells are tolerized while still in an immature state. But why are M+/DC apparently more important for tolerance induction than thymic epithelial cells? Perhaps the simplest possibility is that young T cells bind with particularly high avidity to M+/DC and thus receive a higher dose of the “tolerogenic signal’’ from these cells than from epithelial cells. Firmer T cell binding to M+/DC might be a reflection of (1) a higher density of H-2 molecules cin M+/DC than epithelial cells (see 675,678), (2) a higher density of TCR molecules on T cells at the time these cells reach the medulla (the main site of M+/DC) (675,677), or (3)involvement of some type ofT accessory molecule which binds to a ligand expressed selectively on M+/DC. An
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alternative possibility is that epithelial cells are intrinsically nontolerogenic because these cells release or display a ”tolerance-inhibiting” factor. These highly speculative suggestions sidestep the basic issue of the modus operandi of tolerance induction. Discussing this issue is obviously futile at present because the impact of a tolerogenic signal at the cellular or subcellular level is almost totally unknown. All that can be said with any degree of confidence is that M+/DC (or related cells) seem to be highly tolerogenic for young T cells, whereas thymic epithelium is less tolerogenic. Thymic epithelial cells might play some role in tolerance induction, e.g., by deleting very high-affinity T cells, but direct evidence on this question is still lacking. As a final point, it should be mentioned that although tolerance induction in the thymus probably does lead to functional deletion of T cells, e.g., by clonal “abortion” or “anergy” (709), it is by no means clear that this process is solely a reflection of T-M+/DC interaction. T cell deletion might also be controlled by other bone marrow-derived cells such as veto cells (710-712) and/or I-J-bearing T suppressor cells (690,693). VI. Summary
One of the risks in attempting to give an overview of a subject as mired in controversy as the immunobiology of T cells is that the reader fails to appreciate the distinction between established “facts” and the idiosyncratic views of the authors. With this risk in mind, a short “unbiased” summary of what is known and not known about T cell specificity and function is given below.
A. T CELLSPECIFICITY Perhaps the most striking difference between T and B lymphocytes is that B cells have specificity for free antigen, whereas T cells react with antigens displayed in close association with H-2 molecules on the surface of living cells. The bulk of evidence suggests that the response of T cells to foreign non-H-2 antigens (antigen X) requires that the antigen be “processed” (reduced to peptides or unfolded) by the APC. Processing allows small particles of antigen X to enter into an immunogenic alignment with surface class I or class I1 H-2 molecules; T cells recognize the association of self H-2 X and are triggered. The evidence for antigen processing is quite strong for antigens seen in association with H-2 class I1 molecules, although recent evidence suggests that antigen processing might also be important for certain class I-restricted responses. Although there is continuing debate on whether antigen and H-2 molecules form a true self X complex, two groups have now provided impressive evidence that certain peptide antigens do enter into firm physical association with high responder class I1 molecules; it remains to be seen whether this is a general phenomenon.
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Recognition of associations of self X by mature T cells is controlled largely and perhaps entirely by heterodimers of TCR (Y and p chains; TCR y chains appear to be expressed on a small subset of T cells, but at present, there is no firm evidence that y chains contribute to the specificity of typical self + X-reactive T cells. Each T cell probably uses only one type of TCR molecule to recognize self X epitopes. However, the issue of whether the TCR expresses (1) two binding sites, one for self H-2 and the other for antigen X, or (2) a single binding site specific for altered-self determinants has still to be resolved. In addition to specificity for self X, many T cells have joint specificity for H-2 alloantigens. T cell clones with defined self X specificity show nonrandom patterns of H-2 alloreactivity, which suggests that alloreactivity reflects cross-reactive binding of epitopes shared between allo H-2 and self X. In most instances, recognition of H-2 alloantigens seems to be directed to native H-2 molecules per se, although rare T cells can respond to processed all0 H-2 epitopes seen in association with self H-2 molecules. T cell specificity and/or triggering might be influenced by certain T accessory molecules, especially L3T4 and Lyt-2 molecules. The expression of these markers is mutually exclusive on most peripheral T cells, and the type of T accessory molecules displayed by T cells correlates closely with the class of H-2 molecules that the cells recognize: By and large, L3T4+ T cells are restricted by H-2 class I1 molecules, whereas Lyt-2+ cells are class I restricted. The function of T accessory molecules is still unclear: These molecules might physically associate with H-2 molecules on APC and thereby stabilize binding via TCR molecules; recent data, however, are difficult to reconcile with this attractive idea.
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B. T CELLTRIGGERING For activated T cells or T hybridomas, accumulating evidence suggests that T cell triggering leading to IL-2 production depends largely and perhaps entirely on cross-linking of TCR molecules. In contrast to activated T cells, the induction of resting T cells requires that antigen (self X or all0 H-2) be presented by a specialized class of APC, especially dendritic cells. These cells are presumed to convey some type of activation signal to resting T cells, but the nature of this “second signal” is controversial: Soluble IL-1 seems to be important for T cell induction in some situations, but the evidence on the effects of IL-1 is conflicting, especially for mouse T cells. In attempting to define the s8ignalsneeded for the activation of resting T cells, much attention is being focused on the requirements for inducing purified populations of Lyt-2+ and L3T4+ cells to respond to mitogens in the absence of APC. Although certain cocktails of soluble factors can stimulate resting T cells under thest: conditions, the relationship of these factors to the physiological
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signal(s) provided by intact APC is still largely obscure. Whether Lyt-2+ and L3T4+ cells need the same or different second signals is unclear, although it is of interest that the requirement for viable APC is more easily bypassed for Lyt-2+ cells than L3T4+ cells. Although the function of Lyt-2 cells can show strict dependency on help (IL-2) provided by L3T4+ cells, in certain situations resting Lyt-2+ cells respond well to antigen in the absence of exogenous help; the reason for the marked variability in the response of purified Lyt-2 cells is unknown. The effector functions of Lyt-2+ and L3T4+ cells are still not well understood. Although it is generally assumed that L3T4 + cells function mainly by releasing various lymphokines, whereas Lyt-2+ cells act as cytotoxic cells, it has recently become apparent that, at a population level, both cell types are capable of lymphokine release and cytotoxic activity. +
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C. GENERATION OF T CELLSPECIFICITY It is generally agreed that most typical T cells arise in the thymus. There is now convincing evidence that T cells leave the thymus in a mature state, but in only quite small numbers. Since the production of thymocytes is enormous, it is assumed that most thymocytes die in situ. It seems likely that the large-scale death of cells in the thymus is a manifestation of a stringent process of selection in which only T cells with specificity for self H-2 determinants-cells with potential self X reactivity-are allowed to emigrate to the periphery; cells with irrelevant or unwanted specificity (including cells with high affinity for self-H-2) are destroyed. This scenario is not universally accepted, however, and the issue of whether the thymus plays a decisive role in imprinting T cells with self-H-2-restricted specificity is contentious. Some groups have provided compelling evidence for thymic “education,” whereas others find many examples of T cells displaying specificity for H-2 determinants not encountered in the thymus. Some of these “anomalous” data could be explained in terms of self + X mimicry (T cell recognition of cross-reactive epitopes shared between thymic H-2 X1 and nonthymic H-2 + X2), but the possibility that the thymus does export cells with no apparent specificity for self H-2 cannot be ignored. The data on thymic education are somewhat stronger for class I1 restriction than class I restriction, which has led some workers to propose that a proportion of class Irestricted T cells arises via an extrathymic pathway. Although selection of T cells with self-H-2-restricted specificity is assumed to be controlled by a radioresistant component of the thymus, the identity of the cells which imprint restriction is controversial. Some workers have provided strong evidence that intrathymic restriction is controlled solely by immigrant bone marrow-derived cells, such as dendritic cells; others find no support for this idea and argue that restriction is imprinted by cortical epi-
+
+
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thelial cells. In the case of tolerance induction to self H-2 determinants, most groups agree that tolerance reflects intrathymic contact with marrowderived cells, thymic epithelial cells being poorly tolerogenic.
ACKNOWLEDGMENTS The expert typing skills of Ms. Barbara Marchand are gratefully acknowledged. We are grateful to Drs. M. Bevan, M. Brennan, N. Crispe, D. L., Y. Ron, J. Sheil, and D. Wilson for stimulating cliscussions. The authors are supported by Grants A1 21487, CA 38355, CA25803, CA 35048, and CA 41993.
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Lo, D., and Sprent, J. (1986). J. Znimunol. 137, 1772. Stutman, 0. (1983). Clin. Zmmunol. Allergy 3, 9. DeLuca, D., and Mizel, S . B. (1986). J. Zmniunol. 137, 1435. van Vliet, E . , Melis, M., and van Ewijk, W. (1984). Eur. /. Immunol. 14, 524. Weksler, M. E . , Moody, C. E . , and Kozak, R. W. (1981). A d v . Zmmunol. 31, 271. Glimcher, L. H., Schwartz, R. H . , Longo, D. L., and Singer, A. (1982).J. Zmmunol. 129, 987. Hausman, P. B., Moody, C. E., Innes, J. B . , Gibbons, J. J., and Weksler, M. E. (1983).J. Exp. Med. 158, 1307. Bradley, S. M., Morrissey, P. J., Sharrow, S. O., and Singer, A. (1982).J. Erp. Med. 155, 1638. Chervenak, R., Cohen, J. J., and Miller, S. D. (1983). J. Zmmunol. 131, 1688. Robinson, J. H., and Owen, J. J. T. (1978). Nature (London) 271, 758. DeLuca, D., Mandel, T. E . , Luckenbach, 6. A , , and Kennedy, M. M. (1980). J . Zmmunol. 124, 1821. Good, Fvl. F., Pyke, K. W., and Nossal, G . J. V. (1983). Proc. Natl. Acad. Sci. U . S . A . 80, 3045. Streleirl, 1. W. (1979). Zmmunol. Reo. 46, 125. Wilson, D. B. (1984). Zmmunol. Today 5, 228. Brent, L., Brooks, C., Lubling, N . , and Thomas, A. V. (1972). Transplantation 14, 382. Nossal, G. J. V., and Pike, B. L. (1981). Proc. Natl. Acad. Sci. U . S . A . 78, 3844. Kindred, B. (1978). Zmmunol. Rec. 42, 60. von Boehmer, H., and Schubiger, K. (1984). Eur. /. Immunol. 14, 1048. von Boehmer, H., and Hafen, K. (1986). Nature (London) 320, 626. Jenkinson. E. J., Jhittay, P., Kingston, K.,and Owen, J. J. T. (1985).Transplantation 39, 331. Schuunnan, H.-J., Vaessen, L. M. B., I’os, J. G . , Hertogh, A , , Geertzerna, J. G. N., Brandt, C. J. W. M., and Rozing, J. (1986).J. Zinmunol. 137, 2440. Sprent, J., von Boehmer, H., and Nabholz, M. (1975). J. Exp. Med. 142, 321. Flajnik. M. F . , du Pasquier, L., and Cohen, N . (1985). Eur. J. Zmmunol. 15, 540. Werkele, H . , and Ketselsen, U.-P. (1980). Nature (London) 283, 402. Wick, G . , and Oberhuber, G. (1986). Eur. J. Zmmunol. 16, 855. Jordan, R. K., Robinson, J. H . , Hopkinson, N. A . , House, K. C., and Bentley, A. L. (1985). Nature (London) 314, 454. Morris>ey, P. J., Sharrow, S. O., Kohno, Y . , Berzofsky, J. A , , and Singer, A. (1985). Transplantation 40, 68. Kimoto, M., and Kishimoto, S. (1986). Eur. J. Zmmunol. 16, 835. Villartay, J.-P., Griscelli, C., and Fischer, A. (1986). Eur. J. Zmmunol. 16, 117. Glazier, A . , Tutschka, P. J.. Farmer, E. R., and Santos, G. W. (1983). J. E x p . Med. 158, 1. Cheney, R . T., and Sprent, J. (1985). Transplant. Proc. 17, 528. Nossal, 6. J, V. (1983). Annu. Rec. Zinmuno/. 1, 33. Miller, R. 6. (1980). Nature (London) 287, 544. Crispe, I. N., and Owens, T. (1985). Zmmunol. Today 6, 40. Rammensee, H.-G., Bevan, M. J., and Fink, P. J. (1985). Zmmunol. Today 6, 41.
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ADVANCES IN IMMUNOLOGY, VOL 41
Determinants on Major Histocompatibility Complex Class I Molecules Recognized by Cytotoxic T Lymphocytes JAMES FORMAN Department of Microbiology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235
1. Introduction
Cytotoxic T lymphocytes (CTL) are effector cells that play an active role in the immune response against virus infection, tumors, and allografts (see reviews by Zinkernagel and Doherty, 1979; Doherty et al., 1984). The antigen specificity of these cells is a result of the recognition of class I molecules on target cells that are encoded by genes in the major histocompatibility complex (MHC). These molecules consist of a 40,000-45,000 Da heavy chain noncovalently bound to a 12,000-Da light chain, P,-microgIobulin (Pz-M) (Kimball and Coligan, 1983) (Fig. 1). The heavy chain is an integral membrane protein composed of three extracellular domains (al-a3) of -90 amino acids each, a transmembrane (TM) domain, and a cytoplasmic region that varies in length between 30 and 39 amino acids (Steinmetz et al., 198la) (Table I). These molecules are glycosylated through N-linked oligosaccharides (Nathenson and Cullen, 1974) and have serine, tyrosine, and possibly threonine residues in their cytoplasmic region that can be phosphorylated (Pober et td., 1978; Lalanne et al., 1982; Guild et al., 1983; Guild and Strominger, 1984a,b). The molecule can be fatty acylated to palmitic acid via a cysteine in the TM region (Kaufman et al., 1984). Although class I molecules expressed by an individual are invariant, the alleles of each gene which encode class I molecules are highly polymorphic such that most species possess a large number of antigenically diverse class I alloantigens (Duncan et al., 1979; Klein and Figueroa, 1981). The polymorphism found in class I molecules is not randomly distributed, but rather localized to the a1 and a 2 domains (K.imbal1 and Coligan, 1983). In contrast to the class I heavy chain, &-M is invariant within a species, although in the mouse limited polymorphism has been detected (see Section V). In the mouse, class I genes number between 23 and 37 (Steinmetz et al., 1982; Weis,s et al., 1984; Fisher et al., 1985). They can be divided into two groups: (1)H-2K, D, and L, and (2) genes in the QalTla region including Iimt (Fischer-Lindahl et al., 1983). H-2K, -D, and -L are target molecules for both alloreactive and antigen-specific H-%restricted CTL. These mole135
Copyright 6 I987 by Academic Press, Inc All nghts of reproduction In any form reserved
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JAMES FORMAN
cules are expressed on most cells in varying amounts, the highest density usually found on cells of lymphoid origin where as many as 5 X 105 molecules/cell of each antigen can be detected (Liberti et al., 1979). Qa-1, Qa-2, and Mta antigens serve as target structures for alloreactive CTL, but have not been demonstrated to restrict antigen-specific CTL (Kastner et al., 1979; Fischer-Lindahl et al., 1980; Forman et al., 1982). These latter molecules have a lowered level of cell membrane expression and a more limited tissue distribution than H-2K, D, or L (Flaherty, 1981), which may explain this discrepancy. Although class I molecules within a species are -80-90% homologous, the fact that T cells readily distinguish between class I alloantigens indicates that the immune system focuses on the polymorphic features of these molecules. In order to accomplish this, CTL utilize a and p chains of their T cell receptor to recognize antigen in the context of class I molecules (see reviews by Reinherz et al., 1984; Marrack and Kappler, 1986). Other molecules also play a role in CTL interactions with target cells including Lyt-2/CD8 (MacDonald et al., 1981; Swain, 1981; Meuer et al., 1982) and LFA molecules (Krensky et al., 1983, 1984). Helper T cells have been demonstrated to specifically recognize antigenic peptides in the context of class I1 molecules (Shimonkevitz et al., 1983). Townsend et al. (1986a) has shown that peptides of influenze virus nucleoprotein interact with the membrane of target cells to create determinants recognized by MHC-restricted anti-influenza CTL. Recently, it has TABLE I DOMAIN CIIAHACTEHISTICS OF CLASSI MOLECULES= Domain Characteristic Approximate size Homology with P2-M Homology with immunoglobulin Association with P2-M Phosphorylation site N-linked oligosaccharide H-2Kb mutation sites HLA CTL subtype variant sites CTL epitopes Serological epitopes
a1
a2
a3
TM
CYTO
90
90
90
24
30-39
+?
+?
+?
+b
+b
+ + + + +
+ + + +
+ +
+
-?
+
~~
See text for description and references. In man, only the a1 domain is glycosylated; in mouse, the a2 and in some molecules the a3 domains are also glycosylated. 0
MHC CLASS I MOLECULES RECOGNIZED BY CTL
137
3 are FIG.1. Schematic diagram of a class I MHC molecule, including P2-M. a l - 1 ~domains denoted by different patterns. Potential attachment sites for N-linked oligosaccharides are indicated. POi indicates region for phosphorylation, P indicates site for attachment to palmitic acid. Figure is drawn to indicate that most epitopes in the u l and a2 domains are controlled by the interaction of these domains with each other.
been reported that there is a weak interaction between specific peptides and class I1 molecules (Babbitt et al., 1985). Presumably, this peptide-class I1 molecular complex is recognized by the T cell receptor on helper T cells. Since CTL utilize the same receptors as helper T cells (Rupp et al.,1985; see review, Marrack and Kappler, 1986), it is likely that polymorphic regions of class I molecules not only intereact with CTL receptors, but that these molecules also interact with specific viral peptides. This review will examine the various approaches used to characterize determinants on class I molecules that are recognized by both alloreactive and antigen-specific class I-restricted CTL. Studies attempting to examine the nature of the determinants recognized by CTL have taken advantage of
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variants of normal class I molecules. These variants have arisen by (1)germ line mutation, (2) somatic cell mutation usually induced or detected in uitro, (3) site-specific mutagenesis of class I genes, and (4) exon shuffling of class I genes which results in the expression of domain-shuffled molecules in transfected cells. These topics as well as the role of carbohydrates and P2-M in controlling epitopes recognized by CTL will be discussed. II. Exon Shuffling to Produce Domain-Shuffled Molecules
A. TRANSFECTION OF CLASSI GENES cDNA clones have been isolated that encode class I MHC genes (Sood et al., 1981; Ploegh et al., 1981; Kvist et al., 1981; Steinmetz et al., 1981b). These cDNA clones were used to identify genomic clones, including H-2Kb (Mellor et al., 1982), H-2Ld (Evans et al., 1982a; Goodenow et al., 1982a), S L A d (Singer et al., 1982), and HLA-A2 and -B7 (Barbosa et al., 1983). To test the functional properties of these genes, genomic clones were introduced into murine L cells. The products of these genes were detectable on the L cell plasma membrane (Mellor et al., 1982; Barbosa et al., 1983; Evans et al., 1982b; Singer et al., 1982; Goodenow et al., 1982a). One assumption in using transfectants for functional assays is that the cell membrane product of the transfected gene is identical to the membrane product encoded by the endogenous gene. In general, most evidence supports this, especially when considering the transfection of murine class I genes into murine L cells. Thus, the size and charge heterogeneity of H-2Ld on L cells appear identical to H-2Ld on spleen cells (Goodenow et al., 1982a), the size of H-2Kb is similar on L cells as on EL-4 tumor cells (Mellor et al., 1982), and H-2Kd from spleen cells is similar to H-2Kd expressed on L cell transformants (Goodenow et al., 1982b). The results of serological studies are also consistent with the notion that class I-transfected molecules expressed as a result of transfection are similar to the same molecule expressed endogenously. Thus, transfected class I molecules only react with expected serological reagents (Evans et al., 1982b; Ozato et al., 1983a,b; Allen et al., 1984; Arnold et al., 1985). One exception to these similarities is the difference in glycosylation of the class I QlO molecules secreted by L cells transfected with the 010 gene compared to QlO found in serum (Devlin et al., 1985). However, it cannot be ascertained whether L cells glycosylate this molecule in a unique fashion as opposed to the possibility that serum QlO is altered while in the circulation. Transfection of human class I genes into murine cells could yield an abnormal molecule, since the P2-M is of murine origin. In some studies, human P2-M and human leukocyte antigen (HLA) have been introduced into mouse
MHC CLASS I MOLECULES RECOGNIZED BY CTL
139
L cells (van de Rijn et al., 1984; Bernabeu et al., 1984a; Barbosa et al., 1984). It should also be noted that there is a rapid exchange between the &-M in the medium and endogenously synthesized P2-M (see Section V). Nevertheless, the size and charge heterogeneity of HLA heavy chain in transfected L cells or human cells is similar to the endogenous products (van de Rijn et al., 1984; Bernabeu et al., 1983). While the antigen-specific T cell receptor recognizes products of class I genes, other molecules also play a role in T cell recognition, including Lyt-2/CD8 (MacDonald et al., 1981; Swain, 1981; Meuer et al., 1982) and LFA molecules (Krensky et al., 1983, 1984). It is likely that murine L cells, which are frequently used for transfection studies, do not express all the appropriate ligands for accessory molecules found on commonly used targets in CTL assays (tumor cells or lymphoblasts) (Golde et al., 1985; Naquet et al., 1985), nor is the efficiency of accessory molecule interactions between cells from different species understood (Spits et al., 1986). Therefore, caution must be taken in the interpretation of unexpected data (see Section 111). Orn et al. (1982), Mellor et al. (1982), Forman et al. (1983), Levy et al. (1983), Herman et al. (1983), Margulies et al. (1983), and Ozato et al. (1983a) demonstrated that L cells transfected with exogenous class I genes and expressing the corresponding gene product could be recognized by alloreactive and H-%restricted antigen-specific CTL. Epitopes recognized on the transfected class I molecules by CTL appeared to be similar to those recognized on normal cells, since the transfected antigen could competitively inhibit 1ySi:jdirected against the endogenously expressed antigen (Forman et al., 1983). The ability to exon shuffle genes allowed for the construction of hybrid molecules containing domains from different class I genes. Several approaches have been used, including (1)shuffling the 5’ end of one gene by splicing within the large intervening sequence between exons 3 and 4 with the remaining 3’ end of a second gene using conserved XbaI sites (Evans et al., 198213; Allen et al., 1984), (2) utilization of the conserved BamHI sites in the large intervening sequence and the 3’ end of the gene (Stroynowski et al., 1984), and (3)generation of partial deletions of class I genes followed by ligation of subclones of these truncated genes in the same vector (Arnold et al., 1984). A schematic diagram indicating some of the exon-shuffled class I genes that have been constructed is given in Fig. 2.
B.
EXON-SHUFFLED
GENESBETWEEN H-2Ld A N D H-2Dd 1 . Serology
Evans et al. (1982b) and Ozato et al. (1983a) exon shuffled H-2Ld and H-2Dd between the a 2 and a3 encoding exons so that the domain-shuffled
140
JAMES FORMAN
A. - '5
Es-
1
2
3
4
5 6 7 0 H
KHH,3'+HzNi
HHHJ-U-3'+HzN
a1 a2 a3TMCy I w n w COOH
r
m
FIG. 2. Exon shuffling of class I genes to produce domain-shuffled molecules. The left portion of the figure schematically indicates the exon-intron structure of a class 1 gene while the right portion depicts the protein structure of a class I molecule. Exon 1 encodes the leader; exons 2-4 encode the al-a3 domains, respectively; exon 5 encodes the transmembrane (TM) segment; and exons 6-8 encode the cytoplasmic (Cy)portion of the molecule. A and B represent two class I genes that have been spliced to produce constructs C-E (see text). The resultant molecules have homologous a1 and a2 domains and heterologous a3 domains (C and D) or heterologous a1 and a2 domains and homologous a2 and a3 domains (E and F). See Section I1 and Tables II-IV for explanation of results.
molecules expressed on transfected L cells consisted of Ld/Ld/Dd or Dd/Dd/Ld(referring to the origin of the a l / a 2 / a 3 domains). All monoclonal antibodies (mAbs) specific for H-2Ld or H-2Dd reacted with the domainshuffled antigens. Most mAbs appear to react with epitopes controlled by the a l / a 2 domains of these molecules, while only two mAbs, 28-14-8 and 34-2-12, reacted with the a3 domains of H-2Ld and H-2Dd, respectively. The fact that all mAbs tested retained activity against these domain-shuffled molecules suggests that most serological determinants are not controlled by the interaction of a3 with the a V a 2 domains. Further studies have involved exon shuffling of H-2Ld, H-2Dd, and Q7d (the 27.1 gene described by Steinmetz et al., 1981a) to produce nonhomologous a l - a 2 domain-shuffled molecules. Murre et al. (1984a) constructed a Dd/Ld/Ld molecule. Although the reciprocal Ld/Dd/Dd molecule was not available, they provisionally mapped mAb sites to the a1 and a 2 domains. For example, mAb 34-5-8 reacted with Dd/Dd/Ld, but poorly reacted with Dd/Ld/Ld, suggesting that this defines an epitope in either the a 2 domain of H-2Dd or a conformational determinant controlled by a l / a 2 . Other mAbs were provisionally mapped to the a1 or a 2 domains of H-2Ld or H-2Dd. McCluskey et al. (1986a)constructed the reciprocal domain-shuffled molecule (Ld/Dd/Dd)and was able to further localize monoclonal binding epitopes. Six monoclonal antibodies bound to a 2 of H-2Dd, 5 to a1 of -Dd,
MHC CLASS 1 MOLECULES RECOGNIZED BY CTL
141
and 4 to a1 of -Ld. The binding of 34-5-8 to these domain-shuffled molecules further illustrates the problem in definitively assigning epitopes to particular domains. 34-5-8 binds to Dd/Dd/Dd and Dd/Dd/Ld, but not Ld/Ld/Dd, mapping its reactivity to a determinant in the d / a 2 and not a3 domain of H-2Dd (Evans et al., 1982b). The antibody binds about half as well to Dd/Ld/Ld and Ld/Dd/Dd, suggesting that it interacts with a site controlled by both the a l / a 2 domains or similar sites in a1 and a 2 of H-2Dd. However, the Dd/Q7d/Dd molecule does not bind this antibody (Stroynowski et al., 1985a). Thus, the a 2 domain of H-2Ld, but not Q7d, permits the expression of the 34-5-8 epitope. A similar observation was made by Darsley et al. (1987), who noted that several mAb which were assigned a1 domain reactivity with H-2Dd based on H-2Dd-H-2Ld domain shuffled molecules (Ozato et al., 1985) did not react with the a1 domain of -Dd when this molecule was shuffled with H-2Dp. Therefore, many mAb-defined epitopes currently mapped to a particular domain may later be found to be indirectly controlled by another domain as further domain-shuffled molecules are tested. McCluskey et al. (1986a) noted that molecules shuffled between the a 2 and a3 domains bound mAb at roughly equivalent amounts to the homologous molecules. This was not the case when the a l / a 2 domains were exchanged. Thus, Dd/Ld/Ld molecules bound mAb somewhat less than Dd/Dd/Dd or Ld/Ld/Ld and Ld/Dd/Dd molecules bound mAbs much less than Dd/Dd/Dd or Ld/Ld/Ld. Therefore, constructing molecules from nonhomologous a l / a 2 domains may grossly alter the epitope structure of these antigens.
2 . Cytolytic T Lymphocyte Recognition Structural data indicate that most of the polymorphism found in class I molecules is in the a1 and a 2 domains. Further, studies with H-2 mutants and HLA subtypes (see Section VI1,A) have revealed that point mutation or minor variation in either the a1 or a 2 domain leads to strong alloreactivity. Therefore, it might be expected that the a1 and a2, but not a3 domains control determinants recognized by CTL. Ozato et al. (1983a) used alloreactive anti-H-2Ld CTL to demonstrate that both Ld/Lcl/Ldand Ld/Ld/Dd molecules were recognized effectively. Similar results were seen with anti-H-2Dd CTL and Dd/Dd/Dd and Dd/Dd/Ld molecules. Further evidence indicating that the a3 domain does not control polymorphic determinants recognized by alloreactive CTL was shown by the finding that domain-shuffled molecules expressed on unlabeled cells could block the 1ysis directed against labeled targets expressing the homologous molecules (Ozato et al., 1983a; Stroynowski et al., 1984). McCluskey et al.
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(198613) also noted that cells expressing the a3 domain only of H-2Ld or H-2Dd were not recognized by alloreactive CTL (see Table 11). Antigen-specific CTL restricted by H-2Dd or -Ld were able to interact with domain-shuffled molecules. Thus, antihapten (Ozato et al., 198310) and antiviral CTL (Reiss et al., 1983; Stroynowski et al., 1984) restricted to either H-2Dd or -Ld interacted with Dd/Dd/Ld or Ld/Ld/Dd, respectively, but not the reverse. Unlabeled cells expressing domain-shuffled molecules could block the lysis by antivirus-specific CTL of infected target cells expressing the homologous molecule, demonstrating that few, if any, polymorphic restricting determinants are controlled by the a3 domain (Stroynowski et al., 1984). Levy et al. (1985) demonstrated that both alloreactive and haptenrestricted CTL clones recognize epitopes controlled by the a l / a 2 domains irrespective of whether the a3 domain was from H-2Ld or -Dd. Engelhard et al. (1985) exon shuffled HLA-B7 with H-2Ld and -Dd and showed that the presence of a murine-derived a3 domain had no effect on the recognition of polymorphic HLA determinants in the domains. However, Maziarz et al. (1985) observed that both human ani-HLA-A2 and murine anti-H-2Kb CTL were less efficient at lysing human/mouse and mouse/human hybrid molecules, respectively, than the appropriate homologous molecule. Since the a3 domain of human and mouse class I molecules is -70% homologous in amino acid sequence, it is possible that this domain can affect the conformation of d / a 2 epitopes, or alternatively, the a3 domain could serve as a ligand for an accessory molecule that functions inefficiently between species. Since certain point mutations or minor variations in class I molecules induce strong alloreactive responses (Pimsler and Forman, 1980), it might be expected that class I molecules shuffled between the a1 and a 2 domains would affect most of the CTL epitopes. However, alloreactive bulk CTL and CTL clones specific for H-2Dd and H-2Ld recognize a Dd/Ld/Ld molecule (Murre et al., 1984a; Reiss et al., 1986). H-2Dd- and H-2Ld-restricted antiinfluenza virus-specific CTL also showed lytic activity against this target molecule, but anti-vesicular stomatitis virus (VSV) CTL, which are H-2Ld restricted, did not interact with Dd/Ld/Ld VSV-infected targets (Murre et al., 1984a). In contrast to alloreactive and H-2-restricted recognition of Dd/Ld/Ld, McCluskey et al. (1986a) tested the reciprocal exon-shuffled gene (Ld/Dd/Dd)and found that both alloreactive and antigen-specific anti-VSV CTL failed to react with this molecule. While some anti-H-2Ld and -Dd CTL clones reacted with Dd/Ld/Ld, none reacted with Ld/Dd/Dd. Mouse strain B10. D2-H-2dm1 is a meiotic H-2Dd/Ld mutant resulting from the fusion of the H-2Dd and -Ld genes (Sun et al., 1985). Thus, this fusion molecule represents a “natural” domain-shuffled molecule. The fusion occurred between residues 122 and 155, resulting in the a1 and most of the a 2 domain derived from H-2Dd, while the C-terminal amino acids of the
TABLE I1 EFFECTOF SHUFFLING THE al-a3 DOMAINS OF H-2Dd Derivation of domains
AND
H-2Ld
Alloreactive CTL
Constructh
a1
a2
a3
Anti-H-2Dd
Anti-H-2L"
2C/D 2C/D 2E/F 2EIF
Dd Ld Dd Ld Dd Dd
Dd Ld L'I Dd Q7d Dd/Ld
Ld Dd Ld D" Dd LdY
+'
-
Fig. Fig. Fig. Fig.
-
+
+ +
ON
CTL RECOGNITION^
H-%restricted CTL H-2Dd
H-ZLd
Referencef
+ +
-
1 2 3 4 5 6
+ +
-
-
-
-d
-
+
+
ND
-
-
ND
-
a CTL were generated against H-2Dd, H-2Ld, or antigen (hapten or virus) restricted by H-2Dd or H-2Ld and tested against the domain-shuffled molecules expressed on L cells. See schematic diagram in Fig. 2 for construction of exon-shuffled genes. c indicates cross-reaction of CTL on domain-shuffled molecule, - indicates lack of cross-reaction. CTL were generated against Q7 determinants rather than H-2Ld. p This construct is the H-2dm' molecule (see text for explanation). f References: (l),Reiss et al. (1983), Ozato et al. (1983a,b), Levy et al. (1985), (2), Ozato et al. (1983a,b), Levy e t a / . (1985), Stroynowski et al. (1984), (3), McCluskey et al. (1986a), Reiss et a / . (1986), (4), McCluskey et al. (1986a), (5), Stroynowski et a / . (1985), (6), Burnside et al. (1984), Forman and Klein (1975a), Ciavarra and Forman (1982), Forman (unpublished data).
+
144
J A M E S F ORMAN
a 2 domain are derived from -Ld. Previous studies (Forman and Klein, 1975a; Burnside et al., 1984; Forman, unpublished data) have noted that anti-H-2Dd and -Ld CTL cross-react on H-2dm1target cells and therefore are consistent with the studies of Murre et al. (1984a) showing that H-2Dd/Ld molecules retain alloreactive determinants characteristic of both parents. HLA-Aw69 is another natural domain-shuffled molecule consisting of HLA-Aw68 sequences in the a1 domain and HLA-A2 sequences in the a 2 and a3 domains (Holmes and Parham, 1985). Most CTL clones generated against HLA-AS, -Aw68, and -Aw69 fail to recognize determinants localized to either the a1 or a 2 domain only, but rather recognize epitopes requiring the presence of both a1 and a 2 (Clayberger et al., 1985). However, Wallace et al. (1986) found that anti-Epstein-Barr virus (EBV) -specific HLA-Aw69restricted CTL cross-react on HLA-AS. This finding is similar to that of the Dd/Ld/Ld construct which retained H-2Dd-restricting epitopes for anti-influenza CTL. Stroynowski et al. (1985a) produced a domain-shuffled molecule that contained H-2Dd in the a1 domain, Q7d in the a 2 domain, and -Dd in the a3 domain (Dd/Q7d/Dd).This molecule is not recognized by either anti-H-2Dd, anti-Q7d, or antigen-specific H-2Dd-restricted CTL. Thus, in contrast to the study of Murre et al. (1984a), changing the a 2 domain of the H-2Dd molecule to Q7d, as opposed to -Ld, completely alters the alloreactive and restricting specificity of this molecule. C. EXON-SHUFFLED GENESBETWEEN H-2Kb AND H - 2 D b Allen et al. (1984) constructed hybrid class I genes between H-2Kb and H - 2 D b . These hybrids consisted of Db/Kb/Kb, Db/Db/Kb, Kb/Db/Db, and Kb/Kb/Db. Of 17 antibodies tested, only one (28-14-8) reacted with the a3 domain of H-2Db. This result is not surprising, since 28-14-8 reacts with the a3 domain of H-2Ld, a molecule that has an identical sequence to H-2Db in the a3 domain (Kimball and Coligan, 1983). This antibody bound to H-2Db expressed on transfected L cells to the same extent as the Kb/Db/Db construct, indicating that molecules with nonhomologous a l / a 2 domains (between H-2Kb and -Db) do not necessarily have a reduced cell membrane expression. Although most antibodies were shown to interact with determinants which map to the a1 and a2 domains, their binding was greatly reduced compared to the binding to homologous molecules. Five antibodies lost reactivity with the a U a 2 domain-shuffled molecules. Similar findings were reported by Bluestone et al. (1985). Alloreactive anti-H-2Kb CTL recognized Kb/Kb/Kb and Kb/Kb/Db constructs, but not K ~ / D ~ or / D Db/Kb/Kb. ~ Analogous results were noted with anti-H-2Db CTL (see Table 111). H-2Kb- and -Db-restricted influenza virus-
145
MHC CLASS I MOLECULES RECOGNIZED BY CTL
Derivation of domains Construct”
a1
a2
Alloreactive CTL a3
AntiH-2KI)
AntiH-ZDI)
H-2-resticted CTI,
H-2Ki)
H-2D1>
Data taken from Allen et u1. (1984)and Bluestone et a2. (1985).CTL were generated against H-2K‘) or Hl-2DlJ, or antigen (virus) restricted I)y H-ZKI’ or H-211’) and tested against the domain-shuffled molecules expressed on L cells. b.r See Ta.ble I1 for explanation.
specific C‘TL and CTL clones showed similar patterns of reactivity (Allen et al., 1984). Thus, these results are similar to those noted with the Ld/Dd/Dd and Dd/Q7d/Dd constructs (McCluskey et a l . , 1986a; Stroynowski et al., 1985a) where nonhomologous a l / a 2 domain-shuffled molecules lost all detectable CTL-controlled epitopes. Mouse strains bearing a point mutation in either the a1 or a 2 domains of H-2Kh (H-2Kbmmutant strains, see Section VII,A) were used to generate CTL against H-2Kb. Since an H-2Khm mutant strain differs with respect to H-2Kb in either the a1 or a 2 domain only, these alloreactive CTL were tested to determine if the epitopes they recognized could be localized to that particular domain. Such CTL failed to recognize any molecules shuffled between the a1 and a2 domains of H-2Kh, indicating that both a1 and a2 domains control epitopes recognized by CTL. In a secondary response, (C3H x C57BL)F, mice generated CTL that were Dh/Kh/Ktl specific. However, it was not ruled out that these CTL recognize this hybrid molecule in an H-2-restricted rather than in an unrestricted fashion. D. EXON-SHUFFLED GENESBETWEEN H-2Kd, H-2Kk, A N D H-2Kb Arnold et al. (1984, 1985) constructed hybrid genes using the H-2K alleles,
k, d, and b. These hybrid genes were transfected into IT22-6 fibroblasts or the IC9 fibrosarcoma. Two antibodies reacted with the a3 domain of H-2Kk and one with the a3 domain of -Kd. Most antibodies appeared to be specific for determinants requiring homologous a l / a 2 domains. While some mAbs showed reactivity with the a1 domain on H-2Kk or -Kd, none was reported
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JAMES FORMAN
TABLE IV EFFECTOF SHUFFLINGTHE al-a3 DOMAINS OF H - 2 K d RECOGNITION" Derivation of domains
cxl
a2
Fig. 2C/D Fig. 2E/F Fig, 2C/D Fig. E/F NAe NA NA
Kd Kd Kk Kk Kk Kk Kd
Kd Kk Kk Kd Kk-Kdf Kk-Kb Kd-Kk
H-2Kk
ON
CTL
Alloreactive CTL Anti-
Constructb
AND
a3
Anti-
H-2Kd
H-2Kk
Kk Kk
+'
-
?(5/41)d
*(1/46)
Kd
-
Kd K" or Kk Kk K d o r Kk
-C(6/30)
-
+ -(0/30) + +
-
-
+
H-%restrictedCTL
H-2Kd
+ -
ND
ND ND ND ND
0 Data taken from Arnold et al. (1984, 1985) and Schder et al. (1986). CTL were generated against H-2Kk or H-2Kd or virus restricted by H-ZKd and tested against the domain-shuffled molecules expressed on fibroblasts. b,c See table I1 for explanation. d Number of CTL clones reactive/total number tested. e Not applicable, f Molecule shuffled at residue 142 in a2 domain (Scheller et al., 1986).
to react with the a 2 domain of either of these molecules. Antibodies that reacted with the a1 domain of H-2Kd or -Kk on domain-shuffled molecules showed decreased binding compared to binding to the homologous antigen. Bulk cultured anti-H-2Kk and -Kd CTL recognized Kk/Kk/Kd and Kd/ Kd/Kk molecules, respectively (see Table IV). No recognition was detected when the antigens were shuffled between the a1 and a 2 domains (Arnold et al., 1985). The authors extended these studies by examining CTL clones generated in limiting dilution. Of 76 anti-H-2Kk CTL clones, one was reported to recognize Kd/Kk/Kk. Of 71 anti-H-2Kd CTL clones, 11 recognized either Kd/Kk/Kkor Kk/Kd/Kd.Thus, a small minority of CTL clones generated in alloreactions can recognize determinants expressed on nonhomologous a l / a 2 molecules. H-2Kd-restricted anti-influenza CTL recognized Kd/Kd/Kk, but not Kd/Kk/Kk(Arnold et al., 1984). Domain-shuffled molecules with nonhomologous TM or cytoplasmic domains were efficiently recognized by CTL specific for the a l l a 2 domains. Scheller et al. (1986) shuffled H-2Kd, -Kk,and -Kb genes within the a 2 domain encoding exon to produce molecules with heterologous sequences between positions 142 and 182 of the a 2 domain. Bulk cultured anti-H-2Kk and anti-H-2Kd CTL readily reacted with Kk/Kk-Kd/Kk and Kk/Kk-Kd/Kd (refers to al/a2[91-141]a2[ 142-182]/a3) domain shuffled molecules but not Kd/Kd-Kk/Kd or
MHC CLASS I MOLECULES RECOGNIZED BY CTL
147
Kd/Kd-Kk/Kk. The data above (Arnold et al., 1985)indicate that most antiH-2Kd clones do not react with Kk/Kd/Kd.Thus, it is likely that the carboxyterminal end of the a 2 domain of H-2Kd, when shuffled with H-2Kk, can retain a conformation allowing for the expression of H-2Kd CTL epitopes while the complete a 2 domain of H-2Kd cannot. Since most a l / a 2 domain-shuffled molecules have lost most of the epitopes recognized by CTL, it may be expected that these molecules would acquire new epitopes unique to their structure. Support for this was provided by Horstmann et d . (1986) who studied Kd/Kk/Kk and Kk/Kd/Kd constructs and found that both were specifically recognized in secondary responses by anti-Kd/Kk/Kkand anti-Kk/Kd/KdCTL, respectively. The lysis was H-2 unrestricted. Further, these CTL did not cross-react on either native H-2:Kd or H-2Kk.
E. EXON-SHUFFLED GENESBETWEEN ZAPk AND H-2Dd OR H-2Ld McCluskey et al. (1985) exon shuffled the leader (L) and p l exons of IAkp with the a3, TM, and cytoplasmic exons of H-2Dd and transfected the hybrid gene into 1, cells. A domain-shuffled molecule was expressed on the cell membrane which associated weakly with P2-M microglobulin (McCluskey et al., 1985). This molecule was recognized by anti-IAk CTL lines generated by multiple rounds of in uitro stimulation (Golding et al., 1985). The lysis was partially blocked with the 10-2-16 mAb (anti-IAg,), further substantiating that at least a portion of anti-IAk9 CTL activity is directed against epitopes in the p l domain of IAk. An additional line that was L3T4+ had anti-IAkp, specificity, and the lytic activity of this line was blocked with anti-L3T4 antibody. These data demonstrate that at least some of the epitopes in IA molecules that are recognized by CTL do not require the presence of the a chain or the p2 domain of the p chain. Further, these data suggest that if the L3T4 molecule has a ligand, it need not be in the p2 domain of IAb or the a chain. McCluskey et al. (1986b) deleted the p l exon from the above-described exon-shuffled gene to derive a construct with the L exon from IApkjoined to the a3, TM, and cytoplasmic exons of H-2Dd. A similar construct was also prepared with the 013, TM, and cytoplasmic exons derived from H-2Ld. Both of these shuffled genes would be expected to have their first 4 amino acids contributed by the L exon of IAb. A third mutant gene was also constructed containing the L exon of H-2Dd together with the a3, TM, and cytoplasmic exons from the same genes (the a1 and a 2 exons were excised). Alloreactive anti-H-2Ld or -Dd CTL did not recognize L cells expressing these gene products, consistent with previous findings demonstrating the specificity of alloreactive CTL for the a1 and a 2 domains (see Sections 11,B-11,D). On the other hand, these constructs were recognized if mice were previously
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primed in vivo with the L / a 3 constructs. These a3 domain-specific CTL did not recognize the a3 domain on intact H-2Ld or H-2Dd. It is possible that the recognition of the a3 domain is H-2 restricted by H-2k molecules on the L cells. The lysis did not appear to depend on the amino terminal amino acids controlled by the L exon. F. EXON-SHUFFLED GENESWITH ALTEREDCYTOPLASMIC REGIONS Class I molecules have been constructed with altered cytoplasmic domains. Zuniga et al. (1983) generated two mutant H-2Ld molecules, one containing 7 cytoplasmic amino acids and the other with 25 amino acids, including 19 random amino acids at the C-terminus. Murre et al. (198413) substituted the 3 cytoplasmic domain-encoding exons of H-2Ld for the second cytoplasmic exon of Z-Apd. The resulting protein contained 10 instead of the 31 cytoplasmic amino acids found in the normal H-2Ld product. In both studies, the truncated molecules were expressed efficiently on the surface of transfected L cells. Zuniga and Hood (1986) generated several additional mutants of class I H-2Ld molecules, including some with no cytoplasmic tail, a 4 amino acid tail, and one with a shortened TM domain and a 2 amino acid tail. All of these latter mutants had a reduced cell membrane expression. The constructs produced by both laboratories were recognized by both alloreactive and H-2-restricted CTL. However, Murre et al. (1984b) noted that VSV-specific CTL lysed cells expressing truncated H-2Ld less efficiently than the wild-type molecule. No difference was seen with anti-influenza CTL. In contrast, Zuniga et al. (1983) and Zuniga and Forman (unpublished observations) noted no difference in the lytic activity of anti-VSV CTL directed against any of the truncated H-2Ld versus normal -Ld molecules. Arnold et al. (1984) exon shuffled H-2Kd, -Kk and -Kb genes between the TM and first cytoplasmic exons. No effect on CTL lytic activity was noted. The cytoplasmic domains of class I molecules contain a highly conserved tyrosine at residue 321 and serine at 335, which can be phosphorylated (Pober et al., 1978; Guild et al., 1983). Some of these molecules are also covalently linked to palmitic acid via a cysteine encoded by the TM exon (Kaufman et al., 1984). Analysis of cDNA clones of H-2Kq (Kress et al., 1983) and H-2Dd (Brickell et al., 1983, 1985)indicate that these genes may undergo differential mRNA processing at the 3’ end. McCluskey et al. (198613) noted that an H-2Dd gene construct could give rise to two proteins, one of which represented a deletion of the peptide encoded by exon 7 . Taken together, these findings suggest that this region of the molecule plays an important functional role. However Kranz et al. (1984) and Staerz et al. (1985) have shown that class I molecules may merely play a passive role with respect to recognition and lysis by CTL effector cells. Thus, H-2- target cells bearing an anti-idiotype mAb coupled to their membrane can trigger
MHC CLASS I MOLECULES RECOGNIZED BY CTL
149
CTL clones with the corresponding idiotype to cause their specific lysis. Therefore, the presence of a class I molecule on the target cell membrane is not required for lysis, but rather activates the CTL through binding to its receptor molecules. If the cytoplasmic domain does play a functional role for CTL activity, then it must be at some point earlier in the generation of CTL from their precursors. Ill. Recognition of HLA Class I Antigens on Transfected Cells
LeBoutdler et al. (1982), Lemonnier et al. (1982, 1983a), and Barbosa et al. (1983)lransfected HLA class I genes into murine L cells. The expression was stable for several months. LeBouteiller et al. (1983, 1985) estimated that there were -4 x lo5 HLA molecules/cell, similar to the expression of endogenoiis H-2Kk molecules. One of the unexpected results from studies of HLA expressed on L cells is that in many cases these molecules are not recognized by specific anti-HLA CTL. Herman et al. (1983) transfected L cells with HLA-B7 and -A2 and used these cells as targets for specific mouse anti-HLA-B7 or -A2 CTL clones. Although some CTL clones lysed the L cell transformants, other clones had no activity. Bernabeau et al. (1983) noted that L cells expressing HLA-A2 or -B7 were not lysed using either bulk cultured or cloned human anti-HLA CTL. The L cells did not block specific lysis in a cold target inhibition assay. The ability to lyse HLA appears to be related to the species from which the cells used for transfection are derived. Thus, Barbosa et al. (1984) trarisfected HLA-A2 and -B7 into human cells; murine L, embryonic liver, and B lymphoma cells; and monkey kidney cells. Human anti-HLA CTL clones lysed human transformants, a minority of clones lysed monkey cells but to a lesser extent, and no lysis was observed against transformed mouse cells. These results were not due to a low level of expression of HLA on these cells. Although HLA on mouse cells is not readily recognized by anti-HLA CTL, H-2 antigens are readily detected on human cells by antiH-2 CTL (Maziarz et al., 1985). The failure of CTL to recognize HLA expressed on the surface of transfected murine cells could be due to a number of different factors, including (1)quantiiy of cell membrane HLA molecules, (2) qualitative character of HLA or P2-Mon cultured murine cells, or (3) the interaction of accessory molecules on CTL with ligands on the target cell. Quantitative expression of HLA on murine cells does not account for the lack of lysis, since there is relatively high expression of these molecules on L cells (LeBouteiller et al., 1983, 1985), and human cells transfected with the same HLA gene as L cells and expressing equivalent amounts of cell membrane antigen are readily lysed (Barbosa et al., 1984; Maziarz et al., 1985;
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JAMES FORMAN
Maryanski et al., 1986). There is no serological evidence to indicate that determinants on HLA molecules are altered relative to their endogenous expression on human cells, since mAb typing reagents react with both transfected and endogenous HLA (LeBouteiller et al., 1982, 1983; Bernabeu et al., 1983; Herman et al., 1983; Layet et al., 1984). However, since serological epitopes do not define CTL specificities (see Sections VII and VIII), this type of analysis may not be definitive. Since HLA molecules expressed on murine L cells would not be associated with human &-M, L cells expressing high levels of human P2-M were transfected with HLA (van de Rijn et al., 1984). However, these target cells were not susceptible to lysis either. The role of P2-M was further addressed by Bernabeu et al. (1984a)who examined L cells transfected with both HLA and human P2-M or L cells cultured in human serum. In the latter case, it was shown that serum P,-M rapidly interacts with membrane HLA through an exchange reaction (Bernabeu et al., 1984b). In neither instance were L cells lysed by anti-HLA CTL. Engelhard et al. (1985) and Maziarz et al. (1985) constructed hybrid molecules consisting of HLA in the 01l/a2 domains and H-2 in the 013 and carboxy-terminal domains. This did not alter the inability of anti-HLA CTL to lyse L cells transfected with HLA. No lysis was induced by the addition of lectin to mixtures of human effector cells and transformed L cells (Bernabeu et al., 1983, 1984a), although lysis was reported to have occurred when mouse effector cells were used. Since some accessory molecules may be required for lectin-induced lysis (Springer et al., 1982), it is possible that an interaction between intraspecies accessory molecules and their ligands is required for optimal recognition/lysis of anti-HLA effector cells and their targets. This is consistent with the findings of van de Rijn et al. (1984), who noted that an anti-HLA-A2 CTL clone that lysed human, but not mouse fibroblasts transfected with HLA-A2 was inhibited by antibodies against LFA-1 and CD8. Spits et al. (1986) showed that two human CTL clones could not form conjugates with murine L cells, but did with murine-derived P815 cells, suggesting that L cells lack some accessory molecules that permit effector-target cell interactions. Golde et al. (1985)and Naquet et al. (1985) found that T hybridoma recognition of antigen on class I1 gene-transfected L cells were not inhibited by anti-LFA-1 mAb. Cowan et al. (1985) transfected an HLA-A3 subtype gene from the E l donor (see Section VI1,B) into L cells and showed that human secondary and tertiary anti-HLA CTL lysed these target cells, even in the absence of human P2-M. Antibodies to CD3, CD8, and LFA-1 blocked lysis of both L cell transformants and human PHA blasts expressing the HLA-A3 variant molecule. These data suggest that if CD8 and LFA-1 interact with ligands, they can do so across the species barrier and thus would not account for the
MHC CLASS I MOLECULES RECOGNIZED BY CTL
151
lack of lysis observed in the other studies described above. Greenstein et al. (1986) also showed that Lyt-2+ mouse anti-human CTL could be blocked from lysing specific target cells with anti-LFA-1 antibodies, although the same cells could not be blocked with anti-Lyt-2 antibody. Barbosa et al. (1986) showed that murine L cells lack LFA-3, but that acquisition of this molecule through somatic cell hybridization did not restore recognition by CTL clones incapable of lysing HLA-transfected target cells. Mentzer et al. (1986) noted that most the human anti-HLA CTL clones did not recognize HLA expressed on murine cells, although 2 CTL clones could specifically lyse L cells transfected with HLA. Based on the results of inhibition of target cell lysis using anti-HLA mAb, these two clones appear to have a higher avidity for HLA compared to CTL clones unable to kill mouse transfectants. While these clones could lyse HLA expressing human and mouse cell targets, anti-LFA-1 mAb inhibited lysis to a greater extent when mouse rather than human targets were used. mAb to LFA-2 and -3 inhibited lysis against human, but not mouse targets. These data suggest that either murine cells do not use LFA-2 and -3 accessory molecules or that these receptor-ligand interactions do not occur between human and mouse cells, and as a result LFA-1 interactions become relatively more important for conjugate formation. It should be noted that the lack of lysis observed in the studies of Herman ef al. (1983), Bernabeu et al. (1984a), and Engelhard et al. (1985) involved anti-HLA CTL that were of murine origin where accessory molecules on niurine effector cells should be able to interact with ligands on murine L cell targets. However, some of these ligands may be lacking on murine fibroblasts (Shimonkevitz et al., 1985) and the xenogeneic anti-HLA CTL may be of low avidity and require these ligands for recognition. It is also possible that class I molecules interact with species-specific molecules, both of which control polymorphic epitopes recognized by alloreactive CTL (Matzinger and Bevan, 1977). In addition to the finding that some anti-HLA CTL clones can lyse HLAtransfected L cell targets (Herman et al., 1983; Maryanski et al., 1986), Maryanski et al. (1985) transfected HLA-Aw24 into murine P815 cells and showed that bulk cultured anti-Aw24 CTL readily lysed these cells. Achour et al. (1986) noted that anti-HLA CTL could be generated that recognized transfected L cells, albeit weakly, but that lysis directed against P815-transfected target was highly efficient. Gomard et al. (1986) transfected HLA-A2 into P815 cells and showed that anti-influenza-specific CTL restricted by HLA-A2 lysed infected targets. Since Spits et al. (1986) showed that P815 cells form conjugates with human CTL clones, it would appear that the appropriate ligands are expressed on these cells which can interact with human accessory molecules. Thus, present data suggest that most human anti-HLA CTL require and
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JAMES FORMAN
participation of accessory molecules, possibly including LFA-2 (CD2) and LFA-3, for efficient recognition of HLA by the T cell receptor. Whether murine fibroblast targets express all of these molecules or their ligands is not known, nor is it known whether accessory molecule interactions occur between species. Further, there may be heterogeneity between L cell lines in regard to the expression of accessory molecules, since Mentzer et al. (1986) could block recognition of antigen on L cells with the anti-LFA-1, whereas Golde et al. (1985) could not. Xenogeneic murine anti-HLA CTL responses may be of low affinity and require an accessory molecule interaction that may not occur with fibroblast targets. Achour et al. (1986), Maryanski et al. (1986a), and Holterman and Engelhard (1986) noted that mice primed in uiuo with murine cells transfected with HLA allowed for the generation in uitro of H-%restricted anti-HLA CTL. The results are similar to those for CTL generated against minor H antigens, although in this case the minor H antigen is HLA. Thus, it appears that presentation of xenogeneic class I molecules on xenogeneic cells gives rise to an MHC-unrestricted anti-class I response, while presentation of the same molecules on cells from the same species elicits an MHC-restricted antigen-specific response. Further, in the latter case the recognition of HLA is similar to that described for the nucleoprotein of influenza in that an HLA peptide from HLA-CW3 spanning amino acid residues 171-186 sensitized H-2d target cells to the lytic effects of H-2Kd-restricted DBAIB anti-HLA CTL (Maryanski et al., 1986b). IV. Role of Carbohydrate Moieties in Determining CTL Recognition of Class I Molecules
The heavy chain of class I MHC molecules contains N-linked oligosaccharides attached to the al, the a1 and a2, or the al, a2, and a3 domains. Doberstein et al. (1979) showed that glycosylation of class I MHC molecules is similar to that described for other viral and cell membrane glycoproteins. Class I molecules from all species have a conserved asparagine at residue 86 to which a glycosyl group is attached. In the mouse, an asparagine at 176 also serves as an attachment site for glycans, and in H-2Ld, H-2Db, and H-2Kd molecules there is a third asparagine acting as an attachment site at residue 256 (Kimball and Coligan, 1983). Oligosaccharides have been shown to play a role in the transport of class I molecules to the cell membrane (Ploegh et al., 1981; Landolfi et al., 1985; Miyazaki et al., 1986). For class I molecules, glycans could also (1)comprise a portion of an epitope recognized by CTL, (2) control the conformation of class I epitopes, or (3)affect the interaction of viral peptides with class I molecules. These issues have been approached with drugs that inhibit glycosylation of
MHC CLASS I MOLECULES RECOGNIZED BY CTL
153
proteins. Drugs frequently used are tunicamycin (Tm), which inhibits the attachment of N-acetylglucosamine to dolichol phosphates and thus prevents N-linked oligosaccharide linkage (Tkacz and Lampen, 1975), and 2-deoxy-~glucose (2-DOG), which at low concentrations inhibits glycosylation (reviewed in Scholtissek, 1975). The effect of Tm on the expression of class I molecules varies. Swaminathan and Gooding (1983) removed H-2Kk molecules from C3H cells with papain and showed that their reappearance was prevented by the presence of Tm. Colombo et al. (1983) showed that H-2b antigens were reduced on cells cultured in Tm, although H-2Kb was reduced less than -Db. They also provided evidence that nonglycosylated H-2Kb molecules were present on the membrane of Tm-cultured cells. Carter et al. (1981) did not quantitate H-2 on Tm or 2-DOG cultured cells, but noted that subconfluent SV40transformed C57BL/6 embryo fibroblasts had a reduced sensitivity to lysis mediated hy anti-H-2b CTL. Black et al. (1981) found that P815 cells cultured with Tm for 18 hours expressed normal amounts of cell membrane H-2Dd, but in a nonglycosylated form. Miyazaki et al. (1986) used sitespecific mutagenesis to change the consensus sequences of Asn-X-Ser/Thr by altering the asparagines at position 86, 86 and 176, or 86, 176, or 256. The result was the production of mutant H-2Ld genes which, when transfected into L cells, encoded proteins devoid of oligosaccharides in the al,a1 and a2, or al- a3 domains, respectively. While mutant molecules lacking a glycan in the a1 or a1 and a 2 domains expressed equivalent amounts of H-2 on the cell membrane as native H-2Ld, mutant molecules lacking carbohydrate in all 3 domains were poorly expressed at the plasma membrane. The low level of surface expression of this latter mutant was not due to accelerated degradation or increased shedding of the molecule, but rather to a decrease in intracellular transport. Ploegh et al. (1981) cultured JY cells with Tm and showed that nonglycosylated HLA-A and -B antigens reached the surface of these cells at rates indistinguishable from normal HLA antigens, although the amount of HLA synthesized in such cells was decreased. In general, CTL functional data parallel the effect of Tm on the cell membrane expression of class I molecules. Thus, antiviral-specific CTL directed against SV40 (Swaminathan and Gooding, 1983), herpes simplex virus (HSV) type 1 (Carter et al., 1981), VSV (Black et al., 1981), and Moloney sarcoma-leukemia virus (Colombo et al., 1983; Watson and Bach, 1980) all showed reduced amounts of lysis when the target cells were cultured with Tm or 2-DOG. In contrast, the extent of lysis directed against H-2 by alloreactive T cells was not altered (Watson and Bach, 1980; Colombo et al., 1983) except in one study (Swaminathan and Gooding, 1983).Thus, the lack of lysis in the antigen-specific systems has been suggested to be due to alteration of viral proteins such that these unglycosylated molecules are
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J A M E S FORMAN
either not expressed on the cell membrane or not recognized by CTL. Black et al. (1981) demonstrated that unglycosylated VSV G protein is expressed on the cell membrane of Tm-cultured P815 cells. Recent data have indicated that at least a portion of antiviral CTL reactivity is directed against nonglycosylated viral proteins (Townsend et al., 1984; Abastado et al., 1985). Therefore the effect of Tm in decreasing antigen-specific CTL-mediated lysis could be explained by altering the expression of ligands on target cells that bind accessory molecules on cytolytic T cells; e.g., Lyt-2, LFA molecules. Pimlott and Miller (1986) noted that anti-H-2 conjugate formation was specifically inhibited by glycopeptides extracted from tumor cells. However, these glycopeptides weakly inhibited target cell lysis, and it is not known if these molecules are derived from class I antigens. Jenkins et al. (1985) showed that some of the epitopes on the Qa-1 molecule that are recognized by alloreactive anti-Qa-1 CTL clones are lost with Tm treatment. Since Tm and 2-DOG can have pleiotropic effects (MacDonald and Cerottini, 1979), clearer studies have been performed by Miyazaki et al. (1986) who employed site-specific mutagenesis of class I genes, and Goldstein and Mescher (1986) who prepared liposomes containing deglycosylated H-2Kk molecules. Goldstein and Mescher (1986) showed that the liposomes containing deglycosylated H-2Kk were able to induce alloreactive CTL to the same extent as liposomes with native H-2Kk. Miyazaki et al. (1986) showed that mutant H-2Ld molecules that lacked carbohydrate attachment sites in the a l , a1 and 012 or al-a3 domains, when expressed on the surface of L cells, functioned as target molecules for both alloreactive and VSV-specific CTL. In addition, five out of five alloreactive anti-H-2Ld CTL clones all retained reactivity against the mutant molecules. Thus, it is unlikely that carbohydrates on class I molecules play any more than a minor role in contributing to antigenic determinants recognized by alloreactive or H-%restricted CTL. V. Role of &-Microglobulin in T Cell Recognition of Class I Molecules
P2-M noncovalently associates with the heavy chain of class I molecules (Nakamuro et al., 1973; Natori et al., 1974; Rask et al.,1974; Silver and Hood, 1974;Vitetta et al., 1975). This molecule has a molecular weight of 12,000 and is usually invariant within a species, with the exception of mice where 7 allelic forms have been detected (Michaelson et al., 1980; Gates et al., 1981; Robinson et al., 1984; Gasser et al., 1985; K. Fischer-Lindahl, personal communication). The amino acid sequence homology between human and murine P2-M is -70%, between murine and bovine 68%, and between human and bovine 76% (Gates et al., 1981). Yokoyama and Nathenson (1983) digested H-2 with glycosidases and proteolytic enzymes and found that
MHC CLASS I MOLECULES RECOGNIZED BY CTL
155
fragments associated with the P2-M subunit were derived from the a3 domain, suggesting that the a3 domain predominantly interacts with P,-M. However, the binding of the W6/32 mAb, which reacts with a determinant localized to the a l / a 2 domain of HLA (Maziarz et al., 1985), is greatly reduced if the P2-M is of murine rather than human origin (Ferrier et al., 1985). Further, the a l / a 2 domains of HLA-AS, but not -Cw3, can alter epitopes 011 murine P2-M (Jordan et al., 1983). This indicates that P2-M can interact or affect the conformation of d / a 2 domains of class I molecules. Similarly, McCluskey et al. (1986a) noted that the association of P2-M with H-2 was affected by the a1 domain ofclass I molecules, further supporting the idea that P2-M interacts with heavy chains at more than just the a3 domain. The interaction of @,-M with the heavy chain of class I molecules results in an alteration of the conformation of P2-M. Lemonnier et al. (1983b) and Agthoven et al. (1984) noted that a murine anti-human P2-M mAb reacted with murine P2-M associated with HLA. Apparently the murine P2-M requires a conformational determinant that resembles a human p,-M epitope when the murine P2-M interacts with a human class I molecule. P2-M from serum can readily exchange with endogenous P,-M on the cell membrane. Bernabeu et al. (198413) noted that human cells grown in fetal bovine serum (FBS) had both human and bovine P2-M associated with T6 and HLA-MB. H-2KID antigens exchange murine for bovine @,-M with a t,,, of -2 hours. HLA-B7 expressed on JY cells cultured in FBS associates predominantly with human P2-M, whereas HLA-B7 expressed on murine L cells associates predominantly with bovine P2-M. Thus, the ability of one species of pz-M to exchange with another appears to depend on the source of the endogenous &-M. Kubota (1984) noted that P2-M from FBS exchanged with murine P,-M on L cells and that the acceptor molecules for the exchange were the heavy chain of class I molecules. The association of P,-M with class I molecules could have an effect on the structure of class I molecules themselves. Thus, most anti-HLA antisera do not react with isolated heavy chains (Krangel et al., 1979) and HLA loses some of its @-pleatedsheet structure in the absence of P2-M (Lancet et al., 1979). P,-M may also be required for the transport of class I molecules to the cell membrane. Thus, P,-M-negative Daudi cells make cytoplasmic HLA, but do not transport it to the cell membrane (Ploegh et al., 1979; d e Preval and Mach, 1983). Similar findings have been described for the P,-M- Rl(TL-) mutant cell line (Hyman and Stallings, 1977) which also fails to express membrane H-2. There is little information as to whether P2-M plays a role in controlling epitopes recognized by alloreactive CTL. Although there is only -70% homology of P,-M sequences between species (Gates et al., 1981), Cowan et
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JAMES FORMAN
al. (1985)noted that anti-HLA CTL could lyse murine cells expressing transfected HLA in the absence of a source of human P2-M. It is not known whether a large panel of CTL clones would be able to distinguish class I molecules associated with different species of P2-M microglobulin. An mAb directed against P2-M blocks about 40% of alloreactive CTL activity, while blocking of H-2Db-restricted anti-H-Y-specific CTL was only inhibited -20% (Tomonari et al., 1982). The blocking could be due to steric factors or reflect poor inhibition by a low-affinity antibody. Langhorne and FischerLindahl (1982) failed to detect any alloreactive CTL clones that distinguish between the two murine alleles of P2-M. However, Rammensee et al. (1986) generated a CTL line across the H-3 barrier which includes the P2-M locus. This CTL line was specific for the b allelic form of P2-M and was restricted by H-2Kb. Target cells from &-Ma strains incubated with purified P2-Mb acquired the target antigen. Thus, in this case specific reactivity against a minor H antigen was shown to be directed against a determinant dependent on a P2-M alloantigen. VI. Use of Monoclonal Antibodies to Block CTL Recognition of Class I Molecules
Fischer-Lindahl and Lemke (1979) demonstrated that anti-H-2Kb mAbs could block alloreactive and H-2-Kb-restricted CTL. One antibody, 27R9, was the least effective at blocking, but the other two mAbs blocked lysis almost completely. Epstein et al. (1980) tested eight anti-H-2k mAbs, all of which were able to block CTL activity. In general, the affinity of the mAb for the H-2 antigen correlated with its blocking activity. The use of mAbs directed against particular domains of class I molecules may allow an assignment of CTL reactivity to epitopes localized to one of the three external domains. Hammerling et al. (1982) described a series of mAbs directed against H-2Kb and further defined their specificity based on their reactivity with H-2Kbm mutant strains. Two antigenic clusters were identified, one localized to the a 2 domain based on the fact that the antibodies do not react with the H-2Kbm1 and Kbm4 molecules, which are a 2 domain mutants (see Section VI1,A). The other cluster presumably reacts with the a1 domain, since these antibodies do not react with the a1 mutant, H-2Kbm3. Several of these antibodies were also later described by Allen et al. (1984)and Bluestone et al. (1985) using the technique of exon shuffling. The cluster of antibodies defined by Hammerling et al. (1982) as reacting with the a 2 domain was shown to have reactivity with the a 2 domain of H-2Kb on a domain-shuffled molecule, although it was much weaker when compared to binding to the homologous molecule (Allen et al., 1984; Bluestone et al., 1985). Three antibodies defining the a1 domain were shown to react with the a1domain of H-2Kb only or al/012 using domain-shuffled molecules. Thus, an
MHC CLASS I MOLECULES RECOGNIZED BY CTL
157
antibody (e.g., 10-56) that lost reactivity with an al-domain mutant (H-2Kbm3)and is presumably specific for an epitope in the a1 domain does not necessarily react with a molecule containing H-2Kb in only the a1 domain, supporting the concept that many serological epitopes require interaction of both a1 and a 2 domains. Lemke et al. (1979) described six mAbs that reacted with H-2Kk; three reacted with one region of the molecule (cluster A) while the other three reacted with a second region (cluster B), as defined by inhibition of binding. Antibodies to either cluster blocked a portion of alloreactive anti-H-2Kk CTL activity as well as -Kk-restricted anti-TNP activity (Weyand et al., 1981a). When all six mAbs were used in a mixture, the lysis was completely blocked. Blanden et id. (1979)noted that H-2Dk-restricted anti-influenza, but not antiBebaru virus CTL were inhibited by a -DkmAb. Stringfellow et al. (1983) noted that the ability of mAbs directed against H-2Kk to inhibit influenza virus-specific CTL varied with the mouse strain, suggesting that different selfrestricting epitopes were selected by mice of different background genes. Pircher et al. (1984) described H-2Kb-restricted CTL clones specific for the hapten I-AED [N-iodoacetyl-N-(5-sulfonic-naphthyl)ethylenediamine]. One set of clone:; (type A) did not recognize H-2Kbm mutants that had amino acid substitutions in their a1 domain (H-2Kbm3,8,11),while a second set (type B) did not recognize mutants that had amino acid substitutions in the “loop” region of the a 2 domain (H-2Kbm536,9).Type B CTL clones were inhibited from lysing H-2Kb AED coupled targets by three mAbs specific for the a 2 domain, while type A was not inhibited by the same mAbs. Thus, these type B clones could be predominantly a 2 domain specific. Further support for the existence of domain-specific CTL based on mAb inhibition of CTL activity was produced by Weyand et al. (198lb), who showed in limiting dilution that anti-H-2Kk CTL consist of three populations with different precursor frequencies. rnAbs directed against cluster A block -70% of clones with the highest precursor frequency and only 25% of clones of the lowest precursor frequency. In contrast, antibodies to cluster B do no block high-frequency CTL, but do block 50-70% of clones of the lowest frequency. There are numerous reports indicating a discordance between epitopes on class I molecules defined by mAbs versus CTL. Davignon et al. (1983) generated TNP-specific H-2K“-restricted CTL clones and used mAbs to inhibit their ability to lyse TNP-derivatized targets. Two mAbs, which do not react with the H-2Kbm3mutant molecules, were able to block the reactivity of CTL clones on H-2Kb-TNP targets. These clones also lysed H-2Kbm3-TNP targets, indicating that the antibody specificity does not correlate with the CTL epitope. Rusch et al. (1983) tested CTL generated in bulk culture between H-2Kbm mutant strains and C57BL/6 (H-2Kb). H-2Kbm3anti-B6 (putatively a1 domain specific) and H-2Kbm1anti-B6 (putatively a 2 domain
158
J A M E S FORMAN
specific)were tested with domain-directed antibodies (termed epitope groups A, B, and C) for inhibition of CTL activity. No correlation was observed between the epitope groups that the mAb was directed against (epitope group A corresponds partly to the a1 domain and epitope group C corresponds to the a 2 domain) and the putative domain specificity of the CTL. Similarly, Bluestone et al. (1984a) noted that an anti-H-2Ld CTL clone which did not react against an mAb-selected H-2Ld variant cell could be blocked from killing a cell expressing wild-type H-2Ld using the same mAb. Wraith et al. (1983) showed that an anti-influenza-specific H-2Kk-restricted CTL clone lysed a target cell that expressed an H-2Kk variant molecule which lacked reactivity with an anti-H-2Kk mAb, yet could be blocked from lysing target cells expressing wild-type H-2Kk with the same mAb. Thus, these studies demonstrate that determinants defined by mAbs are different from those recognized by CTL. Since exon-shuffling studies have shown that the a3 domains do not control polymorphic determinants recognized by CTL (see Section 11),it might be expected that mAbs directed against a3 would not block CTL recognition of class- I molecules. Thus, McCluskey et al. (1986b) did not block antiH-2Ld CTL activity with the 28-14-8 mAb directed against the a3 domain, but did note inhibition of a3 domain-specific CTL with the same mAb directed against a domain-shuffled molecule missing the a1 and &2 domains, but expressing the a3 domain on the cell membrane. However, there are several reports indicating that a3 domain mAbs do block both alloreactive and H-2-restricted CTL (Ciavarra and Forman, 1982; Orn et al., 1982; Forman et al., 1983; Levy et al., 1983; Ozato et al., 1983a; Stroynowski et al., 1985b). While these data would support the concept that blocking of CTL activity with mAb directed against the a3 comain is steric, it is possible that the a3 domain contributes a constant portion of a polymorphic epitope or that the a3 domain serves as a ligand for accessory molecules on CTL that assist in binding. Further evidence consistent with this is the findings of Maziarz et al. (1985) who noted that hybrid HLA molecules containing H-2 in the a3 domain and remaining carboxy-terminus were recognized less efficiently by anti-HLA CTL than the homologous molecule. Analogous results were also observed for reciprocal H-2/HLA constructs. VII. Class I Heavy Chains Bearing Defined Amino Acid Changes: Effect on Polymorphic Determinants Recognized by CTL
A. H-2 MUTANTSTRAINS Bailey and Kohn (1965), Egorov and Blandova (1968), Kohn and Melvold (1974), and Melvold and Kohn (1976) skin grafted syngeneic mice to screen
MHC CLASS I MOLECULES RECOGNIZED BY CTL
159
for histoconipatibility mutants. Several mutant mice were identified with a mutation in one of their M H C genes, and coisogeneic strains were derived from these mutant animals (for review, see Klein, 1978). Most of the mutants were detected in (BALB/c x C57BL/6)Fl animals, and of these, most involved the (257BL/G-derived class I H - 2 K b gene. Other strains bearing mutant M H C genes have also been described, including the H-2dm1 and H-2dm2mulants involving H-2D and H-2L molecules encoded by the D end of the H-2d haplotype (Egorov, 1967; Melvold and Kohn, 1976; Sun et al., 1985; see Section 11,B). Nathenson and his colleagues (see Nathenson et al., 1986)have analyzed the structure of 12 of the H-2Kb in vivo-detected mutant genes. These mutations have occurred in either the a1 or a 2 domain, are complex, involving changes in several bases, and are probably the result of gene conversion mediated by other donor genes in the H-2b haplotype (Pease et a/'., 1983). All of these mutant molecules have an alteration in H-2Kb epitopes detected by alloreactive anti-H-2Kb CTL (Forman and Klein, 197513; Melief et al., 1980; Albert et al., 1982; Sherman, 1980) and H-2Kb-restricted CTL (Blanden et al., 1976; Zinkernagel, 1976; Whitmore and Gooding, 1981; Pan et al., 1982; Wettstein, 1982; de Waal et al., 1983b). In contrast, most of these molecules are serologically very similar to native H-2Kb. Since these mutant strains are detected by their ability to elicit skin graft rejection (Bailey and Kohn, 1965), it is expected that all these mutant molecules should elicit T cell responses by altering polymorphic epitopes on H-2Kb. An extensive analysis of these mutants has been reviewed by McKenzie et al. (1977), Klein (1978), and Nathenson et al. (1986). The 12 H-2Kbm mutant molecules that have been characterized either at the protein or nucleotide level are shown in Tables V and VI. Although the number of mutants is limited, several seem to involve the same amino acid changes, e.g., H-2bm5,16and H-2bm6,7,9.There is a clustering of mutations involving certain amino acids. Thus, mutants H-2bm3,11,23have an Asp * Ser interchange at residue 77. This residue is also one of the more variable positions between H-2 alloantigens (Kimball and Coligan, 1983). H-2Kbm8 has a Glu -* Ser interchange at residue 24, which represents another variable amino acid. H-2Kbm1, the mutant least related to H-2Kb at the T cell level (Melief et al., 1980; Sherman, 1980; Clark and Forman, 1983), has alterations at residues 152, 155, and 156, and residues 155and 156 are highly polymorphic within the murine species. HLA-A2 and -A3 subtype molecules also have amino acid changes involving residues 152 and 156 (Krangel et al., 1982, 1983; van Schravendijk et al., 1985). There is a hierarchy of relatedness of H-2 mutants to H-2Kb (Melief et al., 1980; Sherman, 1980; Clark and Forman, 1983), as defined by CTL reactivity, with H-2Kbm1being the least related, while H-2Kbm5,6,7,9,16 are the most similar to H-2Kb. This latter group of mutants has a Tyr * Phe in-
TABLE V AMINO ACID ALTERATIONS ON CLASS I MOLECULES AFFECTING T CELLREACTIVITY~ Amino acid residue a1 domain 1 11 21 31 41 51 61 71 81 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890
c
8
Variable residuesb Mutants H-2Kb KbmE Kbm3 Kbmll Kbm2.3 R8.313 HLA subtypes HLA-A2 M7;DRl
0
0
x
y
0
x
0
0
0
0
0
x
X
X
x x x x X
X
a Data taken from Nathenson et al. (1986), G. Pfaenbach and S. G. Nathenson (personal communication), Nakagawa et al. (1986), Krangel et al. (1982, 1983), Taketani et al. (1984), and van Schravendijk et al. (1985). Residues of murine class I molecules with a variability score >6 (Kimhall and Coligan, 1983) calculated according to Wu and Kabat (1970).
TABLE VI AMINOACID ALTEHATIONS ON CL.\SSI MOI.ECUIXS AFFECTINGT CELLR E A C T I V I ~ Amino Acid Residue a 2 Domain
151 161 171 181 91 101 111 121 131 141 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 12 Variable residues" Mutants H-2K"
0 0
n
00
Kbml
r
x x x
Kbrn5.16
X
Kbrn6.7.9
X
X
Klim4
B
xx x x x
KbrnlO
HLA subtypes HLA-A2 M7;DKl DK1 HLA-A3 El HLA-B7 CF a
See Table V for explanation and references. Indicated region of mutation(s), site not defined
h
. . . .
x
X
x
x
x
x
xx
xx
162
JAMES FORMAN
terchange at residue 116 [mutants H-2Kbm6,7,9have an additional Cys + Arg interchange at 121, but are indistinguishable from mutants H-2Kbm5,16 at the T cell level (Melvold et al., 1982)l. Shiroishi et al. (1984) changed the tyrosine at position 116 of H-2Ld to phenylalanine. Thus, it was to be expected that alteration of this residue would alter this molecule in a fashion similar to what happens with H-2Kbm5,l6.However, alloreactive anti-H-2Ld CTL lysed these target cells like normal -Ld-bearing targets. Since mutants H-2bm5,16are closely related to H-2Kb, as defined by CTL reactivity, a more detailed analysis with anti-H-2Ld CTL clones may be required to reveal differences. C57BL/6 (H-2b) anti-H-2Kbm1 CTL cross-react on H-2Ld expressing targets (Nabholz et al., 1975), and this molecule bears the three mutant amino acids of H-2Kbm1,i.e., Ala, Tyr, Tyr at positions 152, 155, 156, respectively, suggesting that these amino acids are recognized in a linear fashion by antiH-2Kbm1 CTL. Hunt and Sears (1983) demonstrated that H-2b anti-H-2a CTL lysed H-2Kbm1 target cells, and a portion of this activity could be blocked by H-2d, but not H-2dm2 inhibitors, the latter of which lack expression of H-2Ld. Similarly, Mann et al. (1987; and unpublished observations) noted that CTL directed against the QlO molecule, which also bears the three mutant amino acids of H-2Kbm1(Mellor et al., 1983), react against target cells expressing H-2Ld. De Waal et al. (1983a), however, noted that most H-2Kb anti-H-2Kbm1 CTL activity was not cross-reactive on H-2Ld, but was reactive against other unrelated H-2 molecules. Further, H-2dm2 anti-H-2d CTL (anti-H-2Ld) do not distinguish H-2b from H-2bm1target cells (Hunt and Sears, 1983). Thus, definitive evidence is lacking for recognition of linear determinants on class I molecules by alloreactive CTL. It should also be noted that although the assignment of H-2Kbm1cross-reactivity with H-2Ld relies on studies using the H-2dm2mutant strain, H-2dm2mice have deleted three other class I H-2d genes in addition to H-2Ld (Stephan et al., 1986). A recent report by Parham et al. (1987) indicates that anti-HLA-A2 CTL can be specifically blocked from recognizing HLA-A2 targets by a peptide spanning amino acids 94-112 of HLA-A2, albeit at high concentrations. Whether this peptide directly interacts with the receptors on CTL or interacts with class I molecules on the target cell has not yet been ascertained. Most data derived from studies with H-2 mutant mice suggest that CTL recognize determinants controlled by both the a1 and a 2 domains. Thus, B6 anti-H-2Kbm" CTL clones which might presumably be specific for an epitope in the a1 domain (the H-2Kbm11 mutation has altered amino acids at positions 77 and 80) recognize other H-2Kb mutant molecules carrying the wild-type residues at positions 77 and 80, but vary elsewhere in the molecule (Sherman, 1982). Similarly, both a1 domain mutants H-2Kbm3,11and
M H C CLASS I MOLECULES RECOGNIZED BY CTL
163
a 2 domain mutant H-2Kbm1have lost most of their H-2Kb-restricting determinants for Sendai virus and VSV (de Waal et al., 1983; Clark and Forman, 1983). Furthermore, (H-2Kbm3 x H-2Kbm1)F, mice which express H-2Kb molecules with mutations in the a1 and a 2 domains, respectively, are able to generate anti-H-2Kb responses (Apt et al., 1975; Melief et al., 1980). If allodeterminants were solely domain specific, then the F, animals would be expected to be unresponsive to H-2Kb. While the above data suggest that alloantigenic and H-2-restricting epitopes result from the interaction of the a1 and a 2 domains, it is not clear whether the epitopes are localized to one domain as opposed to being formed by the interaction of amino acids from both domains. The description of restricting activity by class I mutant molecules generally correlates with alloantigenic activity. Thus, the H-2Kbm1molecule is the least related to H-2Kb as defined by alloreactive CTL and does not crossreact with H-2Kb with respect to restricting determinants (Table VII). H-2Kbm5,6,’3 molecules are closely related to H-2Kb, as defined by alloreactive CTL, and are highly cross-reactive with H-2Kb-restricting epitopes. Most other class I mutants show a varied retention of allo- and restricting determinants compared to H-2Kb, suggesting that epitopes on class I molecules can be recognized by T cell receptors in many different ways. Shirioshi et al. (1984) used site-specific mutagenesis to change the cysteine at position 101 of H-2Ld to serine so as to interrupt the disulfide bridge in the a 2 domain. This mutant gene was transfected into L cells where marked changes in the binding of H-2Ld-specific mAbs were noted. Not unexpectedly, alloreactive CTL failed to recognize the mutant molecule. Jordan et al. (1983)noted that an HLA gene lacking a cysteine at residue 164 (Malissen et al., 1982), which also should not be able to form a disulfide bridge in the a 2 domain, could not be successfully transfected into L cells. B. HLA SUBTYPESDEFINEDBY CYTOTOXIC T LYMPHOCYTES Many serologically defined HLA antigens can be further subdivided into subtypes using alloreactive or HLA-restricted CTL (Biddison et al., 1982a; Breuning et‘ al., 1982; Horai et al., 1982; Spits et al., 1982; van der Poel et al., 1983a,h; Molders et al., 1983; Goulmy et al., 1984). Thus, these subtypes bear resemblance to the H-2Kb mutant antigens described above. HLA-A2 can be divided into one major and three minor subtypes using both CTL and biochemical analyses (van der Poel et al., 198313). Approximately 11% of Caucasian populations express one of the minor HLA-A2 subtypes. The HLA-A2 minor subtype from donor DK1 is not recognized by anti-influenza HLA-A2-restricted CTL (Biddison et al., 1982a) or an alloreactive anti HI,A-A2 CTL line (Ware et al., 1983). This HLA-A2 molecule has three amino acid changes at residues 149, 152, and 156 (Krangel et al., 1983)
TABLE VII O N C u s s I MUTANT OR SUBTYPE MOLECULES THATCROSS-REACT WITH NATIVE DETERMINANTS~ CTL DETERMINANTS Antigen Class I molecule Parent H-2Kb
Mutantlsubtype
(az)~
~-2~bm5 H-2Kbms.’ (a2) ~ - 2 ~ b m 4(a2) H-2Kbm1 (4 H-2Kbms (al) ~ - 2 ~ b m 3(4 H-2Kbmll (4 HLA-A2 M7, DR1 (a1 and a2) DK1 (4 HLA-A3 E l (a2) (4 HLA-B7 C F
l b 2 TNP VSV
+ +
+ +
+
-
-
*
* +
*
+ -
-
3 4 5 6 SV ECTRO VAC LCM
+ + +
-
+ -
-
+ +
+ + -
-
+
-
-
-
+
+ +
7 SV40
8 HSV
+ +
+ +
9 10 MoLV FLU
11 12 13 14 15 16 EBV H-Y H-1 H-3 H-4 ALLO
+ +
+
-
+ + +
-
-
-
-
-
+
-
-
+ + +
-
* - -
-
+ +
+ + +
-
-
-
+ + 2
-
’’
-
-
-
a Abbreviations: TNP, Trinitrophenylated cells; SV, Sendai virus; ECTRO, ectromelia virus; VAC, vaccinia virus; LCM, lymphocytic choriomeningitis virus; SV40, simian virus 40; HSV, herpes simplex virus; MoLV, Moloney leukemia virus; ALLO, alloreactive. Because of different assay systems used, some variation in the extent of cross-reactivity exists. Therefore, the results are meant to indicate the relative extent of cross-reactivity only. + indicates moderate-to-high cross-reactivity of antigen-specific class I restricted CTL on mutant or subtype targets; 2 indicates weak cross-reactivity; - indicates little or no cross-reactivity. Reference key: 1, Forman (1979), Levy and Shearer (1982), Davignon et al. (1983), 2, Clark et al. (1985), 3, de Waal et al. (1983b), 4, Blanden et al. (1976), Pang et al. (1977), 5, Zinkernagel(1976), Zinkemagel and Klein (1977), Forman (1979), 6, Blanden et al. (1976), Zinkernagel(1976), Zinkernagel and Klein (1977), Byrne et al. (1984), 7, Whitmore and Gooding (1981), Pan et al. (1982), 8, Jennings et al. (1984), 9, Stukart et al. (1984), 10, Townsend et al. (1983), Biddison et al. (1980a,b, 1981, 1983), 11, Gaston et al. (1983), 12, Goulmy et al. (1982), 13, Chiang and Klein (1978), 14, Chiang and Klein (1978), Wettstein (1982), 15, Wettstein (1982), 16, Melief et al. (1980), Sherman (1982), Clark and Forman (1983). c Refers to domain bearing mutation.
MHC CLASS I MOLECULES RECOGNIZED BY CTL
165
(see Table YI). HLA-A2 subtypes from donors M7 and DR1 have a Gln + Arg substitution at residue 43, a change in the 147-157 peptide of the molecule, and could also have an alteration in their carbohydrate (Krangel et al., 1982). M7 is not recognized by either alloreactive, influenza-specific, EBV-specific, or H-Y HLA-A2-restricted CTL (Biddison et al., 1980a,b, 1982; Goulmy et al., 1982; Ware et al., 1983; Gaston et al., 1983; Molders et al., 1983). HLA-A3 subtype from donor E l has an alteration at residues 152 (Glu + Val) and 156 (Leu + Gln) (van Schravendijk et al., 1985). This HLAA 3 subtype can induce an alloreactive CTL response with HLA-A3 cells (Monos et al., 1984). Further, influenza virus-specific HLA-A3-restricted CTL do not interact with virus-infected cells from this -A3 subtype (Biddison et al., 1981). HLA-B7 subtype from donor C F has a change in residue 116 from Tyr to an unidentified amino acid (Taketani et al., 1984) and is not recognized by alloreactive anti-HLA-B7 CTL (Spits et al., 1982). The parallel between the H-2Kb mutants and HLA subtypes is striking, both with respect to the site of alteration and to changes in CTL-defined determinants. The HLA-A2 and -A3 subtypes have an alteration in the molecule which includes the same region as the H-2Kbm1mutant. Clearly, mutation in this region is not readily detected by serological reactivity whereas it is by the T cell system. Similarly, the HLA-B7 subtype from donor C F is similar to H-2Kb1n5,16in that there is a change in residue 116. VIII. CTL Recognition of Monoclonal Antibody-Selected Somatic Cell Class I Variants
The H-2 mutant molecules described above represent variants that have been selected by their capacity to elicit skin graft rejection in uiuo. Subtypes of HLA molecules defined by CTL also represent class I variants that arose in uiuo and are subject to selective pressures. Therefore, alternative approaches have been attempted to characterize CTL-defined epitopes using somatic cell variants selected by mAbs. Rajan (1977, 1980) used anti-H-2 antibodies to select stable heterozygous H-2 variant cell lines. Potter et al. (1983) used this technology to isolate somatic cell variants in (H-2d x H-2b)F, cell lines with mAb 34-2-12, specific for the a3 domain of H-2Dd. Two variants were described that retained reactivity with other H-2Dd mAb, but lacked reactivity with 34-2-12. These variants (selected with anti-a3 mAb) were poorly recognized by anti-H-2Ddreactive CTL. Although this finding appears to contradict studies with exonshuffled mctlecules whereby it was found that the a3 domain does not control polymorphic epitopes (see Section 11), a gross change in molecular weight was noted in one of the mutant molecules which was not due to an alteration in carbohydrate structure. It is possible that the ability of the molecule to interact with P2-M or other proteins could have been affected by this structural change.
166
JAMES FORMAN
Bluestone et al. (1984b) and Geier et al. (1986) used technology similar to that described by Potter et al. (1983) to derive heterozygous somatic cell H-2Kb variants. The variants were selected with anti-H-2Kb mAb specific for epitopes controlled by the a1 or a 2 domains. CTL clones generated in the strain combination H-2Kbm8anti-H-2Kb (H-2Kbm8is a mutant strain bearing mutant residues at amino acid positions 22-24, 30 (G. Pfaffenbach and S. G. Nathenson, personal communication) of the a1 domain of H-2Kb) recognized H-2Kb, but lost reactivity with H-2Kb variants selected for a loss of either al- or &defined serological epitopes. H-2Kbm10anti-H-2Kb (H-2Kbm10is a mutant strain bearing mutant residues at amino acid positions 165 and 173 of the a 2 domain of H-2Kb) -derived CTL clones lost reactivity against H-2Kb variant molecules selected for loss of either al- or a2-controlled serological epitopes. Cell line R8.313, which has a variant H-2Kb molecule with a single amino acid change at residue 82 from a leucine to phenylalanine (Nakagawa et al., 1986), lost reactivity with most anti-H-2Kb CTL clones generated in H-2Kbm strains (Bluestone et al., 1986). These data are consistent with a contribution of both a1 and a 2 domains in controlling epitopes recognized by monoclonal CTL. Clones that were inhibited from recognizing H-2Kb by mAbs could react with loss variants selected using the same mAb, further indicating that mAb-defined epitopes do not necessarily correlate with CTL epitopes. Bluestone et al. (1984a) selected variants of (H-2k x H-2d)F, cell lines with anti-H-2Ld mAbs directed against the a 2 or a3 domains. Of 36 antiH-2Ld CTL clones tested, only 3 did not react with the variants selected with anti-a2 mAbs, and all reacted with those selected with anti-& Similar to the findings with H-2Kb variants (Bluestone et al., 1984b), mAb defined epitopes did not correlate with CTL epitopes. Vohr et al. (1983) selected (H-2k x H-2d)F1 variants with two mAbs directed against H-2Kk. Approximately 40% of H-2Kk-TNP CTL clones generated in a limiting dilution assay lost reactivity with cell lines that were selected with either of the two antibodies. However, these serological epitopes probably do not define CTL specificities, since variant cell lines expressing H-2Kk molecules that lost reactivity with both mAbs retained reactivity with all H-2Kk-TNP CTL clones. H-2Kk-restricted CTL specific for fluorescein isothiocyanate (FITC), Newcastle disease virus (NDV), and influenza virus did not distinguish wildtype H-2Kk from the variants. Although the antibodies used to select the variants block lysis of influenza virus-specific H-2Kk-restricted CTL clones (Wraith et al., 1983), H-2Kk variants lacking these epitopes still were recognized by anti-influenza CTL. Vohr et al. (1983) suggest that since -40% of H-2Kk-restricted anti-TNP CTL clones lose reactivity with the variants, the number of restricting epitopes on an H-2 molecule is limited. While it may be argued that the variants represent gross alterations in H-2Kk, the fact that
MHC CLASS I MOLECULES RECOGNIZED BY CTL
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all NDV, influenza, and FITC clones retain reactivity with the same variants mitigate against this. The authors suggest that since TNP covalently couples to H-2, which probably accounts for the specificity of TNP-H-2Kk CTL (Forman and Vitetta, 1978; Handa and Herrmann, 1985), these variants may not allow relevant covalent coupling of this hapten. Pious et al. (1982) selected HLA-A2 somatic cell variants from the T5-1 cell line with the BB7.2 anti-HLA-A2 mAb. Cell line 8.6.1 was described as having a change at amino acid 161 from Glu + Lys, while variants 8.18.1 and 8.21.1 have an alteration in the region from 98 to 108 (Taketani et al., 1983). These variants have been tested for their ability to react with both alloreactive and HLA-restricted CTL. Ware et al. (1983) and Brenner et al. (1985) found that these variants react with anti-HLA-A2 CTL and Gaston et al. (1984) noted that these variant molecules are recognized by HLA-A2-restricted EIW-specific CTL. Similar to studies with variant H-2 molecules selected by mAbs, deletion of serologically defined HLA epitopes does not lead to readily detectable changes in CTL determinants. IX. Concluding Remarks
MHC molecules may play at least three roles in determining the specificity of T lymphocyte recognition. For class I1 molecules, these roles are characterized by interactions with (1)the a / p chains of the T cell receptor, (2) antigenic peptides, and possibly (3) L3T4 or CD4 receptors from the mouse or human, respectively. Since CTL use the same aY/Pchains to recognize antigen in the context of class I molecules, it is worth considering whether similar interactions might govern class I molecules in determining the specificity of CTL. First, it is clear that CTL use the a and p chains of the T cell receptor heterodimer to recognize antigen (Dembic et al., 1986). These genes are uniquely rearranged in different CTL clones, and antibody directed against clonotypic determinants on these molecules can either block or activate CTL clones, depending on the assay in which they are used (Meuer et al., 1983; Reinherz et al., 1984). T helper cells and CTL clones have also been described that use the same V, and J, segments in their receptor genes (Rupp et al., 1985). The second issue concerns the interaction of MHC molecules with antigen. Recent evidence by Babbitt et al. (1985, 1986) indicates that a peptide of hen egg lysozyme interacts with Ia molecules, albeit weakly. Further, H-2k strains are able to generate an immune response to the peptide and show binding of the peptide to IAk, while H-2d is a nonresponder strain and the peptide does not demonstrate detectable binding to IAd. Similar findings have been reported by Buus et al. (1986)using ovalbumin peptides. Watts et
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al. (1986) found an interaction between an ovalbumin peptide and Ia using resonance energy transfer, although it was reported that this interaction required the presence of specific T cells. Antigen presentation involving class 11 molecules appears to use a pathway whereby molecules from the exterior are internalized, interact with the lysosomal compartment, and then are reexpressed on the cell membrane in a “processed” state (reviewed in Unanue, 1984). In contrast, inhibitors of lysosomal activity do not inhibit the ability of target cells to present antigens restricted to class I molecules to CTL (Morrison et al., 1986). Apparently, internally synthesized polypeptides which do not need to be expressed on the cell membrane as intact molecules are processed and transported to the cell membrane through an unknown mechanism where they can be recognized by CTL (Townsend et al., 198613). Examples include nonenvelope proteins of influenza, VSV, and Friend virus (Townsend et al., 1984; Holt et al., 1986; Yewdell et al., 1986). Since peptides of the influenza virus nucleoprotein and HLA-CW3 can be added to target cells to create antigens recognized by CTL (Townsend et al., 1986a; Maryanski et al., 1986b), it would appear likely that the same type of interaction described between peptides and Ia would also apply to peptides and class I molecules. This would be consistent with class I and I1 molecules having a similar structure and the fact that they interact with the same a/p chains of the T cell receptor. Therefore, the ability of class I molecules to act as restricting molecules for antigen-specific responses may be controlled in part by the ability of these molecules to interact with various antigenic components of internal antigens, e.g. virus and minor H antigens. The third aspect to consider is whether accessory molecules interact with portions of class I molecules. Such interactions have been postulated to account for the functional activity of T cell subsets (Forman, 1984). Lyt-2/CD8 and L3T4/CD4 are nonpolymorphic immunoglobulin-like molecules which could interact with nonpolymorphic portions of MHC molecules (Littman et al., 1985; Maddon et al., 1985; Sukhatme et al., 1985). Lyt-2/CD8 is expressed on cells that interact with class I molecules, while L3T4/CD4 cells interact with class I1 molecules (Swain, 1981; Meuer et al., 1982), and this observation has led to the suggestion that these molecules aid in the binding of T cells to specific antigen controlled by class I or I1 molecules, respectively (Biddison et al., 1982b; Marrack et al., 1983; Dialynas et al., 1981, 1983; Reinherz et d., 1983; Greenstein et al., 1984). L3T4+ T hybridoma cells react to low amounts of antigen more readily than L3T4cells and the presence of L3T4 enhances the ability of cells to become activated when specific antigen is limiting (Marrack et al., 1983).The ligand(s) for L3T4 is not known. Greenstein et al. (1984, 1985) noted that an antiH-2Dd reactive hybridoma that expressed L3T4 was inhibited by anti-L3T4 mAb recognizing H-2Dd on an Ia+ L cell only if the expression of H-2Dd was
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relatively low. Watts et al. (1984) and Gay et al. (1986) showed that hybridomas sensitized against planar membranes or glass beads containing lipid, Ia, and peptide could be inhibited from responding by anti-UT4 mAb. Golding et al. (1985)showed that an L3T4+ anti-class I1 CTL line could be inhibited fxom recognizing a class II/class I hybrid molecule with anti-L3T4 mAb. The molecule lacked the a chain and the (32 domain of the (3 chain of Ia (see Section 11,E). Therefore, while evidence is suggestive that class 11 molecules may be a ligand for L3T4, other molecules may also serve as ligands. Alternatively, L3T4 could interact directly with the T cell receptor or play a role in binding after initial antigen recognition occurs. It should also be noted that mitogen-activated T cell responses directed against class I1 or class I negative cells can be inhibited by anti-L3T4 or Lyt-2 mAbs, respectively (Hunig, 1984; Malek et al., 1985; Tite et al., 1986). I have discussed data demonstrating that most of the polymorphic epitopes recognized by CTL are controlled by both the a1 and a 2 domains. These data were first obtained by studies using H-2 mutant strains where mutation in either the a1 or a 2 domain resulted in a loss of most of the alloreactive and restricting epitopes recognized by CTL (Section VII). These findings have been extended more recently using the techniques of exon shuffling and site-specific mutagenesis. Most class I molecules that bear a1 and a 2 domains derived from different class I genes or alleles lack most of their alloantigenic epitopes recognized by CTL generated against the native molecules (Section 11). A few exceptions exist, e.g., Dd/Ld/Ld and the H-2dm1 mutant, the latter of which represents a natural domain-shuffled molecule similar in structure to Dd/Ld/Ld (Section 11,B). Both of these molecules retain epitopes recognized by alloreactive and H-2-restricted CTL reactive with both H-2Dd and H-2Ld. While it is tempting to conclude that epitopes can be confined to both the a1 and a 2 domains, it is more likely that substituting the a2 domain of H-2Dd with H-2Ld allows much of the native epitope structure of the H-2Dd molecule to remain intact. This interpretation is supported by the finding that changing the a 2 domain of H-2Dd to Q7d completely alters the alloantigenic and restricting H-2Dd epitopes recognized by CTL. Further, the reciprocally shuffled molecule, LJd/Dd/Dd.is devoid of detectable H-2Ld and H-2Dd CTL-defined determinants. Studies with other molecules show that altering either a1 or a2 domains results in a loss of the vast majority of CTL-defined epitopes. In contrast to the role of the a1 and a 2 domains, a change in the a3 domain appears to have no detectable effect in CTL specificity. This has been noted even when the a3 domain is derived from a different species, although in one report it was found that such a molecule was recognized less efficiently. Although current data indicate that the a3 domain does not contribute to polymorphic determinants recognized by CTL, this does not preclude the
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possibility that a conserved segment of the a3 domain either constitutes a portion of CTL-defined determinants or interacts with accessory molecules on the effector cell. A role for P2-M in controlling polymorphic determinants recognized by alloreactive T cells is also lacking; however, an allele of P2-M has been shown to be recognized by MHC-restricted CTL (Section V). Current evidence also indicates that carbohydrate does not play a major role in controlling epitopes recognized by either alloreactive or H-2-restricted CTL (Section IV). The H-2Kbm1 mutant strain as well as several serologically silent CTLdefined HLA subtypes have an alteration in amino acids involving residues 149 to 156. The substitutions frequently involved a change in a charged amino acid, and this type of change appears to be a characteristic of H-2 mutant and HLA subtype molecules (Monos et al., 1984). This region of the molecule has been postulated to comprise a turn of an a helical structure (Vega et al., 1984) and may define a part of a class I antigen important for T cell recognition. In the mouse, CTL generated against these molecules bearing mutant amino acids appear to cross-react on other H-2 molecules bearing the same “mutant” amino acids (Mann and Forman, 1987). However, most data would suggest that this cross-reaction is not directed specifically at these mutant amino acids, but rather at a conformation that they induce elsewhere in the molecule. It would also appear that many mutations have little effect on CTL epitopes. Thus, most mutant class I molecules which have been selected by mAb treatment of somatic cells show no alteration in their CTL epitopes (Sections VI and VIII), and this finding is consistent with a lack of correlation between serological and CTL-defined determinants. As the three-dimensional structure of class I molecules will soon be available, this puzzle may readily be solved (Bjorkman et al., 1985). At present, however, this finding suggests that CTL epitopes may be partially cryptic and revealed following interaction with a T cell. Since conjugate formation precedes antigen recognition (Spits et al., 1986), it is possible that as a result of conjugate formation alterations occur in the conformation of class I molecules revealing previously undetectable alloantigenic sites to the T cell receptor. Changes in the interaction of one mAb directed against a class I molecule have been reported to occur subsequent to the binding of a second mAb (Lemonnier et aZ., 1984; Diamond et al., 1984). This type of observation could be a model for exposure of cryptic CTL sites.
ACKNOWLEDGMENTS This work was supported by NIH Grants AI13111, CA41009, and AI11851. I wish to thank Ms. Betty Jo Washington for her excellent secretarial help and Drs. K. Fischer-Lindahl and B. Loveland for their careful reading of the manuscript together with their valuable suggestions. I
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am grateful for the many discussions I have had with Dr. Stephen Clark and Mr. Don Mann regarding this work.
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ADVANCES IN IMMUNOLOGY, VOL 41
Experimental Models for Understanding 6 lymphocyte Formation PAUL W. KINCADE !minunobiology Laboratory, Oklahomo Medical Reseorch Foundation, Oklahoma City, Oklahoma 73104
1. An Introductory Overview
While many points that can be made in an introduction are arguable and await further experimentation, the outlines are beginning to emerge of a fascinating process which can produce hundreds of billions of B lymphocytes daily and for the lifetime of an individual. This is achieved in a highly regulated manner, with requisite stem cells occasionally being drawn from a common, but very small pool. Increasingly less reversible changes commit them to a differentiation pathway as a series of divisions expand cell numbers. This is coordinated with simultaneous and parallel production of at least eight other lineages of blood cells which will coexist and be functionally interrelated when mature. The name B lymphocyte denotes the origin of these cells in birds (bursa of Fabricius) and mammals (bone marrow), and this review will emphasize events taking place in the latter. Progress in this field is being achieved by rapidly converging technologies and experimental approaches as the same general questions attract molecular, cellular, and developmental biologists. The objective of this chapter will be to highlight some recent developments, with a focus on issues that have been of interest to our laboratory. Studies involving normal and genetically defective experimental animals, monoclonal antibodies, inducible cell lines, soluble mediators, and innovations in long-term culture will receive particular emphasis. Early in gestation, before blood circulation begins, cells destined to become hemopoietic stem cells migrate from the neural crest area to extraembryonic tissues (yolk sac) as well as to the midline of the e m b q o . Both populations are potentially capable of making lymphocytes, but only those within the embryo which are incorporated into the developing liver and spleen norrnally do so (78, 272). Precursor cells sharing some of the distinctive properties of B lymphocyte lineage cells in adult marrow are found within embryonic tissues, and their differentiation potential can be demonstrated with a number of functional assays (review: 186). However, there are a number of notable differences in the initial emergence of lymphocytes in embryonic liver and the steady-state production of B cells within adult bone marrow (192, 197, 223, 326). In addition, early cells make preferential use of 181 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any farm reserved.
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certain families of variable region genes to make immunoglobulin heavy chains and assemble antibody molecules which will not be common in adult life (338, 436, 499, 502). At around the time of birth, hemopoiesis and the production of lymphocytes move from liver and spleen to the bone marrow (272, 457). The models and arguments used in this chapter are mainly based on the adult steady state and may not be completely applicable to the emergence of lymphocytes during ontogeny. While some multipotential stem cells can be found in the circulation, these may have limited proliferative potential, and it has not been proved that they are capable of becoming B lymphocytes (277). More likely, a relatively small pool of stem cells is retained within specialized “niches” within bone marrow and maintained by minimal self-renewal (442). They have finite proliferative potential which can be exhausted by serial transplantation, but aging is not appreciable during a normal individual’s life span (139). Such early stem cells can be considerably enriched experimentally by selective cytotoxic drugs, use of monoclonal antibodies, and electronic cell sorting strategies (17, 122, 151, 462). When injected into lethally irradiated recipient mice, they probably must mature briefly within bone marrow before being detectable as spleen colony-forming cells (CFU-s) (251, 455). While many of the latter are also multipotential, some are committed progenitors of erythroid, myeloid, and megakaryocytic lineages. Few, if any, CFU-s have the option of becoming B cells (187, 328; Fig. 1). The multilineage potential of stem cells was originally demonstrated in mice by use of cytogenetic markers in cloned populations(1). Premalignant expansion of genetically marked hemopoietic stem cells provided evidence for their existence in humans (101, 102). More recently, retrovirus vectors are being used to introduce selectable markers into hemopoietic stem cells (77, 169, 183, 488). This approach should pave the way for eventual gene replacement therapy (14) and provide a more practical means of studying stem cell population dynamics (235). Monoclonal antibodies can be used to identify normal cells which are committed to becoming B lymphocytes, and this has opened many investigative possbilities (43, 195). For example, we know the morphology, size, and frequency of B lineage precursors in late embryonic, neonatal, and adult life as well as in some genetically defective animals (see Section 111). It has also been possible to enrich for them and follow their short-term fate in a number of culture systems. Transformed counterparts of normal B cell precursors can undergo spontaneous or induced changes in culture (Section VII). These cell lines have been particularly useful in learning that functional immunoglobulin (Ig) genes are made by first reconfiguring heavy chain (D-J and then V-D-J) and then light chain (V-J) gene segments. There are several opportunities for errors as Ig genes are rearranged, and this contributes to
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multipotential stem cell (pre-CFU - s )
large small newly early B lineage pre - B pre-B formed cell B cell precursor cell
FIG.1. Lineage relationships between hemopoietic stem cells and B cell precursors. Areas of particular uncertainty are indicated by dotted lines.
potential antibody diversity. Some unknown fraction of lymphocytes may reach a “dead end” because a functional gene is not assembled. The fate of these defective cells is not known, but a mechanism has recently been found through which some may be functionally rescued (Section IV). The first DNA rearrangement event probably occurs soon after a distinctive surface marker [ Ly-5(220)]is expressed on large bone marrow lymphocytes which have basophilic cytoplasms containing numerous polyribosomes (45, 223). Detailed kinetic studies suggest that these cells probably initiate synthesis of the heavy chains of IgM while still large and that light chains will not be made for several more days (Section V). During this interval, replicative activity of the B cell precursors declines, and there is a marked reduction in cell size (Section 111). During an approximately 2day postreplicative period, additional cell surface molecules that are typical of mature I) cells are acquired, and functional receptors for certain soluble mediators can be demonstrated (Section IX). B lineage cells are not influenced by specific antigen or idiotypic networks before surface Ig appears (201, 202, 296). However, their production is affected by nonspecific environmental stimulation (110, 114, 311, 343). An interesting period of hypersensitivity exists just as receptors for antigen emerge on membranes of newly formed small B cells (274, 344, 350). Through an energy-dependent mechanism, cells exposed to small concentra-
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tions of specific antigen are functionally inactivated, and this “clonal abortion” mechanism contributes to immunological tolerance (362, 437); i. e., many clones which have high-affinity receptors for abundant self-antigens are eliminated. It has been estimated that 5 X lo7 small lymphocytes are made daily in the bone marrow of a mouse, and many of them probably migrate via the circulation to the spleen (310; Section V). Depletion of mature B cells does not change the rate of B cell production (112), and we have little idea how a steady mind is maintained. Most of this chapter will deal with recent studies aimed at learning the nature and function of cells and molecules which regulate this process. Soluble mediators that influence several cell lineages have been found to augment the maturation of normal B cell precursors in culture (Section IX). These are now made by recombinant DNA technology and are available in highly purified form, which will permit molecular studies of receptor-factor interactions. Other factors, which seemed like better candidates for local regulation of bone marrow lymphocytes, were identified through studies of genetically defective animals and man (177, 225). However, the origins of these materials are not known, and thus far, only small quantities have been obtained in partially purified form. Lymphocytes in bone marrow are crowded among immature cells of all of the other blood lineages in what might seem a random array. However, they are probably closely associated with specialized cells of the bone marrow stroma, which provide a favorable microenvironment (oxygen, tension, nutrients, soluble mediators, inductive stimuli, etc.) for their proliferation and maturation (444). One or more adhesion molecules are probably involved in precursor cell-stromal cell recognition (Section VI), but this probably does not result in a permanent fusion. Maturing B cell precursors must gradually be carried along stromal cell surfaces to eventually be favorably located for discharge into venous sinuses. Essentially nothing is known about this process, but it presumably involves modulation in the expression of particular adhesion molecules and energy-dependent contractile elements. Replication of early precursors is essential to maintain population sizes within adult marrow, yet it has been difficult to find concentrated collections of lymphocytes. This suggests that a type of “conveyor belt” mechanism must be operating and that daughter calls from a mitosis are moved away from each other even as they mature. Long-term bone marrow culture approaches now make it possible to ask what types of microenvironmental cells interact with B lymphocyte precursors (74, 483). Different variations on the culture system permit long-term propagation of stem cells, B lineage precursors, or intermediate cell types in
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absolute dependence on adherent stromal cells (Section VI). Some possible sources of lab-to-lab and culture dish-to-culture dish variation are considered below, but it is already clear that this is a powerful methodology which can be manipulated in many ways. Our laboratory has characterized the large stronial cells to which lymphocytes are bound in culture and hopes that this will lead to an identification of critical adhesion molecules. Other have reported in workshops that it is possible to establish cloned lines which can replace some of the functions of adherent stromal cells in long-term cultures. These should yield large quantities of at least some of the critical regulators of precursor replication and maturation. Antagonists of this process are probably selected against by culture strategies, and we think that their characterization will also provide an exciting area of investigation. This is important not only for understanding how homeostasis is achieved, but also because of their potential in leukemia therapy. Genetic defects have long been informative about precursor-product and functional relationships between cells (Section VIII). We find it useful to return to the same models again and again because, as our methodology steadily improves, it is possible to appreciate ever more subtle connections and realize new opportunities to exploit particular mutations. A summary of our recent studies of genetic defects which influence B lymphocyte formation will comprise one section of this chapter. Finally, after considering individual cell types, mediators, and approaches, we hope to encourage a more catholic appreciation of relationships between different blood cell lineages. This will certainly be required in order to understand how the balance between massive production, maintenance, and death of cells of the humoral immune system is maintained. II. Organization of Lymphohemopoietic Tissues
The microanatomy of normal adult, embryonic, and regenerating hemopoietic tissues has been described in detail (64, 239, 240, 477-479, 482). However, until recently, it was only known that large collections of lymphocytes were not conspicuous. The precise relationships of differentiating B lymphocyte lineage cells to other cells in mammalian tissues have now become an object of investigation, and this occurs just as in uitro models are becoming better understood. A simplified interpretation of the anatomical studies will hopefully provide a basis for comparison with the culture work described below. Formal descriptions can be found in the many excellent reviews on this subject (240, 479, 478). The volume of hemopoietic tissue is determined by space not occupied by bone cortex, bone trabeculae, and specialized fat cells, while the latter may
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be able to contract in situations of hemopoietic demand. Blood-forming areas of marrow are situated in cords between two types of circulation. The subendosteal area has a rich capillary plexus, and the core of the marrow space is traversed by interconnecting venous sinuses. The latter provides an exit route for newly formed blood cells and are comprised of a minimal basement membrane and overlapping endothelial cells which lack tight junctions. The abluminal surface of these sinuses is largely covered by fibroblastic “adventitial reticular” cells and occasional macrophages. Radiating out from these are processes of reticular cells and fibrils which form the basic spongy scaffolding of the hemopoietic cords. It is generally assumed that bone marrow “stromal” cells provide inductive signals as well as support to hemopoietic progenitors, and we use the word interchangeably with “microenvironment” or “microenvironmental elements.” Two particularly significant types of cellular associations have been noted by experimental hematologists. Maturing erythroid cells can be found clustered around a central macrophage in what has been termed an erythroblastic island (18). Immature granulocytes are physically associated with nonphagocytic adventitial reticular cells, which can express alkaline phosphatase (482).The same relationship has been appreciated between myeloid progenitors and adherent cells in long-term cultures, but in that circumstance, their well-spread appearance has led to such names as blanket cells (4, 5). This is particularly appropriate in the sense that progenitor cells can crawl underneath and proliferate. An analogy will be drawn between this phenomenon in lymphoid cultures and the behavior previously noted for tumor cells, termed pseudoemperipolesis (Section VI). Established bone marrow stromal cell lines can at least briefly support myelopoiesis, and some have the potential of turning into fat cells (preadipocytes) (207, 409, 505, 506). Large flattened cells to which lymphocytes are intimately associated in long-term bone marrow cultures have many similarities to adventitial reticular cells, and it is possible that functionally specialized microenvironmental elements would not be distinct morphologically (85; Section VI). This would be as dificult as resolving B and T cells on the basis of appearance alone. Alternatively, stromal cells capable of supporting myelopoiesis might under different circumstances be converted to components of a lymphoid microenvironment. Precursors of B cells have been identified in sections of fetal and neonatal liver (180). Before birth, they were found in scattered arrays or “star bursts.” As hemopoiesis is shifted from liver to bone marrow during the first days of life, small collections of hemopoietic cells are left. These were shown to be clonal and to contain foci of pre-B cells (368). It is not clear why such collections of lymphocytes can only be found in this special circumstance, but one could speculate that physical limitations are imposed by the rapidly
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developing nonhemopoietic areas of liver. In other tissues, such as bone marrow, the progeny of replicating precursors are permitted to move away from each other. However, within an individual bone, it is still possible to identify clonally related precursors (201). Finding multiple pre-B cells together has prompted speculation about their self-renewal at this stage of differentiation (Section V). Osmond and Batten (319)perfused murine femurs with radiolabeled antiIg antibodies and were subsequently able to identify B cells by autoradiography of marrow cross sections. Some mature, but presumably recently formed B cells were found adhering to venous sinuses. Other B cells were found scattered throughout the hemopoietic cords or in collections of less than four cells. The same laboratory has now utilized this approach to identify earlier cells with a monoclonal antibody to Ly-5(220) (D. G. Osmond, personal communication). Again, large lymphoid foci were not conspicuous, but there was a tendency for a gradient to be seen; i. e., there may have been more immature cells just beneath the bone capsule. Dissection of cells from that region of viable bone marrow and phenotyping also indicated that precursors may be enriched in the well-vascularized endosteal area (218). Earlier studies had revealed that active proliferation and immature myeloid cells are most prominent in this region, suggesting that as cells become closer to the stage of discharge into the bloodstream, they are moved toward the collecting sinuses in the center of the bone (241, 394). Parallel studies are being done with rat bone marrow where a new monoclonal antibody is particularly useful for identifying the pre-B cells (312; D. Opstelten, personal communication). One can expect that distinctive markers will soon be identified on subsets of bone marrow stromal cells, and this should make possible their localization in situ in relation to B lineage lymphocytes. The avian bursa of Fabricius should be used as a frame of reference of mammalian B lymphopoiesis because this represents a centralized and highly specialized site of lymphocyte formation (48, 120, 121). Some myelopoiesis can occur within the bursal mesenchyme during embryonic life, but it is essentially a lymphoid tissue (232). At a discrete stage of development, small numbers of stem cells colonize the bursa and move into epithelial buds. These proliferate and quickly differentiate to form small follicles of B cells, and it is notable that in birds, there is little delay between expression of heavy and light chain immunoglobulin genes (49).Much remains to be learned about the bursal microenvironment, but an interesting type of “secretory cell” has been identified in ultrastructural studies which might ultimately be associated with the production of critical soluble mediators (307). As we move toward an understanding of the nonlymphoid cells which induce andl regulate B lymphocyte formation, the question of their developmental origin recurs. There are clonal assays and other experimental ap-
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proaches for studying the progenitors of fibroblasts, which share at least some properties with marrow stromal cells. These cells are noncycling, more radioresistant than hemopoietic progenitors, and not usually transplanted with marrow grafts. In addition, they can lack, the Philadelphia chromosome which marks all other lineages of cells in chronic myeloid leukemia (review: 490). However, in at least one situation, a clonal relationship was found between cultured adherent cells and premalignant hemopoietic cells (403). It is controversial whether adherent stromal cells are donor type in recipients of bone marrow transplants, and resolution of this question may await better definition of the cell types involved (70). 111. Resolution of B Cell Precursors
Terminology is certainly a problem for newcomers to immunology, and attempts will be made here to be consistent with a previous contribution to this series (186). Cells with potential for extensive self-renewal as well as differentiation into all eight blood cell lineages will be called multipotential hemopoietic stem cells. Cells bearing Ig chains which they made will be referred to as B cells. The immediate precursors of functional B cells in mammals lack surface Ig, but probably contain sufficient quantities of the p. heavy chain of IgM to be detected by immunofluorescence (351). Although the term was originally coined to describe a cell with slightly different characteristics (216), we refer to cells with this particular staining pattern as pre-
B cells (47). There are practical limitations to characterization of cells on the basis of a test which requires their fixation. Also, it is not clear how many cells successfully rearrange and express p. chain genes, but do not make light chains and, consequently, never become B lymphocytes. Many monoclonal antibodies and flow cytometers are now available, and it has been tempting to refer to lymphocytes which display one B lineage marker, but not sIgM, as pre-B cells. However, it is not clear that a single set of surface characteristics defines all murine pre-B cells (see below). The lesson has emerged from studies of tumor lines that cells which have rearranged one or more Ig genes and which display one or more B lineage markers may still retain the option of becoming something other than a B cell. Similarly, although we are learning a great deal about cells that are slightly earlier in the differentiation series, no set of characteristics unambiguously defines them. We refer to lymphocytes which may be irrevocably committed to becoming pre-B cells as early B lineage precursors. Such definitions may have only temporary usefulness as we gain more insight into molecular aspects of differentiation, and it is unfortunately the case that different investigators use the same words to describe different or incompletely overlapping cell populations. An
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attempt will be made in the following sections to summarize the many techniques. antibodies, and functional assays now being brought to bear on these questions.
A. AN OVERVIEW OF CELLSURFACE MARKERS It is easy to appreciate the value of monoclonal antibodies in resolving and manipulating lymphocyte populations. The surface markers most commonly used to study murine B lineage precursor cells are listed in Table I. No known antigen is exclusively expressed on C F + , sIg- pre-B cells. This may mean that pre-B cells represent a transient differentiation compartment rather than a discrete stage, and the extent to which these lymphocytes can self-renew under different circumstances is discussed below. The assumption is generally made that lymphocyte antigens must be receptors with unknown, but presumably important functions. Pre-B cells are unresponsive to specific foreign antigen because they have not yet acquired surface Ig. However, most membrane molecules required for regulation of pre-B cell activities must be shared with cells earlier and/or later in this pathway as well as with cells in other lineages. Indeed, our studies reveal that multiple soluble mediators can influence pre-B cells in culture, and thus far, none has been found which exclusively influences cells at this stage of differentiation (Section IX). Formal demonstration that a marker is on the precursors of B cells requires that it be used to deplete and/or enrich for them before testing their ability to become B cells in a functional assay. This has been shown for only the most widely utilized reagents (Section 111,H). However, a wealth of information has been obtained from studies of established pre-B cell lines and lymphocytes in long-term cultures (Table I). While no single antibody is stage specific, some fairly restricted markers, such as one detected by the recently described BP-1 antibody (50), can be used together with other reagents to define the immediate precursors of B cells. B. Ly-5 FAMILY OF GLYCOPROTEINS Macromolecular antigens encoded by the Ly-5 gene are very immunogenic in rats and many monoclonal antibodies are available to them. These have been extremely valuable as experimental tools, and extensive study of this antigen family has revealed a most interesting pattern of tissue-specific gene regulation. Trowbridge and colleagues (446) first demonstrated macromolecules that were uniquely expressed on T cells (T ZOO), and related antigens of slightly different sizes were subsequently found on all blood cells with the exception of erythrocytes (276, 309, 382, 399, 445). B lineage cells preferentially express the largest member of this family (220 kDa) and intermediate-sized (205 kDa) molecules have be.en demonstrated on myeloid
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TABLE I SOMEMARKERS EXPRESSED BY MURINE B CELLPRECURSORS Demonstrated on some Markera
Pre-B tumors
LTBM cultures
c p + , sIgcells
Functional precursors
Lyb-2 Lyb-8 BP-1 GF-1.2
Yes Yes Yes Yes
Yes
Yes
Yes
?
p
Yes Negative
Yes Yes
? ? ?
Ly-5 (220)
Yes
Yes
Yes
Yes
ThB Ly-1 IL-2R Me1 14 TdT Lym-19
t
Yes t
Yes
Yes ?
2
r + Yes Yes
? Negative Yes
p
p ? ? Neg. Yes
b,c,138,192
? ?
Yes t
AA4.1 Mac-1 (CR-3) LFA-1 Ly-17 (FcR)
Yes Yes Yes Yes
Yes t t Negative
Yes
Yes
6C3 PNA MU75 M 1/69 (HSA) JllD LGPl00
Yes ? t Yes Yes Yes
? ? Yes Yes Yes t
43,44,59,69 198,223,289 57,59,92 1,59,158,228 c
t Negative
Yes -
57,59,195 158,430 50,492 17,262,263
? ? ? ?
2
Qa-2 la
Selected references
b,C
129,265,335,482 c,431 c,57,59,158, 497 b,262,263,330 59,155
? ?
p
b,c
Yes
Yes
b,',59,155 157,158,501 b,158,441,345 318,320,321 bsC,492 b,c , 492 262,263,492 153,228,230,492
?
?
Yes
Yes
?
? ? ? ?
Yes Yes
?
Markers are grouped according to probable restriction to B lineage cells, B and T lineages, or broader representation on hemopoietic cells. In many cases, only a subset of cells was clearly positive for the marker, and in others, the results are equivocal or ambiguous (t). All pre-B cells also express H-2 as well as the Ly-5 common leukocyte antigen, and some stain with a monoclonal antibody to the transferrin receptor (G. Lee et al., Ref. 509). b P. L. Witte (unpublished observations). 6. Lee (unpublished observations). (I
cells (383,445,448).Although the molecules are glycosylated, the major size differences have been attributed to varying lengths of the polypeptide chains (354, 473). The Ly-5 alloantigen system appears to identify virtually (380) all of this group of molecules in mice, regardless of their cellular origin, and we refer to them collectively as the Ly-5 family.
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A single structural gene has been cloned from rats and mice which corresponds to this family of macromolecules (395, 439). The nucleotide sequence predicts a transmembrane protein which has a very large intracellular domain. Northern blot analysis of RNA from B and T cells revealed differentsized messages, and recent studies suggest that RNA processing determines which type of molecule will be synthesized (374, 439). The largest message corresponds to the 220-kDa protein, whereas the shortest message results in the 200 kDa molecule preferentially expressed by T cells. Coffman and Weissman (44) found that one of their monoclonal rat antimouse B lineage antibodies (RA3-3A1)precipitated a 220-kDa antigen. Subsequent studies revealed that five of our independently developed antibodies, additional antibodies prepared by Coffman and Weissman, and a similar one to a human lymphocyte antigen all recognized molecules of this approximate size (42, 56, 383). We know from typing lymphocytes of different species that at least several epitopes are recognized by our antibodies. For example, only two of the five bind to human cells (222). Therefore, the N-terminal portion of the largest Ly-5(220) molecules must carry several unique antigenic determinants which have not yet been characterized in molecular terms. Essentially all peripheral B cells, all precursor cells that can quickly become B cells in culture, and a majority that do so within 10 days of transplantation express the Ly-5(220) molecule (43, 195). Most of the cells enriched from fetal liver and adult bone marrow with anti-Ly-5(220) antibodies have rearranged at least one allele of Ig heavy chain genes (45; Section IV). At the other end of the differentiation pathway, only some of the antibody-secreting cells in a primary immune response or plasma cell tumors are Ly-5(220)+ (42, 195). The WEHI-3 myelomonocytic leukemia cell line is recognized by our antibodies, but myeloid progenitor cells (CFU-c) and multipotential hemopoietic stem cells (CFU-s) are uniformly negative (195). Similarly, when we have used these reagents to enrich cells from fetal, neonatal, and adult hemopoietic tissues as well as from human bone marrow, they have been virtually all lymphocytes (222, 223). In one study, electronically sorted cells included some granulocytes, especially when the suspensions were obtained from neonatal liver (457). We know that autofluorescent nonlymphoid cells are problematic in that circumstance (unpublished observations). However, given the result with WEHI-3 cells, it remains possible that a very small number of myeloid lineage cells express the Ly-5(220) marker. Significant binding to platelets and macrophages as well as partial inhibition of CFU-s was reported with one, but not all antibodies to this antigen family (353). While th ymocytes and thymus-seeking cells in bone marrow are Ly-5(220) negative, the marker is detectable on a subset of peripheral T cells (195, 236). These are most conspicuous in lymph nodes of mice, where they
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normally are also positive for Lyt-2 (195, 211, 383). Our two human reactive antibodies bind to a subset of T cells in peripheral blood, as do antibodies made against human and rat antigens of this size by others (55, 56, 222, 231, 412). The size of the molecule recognized by these antibodies on the normal peripheral T cells of mice has not been determined. However, this has been studied by several groups in the case of MRL/Lpr mice, where a lymphoproliferative disorder causes expansion of T cells with a very unusual phenotype (60, 88, 286, 383). In that circumstance, Lyt-2-, Thy-l+ lymphocytes make the 220 form of Ly-5 antigen. We know of only one situation where monoclonal antibodies to the Ly-5(220) molecule do not recognize at least small numbers of peripheral T cells. This is said to be the case for the RA3-6B2 antibody (42). If at least some T cells can transcribe the entire Ly-5 gene, special RNA processing or postsynthetic modification mechanisms would have to be envoked to explain how the 6B2 epitope is not expressed on those T cell Ly-5 molecules. For example, alternate patterns of glycosylation could yield different epitopes on these macromolecules (299). By analogy to other markers that were initially thought to be lineage restricted, the term B-220 was coined (44). However, until it is formally shown that any unique molecule of this size is exclusively made by B cells, we prefer the designation Ly-5(220). This is used throughout this chapter to refer to antigens recognized by our monoclonal 14.8 antibody and other antibodies with related specificities (195). While the Ly-5 family antigens are relatively abundant on leukocyte surfaces (445), their function remains unclear. Hemopoietic stem cells must synthesize the smallest member of this family of “common leukocyte antigens” and at some point early in the B lymphocyte differentiation lineage, a decision to change the pattern of transcription must be made. Early B precursors, pre-B cells, B cells, and a subset of peripheral T cells all make the largest form of these antigens. The Ly-5(220) molecule bears several unique epitopes that presumably correspond to functions most required by cells of the humoral immune system. It will be interesting indeed to learn what those functions might be. Antibodies to Ly-5 inhibit antibody responses to particular antigens in vitro, possibly by interfering with macrophage-B cell communication (495). It has been shown that antibodies to this family or antigens of similar size on human cells can depress T cell and NK cellmediated cytotoxicity (234, 298, 299, 380). Mitogen-driven proliferation of human T cells was augmented by trace amounts of antibody to a 220-kDa glycoprotein (231). It is important to stress that cultured or transformed B lineage lymphocytes do not always express the 220 form of Ly-5 antigens. This was first obvious in the case of established B lymphoma cell lines such as WEHI-231, which is Ly-5+, but lacks epitopes associated with Ly-5(220), (195, 228). All
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of a series of human pre-B leukemias were negative for Ly-5(220), and we have found that this is often the case with pre-B cells in long-term bone marrow cultures (198, 219, 231, 493; Section IV). Although from 16 days of gestation, all pre-B cells are Ly-5(220) , pre-B cells and functional precursors of B cells in earlier embryos can be negative (195, 223, 265, 457). Therefore, while expression of Ly-5(220) probably precedes rearrangement and expression of Ig genes in normal circumstances, this is not an obligatory sequence of events. Use of this or any other marker alone to detect B lineage cells could be problematic, and especially so when working with early embryos, cultured cells, or transformed cells. +
C. MARKERSMERITING MOREATTENTION
The BP-1 marker is interesting because it represents a new approach to preparing monoclonal antibodies as well as a unique cellular representation (50).Reasoning that many genetic polymorphisms might not be appreciated by inbred strains of mice, Cooper and colleagues immunized wild mice with a pre-B cell tumor. One of the resulting antibodies detects a 140-kDa disulfide-linked heterodimer which is expressed on pre-B and some B cells in bone marrow, but not on any lymphocytes in peripheral tissues. Studies in our laboratory have shown that the BP-1 antigen is expressed on long-term cultured lymphocytes and that its density changes with culture age (492). It has also been found that the density of this marker on an inducible pre-B tumor can be regulated up or down by coculture with particular factors or residence in uivo (342; G . Lee, unpublished observations). The receptor for interleukin 2 (T cell growth factor) was initially thought to be restricted to T lymphocytes, but it is also present on activated B cells (171, 248, 352, 313, 465). Large, cycling pre-B cells might be considered to be in an “activated” state, and it therefore seemed possible for them to be IL-2 receptor positive (509). Pre-B cell lines were indeed stained with a monoclonal anti-IL-2 receptor antibody, and it will be interesting to learn if these receptors mediate functionally significant signals on normal pre-B cells. Similarly, all dividing cells express receptors for transferrin (237), and this could also be demonstrated on at least some normal and transformed pre-B cells (G. Lee et al., unpublished observations). Evidence has recently been presented which suggests that a close functional relationship may exist between the receptor for B cell stimulating factor-1 (BSF-1) and the murine Lyb-2 alloantigen (497). This marker appears to be exclusively expressed on B lineage cells, including pre-B cells in adult mice, but it was difficult to demonstrate by cytotoxicity on functional B cell precurs,ors in fetal liver (195, 193; see also below). It also appears to be most easily detectable on pre-B cells which are small (509). A recently described antibody to the ci chain of the LFA-1 antigen has
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similar effects to BSF-1 on mature B cells (281); i.e., it causes increased Ia antigen expression, augments proliferative responses, and influences isotype switching in culture. LFA-1 is a member of a very interesting antigen family that has been implicated in leukocyte adhesion and function (215, 414, 416). Patients who are unable to make the common p chain of this family do not express surface LFA-1, Mac-1, or p155, 95 and have recurrent infections (367, 415). Some studies suggest that the function of LFA-1 is to stabilize weak recognition bonds between cells of the immune system (407). At least small amounts of LFA-1 are detectable on murine pre-B cell lines (509). Recent evidence that pre-B cells can receive signals via the BSF-1 receptor will be discussed below. Previous studies suggested that newly formed B cells in bone marrow must acquire MHC class I1 (Ia) antigens at around the same time as surface IgM (195, 217). This question has recently been reexamined by direct staining of sIg- ,Ly-5(220) cells, and approximately one-third of them had detectable Ia. Moreover, it was possible to influence the density of both class I (H-2) and Ia antigens on established pre-B cell lines (349, 509; Section IX). An association has been found between a lymphocyte alloantigen and one type of receptor for the Fc portion of Ig molecules (157). Normal pre-B cells, as well as pre-B cell lines stain with monoclonal antibodies to this molecule, but long-term cultured lymphocytes usually do not (59, 155, 156, 158, 509; P. L. Witte, unpublished observations). Several types of Fc receptors have been described and at least three different ones can be simultaneously expressed by macrophages (104, 452, 458). The monoclonal Mac-1 antibody detects a C 3 receptor-related molecule on pre-B cell tumors (13, 155; see below). However, it is usually very low on long-term cultured lymphocytes, and there is no evidence that normal pre-B cells express it (492). The nuclear enzyme terminal deoxynucleotidyltransferase (TdT) has been frequently utilized as a marker for differentiating lymphocytes. There are indications that TdT might help to diversify antibody and T cell receptor diversity by introducing non-germ line-encoded nucleotides into the functionally configured genes (11, 68, 212, 400). Much of the recent information regarding TdT expression has come from studies of rats and humans, and in those circumstances, it appears that the marker is associated with early B lineage precursors in bone marrow (29, 146, 166, 312). Some murine lymphocytes in a long-term bone marrow culture system simultaneously expressed Ly-5(220) and TdT (129, 266). A detailed kinetic study of lymphocytes in murine marrow has just been completed and it included an assessment of TdT expression (335). Approximately one-half of the TdT+ cells were medium sized and also positive for Ly-5(220). Such cells would appear to be good candidates for the immediate precursors of large pre-B cells. Many other markers are listed in Table I which have not received detailed +
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study or which have relatively broad tissue representation. However, most have unknown functions and might be invoked to explain any of the soluble mediator responses, adhesion, and cellular recognition processes needed by B lineage precursors.
D. TUMORCELLLINESAND LINEAGEFIDELITY Spontaneous pre-B cell tumors are not common in mice, but they can easily be derived with Abelson and other transforming retroviruses (59, 156, 158, 470, 471). Established in uitro lines of these tumors have been extremely important for understanding the process of Ig gene rearrangement (7). Some of the differentiation events associated with normal B cells occur spontaneously or following stimulation of murine pre-B cells, and this is providing insight into receptor-factor interactions (Sections VII and IX). By typing large numbers of B lineage cell lines with the available monoclonal antibodies to lymphocyte antigens and correlating this with Ig gene rearrangements, a possible order of normal differentiation events can be inferred (59). Schemes have been similarly derived from characterizing large numbers of human tumor cells (9). However, it remains to be formally demonstrated that such sequences of marker acquisition occur with untransformed cells. A variety of experimental evidence has been used to conclude that progenitors of ‘rand B lymphocytes do not descend directly from cells which can become granulocytes and macrophages (186, 187). However, some studies with transformed cell lines have revealed an unexpected kinship between pre-B cells .and macrophages. A pre-B cell line mutagenized with 5-azacytidine gave rise to cells with macrophage characteristics, and there is precedent for a similar transition with human tumor cells (23, 98, 405, 426). Holmes and colleagues (12, 155, 158) recently provided definitive evidence that rearrangement of one Ig heavy chain allele and expression of some B lineage antigens does not preclude transformed cells from taking on macrophage properties. We need to know if this is an anomalous process which only occurs in tumors, and it will be interesting to learn if Ig gene rearrangements can be found in normal macrophages. However, together with the Ly-5(220) findings discussed above, these observations suggest that caution should be exercised in extrapolation from cell lines to the normal situation in uiuo. They also demonstrate that no single criterion can be used to assign cells to and position them within the B lymphocyte lineage. Ly-1 is a particularly interesting marker inasmuch as it is preferentially expressed by a B cell subset with a unique pattern of tissue localization and autoreactivity (144, 145). A series of transplantation experiments indicate that in adult mice, Ly-1+-bearing B cells may derive from a self-renewing pool of stem cells which are independent of bone marrow (136). It has been
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difficult to demonstrate Ly-1 on significant numbers of lymphocytes in longterm bone marrow cultures or by direct staining of pre-B cells (492, 509). However, pre-B tumor lines often express this marker (137). Ly-1 B cells are most frequently among mature populations in the peritoneal cavity (145). However, sublines of a pre-B tumor which expressed increased amounts of Ly-1 after LPS stimulation did not have a notably different pattern of tissue localization when injected intravenously; i.e., many of them were still recovered in the bone marrow (342). It remains unclear if Ly-l+ tumors represent transformed equivalents of a particular lineage of functionally specialized B cells. E. TECHNICAL CONSIDERATIONS Most antigens are expressed in variable densities on murine B cell precursors and often at the limit of detectability. The sensitivity of fluorescence microscopes can be optimized with frequent bulb changes, proper alignment, and use of high numerical aperture optics. Even then, it is usually possible to detect more positive cells by flow cytometry. However, the difference in sensitivity could be more apparent than real. With the microscope, phase contrast is used to select “nucleated” cells for scoring, whereas low-angle forward and 90” light scatter is used in flow cytometry. It is not certain that identical populations of hemopoietic cells are evaluated with the two methods. We are very concerned about experimental protocols that employ hypotonic shock to prepare cell suspensions. Recovery of murine bone marrow nucleated cells is often 50% or less with many NH,Cl lysis procedures, and occasionally, the ratio of small-to-large cells appears to be skewed. Our best results have been obtained with Gey’s solution (280), and we use it only when absolutely necessary (such as with particular sorting protocols). It might appear that clear-cut staining results would always be obtained with recently cloned lymphoid tumor cells. This is the case with some antigens and cell lines. However, with some others, the cultures include both positive and negative cells. The density of some antigens is markedly influenced by growing the cells in serum-free medium or different concentrations of fetal calf serum ( G . Lee, unpublished observations). Variant subsets might grow out under the different conditions or this might also reflect the influence of hormones or other soluble mediators in serum. However, it points out the difficulty in obtaining consistent results from laboratory to laboratory, especially when dealing with normal, heterogeneous cell populations from different strains of mice. The relative abundance of the different sets of B lineage precursor cells is reasonably predictable in any given mouse strain maintained under the same conditions. However, it should be noted that the kinetics of production of
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these cells is sensitive to nonspecific environmental stimulation (110, 114, 311, 343). Animals from a given conventional breeding room at times give higher or lower than average expected values. It is easy to imagine that some of the systemic, non-lineage-specific mediators such as IL-1, IFN gamma, and BSF-1 participate in such fluctuations (see below). The effect of corticosteroids on the bone marrow merits further investigation because preliminary studies in this laboratory suggest that stress can have major effects on precursor population sizes (P. L. Witte, unpublished observations). One should also be aware that in addition to polymorphism of alloantigens, amounts of markers such as Qa and ThB vary greatly on different pedigrees. In these two examples, the regulation has been mapped to an MHC-linked gene and the Ly-6 locus, respectively (92, 275). The aim of these comments is not to diminish confidence in published descriptions of antigens on murine B lineage cells or to suggest that our approaches are superior to those used by others. Rather, it is hoped that the reader will appreciate the difficulty in categorizing low densities of antigens on small subsets of hemopoietic cells and be aware of some causes for differences between studies. In our experience, it has been difficult to “nest” all defined populations into a single lineage scheme. This may reflect branching avenues of differentiation or the limits of our present technology.
F.
PHOSI’HATIDYLINOSITOL-LINKED
LYMPHOCYTE ANTIGENS
Most of the known lymphocyte antigens are probably anchored in the membrane by means of a stretch of hydrophobic amino acids embedded in the lipid bilayer. However, it has recently been appreciated that a novel form of membrane attachment is used by a subset of lymphocyte markers, and this may be important for understanding the function of these molecules. Tissue-specific control over this attachment mechanism could permit utilization of a single structural gene to produce a secreted protein, a releasable membrane protein, or a permanently anchored surface molecule. Investigation of this question has also provided a practical means of distinguishing and independently studying molecules which are similar in other respects. Alkaline phosphatase was the first molecule shown to be attached to cell membranes via phosphatidylinositol (PI) (245). A similar linkage mechanism was subsequently discovered for 5’-nucleotidase, the VSG antigen of trypanosomes, a form of acetylcholinesterase, decay-accelerating factor, and putative second messengers associated with the insulin receptor (62, 99, 115, 244, 376, 377). Where it has been examined in detail, the PI is attached via a short carbohydrate spacer and ethanolamine to the C-terminal amino acid of the protein (52, 99, 247; review: 243). The lymphocyte Thy-1 antigen usually utilizes this form of membrane attachment (246, 447). While the nucleotide sequence for this molecule can
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encode hydrophobic C-terminal amino acid residues, none were found by direct sequencing of the purified protein (31, 391). A staphylococcus aureus-derived phospholipase is specific for the PI linkage, and this selectively removed Thy-1 from viable thymocytes or thymoma cells (242, 246). An extension of those studies revealed that ThB and some of the Qa-2-related antigens, but not TL, Qa-1, µglobulin, or other class I or class I1 molecules are released from lymphocytes by this enzyme (424). Spleen cell responses to LPS were only slightly affected, whereas those to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) were nearly abolished by this treatment. These results demonstrate the selectivity of the enzyme and indicate that some PI-linked molecules may be important in mature lymphocyte responses. Activated T cells have been shown to release intact Qa-2 molecules (406). It is not known if these were derived from PI-linked cell surface molecules, but we previously speculated that endogenous phospholipases might allow molecules that are attached in this way to be shed under appropriate circumstances (246). Some cell types, exemplified by L cells, seem not to be able to express surface Qa-2 when transfected with the unmodified gene, and this is the case even when RNA transcription is achieved (123, 427; G. Waneck, personal communication). It seems possible that such cells might make and secrete soluble forms of potential membrane molecules, as has been found for a mutant T cell line that secretes Thy-1 antigen (97). The antigens on some lymphocytes out of a population seem unaffected by treatment with PI-PLC (424; unpublished observations with G. Waneck). While there is precedent for the PI linkage being cryptic on some cells (62, 115, 363), it could also be that those lymphocytes synthesized molecules with conventional hydrophobic C-terminal residues. Further study may show that sensitivity of an antigen to PI-PLC reflects the maturation state or functional capability of the cell type on which it is expressed. It would then be important to understand the RNA processing and/or postbiosynthetic modification mechanisms responsible for such selective use of a PI anchor. As with the Ly-5 family of common leukocyte antigens discussed above, it provides considerable flexibility in use of a single structural gene. The functional significance of this for B lineage precursors and other lymphocytes remains unknown. However, preliminary results indicate that a PI-linked molecule may contribute to the adhesion of maturing pre-B cells to bone marrow stromal cells (Section VI,C).
G. OTHERDISTINCTIONS In addition to monoclonal antibodies and surface markers, there are other approaches to identifying and characterizing B lymphocyte lineage precursors. Forman and colleagues (103) described an IgH-linked alloantigen sys-
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tem (H-40) which is defined only by cytotoxic T cells and whose expression on B cells seemed to correlate with their initial acquisition of surface Ig. O’Toole and Bosma (323) discovered a broader range of specificities encoded by genes of this general region (Lm-1) and also recognized by T cells, but expressed on cells other than B cells. They noted several important implications for such alloreactivity in bone marrow transplantation as well as experimentation involving allotype congenic animals. Storkus and Dawson (425) found that B lineage cells become sensitive to recognition and lysis by natural killer (NK) cells at a particular stage of differentiation. Earlier studies showed that some bone marrow cells and subsets of thymocytes are potential NK-sensitive targets (133, 134, 304). Molecules recognized in these circumstances could play a role in normal B lineage differentiation and function. Subtractive RNA hybridization techniques have been extensively used to isolate genes which are expressed in a lineage-restricted manner (61). For example, this approach was used to clone the first T cell receptor gene and an X chromosome-linked gene family associated with murine immunodeficiency (46, 147). A series of genes have recently been identified which are utilized only in B lineage cells and which may correspond to different stages of maturation (148, 374b, 374c). On the basis of size and partial sequences, some of these have been assigned to previously known B cell products (Ig light and heavy chains, etc.). Others appear to encode molecules with unknown characteristics and functions (148). Studies now under way should reveal if these are inducible with any of the known lymphokines and B cell stimuli and if they are aberrantly expressed in mutant animals. While protooncogenes can be expressed in many embryonic and dividing cells, there is tissue specificity, and certain ones of these might be used as an additional criterion for resolving B lineage cells which otherwise appear to be similar (503). It is also interesting that Myc expression in transgenic mice causes preferential expansion of a population of B lineage precursors in prelymphomatous animals (2, 226).
H. RELATIONSHIPOF MARKERS TO FUNCTIONAL ASSAYS Until recently, the transition of uncommitted stem cells to functional B cells could only be studied by transplantation to irradiated or immunodeficient animals in a maturation process which required 6 to 8 weeks (186, 187, 195). Fortunately, a variety of systems are now available for demonstrating that murine cells expressing a particular set of surface markers can give rise to B cells. For example, the immediate progenitors of B cells mature quickly and have traditionally been characterized with a variety of short-term culture procedures. Furthermore, innovations in long-term culture methodology may not only facilitate a detailed study of the characteristics of B lineage precursors, but allow their emergence from multipotential stem cells
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to be followed in culture (Section VI). Soluble mediators now known to augment differentiation steps in culture will be discussed later (Section IX). Representative studies of early and late B lineage precursors will be summarized here. A semisolid agar cloning system has been useful for monitoring the emergence of functional B cells in a variety of experimental circumstances. Cells bearing surface IgM proliferate in response to mitogens intrinsic to laboratory agar, and colonies are scored after 6-7 days of culture (190, 273). Inclusion of anti-p antibodies in the medium of semisolid agar cultures completely prevents colony formation (193).Therefore, cells must mature before being capable of replication in this system, and the maturation of sIg- cells after being dispersed in agar is inefficient. However, this transition can be augmented by hyperactive regulatory cells and soluble mediators (Sections VII1,D and IX). Partially immunodeficient CBA/N mice (Xid mutation) totally lack B cells that can respond in this assay (185). This is not because CBA/N B cells are mitogen unresponsive, but seems linked to their inability to be diluted in culture. For example, Xid B cells divided in crowded liquid cultures in response to agar-associated mitogens or LPS, but the blasts quickly died when plated in agar. Normal histocompatible hemopoietic cells almost completely reconstitute this functional deficiency, and this facilitated many studies of the characteristics of B lineage precursors (review: 186). Distinctions between early stem cells and late progenitors have been made on the basis of size, surface marker expression, and the time required to yield clonable B cells after transfer to CBA/N mice. For example, cytotoxic elimination of cell suspensions with the 19B5 monoclonal antibody greatly diminished precursors which could generate functional B cells within 6 weeks after engraftment into irradiated CBA/N recipients (195, 328). Particularly significant was the finding that multipotential cells (CFU-s) capable of spleen colony formation were unaffected by 19B5. This provided one of the first indications that early B lineage precursors are not closely related to myeloid stem cells (Fig. 1). Numbers of clonable B cells found per recipient spleen 10 days after transplantation are directly related to numbers of injected precursors. At this interval, -70% of the precursors could be depleted with monoclonal antibodies to Ly-5(220) (195). In addition, cells bearing this marker, but lacking surface Ig, were enriched by positive selection with the same antibodies and shown to give rise to functional B cells (223). Cells that spontaneously matured during 2-4 days of conventional culture to express sIgM and clone in agar were found to be Ly-5(220)+ (43, 195). Similar findings were recently made when cells with these characteristics were placed on preestablished layers of stromal adherent cells (289).
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Paige and colleagues developed conditions which permit relatively early cells to be cloned in agar and followed to an Ig-secreting stage (119, 324, 331b). Using an adherent underlayer of fetal liver cells, they found a linear relationship between numbers of precursors plated and immunoglobulinsecreting clones which developed. At 12 days of gestation, the precursors did not bear B lineage-associated antigens (330, 331). One day later, AA4.1 was acquired, and still later, Ly-5(220) was expressed. Finally, by 15-16 days of gestation, it became possible to enrich for the precursors with monoclonal antibody to Lyb-2. Precursors taken from late-stage embryos required the least amount of time in culture before giving rise to plaque-forming colonies. Osmond and Owen found that the lectin peanut agglutinin (PNA) preferentially bound to c p + , sIg- pre-B cells in adult murine bone marrow (320). The lectin was then used to enrich for large and small pre-B cells by electronic cell sorting (321). Both populations seemed to give rise to sIgM B cells and clonable B cells when held in liquid culture, and this was most convincing for the large fraction, which initially contained very few B cells. Many noa-lineage-specific receptors are acquired by differentiating B cell precursors (Table I). For example, the PI-linked Qa-2 family of antigens is demonstrable on pre-B cells and other hemopoietic cells in adult marrow, and developmental age rather than position in the lineage determines their expression i(192).Yang et al. (501) used a double-labeling procedure to associate the expression of sIg, receptors for the F c portion of Ig, and complement receptors with the postmitotic age of bone marrow lymphocytes. The results would be consistent with sequential acquisition of sIg, Fc, and C’ receptors by some cells within bone marrow, and when newly formed, radiolabeled, marrow lymphocytes were transferred to recipient mice, expression of these markers increased. However, it was pointed out that subsets of maturing B cells could acquire Fc and C’ receptors at different times. This may explain in part why numbers of B lineage cells bearing the Fc receptor detected by monoclonal 2.4G2 are particularly variable (509). A splenic fragment cloning assay developed by Klinman has been invaluable for studying some aspects of B lymphocyte lineage differentiation (202). Cells to be assayed are allowed to home to spleens of carrier-primed, irradiated recipient mice. The spleens are removed, diced, and cultured with antigen, and antibody secretion is subsequently measured. It is clear that sIg - precursors are capable of quickly maturing and responding to antigen in this system, and this has formed the basis for several studies of “prereceptor” B cell characteristics (201). Small marrow lymphocytes which are phenotypically Ly-5(220) , c p , sIg - comprise a majority of the cells which give rise to functional B cells under these conditions (296). Although other markers and physical characteristics of prereceptor B cells h a w not been +
+
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described in detail, several interesting aspects of their potential antibody specificities have been reported. As a population, they already reflect certain biases found among the repertoire of mature splenic B cells, and it seems clear that they have not been specifically influenced by idiotypic networks or environmental antigens. I. RELATIONSHIP OF MARKERS TO CELLSIZE CHANGES Transformed cell lines consist of very large blast cells which are continuously replicating. This is true regardless of whether the lymphomas are classified as representative of mature B cells, pre-B cells, or earlier B lineage lymphocytes. However, small size has been repeatedly stressed as a distinguishing feature of the immediate precursors of B cells, newly formed B cells, and unstimulated B cells in peripheral lymphoid tissues. Kinetic studies of bone marrow lymphocyte populations are consistent with large pre-B cells giving rise to small pre-B cells (Section V). The significance of this change is not clear because lymphocytes in long-term bone marrow cultures are relatively homogeneous and small to medium in size despite the fact that they are cycling (493). Low fonvard-angle light scatter as determined by flow cytometry is being frequently used as a convenient way to discriminate small and large lymphocyte populations, but there is no agreed upon criterion for distinguishing the two. The upper size threshold for small cells in smears has traditionally been 8 p, and for cytocentrifuged lymphocytes 10 p has been used (221, 310, 316). These are experimentally convenient criteria for monitoring what must be a gradual shrinking process. It has been proposed that the ThB antigen is preferentially associated with small bone marrow lymphocytes (42). However, recent studies in this laboratory suggest that while this is true for unfractionated (B6 x DBA)F, bone marrow, most of the ThB-bearing cells are actually small B cells, and the marker is certainly expressed by some large sIg- lymphocytes (G. Lee, manuscript in preparation). The magnitude of ThB expression is governed by genes linked to the Ly-6locus, and the ThB molecule is linked to the lymphocyte surface via PI (92, 424). It is possible that differences in strains and/or husbandry of animals will provide explanations for discrepancies between results obtained by different laboratories. It is clear that cells destined to become B lymphocytes already express a variety of distinct surface markers. Although a few of the available monoclonal antibodies have been used in conjunction with functional assays, no definitive picture of the order of acquisition or significance of these putative receptors has emerged. Particularly useful would be any correlations that could be found between cell surface marker expression and changes in proliferative activity, rearrangement of immunoglobulin genes, association with
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particular marrow stromal cells, and immigration from this central lymphoid tissue. IV. Rearrangement and Utilization of Immunoglobulin Genes
Antibody molecules represent the most distinctive and important product of the B lymphocyte lineage. Their synthesis and display in membranebound and secreted forms provide a most complex and interesting system of coordinate gene expression during development. The NH,-terminus of an Ig heavy chain is encoded by just one of at least 100 variable region gene segments which, in the mouse, are grouped into seven families on chromosome 12. Selection of this gene segment is done during the second of a twostep rearrangement process. The components of an Ig gene are separated (in germ line configuration) in nonlymphoid cells and early embryonic tissue. An initial event involves alignment of 1 of 4 J segments with 1 of 12 D region segments and deletion of the intervening DNA. This is done via nonamer and heptamer recognition sequences which flank the J and D segments and which are separated from each other by 12 or 23 base pairs. A second and presumably similar process involves deletion of the DNA between a V region gene and the D-J complex. Use of this fully assembled gene to synthesize heavy chains then precedes rearrangement of gene segments for K light chains on chromosome 2. If this results in a functional Ig molecule, the process is usually terminated. However, if both alleles of K light chains are abortively rearranged, rearrangement of h light chains is attempted (chromosome 22). All of these events have been described in detail (7, 26, 90, 159). Only recent developments and particular aspects that are relevant to B cell precursors will be considered here. The alignment of V and D segments during rearrangement commonly does not result in a correct reading frame (91, 132). In addition, extra nucleotides (non-germ line elements) can be randomly inserted at this junction through the action of TdT (11, 68, 212). Since the D segment and V-D junction encode portions of the third hypervariable region of antibody molecules, errors contribute to the diversity of antibody-combining sites (90, 91). The process requires that as many as half of all B cell precursors generate nonfunctional heavy chain genes on both chromosomes (7). Until recently, it was assumed that all such precursors would represent abortive or “dead end” clones (132). However, a mechanism has been found through which some of these may be rescued. Studies with two transformed cell lines indicate that V-D-J rearrangement, regardless of whether it yields a functional Ig gene, can be followed by a second rearrangement which essentially exchanges a second upstream V region for the first (6, 200, 360). This seems to be possible because a heptamer, which is part of most V region genes, can
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be used as a recognition sequence for further rearrangement. The net result is that most of an initially selected V gene is replaced by a second choice. Particular antibody specificities appear in a predictable sequence during ontogeny, and recent studies indicate that this is mediated by a nonrandom selection among the seven families of variable region genes (338, 499). Some aspects of the orientation and chromosomal location of these gene families are controversial, and the order of expression may not be identical with all strains of laboratory mice (P. Tucker and C. J. Paige, personal communications, 495b, 495c). However, families situated nearest the constant region gene segments tend to be used first (7, 388, 499). In contrast, B cells in mature animals appear to utilize all of the gene families approximately the same. The model proposed above for V, replacement would only permit progression in an upstream (5’)direction, and it was suggested that this may contribute to the order of gene utilization (200). A common rearrangement mechanism may be involved in configuring light and heavy Ig chains as well as antigen receptors on T cells (500). The signals that initiate this process are not known, but it has been hypothesized that access of a recombinase enzyme to Ig gene segments controls the order and rate of rearrangement events (7). This is based in part on findings that Ig genes are transcriptionally active prior to assembly (184, 359, 467). Short transcripts of one of the variable region gene families have been detectable in fetal liver, and actual synthesis and release of “truncated” p chains has been demonstrated in adult animals (258, 498). The latter are made without variable regions by bone marrow cells and even by plasma cells which synthesize complete Ig molecules. Transcripts or products of incompletely assembled Ig genes may or may not have any functional significance, but it is clear that chromatin is “open” at a time when the rearrangement steps are proceeding. Most of the available information on Ig gene rearrangements has come from studies of tumor cell lines. Coffman and Weissman (45) addressed this question with sorted bone marrow cells which lacked surface Ig, but which were positive for Ly-5(220). All of these cells had rearranged both alleles at least to the D-J stage. Many small cells, but no large cells, had begun rearrangement of light chain genes. In a similar study with Dr. S. Akira, we found that Ly-5(220) cells in fetal liver were already undergoing D-J rearrangement by 16 days of gestation (unpublished observations). However, these and phenotypically similar cells isolated from adult marrow still retained some germ line IgH genes. Essentially all of our cells had a lymphoid morphology, and it may be that the different monoclonal antibody and selection techniques that we used permitted isolation of some earlier cells in the B lineage series which were not included in the Coffman and Weissman analysis. Early Ly-5(220) cells, which possibly express TdT (see Section +
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111), could be comparable to B lineage leukemias which have only one rearranged heavy chain allele (209). It is also possible that D to J joining has not occurred on either chromosome in some of these cells, and we need better ways to isolate them from normal tissues. In contrast to the situation with light chain genes, both Ig, alleles are rearranged in B cells in peripheral lymphoid tissues (161, 303). It would be advantageous to have additional information about the Ig gene configuration of normal bone marrow cells resolved on the basis of various markers. Long-term bone marrow cultures can be manipulated such that precursors with Ig genes in germ line configuration undergo most or all of these rearrangement steps (Section VI), and it will be interesting to learn if this can be controlled with particular stimuli. Other issues include the necessity for rearrangement of both heavy chain alleles before light chain genes and the frequency of abortive clones among unselected populations. Bias in the use of 1’ region gene families might also be seen in cells with an “early” rather than a “late” pre-B phenotype. Expression of a fully assembled Ig gene is thought to be regulated by cisand trans-acting nuclear factors and control sequences within the gene (268, 392, 422, 476). The latter include at least the promoter and enhancer regions and possibly an additional “facilitator” segment (117, 128, 130). Initiation of transcription may depend on factors already present in the cell, but whose activity is blocked by labile repressor molecules (469), e.g., cyclohexamideinduced transcription of a human heavy chain which had been transfected into mouse fibroblasts (164). Brief interruption of protein synthesis also induced light chain gene expression in a pre-B cell line (393, 469). Nuclear factors from heavy chain-synthesizing pre-B and B cells have also been used to signal transcription after microinjection (250). High-resolution DNA “footprinting” techniques, retardation of mobility of DNA fragments, and functional assays are being used to obtain detailed information about regulatory proteins and regions of the Ig genes with which they interact (10, 96). V. Population Dynamics
Depending on the species and age of an animal, bone marrow contains small numbers of T lymphocytes, some long-lived B lymphocytes of peripheral origin (presumably memory cells), and some cells involved in active immune responses (204, 205, 296, 369). However, the vast majority of lymphocytes are made locally and within a %day time span (315). Much of our understanding of the proliferative events which generate B lymphocytes within this #centrallymphoid tissue has derived from radioautographic studies of tritiated thymidine incorporation into B lineage precursors. A number of generalizations can be drawn from those reports and some will be briefly
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summarized here. However, it is important to realize that the arguments made are essentially statistical and, while one can say that the data are compatible with particular hypotheses, it is not possible to conclude that they are proved. For example, it has been frequently stated that lymphocytes leave the pool of small marrow cells randomly rather than as a function of postmitotic age (221, 317, 369). This would make lymphocytes different from other blood cells, which normally exit the marrow at maturity (348). However, it is difficult to distinguish between those cells physically leaving via the circulation and those dying locally. As marrow cells are resolved into smaller and smaller subpopulations (Section 111), it becomes ever more difficult to determine the rates at which each compartment self-renews, is replenished with less differentiated cells, and relinquishes cells to the next compartment in sequence. The analyses become almost hopelessly complex if the issue of separate lineages of B cells is raised (136). For purposes of this brief discussion, it will be assumed that there is but one linear differentiation pathway in marrow, with a series of obligatory steps through which all B cells are made. A delay of -12 hours between 3H-labeled TdR infusion and the appearance of labeled small (<8 pm) lymphocytes demonstrates that these cells are not actively dividing (317, 369). However, the number of labeled small cells ‘increases exponentially over 72 hours of continuous radiolabel infusion, indicating that they derive from cycling precursors. In one study, large pre-B cells became labeled before small pre-B cells, and grain counts for the large cells were approximately twice those of their small progeny (221). Landreth and colleagues concluded from these data that large pre-B cells probably divided only once before giving rise to small pre-B cells, which in turn matured without further division (221).The kinetic behavior of large Ig-negative lymphocytes, which would be the presumptive precursors of large pre-B cells, suggested that while they are cycling, this is also not an extensively renewing stem cell population. It was proposed that in the steady state, identifiable B lineage populations are continuously replenished by cells from earlier compartments. Replication is minimal for large pre-B cells and essentially nil for small pre-B and B cells. Opstelten and Osmond (310) showed that over a 4-hour period following vincristine injection, large pre-B cells steadily accumulated in metaphase while numbers of small pre-B cells declined, suggesting again that small preB cells derive from the large cell population. They used these and other data to construct a mathematical model for bone marrow lymphocyte proliferation and maturation. A total daily production of 5 x lo7 pre-B cells per day was calculated. The presumptive precursors of pre-B cells have been similarly studied by Park and Osmond (335). Large lymphocytes marked by the surface expression of Ly-5(220) and absence of cytoplasmic or surface IgM
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were found to have turnover times (7.4 hours) and production rates (13.5% /hour) similar to those previously found for large pre-B cells (7 hours turnover and 15.3%/hour production rate) (310). A higher apparent daily production of large pre-B than small pre-B cells was interpreted by Opstelten and Osmond to mean that half of the newly formed cells may be lost (310). One could speculate that these represent cells which have abortively rearranged Ig heavy chains at both alleles and are potential dead-end clones (7, 132). There could be specialized mechanisms in marrow for sensing and eliminating such intrinsically defective lymphocytes (Section VIII,B), and recent molecular studies have shown it is possible for them to be salvaged (Section IV). An additional stage at which newly formed lymphocytes may be lost is just as surface Ig molecules are acquired. It was first learned in experiments with chickens that differentiating B lineage cells are sensitive to anti-p antibodies (188). Subsequent studies showed that the same was true for mammalian lymphocytes and that encounter with specific antigen could functionally inactivate them at an early stage (274, 344, 350). The Klinman splenic focus assay has provided an excellent model for determining the requirements for tolerizing newly formed B cells (202, 437). The potential and steady-state proliferative activities of particular B cell precursors are related and unresolved issues. In contrast to the model discussed above, it has been argued, but not directly shown, that pre-B cells in marrow might be able to divide 6-8 times (322). Cooper and colleagues (180, 368) found collections of pre-B cells within the enclosed spaces of neonatal liver and showed that they were clonally derived. This was only seen at a time when hemopoiesis was waning, and in fetal liver, the pre-B cells were scattered. In what might also be a special case, Klinman and Stone (201) found evidence for expanded clones of B cell precursors within particular marrow shafts of immunodeficient CBA/N mice. The potential for extensive replication of pre-B cells can be demonstrated with long-term culture (Section VI), and particular memory cell clones are able to divide extensively (489). Potent selection processes have no doubt operated in the latter two cases, and this may only reflect the proliferative capability of a small fraction of pre-B and B cells. Further information concerning the average replicative behavior of normal B cell precursors would be helpful, especially if it could be correlated with the selection of heavy and light chain variable region genes. We could then begin to consider the production rates and clone sizes of cells utilizing particular variable region genes and ask if their emergence is purely random. While the production of lymphocytes in mammalian bone marrow is large, it may not be comparable to the great overproduction of T cells thought to occur within the thymus (259, 390). Most lymphocytes leaving the marrow
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appear soon after in the spleen, a majority probably die there within a few days, and few are able to recirculate (106, 108, 369). The most direct proof for these statements was obtained by specific local labeling of cells within the single femur and following their subsequent dissemination (24). In other studies, the migration of labeled bone marrow cells was followed at intervals after transplantation (369, 373). While a portion of splenic B cell populations must represent recent immigrants from marrow, it is not clear what fraction they represent and how this changes with age, immunization, disease, etc. Ablation of marrow with radiostrontium reduces numbers of splenic B cells to approximately half and the same result has been obtained by injection of hydroxyurea (107, 189, 371). Marrow cells are quickly destroyed by a single injection of 5-fluorouracil and again, B cell numbers in spleen are halved (459). However, in this case, direct toxicity of the drug on spleen cells was found to be a possible complication. The lymphoproliferative activity of mammalian bone marrow is greatest in young adolescents and adults, the thymus markedly declines with age, and the avian bursa of Fabricius involutes at sexual maturity (272, 279). However, little is known about primary lymphocyte formation within marrow very late in life. The total rate of lymphocyte formation is not increased in B celldepleted animals, but early studies suggested that it might be elevated by experimental polycythemia (311, 322, 369). If there is a clear relationship between peripheral B cell numbers and marrow activity, it remains to be shown. There is reason to believe that B lymphocyte production can be modulated by systemic physiological processes. Injection of foreign antigens stimulates lymphocyte formation within bone marrow and, in the case of sheep erythrocytes, splenic macrophages seem to mediate the effect (343). This is interesting in light of other findings that the monokine IL-1 augments the final steps of B cell formation (118, 420). A systemic inflammatory response induced by intraperitoneal injection of Corynebacterium paruum resulted in a dramatic decline in functional B cells in bone marrow, and this was attributed to prostaglandin-secreting adherent cells (173). Other potential regulatory molecules and their roles in maintaining homeostasis are discussed below (Sections IX and X). VI. long-Term Bone Marrow Cultures
Dexter’s development of conditions for long-term bone marrow culture (74) represented a significant advance in experimental hematology. It permitted particular elements of the bone marrow microenvironment as well as hemopoietic progenitor cells to be isolated and studied in detail. For the first time, relationships between these could be followed in normal situations,
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with genetically defective animals, and following addition of transforming viruses. C. Whitlock and 0. Witte discovered that a few critical changes in the Dexter culture system resulted in selective outgrowth of pre-B cells (483), and this innovation is now being utilized in a number of laboratories. Many important aspects of bone marrow lymphocyte culture have been described in original articles, technical papers, and reviews by the originators of the method (484-486). Therefore, only new developments and the personal experiences of our laboratory will be emphasized here. A.
GENERAL
FEATURES OF
THE SYSTEM
The original approach utilized by Dexter involved establishment of adherent bone marrow stromal cells in preconditioned glass flasks (71, 72, 74). When most of the hemopoietic cells from the initial inoculum declined, he recharged the cultures with fresh bone marrow. This resulted in sustained growth of multipotential stem cells and myeloid cells, which was optimized by use of a 33" incubation temperature and selected batches of horse serum. Others found that inclusion of corticosteroids in the culture medium made it possible to use fetal calf serum and to establish long-term cultures with a single bone marrow inoculum (127). Many transplantation studies have demonstrated that cells with potential for forming B lymphocytes are present in Dexter cultures (81, 82, 168, 341, 386). However, a significant interval was required before they emerged after transfer and, in one study, it was suggested that expansion in marrow was required for functional maturity to be achieved (301). Cells with rearranged Ig genes, targets for Abelson virus transformation, or cells with defined lymphocyte characteristics are not readily demonstrable in Dexter-type myeloid cultures (67, 80, 82). An exception to this statement is one report demonstrating small numbers of TdTpositive cells (388; Section 111,C). While trying to find suitable conditions for studying early events in Abelson virus transformation, Whitlock and Witte discovered that by omitting steroids, using a relatively low concentration of fetal calf serum, adding 2-mercaptoethanol, and using physiological temperature, normal lymphocytes could be selectively grown (483). An adherent layer formed as in the Dexter system, and during the first weeks of culture, virtually all nonadherent and granulocytic cells disappeared. This was followed by the appearance of lymphoid foci and, most significantly, these lymphocytes maintained an absolute dependence for growth on the adherent stromal layer.
B. STRUCTURALORGANIZATION I N THE CULTURES Dexter and his colleagues, as well as many others, have described the composition and structural organization of myeloid long-term cultures (3-5, 15, 76, 141, 408, 435, 491). The adherent stroma can be multilayered, and it
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includes at least some of the stem cells which have long-term proliferative and differentiative potential (168). Adipocytes are conspicuous, but not essential in Dexter cultures, and several other cell types are present (443). Large cells which are thought to be critical to hemopoietic cell growth have been described as epithelioid, fibroblastoid, reticular, adventitial reticularlike, polygonal, stellate, and blanket-like cells. Established lines of such stromal cells have been described, but subclones of them are less effective in supporting hemopoiesis than the parent lines (207, 409, 505, 506). It may be that interaction between more than one cell type is required for efficient hemopoiesis to begin in uitro. The assumption has been that stromal cells in long-term culture are the functional equivalents to cells found within hemopoietic cords of bone marrow (239, 240, 477, 479). Less information is available about stromal cells in lymphoid long-term bone marrow cultures, and this is an active area of investigation in several laboratories. While Whitlock-Witte-type cultures are generally simpler in composition and less multilayered than Dexter cultures, a three-dimensional array is apparent in many areas. This is most easily appreciated by staining with fluorescein diacetate or labeled lectins and then examining the cultures without fixation (see Fig. 3A and B below). Reticular and overlapping cell processes can then be seen to provide niches for lymphocyte growth. Much of our characterization of lymphoid cultures has depended on subculture to glass chamber slides or onto coverslips following trypsin-EDTA treatment (493). This affords an excellent opportunity to observe the organization of stromal cells and follow the reappearance of lymphoid foci. The assumption is made that conditions pertaining in this situation accurately reflect those in the original plastic culture dishes. The emphasis of our experiments is on the cells which are in close proximity to lymphocytes, with the assumption that these provide essential replicative and differentiative stimuli, and the following is a brief account of those studies (510). Some lymphocytes are free in the culture medium or can be mobilized by gentle pipetting. Others are tightly bound to stromal cells, and still others are pressed beneath large cells (Fig. 2G). Proliferating foci of granulocyte progenitors form a ‘‘cobblestone” appearance in myeloid long-term cultures (76, 141); i.e., the cells are pressed tightly together and constrained from FIG.2. Structural organization in long term bone marrow cultures as seen by phase contrast (A, C, E), bright field (B, D, F), and transmission electron (G) microscopy. Foci of lymphocytes are recognized by immunoperoxidase staining with monoclonal antibodies to an antigen found on virtually all hemopoietic cells (B and F) and antibodies to Ly-5(220),which detects B lineage cells (D). Stromal cells which support lymphocyte growth completely lack these markers. A “cobblestone” area is shown in E and antibodies only partially permeated this area when fixed (F). Two lymphocytes are shown beneath the thin membrane of a stromal cell in G while another is attached to the surface and part of a third cell is seen detached.
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above by stromal elements. Similar areas were found in our lymphoid cultures, and, in some instances, it appeared that there was continuity to the outside (Fig. 2E,F). An antibody that recognizes all hemopoietic cells stained the adherent surface lymphocytes (Fig. 2B), but seemed to only partially permeate many of the cobblestone areas (Fig. 2F). Experiments with enriched stromal cells indicate that lymphocyte precursors actively crawl beneath the extended membranes of stromal cells (see below). This phenomenon has been previously observed and termed pseudo-emperipolesis (150, 300; see below). Dorshkind isolated and sectioned collections of lymphocytes that appeared to be completely encapsulated by the thin membrane of stromal cells (85). An analogy was drawn to previous descriptions of T lymphocytes found within thymic “nurse” cells (480). However, it is easy to imagine this resulting as the spread out stromal cells with bound lymphocyte foci are removed from culture dishes. Indeed, this may have previously been seen as aggregates in fresh bone marrow suspensions and described as phagocytosis of lymphocyte nuclei (314). We hypothesized that a gradient might exist in long-term lymphocyte cultures such that cells which were tightly associated to the stroma would be more actively proliferating and less differentiated. However, cell cycle activity, expression of the transferrin receptor, and display of several surface markers were similar when tightly bound and free lymphocytes were separated and characterized (493). This question has not been extended to include the cells which are imbedded or covered by stromal cells. The degree to which the cycle activity of lymphocytes changes following feeding and depopulation of the cultures should also be investigated, as it has with myeloid cultures (35). It has been said that a steady-state equilibrium is reached between newly formed and dying lymphocytes in long-term culture (486). By staining with propidium iodide, small numbers of dead cells can be seen, and they appear to be randomly distributed (unpublished observations). Rare phagocytosed lymphocyte nuclei were seen within macrophages by electron microscopy, and this would not be detectable by viable staining of intact cultures. Allen and Dexter have emphasized that myeloid growth in long-term cultures is nonuniform (5). Similarly, lymphocytes in culture are found in particular areas and only in association with certain stromal cells. Macrophages typically comprise half or more of the adherent layer and can be identified by immunoperoxidase staining (with F4/80, Mac-1, Mac-2, and M a c 3 antibodies), pinocytosis, phagocytosis, and staining for esterase and acid phosphatase (493). In addition, they take up the red fluorescent label, acetylated low-density lipoprotein (LDL), which also marks endothelial cells (346, 423, 510). Macrophages can be part of complex lymphocyte foci, but lymphocytes do not adhere directly to them. Also, lymphocytes are notably
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absent in areas of the culture that are heavily overgrown by macrophages (see also Section VII1,G). Lymphocytes are found adhering to or covered by a subset of large cells with thinly spread cytoplasms, which are similar in many respects to cells that have been frequently described in Dexter cultures (408; Fig. 2). These usually lack esterase and acid phosphatase, but many stain for alkaline phosphatase (493). They are never pinocytotic or phagocytic, and they can contain small lipid droplets. The cells are occasionally binucleated and the nuclei are large and oval, with distinct nucleoli, but unlike endothelial cells, they do not take up acetylated LDL (510). We characterized the large, lymphocyte-binding stromal cells by immunoperoxidase staining with a large number of monoclonal antibodies (510). The stromal cells bear H-2, but express few, if any of the markers commonly found on lymphoid, myeloid, and erythroid cells. A typical result is shown in Fig. 2B and D. The heat-stable antigen detected by M 1/69 antibody and Ly-5(220) antigen detected by 14.8 antibody stain the lymphocyte foci. However, cells underneath do not have reaction product and can only be visualized by phase contrast optics (Fig. 2A,C). More than 90% of the cells in a typical bone marrow cell suspension would be collectively detected by the monoclonal antibodies we used. Some of the stromal cells are probably lost when marrow is expelled, dispersed in liquid medium, and washed. However, we expect that large lymphocyte-binding cells are not very abundant in situ . The distinct absence of surface antigens, light density, resistance to drugs, and other properties of lymphocyte-binding cells can be used to enrich for them (510). It is clear that lymphocytes can bind to one cell and not to a neighboring one that is morphologically indistinguishable from it. Subsets of stromal cells can be appreciated on the basis of alkaline phosphatase staining (493), but this does not correlate with ability to bind lymphocytes. These observations suggest that functionally distinct microenvironmental elements may have a common origin. One could speculate that individual cells which look identical are in fact restricted to support of myelopoiesis or lymphopoiesis. It should also be noted that published descriptions of thymic stromal lines suggest similarities in morphology to bone marrow stromal cells (282). We have recently been able to isolate cloned stromal cell lines which bind normal and transformed B lineage lymphocytes. Several other laboratories have informally described lines which have at least some of the functions of adherent cell layers. These will be critical in determining the molecular requirements for B lineage precursor replication and maturation. In addition, we have found that normal or transformed B lineage lymphocytes bind to and can crawl beneath stromal cells within 2 hours in culture (Fig. 3F in 511). A seemingly identical interaction between T lineage precursors and a
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thymic stromal line has been described and termed pseudo-emperipolesis (150, 160, 300). It should also be noted that adherent stromal cells can interact with and provide growth assistance to tumor cells in culture (149,
179, 504, 506). C. LYMPHOCYTE ADHESIONTO STROMAL CELLS Adhesion molecules play critical roles in pattern formation during development, in controlling cell migration, and in determining the organization of cells within tissues (94, 142, 181, 361, 378). Particular ones of these have already been implicated in determining lymphocyte trafficking patterns (38, 116, 494), and it seems likely that special adhesion mechanisms would be a critical feature of hemopoietic microenvironments. Spaces between overlapping endothelial cells may physically govern when blood cells can enter venous sinuses and thereby leave bone marrow (433, 434). However, other factors, such as the surface density of particular differentiation antigens, might dictate the positioning of newly formed B cells for marrow egress. We have recently studied the bond(s) that exists between lymphocytes and stromal cells in long-term cultures (510). The strength of lymphocyte adhesion seemed to vary somewhat with the age and condition of the cultures. At times, cultures were largely depopulated of lymphocytes by pipetting alone, and in other situations, some stromal-associated cells remained bound even when the cultures were vigorously shaken on a vortex mixer. When all of the nonadherent lymphocytes were removed with complete medium, an additional population could be liberated by treatment with EDTA. Divalent cations may therefore contribute to the lymphocyte-stromal cell interaction. This is interesting in light of Ca2 (Mn2+)-dependent adhesion molecules which have been described in other systems (142). Stromal-associated lymphocytes have also been liberated by treatment with PI-specific phospholipase C (PI-PLC). This S . aureus-derived enzyme has been very beneficial in studies of molecules which utilize a unique form of membrane attachment (243, 246). As discussed in Section III,F, a similar +
~~~~~
~
FIG. 3. Overlapping cell bodies support foci of lymphocytes in long-term bone marrow cultures. Fluorescent Con A was used to stain cell membranes in (A) and the nuclei were revealed by counterstaining with propidium iodide. Viable cells take up fluorescein diacetate and convert it to free fluorescein (B). Many adherent cells, including those bearing lymphocytes, were recognized by a rabbit antiserum to NCAM as shown by phase contrast (C) or bright field microscopy (D). The normal rabbit serum control is shown in (E). Pre-B lymphoma cells can avidly bind to stromal cells and often crawl beneath them (F). In this example of pseudoemperipolesis, more than 100 702/3cells were under a single large stromal cell after 2.5 hours of coculture. Ten surface adherent lymphocytes are more refractile in this phase contrast photomicrograph.
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linkage can be utilized by some lymphocyte antigens, including Thy-1, RT-6, Qa-2, J l l D , and ThB (203, 424). Treatment of lymphocytes with PIPLC removes such markers in a highly selective way (424). Experiments are under way to establish if any of the known lymphocyte antigens participate in bone marrow lymphocyte adhesion (P. L. Witte et al., unpublished observations). It has been difficult to demonstrate the expression of any surface antigens other than H-2 on the large stromal cells to which lymphocytes are attached in long term culture. One potential exception is illustrated in Fig. 3 (C and D) with a conventional rabbit antiserum to NCAM and a very sensitive immunoperoxidase assay (unpublished collaborative studies with Dr. Melitta Schachner). Cells other than stromal cells were positive to a lesser extent and this will have to be further investigated with monoclonal antibodies. However, it is noteworthy that a form of this neuronal adhesion protein family has been recently shown to be membrane anchored via phosphatidylinositol (146b, 147b, 373b). Fibronectin has been found in Dexter cultures, and stromal cell layers become quickly established in dishes that have been precoated with bone marrow extracellular matrix (15, 30, 357, 508). We were able to identify fibronectin in long-term lymphocyte cultures, but in preliminary experiments, the fibronectin-related peptide Gly-Arg-Gly-Asp-Ser (32, 143) did not interfere with the reattachment of cultured lymphocytes to established stromal cells. These observations indicate that while fibronectin molecules may contribute to the adherence of cells to the culture dish, other mechanisms may be involved in lymphocyte binding. To our knowledge, these studies are the first to consider adhesion mechanisms which might retain differentiating B lymphocyte precursors within microenvironmental niches. However, it is important to remember that lymphocytes probably move within marrow hemopoietic cords. If proliferating B lineage cells were rigidly held for a few days in association with a stromal cell, we would expect to find follicular arrangements, and these have not been described in intact mammalian bone marrow (Section II). We can hypothesize that one or more PI-linked and divalent cation-dependent structures on lymphocytes recognize heterotypic structures on stromal cells and that the density of these molecules is subject to change. Chemical modification (glycosylation) of surface structures is thought to he important in controlling the strength of neuronal adhersion (94), and we already know of antigen families, such as Ly-5, which can be expressed in different size forms (Section 111,B). By analogy to lymphocyte-endothelial cell recognition studies (33, 37, 38, 355), there might be soluble substances which compete with or enhance the adhesive bond. There is reason to believe that the same structural gene
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might be used by different cell types to make molecules which are secreted, premanently membrane anchored, or linked by PI. In the case of an adhesion protein, this would seem to provide considerable flexibility. For example, a PI-linked structure could be released through endogenous phospholipase action or simply made as a secreted, rather than membrane-bound form. Thus, the soluble form of a receptor/adhesion molecule could become an inhibitor of binding, and the relative strength of adhesion would be determined by the balance of these reactions. It is hoped that standardized assays can be constructed to study the specificity, strength, and molecular nature of adhesion of lymphocyte precursors to bone marrow stromal cells, as has been done in other systems (94, 407, 494). D. CHARACTERISTICS OF CULTURED LYMPHOCYTES Lymphocytes grown in Whitlock-Witte cultures are uniformly small to medium in size, but can be heterogeneous in other respects. Considerable lab-to-lab differences are apparent in the types of cells which can be used to initiate the cultures and can subsequently be maintained. However, technical explanations, such as variability in calf serum batches, can be applied in some instances, and it is already possible to make some generalizations.
1. Immature B cells and pre-B cells can extensively self-renew in longterm culture. This was first demonstrated by Whitlock et al., who isolated clones of these cells from established cultures and studied their subsequent behavior on repeated passage and after transformation (484). Dasch and Jones were able to initiate cultures with enriched populations of small pre-B cells taken directly from marrow (57). In another recent study, cells with definitive B lineage characteristics were not effective in initiating cultures, but once these emerged from putative earlier precursors, they could be separated and maintained (289). 2. Progenitors that can be established in culture are present in fetal, neonatal, and adult tissues. They are probably also representative of different stages of B lineage differentiation. Bone marrow from young weanling mice of a particular strain was originally found to be optimal for starting Whitlock- Witte cultures (483, 485). However, with an appropriate adherent stromal layer, cultures can be established from fetal liver or 12-week-old adult bone marrow (57, 66). Examples of “early” cell types which are capable of establishing in culture and giving rise to B lineage cells include those present in mice with severe combined immunodeficiency disease (492), cells maintained under Dexter culture conditions (67, 80), and sorted Thy-1 antigen-positive cells (289). In one laboratory, relatively ‘‘late’’ precursors that were ThB , sIg- could be sorted and shown to immediately replicate when placed with stromal cells (57). However, in a similar study, it appeared +
+
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that cells with long-term replicative potential had to be selected from a more immature compartment before self-renewal of the more differentiated cell types could proceed (289). One recent report indicates that some cells with T lymphocyte lineage differentiation potential were maintained in a long-term culture (86). Further improvements in culture methodology could someday make it possible for any B lineage cells to be established in long-term cultures. There is no direct evidence for the existence of lymphoid stem cells (187; Fig. I), but this might provide an approach to their identification. 3. Once established, at least some progression of immature cells through the lineage occurs. This was first demonstrated by Whitlock and colleagues with cloned lymphocytes that were transformed by Abelson virus (484). More recently, emergence of pre-B and B cells has been documented in cultures initiated with early types of cells (67, 80). In our experience, the composition of established cultures has been similar regardless of whether fetal, adult, B cell-depleted, Ly-5(22O)-depleted, or whole bone marrow has been used to start the cultures (198). As previously stressed by Whitlock and colleagues (486), numbers of B cells tend to increase with time. Also, large amounts of the BP-1 antigen are initially expressed, but eventually lost by most cultured lymphocytes (492). This would be consistent with a maturation step, since the BP-1 antigen is found on bone marrow, but not peripheral lymphocytes (50). 4. Young cultures are polyclonal, but they become less heterogeneous with time. This was first demonstrated in two-dimensional gel analyses of Ig made by long-term cultured cells (483). A similar conclusion results from Southern blot analyses of Ig heavy chain gene rearrangements (66, 492). While some germ line DNA can be retained for a considerable period, relatively few rearrangement products are often found in individual cultures. It might be instructive to sequentially follow large numbers of individual cultures and especially interesting to learn if V, gene selection is random. 5. Replicate cultures established from a common bone marrow suspension can differ considerably. As stressed for both the Dexter and Whitlock-Witte culture systems, cell growth occurs in focal areas within dishes. Many of the observations discussed above indicate that a selection process is probably operative initially as well as in favoring clonal dominance with time. This is compatible with our finding of considerable interculture variability and indications that the characteristics of individual culture dishes were determined during a discrete period, early in culture (493). 6. Cells with unusual characteristics can be found in long-term bone marrow cultures. Spontaneous transformation has only been described for clones of long-term cultured cells that had apparently been maintained for some time (485). Neither Dexter nor Whitlock-Witte cultured cells cause tumors in viuo; the hemopoietic cells maintain an absolute dependence on stromal
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elements for growth, and it has been customary to stress how “normal” they are. However, this does not mean that possibly rare cell types in bone marrow cannot expand in culture. As discussed above, expression of the Ly-5(220) antigen can be quite variable and frequently absent on cultured pre-B cells. In contrast, virtually of of the c p + , sIg- cells and immediate precursors of functional B cells in normal adult tissues express this marker (195). Also, our studies with SCID mice indicate that cells with defects in the process of rearrangement or utilization of Ig genes are not selected against in culture (Section VII1,B). 7. Long-term cultures contain at least some cells with potential for normal differentiation. Cells taken from Whitlock-Witte cultures have given rise to antigen-responsive cells when placed in irradiated and immunodeficient recipients (213, 292, 341; unpublished observations). The cultures also contain small numbers of cells which can respond to mitogens in semisolid agar cultures (CFU-B). However, most descriptions indicate that the majority of cells arrest at or before a relatively immature B cell stage, and it has generally been difficult to make them respond in culture to any well-defined, normal differentiation stimuli (57, 292; unpublished observations by G. Lee and P. L. Witte). It has also been emphasized that the pattern of reconstitution of immunodeficient animals obtained with cultured cells is quite different from that observed with freshly isolated bone marrow (341). These observations probably indicate that the conditions of culture do not provide a complete marrow microenvironment. It may also be that long-term propagated lymphocytes represent unusual populations that have not experienced the selective pressures normally operating in vivo. This is an advantage for studies of abortively rearranged or genetically defective lymphocytes (see below), but a potential limitation to studies of normal functional progenitors. 8. Undefined components of fetal calf serum are critical in establishing long-term cultures. While most culture systems can be optimized by careful selection of fetal calf serum, the requirements for establishment of Whitlock-Witte cultures must be unusually stringent. We found that while some serum lots supported macrophage outgrowth and others encouraged granulocyte production, relatively few consistently allowed lymphocyte cultures to be established (493). However, two additional serum lots were obtained at different times from the same supplier, who attempted to select one that best matched the qualities of the original batch. Essentially identical results have been obtained with this material, and once cultures were established, it was possible to hold them for short periods in serum-free medium or for longer periods in unselected serum. Until the serum requirements for lymphocyte growth become better defined, it would be helpful to exchange assay results on lots that have been successfully employed.
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E. RECE:NTINNOVATIONS In addition to developing the method for long-term lymphocyte culture, Whitlock and colleagues described a number of ways of manipulating this system. For example, initial low cell densities were utilized to advantage to produce stromal cell layers which lacked lymphocyte precursors (485). These were then used for lymphocyte subculture, for recovering frozen cells, and for cloning. More recently, it has been possible to produce hemopoietic progenitor-depleted stromal cells from Dexter or Whitlock-Witte cultures by treatment with mycophenolic acid in uitro (87, 167). In our laboratory, lymphocytes have been preferentially killed by treatment with deoxyguanosine or 5-fluorouracil (P. L. Witte, unpublished observations). Preparation of lymphocyte suspensions which lack stromal cells has been achieved by passage through nylon wool or Sephadex G-10 columns (167; P. L. Witte, unpublished observations). However, the most important technical innovation involves shifting of cultures from Dexter to Whitlock-Witte conditions. Dorshkirid initiated Dexter cultures and showed that recognized progenitors of B cells were absent (80). H e then changed the cultures to Whitlock- Witte conditions and monitored the emergence of B lineage lymphocytes and clonable B cells, which occurred together with loss of granulocytes and their progenitors. A change in the morphology of the adherent layer was also described. Denis and Witte (67) studied population changes when cells maintained in Dexter cultures were transferred to adherent stromal layers under Whitlock- Witte conditions. As myeloid stem cells declined, there was an appearance of pre-B and B cells as well as targets for Abelson virus transformation. They also demonstrated by two-dimensional gel and Southern blot analyses that multiple clones of B lineage cells must have been formed in the secondary cultures. Adherent layers maintained under Dexter culture conditions release at least one type of myeloid colony-stimulating factor (CSF), whereas stromal cells in Whitlock-Witte cultures do not (167). However, CSF was made when the latter was shifted to Dexter conditions and the stroma was functionally capable of supporting myelopoiesis when recharged with nonadherent hemopoietic cells. These experiments are consistent with the possibility that multipotential stem cells and/or very early B lineage progenitor cells are maintained in Dexter cultures. These appear to become irrevocably committed to the B lineage when steroids and horse serum are removed, the temperature is raised, and a low concentration of a very carefully selected batch of fetal calf serum is added. It will be extremely important to learn what changes must take place in microenvironmental cells to bring about this critical transition and whether individual stromal cells can alternatively
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support myeloid and lymphoid differentiation in the two situations. Recent studies with cloned stromal cell lines suggest this may indeed be the case (K. Dorshkind, personal communication). Goldschneider and colleagues have devised culture strategies for propagating cells that seem to be intermediates between those found in Dexter and Whitlock-Witte cultures (129, 146, 266). In this situation, TdT+ lymphocytes, which are normally infrequent in Dexter cultures (388), expand to become the predominant lymphocyte population. Successful growth of TdTbearing cells seems to require a particular type of accessory cell which is conspicuous in rat bone marrow. The Ly-5(220)marker is expressed by many of these lymphocytes, but unlike the situation with Whitlock-Witte cultures, pre-B and B cells are never seen (266; I. Goldschneider, personal communication). It would be interesting to learn if further differentiation would occur if such cultures were established and then shifted to WhitlockWitte-type conditions. In summary, long-term culture methods offer feasible approaches to a number of questions. We should not only be able to identlfy stromal cells which contribute to the microenvironment required for B lymphocyte formation, but study adhesion mechanisms which make short-range interactions with them possible. The lineage derivation, life span, and regulation of stromal cells themselves is an interesting issue, and long-term cultures provide a place to test the differentiation potential of selected populations of progenitor cells. There are still a number of uncertainties about the selective pressures and inductive stimuli which influence B cell precursors in these cultures. Also, the lymphocytes that grow may not be uniformly representative of cell sets found in normal hemopoietic tissues. However, the artificial nature of the cultures can be used to advantage in studies of genetically determined abnormalities (Section VIII,B), and there are likely to be many other experimental applications for these methods. VII. An Inducible Cell line
Myeloid leukemia patients frequently have expanded clones of apparently normal hemopoietic cells. These presumably all derive from one stem cell which achieved a proliferative advantage during a premalignant period (101, 102). Also, patients with myeloma have normal-appearing pre-B cells which are clonally related to the terminally differentiated tumor (210). Such observations indicate that transformed or partially transformed cells retain differentiation potential and that they are responsive to normal physiologic stimuli. The same conclusion results from many studies of the effects of hemopoietins on myeloid progenitor cells from leukemic patients or transformed cell lines which represent them (271).
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Transformed clones of B lymphocyte lineage cells can spontaneously undergo rearrangements of Ig genes, and this fact has been useful in understanding the order in which these critical differentiation events normally take place (7, 208, 359, 429). Other changes have been inducible in a variety of hemopoietic tumors and cell lines with natural and artificial stimuli (22, 51, 109, 137, 227, 229, 290, 291, 325). Responsive clones provide homogeneous models for studying factors, receptors, transmembrane signaling, and gene regulation mechanisms. A cell line developed in our laboratory has been useful in all of these respects, and it provided one of the first pieces of evidence that c p + , sIg- pre-B cells were the immediate precursors of sIgM+ B cells. The 70213 tumor arose in a thymectomized, methyl-nitrosourea-injected BDF, mouse and readily adapted to growth in culture (325). Although all of the cells contained p heavy chains of IgM, very few had sufficient amounts of complete surface molecules to observe by immunofluorescence microscopy. However, this was reversibly induced in culture with lipopolysaccharide (LPS), and there was reason to believe that the line was particularly responsive to stimulation during certain phases of the growth cycle (325, 375). Subsequent studies revealed small amounts of p heavy chain on the surface of 70213 cells (254). This can be appreciated by flow cytometry on current versions of the line and the p chains apparently coalesce in the absence of light chains into a microscopically visible spot when incubated with dextran sulfate (327). Surface K light chains are present on small numbers of uniriduced 70213 cells and in insufficient quantities to be directly demonstrable by immunoprecipitation (253, 254). As in other pre-B cell tumors, RNA and protein synthesis for both secreted and membrane type p chains occur (339). However, no is secreted and small normal pre-B cells in bone marrow probably only make the membrane form (440). Expression of K light synthesis and surface display has been followed in many studies. While one of the K light chain gene-bearing chromosomes is functionally rearranged, the other allele is in germ line configuration (255). However, both genes are activated for transcription on exposure of the cells to LPS (339). An early change occurs in chromatin structure such that nuclease-sensitive sites are exposed in the K enhancer region of the gene (336, 337). In 702/3 cells and other pre-B cell lines, protein synthesis is not required for initiation of K messenger RNA synthesis, even when both alleles are unrearranged (297). In fact, brief interruption of protein synthesis induces transcription, and this suggests that K synthesis may be down-regulated in pre-B cells by a labile repressor protein (469). Molecules that may mediate positive signals for Ig transcription have recently been identified (250, 392; Section IV). These bind specifically to enhancer andlor promoter regions, and at least one dramatically increases following LPS induction in
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70Z/3 cells (393, 422). A trans-acting factor(s) which signals heavy chain transcription is constitutive in 70Z/3 cells and increased by LPS (250). Some studies of the mechanisms of transmembrane signaling have utilized the 70Z/3 cell line. The Na+ /H transport inhibitor, ameloride, inhibited K induction by LPS, and some ionophores either enhanced expression or synergized with other stimuli (364, 421). With the particular clone and fetal calf serum utilized by Rosoff and Cantley (365, 366) phorbol ester induced unique early changes in free intracellular Ca2 , PI turnover, and K synthesis. Cells maintained here are not consistently induced by phorbol alone, but phorbol enhances stimulation when added together with LPS (unpublished observations). It seems likely that changes in free Ca2+ concentration, protein kinase C activation, and transmembrane ion fluxes are all part of the initial response of 70Z/3 cells to stimulation. At least two T cell-derived lymphokines induce changes in the 70Z/3 line. Several studies have employed T cell-conditioned medium to stimulate sIgM expression, and, in some of those circumstances, interferon-y probably mediated that effect (118, 329, 401, 420, 475). Recent studies with B cellstimulating factor-1 (BSF-1 IL-4) have shown that this T cell product is not limited in its action to mature B cells (100, 125, 233). We have found that while K expression is not induced by BSF-1 in 70213, quantitative changes in the density of several other surface antigens is dramatically affected (509). This indication that pre-B cells can recognize BSF-1 (IL-4) is consistent with a recent report describing induction of class I1 expression in another pre-B cell line (349). 70Z/3 cells have at least two additional functional receptors. Native interleukin 1, recombinant IL-la, or IL-lP stimulate K expression (118, 420, 509). The same is true for at least one of the factors found in serum from young NZB mice (177; see below). In contrast, factors excreted by a patient with cyclic neutropenia seem to act only on earlier precursor cells, and there was little, if any, induction of the 70213 line (225; Sections VII1,E and IX). Variant subclones of 70Z/3 cells have been described which are selectively unresponsive to stimulation by LPS and/or T cell-derived mediators (475). This again suggests that multiple receptors and pathways of induction are available to pre-B cells. Premanent induction of K synthesis and surface IgM expression has not been induced in this line with any stimulus. Cloned progeny of LPS or IL-1-induced cells had only background levels of sIgM when grown out in the absence of stimuli (118). The cells share many markers and characteristics with normal pre-B cells that are just at the threshold of becoming B cells. However, they are more like B cells with respect to nonexpression of the N-myc cellular oncogene protein (503). As a technical precaution, it should be mentioned that K synthesis is induced by a mycoplasma product (404). +
+
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Studies with this line may be informative about signals mediated via direct cell contact. Coculture with a T hybridoma cell induced an apparent isotype switch to membrane IgG expression (165).This seemed to require direct cell contact and did not result in Ig secretion. We have recently observed changes in the density of several lymphocyte antigens during coculture of 70Z/3 cells with bone marrow stromal cell lines (C. Pietrangeli and P. L. Witte, unpublished observations). In uiuo localization and growth of 70213 cells has recently been studied (342). A portion of the cells localized to bone marrow and half as many were found in the spleen 24 hours after intravenous injection. There was also preferential growth in bone marrow, and this eventually displaced all normal hemopoietic cells. In contrast to normal lymph node cells, essentially no labeled 70Z/3 cells were recovered in lymph nodes or Peyer’s patches. They also did not seek the thymus and did not recirculate in blood. It seemed possible that 70Z/3 cells would respond to some of the normal differentiation stimuli in oiuo. However, although there were changes in expression of some surface markers, no K induction was observed. It is possible that the tumor cells we recovered and characterized were not correctly positioned in microenvironmental niches, and there might also be competition for rare inductive signals. Another explanation relates to negative feedback control mechanisms, and we now know that normal mouse serum will prevent induction of K expression in uitro (S. I. Hayashi and G. Lee, unpublished observations). It is interesting to consider that B lymphocyte formation might only proceed within areas where concentrations of putative inhibitors are low. VIII. Some Genetically Determined Defects
Genetic defects have been extensively exploited in biology, and it is clear that some aspects of B lineage differentiation may only be revealed in this manner. For example, functional and developmental relationships between cells can often be inferred from genetically determined abnormalities. We have also found it possible in studies of patients and experimental animal models to detect biologically active substances which are normally probably made in only trace quantities. Some of those experiences will be recounted in this section, but before doing so, it is important to define our use of the words defective and abnormal. We make liberal use of these terms to describe mice that differ dramatically from many other common strains of laboratory animals. However, the distinction is not always clear between a simple genetic polymorphism, which might even have survival value, and a defect resulting in disease. For example, we can catalog a list of unusual features in New Zealand strain mice, but it is not clear if any or all of them
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contribute to autoimmune disease. Nonresponsiveness of C3H/HeJ mice to endotoxin could be beneficial to the animal, but subtle deficiencies in the immune system become serious when a second mutation is introduced. Although it does not diminish their practical usefulness for experimentation, we should also acknowledge that many of our animal models probably do not have exact parallels in human disease.
A. W/W ANEMIC MICE In homozygous form, semidominant mutations at the W locus of chromosome 5 cause macrocytic anemias of varying severity, and this has long been a model for deficiencies affecting multipotential stem cells (372). Isolation and expansion of clonally marked stem cells depended on use of these animals (1, 77). Also, the progeny of normal cells injected into WIW recipients without irradiation eventually replace most blood cells, including B and T lymphocytes (140). While it was known that adult mutant mice have normal immune responses (267), little information was available about the immediate progenitors of B cells. We found that phenotypically defined B lineage cells were normal in marrow of adult W/Wv mice (224). Also, functional B cells were formed in normal numbers when B ceil-depleted marrow was held for 48 hours in liquid culture. The kinetics of small lymphocyte production were also normal (311). However, deficiency of a presumably earlier type of B cell precursor was apparent when B cell formation was assessed by transfer of WIW" marrow to irradiated recipient mice. Studies with embryos revealed that although the incidence of B lineage cells was normal, their total numbers in liver were, like other hemopoietic cells, very diminished by the W mutation. We concluded that the immediate progeny of stem cells may be infrequent in these mice, and especially so during embryonic life. If so, compensatory mechanisms must allow cells further along the differentiation pathway to reach normal numbers. Mutations at the W locus differentially affect the mature cells of various lineages. Mature mast cells are strikingly absent, erythrocytes are present but reduced in number, and completely normal B lymphocytes are maintained (199, 372, 410). This could reflect the number of intermediate stages and replicative possibilities in the different lineages. A quantitative deficiency in production of myeloid cells has been observed in Dexter-type long-term bone marrow cultures (73, 182). Many interesting questions remain about relationships between multipotential stem cells and cells which are committed to the B lymphocyte lineage (Fig. 1). W/W" mice should continue to be useful for addressing those issues.
B. SCID MICE Bosma and colleagues developed a murine model for severe combined immunodeficiency disease (SCID). This recessive, single gene mutation
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arose in C.B-17 mice and has now been mapped to chromosome 16 (21; M. Bosma, personal communication). Homozygous defective animals have a very small thymus and virtually no B or T lymphocytes (21, 53). Natural killer cells are present in normal numbers, and it is noteworthy that Ig and T cell receptor genes are unrearranged in those cells (84, 131, 450). Myeloid progenitor cells and antigen-presenting cells are not affected by the mutation, while stimulator cells for Lm-1 responses and truncated p chains are undetectable (54, 83). Transplantation results are consistent with the mutation causing intrinsic abnormalities of hemopoietic cells (21, 53); i.e., engraftment of T and B lymphocytes and restoration of immune functions result from transplantation with normal hemopoietic cells and, reciprocally, mice irradiated and given SCID bone marrow cells are immunodeficient (83, 111). SCID mice must have small numbers of B lymphocyte lineage progenitor cells. This conclusion follows from our findings of a few cells in SCID marrow bearing B lineage antigens detected by monoclonal 14.8 [Ly-5(220)]and BP-1 antibodies (492). Furthermore, a majority of long-term cultures prepared with SCID bone marrow produced lymphocytes and, once established, the cells proliferated at a normal rate. Cultured SCID lymphocytes were indistinguishable from normal cells by morphology, ultrastructure, or staining with 13 monoclonal antibodies to lymphoid and hemopoietic cell antigens. Individual BALB/c cultures typically undergo population changes which could reflect either clonal succession or differentiation (493). This was also the case with cultured SCID lymphocytes. For example, the BP-1 marker is initially expressed on BALB/c cells in long-term culture, but then often lost after 9 to 15 weeks. A relatively high density of BP-1 was found on SCID lymphocytes and this was also lost with time (492). The incidence of cells in SCID marrow which can be transformed by Abelson virus and give rise to B lineage cells is relatively normal (512). Also, engraftment of normal donor lymphocytes in SCID marrow is minimal unless the transplant is preceded by sublethal irradiation (111).A small subset of SCID mice eventually synthesize Ig, and some are able to reject allogeneic skin grafts (21). All of these findings suggest that multipotential stem cells are present in SCID mice and that some of them give rise to lymphocyte-committed progeny cells. Accurate estimates of the population sizes in these compartments are not available, and this might be influenced by environmental antigens (M. Bosma, personal communication). Four approaches have been used to study Ig and T cell receptor gene rearrangements in the rare lymphocytes of SCID mice. The results indicate that these processes can successfully occur, but only rarely do so. No rearrangements of receptor genes were found among fetal liver and bone marrow hybridomas or thymus cells of SCID mice (389). In contrast, both IgH alleles had undergone rearrangement in nine Abelson-transformed cell lines that
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were studied. Similarly, both copies of T cell receptor p chains had rearranged in thymic lymphomas from SCID mice. Deletions of J H and TP segments were common in these lines. Our results with nontransformed SCID-cultured bone marrow lymphocytes revealed an intermediate situation (492). While at least the first rearrangement step had taken place in most SCID cultures, cells from all but two retained some hybridizable J H segments. The patterns were consistent with there being limited population diversity in lymphocytes from most SCID and some BALB/c cultures. No evidence of K light chain rearrangements were seen. B cells and pre-B cells were found in all of more than 100 long-term cultures of normal BALB/c mice. In contrast, in 11 independent experiments involving SCID bone marrow cells, cytoplasmic p mice positive pre-B cells were found in only 1of more than 40 cultures examined (492). It has been hypothesized that the product of the mutant SCID gene affects a recombinase mechanism which is commonly used by differentiating T and B lymphocytes (389). The data are consistent with errors occurring during or subsequent to an attempt to configure D and JH segments for transcription. This step and the subsequent joining of VH and D gene segments is normally error prone, and one of two newly formed B lineage cells may lack an effective receptor for antigen (7). It is interesting to speculate that special mechanisms exist for sensing and eliminating such abortively rearranged clones. They might expand along with other lymphocytes in long-term culture because such selection processes are not operative in vitro. C. CBA/N MICE The X chromosome-linked immunodeficiency of CBA/N mice has been extensively studied and discussed (384). Comments here will be restricted to effects on B cell precursor populations. Numbers of B cells, pre-B cells, and Ig-negative, Ly-5(220)+ cells are all normal in the bone marrow of CBA/N mice (196). This has been confirmed and extended in a study showing that the rate of production of small B cells is also within the normal range (356). However, the development of B cells in these animals is strikingly dependent on thymus-derived cells. This conclusion results from studies with doubly defective nude.Xid mice and adult thymectomized mice given CBA/N bone marrow (284, 413, 495, 180b). This is surprising, since the humoral immune system develops relatively normally in athymic mice which do not have other defects. One explanation would be that B cell precursors are normally responsive to multiple differentiation stimuli, including T cell-derived factors. Receptors for non-T cell-derived factors may be nonfunctional or absent from the abnormal B lineage cells of CBA/N mice, giving them no alternative pathways for development. We have recently initiated long-term bone marrow cultures from Xid
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mice. Cultures presumably provide a relatively T cell factor-independent environment, but no B lineage deficiencies were noted. On the contrary, foci of lymphocytes appeared earlier and expanded more rapidly than in cultures of control CBA/H, BALB/c, NZB, or C57BL/6 marrow (513). Experiments are now being done to determine if this characteristic relates to lymphoid progenitors or stromal cells on which their growth depends. D. NZB MICE Production of autoantibodies is a key feature of New Zealand strain mice, and it was known before we began our studies that hypersecreting cells appear early in life and in a thymic-independent manner (256, 288, 438). Dysregulation of bone marrow events might therefore not be surprising. However, our initial and unexpected finding was that adult NZB mice were extremely deficient in phenotypically defined pre-B cells and cells which could give rise to B cells in short-term cultures (175). It was only in very young NZB mice that we could demonstrate elevated numbers of B lineage precursors detected in this way. Dysregulation of the humoral immune system was apparent as early as 1415 days of gestation in NZB embryos (172). At that time, substantial numbers of committed progenitors that could give rise to functional B cells were already detectable. Several more days of gestation were required for these numbers to be attained in CBA/H embryos. A series of studies done with 4-week-old NZB mice suggested that microenvironmental elements might be hyperactive at that time (176). At the cellular level, Sephadex G-10 column-depleted bone marrow cells from young NZB mice appeared to augment the maturation of normal B cell precursors in cocultures. Serum from 4-week-old NZB mice contained factors which have the same effect. U p to a 1OOO-fold enrichment of this biological activity was achieved, and two similarly acting glycoproteins were resolved. One has an isoelectric point of 3.5 and an apparent size of 17 kDa, whereas the second is 15 kDa and has isoelectric points of 7.8 and 8.4. Both were stable to low pH and denatured by heat, trypsin, or urea (177). The biological effects of NZB serum factors were investigated in some detail (177). It is possible that they interact directly with precursors which are relatively close to becoming functional B cells (Section IX). This conclusion results from the finding that at least one of them can induce K expression in the cloned 70Z/3 pre-B cell line (177; G . Lee, unpublished observations). The factors were effective at increasing the emergence of functional B cells when placed in culture with sIg-, and G-10 column passed bone marrow. However, this did not happen when recognizable B cell progenitors were removed from the cell suspensions with monoclonal I,y-5(220) antibodies (177; H. Jyonouchi, unpublished observations). The latter result provides a
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marked distinction from a quite different type of activity isolated from a cyclic neutropenic patient (see below). Short-term exposure of bone marrow cells to NZB serum factors had no effect and their continuous presence may be required (176). Enriched preparations of factors did not have significant activity when placed in standard assays for IL-1, IL-2, IL-3, CSFs, or interferon (177). Also, no effects were seen on the replication of mature B cells in mitogen-containing semisolid agar cultures. However, there was some indication that one of the factors might stimulate splenic B cells which had been activated by anti-b-coated beads to secrete Ig. The NZB serum factors were not detectable in serum of adult NZB mice or in serum from several other strains of mice (177).This was true even when the serum was put through a purification procedure that should have enriched for it. Such factors may normally be made in trace quantities and in immediate proximity to cells which are responsive to them. This would make them excellent candidates for mediators of local regulation and possibly lineage-specific regulation. However, it must be stressed that these substances have not been purified to homogeneity, and the experimental designs in which they were studied do not rule out their possible influences on non-B lineage cells. Adult-irradiated recipients of young or adult NZB bone marrow briefly had numerous B cell precursors and detectable serum factors (H. Jyonouchi, unpublished observations). However, this was transient, and long-term normal recipients of NZB transplants were like adult NZB mice, i.e., deficient in pre-B cells and not having serum factors (174, 196). These observations indicate that the processes of B cell formation are precocious and excessive for a time in mice of this strain. This is followed by a period of apparent inactivity in the bone marrow, but this can be briefly reactivated by transplantation. The Xid mutation of CBA/N mice diminishes autoantibody production when congenic on NZB background mice (432). Young NZB.Xid mice showed no indication of hyperactive bone marrow regulatory elements and did not have detectable serum factors (176). We interpreted these results within the context of other studies which suggest that the Xid mutation has subtle effects on the hemopoietic microenvironment as well as the B lymphocyte lineage (40, 124, 305, 340, 385). A series of transplantation experients revealed that age-related environmental influences were also critical in determining when autoantibodies are made in NZB mice (25). Serum factors like those found in young NZB mice were not demonstrable in autoimmune MRL/Lpr or motheaten mice, suggesting that these diseases have a very different basis (H. Jyonouchi, unpublished observations). Some similarities were found between NZB and BXSB mice, but in the latter case, a very strong influence of sex was seen (178). Pre-B cell numbers quickly
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became subnormal in male BXSB mice, whereas an apparent hyperactivity of lymphopoiesis was characteristic of female mice of this strain. The results of a series of transplantation experiments are consistent with there being intrinsic hemopoietic abnormalities related to the Y chromosome-linked accelerating factor and not simply to sex hormones. Studies with NZB mice revealed a very interesting genetic polymorphism associated with myeloid progenitor cells. We first found that bone marrow cells from these mice responded very poorly to one form of myeloid CSF (191). Those results have now been repeated and extended with purified and recombinant IL-3 (509). A number of strains of mice were surveyed and found to range widely in responsiveness to this multipoietin. NZB mice were repeatedly backcrossed to CBA/H, with selection at each generation for mice which gave low colony responses. At each step, a range of responses was observed and a very low responder phenotype was not successfully transferred to the CBA/H background. These results suggest that there must be multigenic control over this characteristic. Low IL-:3 responsiveness pertained to formation of colonies in semisolid agar cultures and proliferation in liquid medium, but there was no correlation with the ability of spleen cells to make IL-3 when stimulated with Con A. There was conspicuous absence of colonies with neutrophil granulocytes in NZB cultures, and responses to recombinant G/M CSF or to CSA-1 were unusually high (509). The mice have relatively normal blood leukocyte counts, and these results suggest that alternative pathways of neutrophil formation are being utilized. A single multipoietin such as IL-3 is probably not the sole regulator of all hemopoietic lineages, and the relative abundance of different types of progenitor cells can differ markedly in various mouse strains (Section IX). NZB mice have recently been studied with long-term bone marrow cultures (493). There was a tendency for fewer lymphocytes to be made in NZB than BALB/c cultures and lymphoid foci were less frequent. However, surface marker expression on long-term cultured NZB lymphocytes was not remarkably different from that seen with other strains of mice. It has been possible to extensively subculture adherent, nonlymphoid cells from NZB cultures, and clones of these “stromal” cells are now being characterized (198; P. L. Witte, unpublished observations). Cloned microenvironmental elements from such genetically abnormal mice could provide very important models for understanding the regulation of B lymphocyte formation.
E. CYCLJCNEUTROPENIA A 2-yearold child with cyclic neutropenia was found, as expected, to have cyclic oscillations of myeloid progenitor cells (95). However, a novel finding was that absolute numbers of bone marrow lymphocytes cycled from normal
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to greatly elevated values. These were c k + pre-B cells, and their increases correlated with CFU-c regeneration. Myeloid CSFs and transforming growth factors were known to be excreted in human urine, and serial samples of urine could be obtained without threat to this patient. The hypothesis was tested that dysregulated lymphopoiesis might be correlated with a soluble mediator. Indeed, biologically active material was periodically excreted, and this was initially shown to augment formation of B cells in cultures of normal, B cell-depleted human bone marrow (225). Fortunately, the factors were also active on murine cells, and this permitted a more in-depth study. At least two factors were excreted by the patient, and their presence corresponded to times when bone marrow pre-B cells would be expected to be increasing (225, 514). The factors had isoelectric points of -6 and were resolved by gel filtration chromatography into two size ranges (45 kDa and 16 kDa). None of this material significantly induced K expression when added to the 70Z/3 pre-B cell line, and did not promote survival or differentiation of enriched B cell precursors (sIg-, Ly-5(220)+ cells). In contrast, they caused substantial production of Ly-5(220) cells and pre-B cells when added to cultures of 14.8-depleted marrow. These findings suggested that the factors were unique in supporting the growth and maturation of early B lineage committed progenitor cells. Screens of various preparations containing IL-1, IFN-y, IL-3, myeloid CSFs, or NZB serum factors did not have this effect. The two sized fractions of cyclic neutropenia factors differed somewhat in biological activity (514). While the larger induced pre-B cell formation in cultures which had been depleted of adherent regulatory cells by Sephadex G-10 column passage, the smaller did not. The 16-kDa factor could be a functionally incomplete breakdown product of the larger mediator or be an entirely different molecule. There is at present no explanation for the coordinate dysregulation of neutrophil and pre-B cell formation in this patient. However, it is interesting that a granulocytosis-producingtumor depresses pre-B cell formation in murine bone marrow (113).One could propose that competition for space within a shared microenvironmental niche and/or soluble mediators caused reduction in one progenitor cell type as another is increased. +
F. C3H/HeJ MICE The principal use of C3H/HeJ mice has been as a model of LPS unresponsiveness. A codominantly expressed gene on chromosome 4 prevents recognition of the lipid A moiety of endotoxin by macrophages, T cells, B cells, and fibroblasts (474). Nuclear transplantation studies suggest that some transmembrane signaling events do not occur when HeJ lymphocytes are
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23 1
exposed to LPS (93, 472). However, evidence has gradually accumulated for immunological defects unrelated to LPS (20, 283, 285, 464). We found that B cells from HeJ mice have a poor cloning efficiency in semisolid agar cultures (185).This was especially true of young mice and lymphocytes from bone marrow. Cell separation experiments showed that this characteristic is intrinsic to B lymphocytes (460). Macrophages can facilitate mitogen-dependent proliferation of B cells, and this function was also abnormal in HeJ mice. Purified HeJ B cells did not respond normally to stimulation with anti+coated Sepharose beads in the presence of a lymphokine preparation (460). The lymphokine BSF-1 is thought to act early by preparing B cells for a replicative cycle (302, 306, 463), and it seemed possible that recognition of this factor might be abnormal in HeJ mice. However, hyperexpression of Ia antigen occurred normally during an 18-hour incubation with recombinant BSF-1 (281; G. Lee, unpublished observations), and the density and function of other B cell receptors should be investigated. These observations with mature B cells contrast to the unremarkable production of B cells in C3H/HeJ bone marrow (279).
G. MOTHEATENMICE An autosomal recessive mutation on chromosome 6 leads to fatal autoimmune disease and marked immunodeficiency (58, 126, 396, 397). The least severe of two known mutations at this locus results in death at around 9 weeks of age (398). B cell, T cell, and NK cell functions are all impaired, while plasma cells secreting a wide variety of autoantibodies are very abundant (397). Among these is at least one autoantibody which binds to B lymphocyte precursors (513). Overproduction of a B cell-derived differentiation factor has also been reported in motheaten mice (402). The bone marrow and thymus of these animals is deficient in TdT+ cells, and numbers of B and pre-B cells are also reduced (129, 220, 513). Motheaten bone marrow cells have recently been studied in long-term culture systems. Cheiner and colleagues found that they do not generate TdT+ lymphocytes, and in our laboratory, B lineage lymphocytes were not made under Whitlock-Witte conditions (129, 513). In both situations, addition of motheaten marrow cells to cultures of wild-type cells suppressed lymphopoiesis. Focal growth of lymphocytes does not occur in areas of long-term cultures with a very high density of macrophage-like cells. The adherent cell layer of motheaten cultures was very rich in cells of this type, and typical lympocytebinding stromal cells could only be demonstrated after a series of subcultures (513). This is consistent with previous reports of exuberant macrophage growth in cultures of hemopoietic cells from motheaten mice (260,
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261). However, preliminary studies suggest that the cells had little dependence on macrophage growth factor (CSA-l), and additional studies are needed to learn how their proliferative activity is regulated (S. I. Hayashi, unpublished observations). It is interesting to compare these results to a study of osteopetrotic mice (487). That defect caused a deficiency of macrophage growth relative to stromal cells in culture, and it seems possible that the two cell types may be functionally and developmentally interdependent. Aberrations reflected in these mutations should continue to be instructive in this regard. The motheaten model could be one in which severe and fetal disease results from defective components of the hemopoietic microenvironment. IX. Soluble Mediators
When the last contribution to this series was prepared, virtually nothing was known about soluble mediators which influence the replication and differentiation of B lineage progenitors (186). It is now clear that a number of molecules are potentially involved in this process. A highly speculative model is outlined in Fig. 4 to indicate that functional receptors for particular molecules may be expressed at different stages. By analogy to findings with myeloid cells (270, 271), a heirarchy must exist such that responsiveness to certain factors is attained while functional receptors for other stimuli are lost. However, these are not likely to be abrupt changes, and no single factor and receptor combination would be characteristic of a given stage. Indeed, there are many indications that reception of one stimulus causes up- or downregulation in the expression of receptors for other molecules (34, 105, 252, 466). It would be ideal if the appearance and loss of functional receptors could be correlated with some of the known cell surface markers. However, that remains an objective for future research, and expression of some of these is indicated in the figure merely to show milestones in the differentiation lineage. Relationships between multipotential stem cells and committed B lineage precursors are still very poorly understood (186, 187). However, progress is being made in identifying factors which are likely to influence replication and differentiation at this very early stage. Interleukin 3 (IL-3) is a very welldefined candidate with a molecular weight of -28,000 (135, 162, 358). It can stimulate formation of multilineage colonies in agar and receptors for it are retained and functional on more differentiated cells in several hemopoietic lineages. For example, mast cell progenitors are particularly good responders to IL-3, and many long-term IL-3-dependent lines of myeloid cells have been described (75, 387). There have been several reports that committed progenitors of the B
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Hemopoie t ic Stem Cell
Lymphoid Stem Cell?
Early 6 Lineage Precursor
Large Pre-B Cell
Small Pre-B Cell
< CYCLIC NEUTROPENIA FACTORS > ...----(STROMAL
Newly Formed B Cell
..
CELL FACTORS?>
._
<M-m7>
1 ?>
3 ?>
- - *ZB ..4
SERUM FACTORS
>
M M U N E INTERFERON
..
INTERLEUKIN 1
..
BSF - 1
FIG. 4. Soluble mediators differentially influence various replication and differentiation steps in the B lymphocyte lineage. Potential participation by some of these is indicated along with phenotypic changes which seem to be distinctive for particular stages of differentiation (Section 111).
lineage can be isolated and maintained in sole dependence on IL-3 (264, 332, 334). However, some inconsistencies between those studies and the experience of several laboratories indicate that it may be easier to show functional IL-3 receptors on nonlymphoid cells and uncommitted stem cells than on lymphoid progenitors. For example, IL-3 was useful in establishing retrovirus-transformed myeloid, but not B lineage tumors in culture (156, 163). The clonable pre-B cell assay of Paige and colleagues can be influenced by IL-3, but the evidence suggests that this is not a direct effect on B cell precursors (331). In our laboratory, IL-3 was ineffective in inducing the emergence of pre-B cells in cultures of bone marrow which had been depleted of Ly-5(220)+ cells (514). Also, it did not support the growth of lymphocytes taken from long-term bone marrow cultures (G. Lee, unpublished observations). It may be that with cofactors present in an appropriate fetal calf serum, IL-3 permits selective outgrowth of early B lineage cells with high replication potential, but many aspects of the role of IL-3 at this stage remain controversial. T cells and the WEHI-3 myelomonocytic leukemia cell line have been extensively used as sources of IL-3, but at least one murine B cell tumor appears to make it (39). Also, IL-3 production occurs in athymic mice and in
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keratinocytes (249, 333). It seems clear that IL-3 cannot be the only multipoietin because responses of some mouse strains to it are extremely poor (see Section VIII, D). Furthermore, stimulation of multilineage colonies can occur in the apparent absence of this factor (238). Some other recently described factors appear to function at least as costimulators on multipotential stem cells (170, 347, 481). For example, hemopoietin 1 (HP-1) is a 17kDa molecule which synergizes with IL-3 and macrophage growth factor (CSF-1) to influence multilineage progenitors (419). As these become better defined, it will be interesting to learn if they influence cells destined to become lymphocytes. The discovery of -45-kDa factor in the urine of a cyclic neutropenia patient is significant for several reasons (95, 225; Section VIII, E). While large amounts of this material are not available to permit sequencing and other molecular studies, it provided a prototype for an early-acting factor which promotes pre-B cell formation, but not sustained pre-B cell replication. Bone marrow cells which respond to it lack Ly-5(220) and are induced to acquire this marker and cytoplasmic p, chains without obvious participation of other regulatory cells. This distinguished it from a smaller factor in the same samples which was active only in the presence of adherent bone marrow cells. The cyclic neutropenia factors have had some influence on lymphocytes taken from long-term bone marrow cultures, but it has always been clear that they do not stimulate pre-B cell division (225). Unpublished studies suggest that bone marrow stromal cell lines make a similar activity, and this should considerably facilitate their characterization (K. Landreth, K. Dorshkind, and P. Quesenberry, personal communication). It seems possible that there are additional stromal cell factors which maintain pre-B cell growth in Whitlock- Witte-type bone marrow cultures, as indicated in Fig. 3. However, extensive proliferation of cells at this stage may not be typical of normal steady-state B cell production (see Section V). As cells near the end of the differentiation sequence, they can be influenced in culture by at least four distinct molecules. The first examples of factors which may be important in local control of this lineage came from studies of young NZB mice (176, 177; Section VII1,D). At a time when bone marrow regulatory elements appeared to be hyperactive, two small (15-17 kDa) molecules were isolated from the serum. These augmented the functional maturation and surface IgM expression of normal B cell precursors, and at least one of them induced K synthesis in our pre-B cell line. Immune IFN, I L - l a and IL-lP have a similar effect, but these are known to influence a wide variety of other tissues (118, 257, 420, 421). As previously discussed (Sections III,D and H), functional receptors for the T cell-derived factor, BSF-1 (IL-4) are at least displayed on pre-B cell lines (349, 509). While this factor alone has not induced expression of surface IgM, it has striking effects
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on the density of class I1 and Ly-1 antigens. BSF-1 (IL-4) is an interesting multifunctional stimulus for several cell lineages (125, 302, 306, 463). Tumor necrosis serum was the first native substance shown to influence B cell precursors (152). This material is named for its ability to regress certain tumors, and several well-defined substances have been defined which account for many of its biological activities (308).I L - l a and IL-lP were probably present in tumor necrosis serum in addition to tumor necrosis factor (TNF, cachectin), which is now available in highly purified, recombinantderived form (19). TNF is lytic for some cell types, has no effect on others, and stimulates the growth of still others (428, 461). TNF activates osteoclasts and elicits production of IL-1 and a procoagulant activity by vascular endothelial cells (16, 79, 294, 295). Preliminary studies suggest that TNF may influence B lineage precursor cells, and it will be important to learn the extent of those effects (G. Lee, unpublished observations). B cell growth factor I1 (BCGF 11) also merits further investigation (89). Like BSF-I , this is a T cell-derived substance which was discovered on the basis of its effects on mature B cells. However, it has recently been found to stimulate eosinophil growth (379), and it seems reasonable to propose that receptors for it might be expressed on pre-B cells. Mature B cells have been shown to elaborate various B cell growth and differentiation factors (8, 27, 65, 293, 451). However, it is not yet known if these influence B lymphocyte formation. X. Synthesis and Conclusions
It seems appropriate to conclude this review with some overall impressions about B lymphocyte formation and suggestions for the emphasis of further studies. Precise descriptions of cells and mechanisms are beginning to come forward, and the rapid progress being made results from several highly successful approaches. Use of monoclonal antibodies, short- and longterm bone marrow techniques, and genetic defects has been emphasized in this chapter. However, equally important advances in recombinant DNA technology have opened the possibility of defining differentiation in molecular terms and provided increasing numbers of homogeneous mediators of intracellular communication. It is already possible to propose complex interrelationships between cells and factors which influence B lineage precursors, and a few of them will be briefly considered here. Macrophages and large, fibroblast-like cells comprise the majority of the adherent layer which supports long-term growth of B lineage precursors, and it seems likely that a balance between the two populations is important in establishing hemopoiesis in the cultures (Section VI). One can imagine an interdependence between the two for survival because fibroblasts make
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macrophage growth factor (CSF-1) and macrophages make tumor necrosis factor (TNF), which can be a fibroblast growth factor (418, 461). Lymphocytes bind directly only to a subset of the fibroblast-like cells, but it remains unclear that these alone can provide a complete microenvironment for the formation of B lineage precursors. Indeed, while cells adapt to long-term growth in such cultures, they only gradually progress toward a mature phenotype. Macrophages can elaborate IL-1, which augments the final steps in this process, and it is interesting that they have been found situated on the abluminal surface of venous sinuses within bone marrow (240, 478, 479). This is the exit point for newly formed B lymphocytes, and the endothelial cells, through which they must pass, can also make IL-1 in response to TNF (295). It should eventually be possible to define most of the stimuli required for B lymphocyte formation with the methodology now available. This optimism is particularly striking when compared to a previous contribution to this series (186). We knew then that close cellular communication and participation of two cell types, including macrophages, was important (194). However, no soluble mediators had been clearly implicated in the process, and regulatory cells which presumably make them were undefined. It is now certain that B lymphocyte precursors display functional receptors for a variety of substances, and the challenge for future studies will be in determining which ones of these are important for normal homeostasis. A number of the soluble mediators known to influence lymphoid and hemopoietic cells are made by T lymphocytes (Section IX). These include CSFs, immune IFN, IL-3, BSF-1 (IL-4) BCGF-11, and TNF. However, T cells are a small minority of murine bone marrow, and there is no evidence that primary B cell formation is thymus dependent. On the other hand, full maturation of B cell functional capability may well depend on T cells (36), and this influence is most conspicuous when the B cells are partially defective, as in CBA/N mice (Section VIII). Many of the known T lymphocytederived factors are produced by other cell types, although there is reason to suspect active T participation in some aplastic anemias and other diseases (41, 507). Virtually nothing is known about antagonists of B lymphocyte formation. Normal serum inhibits induction of surface K expression on a pre-B cell in culture (Section VII). Serum components have been described which block a variety of other culture responses, and one must presume that events in marrow take place in sequestered locations protected from high concentrations of such substances (269,453,456,468). Many substances, such as TNF, prostaglandins, steroids, a-fetoprotein, a-1-antitrypsin, suppressor factors, and lymphotoxins should be investigated as potential inhibitors of B cell precursor replication and differentiation. Natural killer (NK) cells were depicted in Fig. 1 as an independent lym-
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phocyte lineage. This is because of recent findings that NK cells do not rearrange T cell receptor or Ig genes, they are intact in SCID mice, they are not closely related to multipotential myeloid stem cells (CFU-s), and they are unique in their developmental dependence on bone marrow (84, 131, 450). Functional NK cells are present in bone marrow, some marrow cells are targets for N K lysis, and there is evidence that N K cells can participate in regulation of hemopoiesis and immune responses within marrow (154, 278, 287). TNF is a well-defined substance through which NK cells can regulate hemopoiesis (28, 63), and it will be important to determine the effects of this molecule on B cell precursors. Unlike the thymus and bursa of Fabricius, bone marrow is known to be an important site for secondary immune responses (204, 205, 206). This imposes some unique regulatory requirements, and it is interesting to consider that many of the soluble mediators released by mature lymphocytes can influence the generation of newly formed B cells (Section IX). Some effects of nonspecific environmental stimulation on lymphopoiesis were discussed in Section V. There is clearly much more to be learned about the impact of systemic inflammatory responses on this primary lymphoid organ. B lineage cells are often depicted as passive recipients of stimulation by mediators from other cells. However, it is now clear that at least in some circumstances, B cells can make myeloid CSFs, IL-1, IL-2, IL-3, a T cell receptor-inducing factor (BEF), and B cell growth factors (8, 27, 39, 65, 293, 381, 451). The possibility is open that differentiating precursors of B cells elaborate substances which influence neighboring cells. Soluble mediators often elicit responses in multiple tissue types, and B lineage precursors are formed within marrow among cells of seven other hemopoietic lineages. This suggests that regulatory balance might be maintained by a competition for space within microenvironmental niches and/or stimuli and could in part explain the coordinate dysregulation found in cyclic neutropenia (Section VII1,E).,Absorption and degradation of a factor by one cell type would deprive other precursors from being stimulated (449). However, if hemopoiesis were solely controlled by diffusible, multifunctional mediators, the marrow would have to be a poorly regulated malange. Interleukin-1 provides an example of a mediator which can be “presented” to responding cells in membrane form, and a stromal cell line stimulates hemopoiesis only when in direct contact with stem cells (214, 238). A pre-B tumor line was induced to express membrane IgG only after cocultivation with T hybridoma cells (165). Short-range communication of this kind would seem to be ideal for regulating events within bone marrow. Lymphocyte precursors are intimately associated with bone marrow stromal cells in culture, and a definition of the adhesion molecules which make this possible must receive a very high priority. B lymphocytes and their precursors could be subdivided into an almost
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infinite number of sets on the basis of surface marker expression, physical properties, responsiveness to various stimuli, etc. Some of these may represent branching or parallel differentiation pathways. Incomplete representation of an antigen such as GF-1 on pre-B cells might be meaningful in this context, and there is considerably more evidence that Ly-1-bearing B cells are special (136, 263). In the latter case, it has even been suggested that the usual rules for Ig gene selection and utilization might not be obeyed (137). There are clonal procedures for studying single B lymphocyte precursors, and these might be employed as successfully as with mast cell precursors; i.e., a single cell was shown to make multiple types of mast cell progeny (199, 410). The diagrams and logic of this review have assumed that a simple linear sequence of events results in formation of all B cells. However, we find it very difficult to “nest” all small populations of bone marrow lymphocytes into such a scheme, and we know that expression of one particular antigen, Ly-5(220) is not obligate in order for other events to proceed (Section 111).Regulation of the production of different sets of B cells might have to be considered in the near future. A number of questions about the origin and regulation of bone marrow stromal cells now seem timely. There appear to be morphologic similarities between thymic stromal cells, cells in marrow which support B lymphopoiesis, and fibroblast-like cells related to granulocyte and macrophage production. It will be interesting to learn the extent to which they express common and distinctive genes. Marrow stromal cells might be very closely related inasmuch as their function can be manipulated with different culture conditions (Section VI). It remains to be learned if the inclusion of steroids, etc. merely biases against lymphocytes or causes a fundamental change in stromal cell activity. Monoclonal antibodies to marrow stromal cells will probably be developed, as they have for elements of the thymus microenvironment (454). These would make it possible to localize the stromal cells in situ and investigate possible developmental relationships to hemopoietic stem cells. While spleen and marrow can both support primary B lymphocyte formation, it normally occurs only in the latter (189). It would be interesting to know if microenvironmental elements are always present in the spleen and what factors might lead to their reactivation (417). Unlike lymphocyte progenitors, stromal cells may not normally be renewed at a high rate. However, an understanding of their origin and growth regulation could be very important experimentally and significant in terms of human disease. Platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor have been shown to influence long-term bone marrow cultures (146, 370), and more studies of this kind could be very informative. It is also important to clarify the relationship between fibroblast colony-forming cells in bone marrow (490) and stromal cells which support lymphopoiesis.
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The versatility of biological systems is apparent in several sections of this review. A family of common and lineage-restricted cell surface molecules is encoded by the Ly-5 structural gene, and their expression is both conserved in evolution and carefully regulated (Section 111,B). Thy-1 and several other lymphocyte surface antigens were found to be potentially linked by PI, and cells might be able to shed such molecules through the action of endogenous phospholipases (Section 111,F).However, there is also reason to believe that at least some of the PI-linked proteins could be made as permanently membrane-anchored or secreted forms. This perspective will hopefully aid in identifying functions for such structures. For example, advantages might accrue to adhesion molecules whose structure and cell association could be modulated during differentiation (147b, 373b, 146b).
ACKNOWLEDGMENTS Sincere appreciation is expressed to the many colleagues who freely shared unpublished observations and ideas utilized in this chapter. Discussions with Dr. Carolynn Pietrangeli and her critical comments on the manuscript were especially helpful. Studies alone in our laboratories were supported by NIH Grants AI-19884 and AI-20069, as well as by fellowships from The Leukemia Society of America and the Damon Runyon-Walter Winchell Cancer Fund.
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ADVANCES IN IMMUNOLOGY, VOL 41
Cellular and Humoral Mechanisms of Cytotoxicity: Structural and Functional Analogies JOHN DING-E YOUNG AND ZANVIL A. C O H N Laboratory of Cellular Physiology and Immunology, The Rockefeller Universiiy, New York, New York 10021
1. introduction
Cell killing by immune cells represents an important natural defense barrier against proliferation of transformed cells, virus-infected cells, parasites, and other foreign invaders. Target cell (TC) killing, however, is not restricted to immune cells and has been documented for various cell types, including bacteria, fungi, yeast, plant cells, and protozoan parasites. Perhaps one denominator common to all these different cytotoxic cell types and their killing machineries is the involvement of soluble cytotoxic mediators which are secreted by the killer cell and used to lyse the target. The increased interest in these soluble mediators of cell killing, generally referred to as cytotoxins, has been greatly stimulated in part by the feasibility of their isolation in the laboratory in high yields and their further characterization by conventional biochemical techniques. The most widely studied soluble cytotoxins, for example, are the bacterial toxins. Cell-mediated killing by immune cells differs from other forms of TC killing in that it usually requires contact between effector and target cells. The surface contact may be initiated by binding of effector cell to antibodycoated TC through surface Fc receptors. This type of antibody-dependent cell-mediated cytotoxicity (ADCC) has been described for macrophages, neutrophils, eosinophils, and natural killer (NK) cells. Alternatively, the surface contact may be mediated by specific surface receptors on effector cells as exemplified by cytotoxic T lymphocytes (CTLs). Finally, in the case of activated macrophages, binding to TCs occurs by still poorly understood mechanisms. In all instances, it is thought that binding to lysable targets elicits the cytotoxic reaction. The secretion model for killing, discussed in detail later on, argues that specific surface binding triggers release of cytotoxins by the effector cell into the intercellular space of cell contact. The search for such mediators of cell killing has been an intensive area of research in a number of laboratories over the past few years. In this review, we will focus on the molecular mechanisms of membrane
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damage inflicted by CTLs and N K cells. In particular, we will review recent studies that have led to the isolation and characterization of a cytolytic poreforming protein (PFP) found in the cytoplasmic granules of these cell types. Information pertaining to TC recognition as well as surface antigens and receptors of CTLs and NK cells will not be reviewed in detail. These topics have been discussed in other recent reviews (Berke, 1983; Burakoff et al., 1984; Goverman et al., 1986; Ortaldo and Herberman, 1984; Trinchieri and Perussia, 1984; Goldfarb, 1986). Instead, only cellular events which occur after formation of effector-TC conjugates leading ultimately to TC lysis will be considered in depth. We will attempt to draw analogies between killing mediated by lymphocytes, by other cell types, such as eosinophils and the protozoan parasite Entameba histolytica, and by complement. Finally, recent studies made on cytotoxic PFPs found in bacteria, fungi, yeast, insects, and other lower organisms will also be outlined, particularly when mechanistic information is available that is judged of overlapping interest to studies on cell-mediated cytotoxicity. It will become apparent to the reader that proteins with closely related functions have become implicated as general mediators of cell killing in nature. It will also become apparent that conceptual problems relating to the mechanism of action of lymphocyte PFP and complement proteins have similarly been raised in the past with other wellknown cytotoxins. The surprising functional analogies between the cytotoxin released by lymphocytes and the better known toxins found in certain bacteria and insects, for example, should greatly stimulate and aid future studies on the cytotoxic reaction mediated by immune cells. II. Nature of Cytotoxicity Mediated by CTL and NK Cells
CTLs, capable of specifically lysing TCs in uitro, have long been implicated as the principal effector cells in allograft rejection (Cerottini and Brunner, 1974; Berke, 1980), tumor immunity (Germain et al., 1975; Schrader and Edelman, 1976), and lysis of virally infected cells (Zingernagel and Doherty, 1974; Glaser and Law, 1978). CTLs capable of killing autologous transformed cells in vivo have also been demonstrated in tumor-bearing hosts (Herberman, 1974). CTLs recognize lysable targets by binding to specific TC surface antigens and class I proteins of the major histocompatibility complex (MHC) (H-2K,D in mice and HLA-A,B,C in humans). NK cells, on the other hand, are bone marrow-derived mononuclear cells functionally defined for their capability of lysing certain allogeneic TCs without prior sensitization. Unlike CTLs, N K cells lyse a wide spectrum of TCs without any restriction imposed by or associated with expression of MHC antigens on their TCs. NK cells bear a characteristic combination of surface differentiation antigens that serve to define them as a discrete and homogeneous leukocyte subset distinct from B and T lymphocytes and myelomonocytic
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cells (Perussia et al., 1983; Trinchieri and Perussia, 1984; see also Kaplan, 1986, for a discussion of their cell lineage). Nevertheless, the lineage of N K cells has not been firmly established. Recent data on the rearrangement and expression of T cell receptor genes in NK clones appear to reflect the heterogeneity of these cell types, showing that N K clones may either express functional T cell receptors or truncated Ti-P transcripts, accompanied by lack of a Ti-a transcript and no detectable surface Ti protein (Hercend et al., 1983a; Ritz et al., 1985). The Ti molecules represent a family of markers previously identified only on antigen-specific T lymphocytes. Morphologically, peripheral blood NK cells resemble a population of large granular lymphocytes (LGL) that can be distinguished and separated from conventional T cells by a number of differing physical and biochemical characteristics, including their low buoyant density due to high cytoplasmic : nuclear ratio, the presence of numerous azurophilic cytoplasmic granules, the presence of surface Fc receptors for IgG, and a number of distinct surface markers (Saksela et al., 1979; Timonen et al., 1981; Trinchieri and Perussia, 1984). Although it is not clear yet to what extent CTLs and N K cells differ in their TC antigen recognition, nevertheless these cell types share a number of similarities in post-membrane-binding cellular events which ultimately lead to TC lysis, as described later. For this reason, the cytotoxic mechanisms of CTLs and N K cells will be discussed together in this review, and the cytotoxic reaction common to both will simply be referred to as the mechanism of lymphocyte-mediated killing. Extensive work performed in several laboratories over the years has helped to identify and define the various intermediate stages of cell killing. Following cell contact, killing of TCs by CTLs has been resolved into at least three discrete stages: (1) specific binding, with the rapid formation of a strong adhesion between the two cell membranes, thought to occur within 2 minutes of cell contact; (2) the delivery of lethal hit or the temperature-and calcium-dependent programming for lysis stage, which occurs in the next 10 minutes; and (3) the slower stage of killer cell-independent lysis, during which TC dissolution and lysis occur, and the CTLs are capable of initiating a new lytic interaction (Martz, 1977; Golstein and Smith, 1977; Berke, 1980). Similar binding, programming, and killer cell-independent stages for cell killing have been identified for human N K cells (Hiserodt et al., 1982). The prelytic adhesion of CTL to TC is absolutely dependent on magnesium, but not on calcium, and occurs optimally under defined conditions of temperature and pH (Stulting and Berke, 1973; Martz, 1980). Several antigens on T cell surface have been implicated in the adhesion process (Meuer et al., 1982a; Burakoff et al., 1984). These include T8 in man or Lyt-2 in mouse (Fan et al., 1980; MacDonald et d., 1982a), Leu3, T4 in man or L3T4 in mouse (Biddison et al., 1982, 1984), T11 (Krensky et al., 1984; Schmidt et al., 1985), and LFA-1 (Davingnon et al., 1981; Krensky et al., 1983; Martz et
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al., 1983). Anti-LFA-1 and anti-Lyt-2 antibodies, for example, block killing mediated by CTL bearing these markers (Davingnon et al., 1981; Pierres et al., 1982; Nakayama et al., 1979; MacDonald et al., 198213; Meuer et al., 1982a). LFA-1 and T11 have also been implicated in NK-TC adhesion (Martz et al., 1983; Krensky et al., 1984; Schmidt et al., 1985). It has now been established that T8/Lyt-2 is mainly expressed by class I-reactive T cells (Cantor and Boyse, 1975; Swain, 1981; Krensky et al., 1982; Spits et al., 1982; Meuer et al., 1982b; Biddison et al., 1982; Marrack and Kappler, 1986). These results were mainly obtained by preincubation of representative TCs with either anti-class I or anti-class I1 blocking antibodies. This type of nonspecific adhesion is thought to be distinct from the binding of T cell receptors of CTLs to class-specific antigens on TC surface. Thus, cells which have been transfected with class-specific antigens by gene transfer experiments are not lysed by the appropriate antigen-restricted CTL, supporting the notion that the presence of specific antigen on TC is not sufficient for cytolysis (van de Rijn et al., 1984). A follow-up study by Spits, van de Rijn, de Vries and their colleagues (Spits et al., 1986) has demonstrated that the T cell receptor and the TC antigen cannot interact unless there is conjugate formation. Nonspecific conjugate formation involving LFA-1, T8, and possibly T11 is thought to precede the recognition of specific antigen by the T cell receptor. Moreover, the authors suggest that if the relevant target antigen is not found after conjugation with TC, the CTL simply detaches from the bound TC without inflicting any injury on the target. On the other hand, T3 is thought to be closely associated with the antigenspecific T cell receptor in the membrane of T cells and to play a direct effector function in the transmembrane signaling of the T cell response (Meuer et al., 1984; reviewed by Goverman et al., 1986; Marrack and Kappler, 1986). Thus, antibodies specific for T3 block CTL killing at a postbinding step that precedes the calcium-dependent programming stage, but do not block the nonspecific conjugate formation (Landegren et al., 1982; Tsoukas et al., 1982a,b; Moretta et al., 1984). T3 is now known to consist of a trimolecular complex of 25-kDa (y), 20-kDa (ti), these two being glycosylated, and 20-kDa (e) proteins (Reinherz et al., 1979; Ledbetter et al., 1981; Borst et al., 1983), thought to be extensively buried in T cell membranes because of their inaccessibility to labeling on intact cells with aqueous reagents and 1251(Meuer et al., 1984; Oettgen et al., 1984). Recent crosslinking experiments have shown that the T3 complex is bound to T cell receptor mainly through the y chain (Brenner et al., 1985). The effector role of T3 is indicated by recent experiments using T3-specific antibodies which reveal that binding to T3 exerts potent mitogenic (Wauwe et al., 1980; Chang et al., 1981) and secretory (interferon-y) (Von
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Wussow et al., 1981) activities. Moreover, anti-T3 reagents can cause nonspecific killing of bystander cells (Leeuwenberg et al., 1985; Schrezenmeir et al., 1985; Spits et al., 1985). The early signaling event transmitted to the cell by T3 binding is not yet clear, but there is presently an accumulating body of evidence that suggests that anti-T3 antibodies induce membrane depolarization and calcium influxes in T3-bearing T cells (O’Flynn et al., 1984, 1985; Oettgen et al., 1985; Weiss and Stobo, 1984; Weiss et al., 1984). In mice T cells, activation via the receptor has been shown to result in the early phosphorylation of a protein thought to be analogous to human T3 6 chain (Samelson et al., 1985). These early signaling events may be involved in the triggering of the lytic machinery of CTLs, and in particular, in the triggering of granule exocytosis, as discussed later in this review. The relevance of T3 in NK cell-mediated killing, however, is unclear. Only a portion of N K cell clones derived in vitro present surface T3 and other mature T cell phenotypic antigens (Hercend et al., 1983b), and peripheral blood NK cells, purified and defined as T3-negative lymphocyte populations, mediate both spontaneous and antibody-dependent cytotoxicity (London et al., 1985). The involvement of other T3-like surface structures in NK cell killing awaits further studies. Ill. Cytolytic Mechanisms Proposed in the Past and the Concept of Secretion and Colloid Osmotic Lysis
A. POLARITY OF CELL KILLING A N D CYTOPLASMIC REARRANGEMENTS
After a CTL has damaged its TC, it is known that the same CTL can recycle to lyse new targets (Berke et al., 1972; Berke and Amos, 1973; Zagury et nl., 1975; Martz and Benacerraf, 1976). Throughout the expression of this repetitive lytic activity, the CTL spares itself from lysis. Classic studies by Golstein (1974) and Kuppers and Henney (1977) first demonstrated the unidirectionality of the killing process. These investigators showed that a given CTL A is not immune to lysis mediated by a second, appropriately sensitized CTL B anti-A. Furthermore, when A anti-B cells were incubated with B anti-C cells, only B cells were lysed. This unidirectionality concept was further extended by experiments in which CTL A and CTL B, mutually sensitized against each other, were incubated together in cytotoxicity assays (Fishelson and Berke, 1978). Results of such experiments showed that the interaction of CTL A anti-B and CTL B anti-A did not result in simultaneous lysis of both cell types. The ability of CTL to lyse and to be lysed by other cells, but not to lyse themselves, was recently confirmed for long-term CTL cell lines (CTLL) (Luciani et al., 1986).Recent observations made by William R. Clark and his colleagues (personal communication),
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however, suggest that cloned CTLs and T helper cells show an unusually high resistance to lysis by effector CTLs. Their recent studies suggest the resistance of cloned CTLs to lysis is governed by a still undefined postbinding step. In view of the implications that such important observations may bear on the mechanisms of lysis and self-protection mediated by CTLs, further studies of this type are needed to clarify this issue. Another interesting finding related to the directionality of killing is that a CTL, which has bound simultaneously to several TCs, will only lyse one TC at a time (Zagury et al., 1979). These results appear to indicate that the delivery of the lethal hit requires not only intimate intercellular surface contact, but also that it is restricted to discrete regions of the plasma membrane of the CTL. It is interesting that in this regard, proposals have been made in the past (Berke, 1983), implying that the T cell receptors may play not only a signaling role, but also a direct cytotoxic function by changing the fluidity and permeability of the TC membrane during antigen binding. Since CTL-TC binding can occur without lysis, at low temperatures (Berke and Gabison, 1975), or in the absence of calcium ions (Martz, 1977), it is now clear that binding and lysis are distinct cellular events, as outlined above. Ultrastructural analysis of CTL-TC conjugates performed in several laboratories (Kalina and Berke, 1976; Sanderson and Glauert, 1979; Rosen et al., 1981) has revealed the extensive apposition of plasma membranes at the sites of cell contact that occur in the form of a network of membrane interdigitations. The close apposition of membranes provided the basis for the past notion that a tangential shear force is generated during CTL-TC binding which results in direct mechanical rupture of TC membrane (Seeman, 1974; Grimm et al., 1979). This close attachment of membranes also led to observations that effector cell membranes themselves, isolated from CTLs, could lyse TC (Ferluga and Allison, 1975). These observations have not been confirmed since then, and it turns out that membrane preparations isolated from resting lymphocytes and nonlymphoid cells also exert a certain level of cytotoxicity (Kahn-Perles and Golstein, 1978). Recently, a subcellular material (membranes) from CTL was reconstituted into proteoliposomes which were subsequently fused with noncytolytic cells (Harris et al., 1984). This procedure has been claimed to confer cytolytic capability to the host cells (Harris et al., 1984). These results, which would favor a role for a plasma membrane component in cell-mediated cytotoxicity, however, have also not been confirmed. In addition to the plasma membrane interdigitations, morphological observations of CTL-TC pairs have revealed asymmetrical distribution of several other subcellular elements. Granules and Golgi stacks have been shown to accumulate in the contact region of the effector cell within 15 to 30 minutes after conjugate formation (Zagury et al., 1975; Bykovskaya et al.,
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1978a,b). The CTL contact area has also been shown to be enriched for actin, but not myosin, as demonstrated by immunofluorescence microscopy (Ryser et al., 1982). Interestingly, the asymmetrical distribution of actin appears to occur immediately after conjugate formation. Using anti-tubulin antibodies and immunofluorescence techniques, Geiger et al. (1982) have found that the microtubule organizing center (MTOC) within the effector cell is also polarized toward the area of cell contact. They concluded, however, that the TC binds to an already polarized region of the CTL and ruled out the possibility that the cytoskeletal reorientation may have occurred as a result of conjugate formation. Kupfer and Dennert (1984) have made similar observations by double indirect immunofluorescence microscopy with antibodies specific for membranes of the Golgi apparatus and for tubulin. Unlike Geiger et al. (1982), the latter investigators concluded from their studies that a rapid cytoplasmic reorientation of the MTOC and the Golgi apparatus must have occurred following conjugate formation. The MTOC/Golgi reorientation is not observed in the presence of the microtubule-disrupting agent nocodazole. Moreover, cytoplasmic polarization is not observed when CTLs bind to nonlysable targets. Similar observations arguing for asymmetric distribution (Carpen et al., 1982) and reorientation (Carpen et al., 1983; Kupfer et al., 1983) of the Golgi and the MTOC have been made for NK cell-TC conjugates. Previous studies with cells involved in ADCC also showed a rapid asymmetrical rearrangement of the cytoplasm that is observed upon cell binding to substrates coated with immune complexes (Alexander and Henkart, 1976). Recent observations using high-resolution cinemicrography (Yannelli et al., 1986) have provided additional support to the cytoplasmic reorientation concept proposed by several investigators. Yannelli and his colleagues (1986) were able to monitor continuously the interaction between a CTL and its TC. The CTL has a polar cytoplasm, with the leading edge containing the nucleus and a tapered tail carrying the cytoplasmic granules. Upon contact with TC through the leading edge, the nucleus immediately moves away from the region of contact and is replaced rapidly by granules. As early as 4 minutes after binding, the polarized granules are seen to fuse with the plasma membrane of CTL in the vicinity of the area of its contact with TC. B. HYDROLYTIC ENZYMES
A role for proteolytic and lipid-active enzymes in cell-mediated killing has also been proposed in the past. The activation of a membrane-bound phospholipase A2 activity has been suggested that would convert membrane phosphatidylcholine into the detergent-like and cytolytic lysolecithin (Frye and Friou, 1975). This possibility was suggested by experiments in which phosphatidylcholine and its analogs were shown to block the CTL function
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(Frye and Friou, 1975). Previous observations supportive of this contention included studies that showed an altered phospholipid metabolism in TC, particularly an increase in TC phospholipase A activity, which occurred during cell-mediated killing (Koren et al., 1971). Berke (1977) has argued convincingly against an involvement of phospholipases in cell-mediated reactions by demonstrating that the effects of phosphatidylcholine and its analogs and of a phospholipase A inhibitor (known as Rosenthal’s inhibitor) on cell-mediated killing could be explained by their inhibitory effects on conjugate formation rather than lysis itself. However, the possibility of an involvement of detergent-like phospholipid metabolites cannot be excluded, especially in view of more recent results that also suggest a role for phospholipase A2 in N K cell-mediated lysis (Hoffman et al., 1982). Proteolytic enzymes, presumably localized on effector cell surfaces, have recently been implicated in the cytotoxic reactions mediated by activated peripheral blood lymphocytes (Grayzel et al., 1975), CTLs (Chang and Eisen, 1980; Redelman and Hudig, 1980, 1983), and N K cells (Hudig et al., 1981, 1984; Quan et al., 1982; Hiserodt et d., 1983a; Lavie et d., 1985; reviewed by Goldfarb, 1985). These studies mainly determined the effect of various protease inhibitors on cell-mediated killing. Based on these studies, the involvement of effector cell serine esterases in the cytotoxic reaction has been implicated. A neutral proteinase of 30 kDa was isolated from subcellular extracts of unstimulated human peripheral blood lymphocytes that was shown to be cytotoxic to bladder carcinoma cells (Hatcher et al., 1978). Since follow-up observations on this proteinase have not become available, it would be premature to assign any definitive role to this proteinase in lymphocyte-mediated killing. Zagury (1982) has similarly proposed a role for hydrolytic enzymes of a lysosomal nature (such as acid phosphatase) in cell killing. This contention was based on morphological evidence that suggested release of lysosomal enzymes after CTL-TC conjugate formation (Zagury, 1982). A recent study using the affinity label specific for serine esterases, diisopropyl fluorophosphate (DFP) (Kraut, 1977), showed that NK cells contain a DFP-labeled polypeptide of 55 kDa which appears only after conjugate formation (Petty et al., 1984). A parallel study carried out with CTL (Pasternak and Eisen, 1985) demonstrated that DFP labels a polypeptide of 28 kDa of CTL present at levels which are at least 300-fold higher in CTL than in helper T cells and resident thymocytes. The specific expression of serine esterase by CTL has recently been confirmed and extended by at least three laboratories that have succeeded in isolating CTL-associated cDNA clones (Lobe et al., 1985, 1986; Gershenfeld and Weissman, 1986; Brunet et al., 1986). The strategies used by these investigators consisted of generating subtractive cDNA libraries and of searching for those transcripts which are specific for CTL by Northern blot hybridization. This collective effort has
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resulted in the elucidation of three clones now thought to encode for key sequences characteristic of serine proteinases. The relevance of these specific transcripts and their protein products to cytotoxicity is not known. We and others (Masson et al., 1986; Pasternak et al., 1986; Young et al., 1986a) have recently isolated two species of serine esterase from the granules of CTL and of NK-like lymphocytes. Some of their properties and a discussion of their putative role in cell-mediated killing will be given in a later section of this review. C. MEMBRANEDAMAGE A N D COLLOIDOSMOTICKILLING OF TARGETS One of the great advances that enabled experimental quantitation of TC lysis was the standardization of the 51Cr release assay (Wigzell, 1965; Walker and Demus, 1975; see also Berke, 1980, for a review on other cytotoxicity assays). As the release of this and other labels measures the integrity of TC membrane, it became apparent early on that membrane damage and increase in membrane permeability must occur at some stage during cellmediated killing. That membrane damage might represent the primary site of cell injury, however, was later suggested by the following series of observations. Time-lapse cinematography of lymphocyte-mediated cytolysis revealed lysis as a “ballooning phenomenon” in which the TC becomes swollen and ruptures after contact with a lymphocyte (Rosenau, 1968). Such changes and the subsequent lysis are not observed when the effector and target cells are suspended in a viscous medium. Similarly, Biberfeld and Perlmann (1970) have observed that chicken erythrocytes swell during ADCC. Henney (1973a) demonstrated that the release of the low-molecular-weight markers ATP and 86Rb from TC during lymphocyte-mediated lysis precedes the release of the larger markers, 51Cr-labeled protein and [3H]thymidineDNA. The inverse relationship between the effective molecular size of the marker and the time of its observed release from injured TCs suggested the possibility that lymphocyte-mediated injury confers a new diffusion-limited permeability pathway to TC membranes. Henney (1974) then proceeded to show that both the efflux of large markers from the damaged TC and the plasma membrane destruction could be prevented by the addition of exogenous high-molecular-weight dextrans. The minimal size of dextran molecules which could afford such protection was determined to be -40,000 in molecular weight, leading Henney (1974) to suggest that the initial T cellinduced lesion is at least 90 A in diameter. The author proposed that water must have entered the TC during cytolysis and the eventual death of the target cell is caused by “colloid osmotic” forces resulting from this water influx (Henney, 1974). At the time, Henney (1974) also pointed out that his findings on T cell-mediated cytolysis revealed “striking similarity to the
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nature of the antibody induced complement lesion in erythrocytes.” These observations were confirmed and extended by Martz and his colleagues (1974), who used [14C]nicotinamide as a label for TCs, and by Sanderson (1976) using other small markers. Sanderson and Thomas (1977) subsequently extended these observations to ADCC. Martz et al. (1974) also found that lowering the temperature of incubation to 0°C after conjugate formation interrupts the release of markers from TCs, an observation which is consistent with the temperature-sensitive programming stage of cell killing. In fact, kinetic analysis of marker release performed later by Martz (1976) revealed that the electrolyte permeability increase in the TC membrane occurs concomitantly with the onset of the calcium-programming stage for lysis. Later attempts at sizing the “holes” produced during ADCC using resealed erythrocyte ghosts and sieving macromolecules of known Stokes radius resulted in the estimation of a maximal diameter of 15 nm for the pore (Simone and Henkart, 1980). As noted by these authors, the sizes observed for lymphocyte-mediated lesions were considerably larger than those associated with the complement-mediated pores. Cytolysis by lymphocytes has been examined with respect to the one versus multihit issue. The one-hit theory has been substantiated for complement-mediated lesions and has been taken to support the transmembrane channel hypothesis elaborated earlier by Mayer and his colleagues (Mayer, 1972; Rommel and Mayer, 1973; Kitamura et al., 1976). Accordingly for complement (C), a multihit response would support a detergent-like or enzymatic lytic mechanism. The discrimination between a one-hit and a multihit or cooperative process is based simply on the shape of the dose-response curve. In the case of lymphocyte-mediated killing, Ziegler and Henney (1975) studied the extent of lysis of Chang cells by antibody and human peripheral lymphocyte populations as a function of the number of lymphocytes. The slope of the dose-response curve approaches unity. From these data, the authors concluded that this cytotoxic reaction also conforms to a one-hit mechanism. Similar results were later confirmed by Mayer (1977), who on replotting previous lysis kinetics data reported by Gerottini and Brunner (1974) also demonstrated that the initial lysis velocity varied 1 : 1 with the lymphocyte : target cell ratio. An important difference between the one-hit behavior observed for the C system and that for lymphocyte-mediated reactions is that in the case of lymphocytes, the one-hit characteristic is observed in terms of number of cells rather than the amount of protein, as in the case of C. This result only means that TC lysis requires attack by only one effector lymphocyte. The complexity of lymphocyte-TC interactions stimulated several laboratories to use model lipid membranes as targets. The use of proteoliposomes or artificial planar bilayers would enable one to address the issue of mem-
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brane damage directly, without the interference from other subcellular components derived from the TC. The experiments performed by Henkart and Blumenthal (1975) are such an example. Using dinitrophenylated planar lipid bilayers, they showed that in the presence of anti-DNP antibodies, human lymphocytes rapidly induce increases in membrane conductance. Furthermore, such ionic permeability increases occur only when the membrane voltage is made positive on the lymphocyte side. The authors concluded from these observations that the primary event in lymphocyte killing via ADCC relies on the creation of ion-conducting channels in the TC membrane. However, such experiments are not conclusive, since the authors did not rule out the possibility that the conductance increase might have only reflected the effect of clustering of surface antigens by the interaction of lymphocyte Fc receptors with immune complexes. The use of model membranes can also be exemplified by experiments designed to verify the allogeneic cytolysis of reconstituted membrane vesicles mediated by lymphocytes (Hollander et al., 1979). These investigators prepared lipid vesicles from a mixture of defined lipids and reconstituted into them H-2 antigens and TC surface proteins. Vesicle damage monitored by release of 51Cr was produced by thymus-derived lymphocytes previously sensitized against the antigen-bearing allogeneic cells in mixed lymphocyte cultures. A separate piece of evidence implicating the lipid bilayer as the primary target of lymphocyte-mediated damage came from experiments by Willoughby and Mayer (1980) in which it was shown that incorporation of additional cholesterol into sheep or chicken erythrocyte membranes causes a substantial decrease in the susceptibility of these cells to ADCC. The same effect has also been observed in experiments on complement-mediated hemolysis. The authors interpreted these findings as suggesting that the exogenously added cholesterol would tighten the packing of the lipid bilayer, which, in turn, would be expected to decrease the probability of insertion of “hydrophobic channel-forming peptides. ” As early as 1977, in his presidential address to the American Association of Immunologists, Mayer had the foresight to draw analogies between the cytolytic reactions mediated by lymphocytes and those produced by C, in spite of lack of any solid or structural data to support such claims (Mayer, 1977, 1982). He proposed that channel-forming proteins from lymphocytes, possibly C components or C-like polypeptides, could be involved in the delivery of TC membrane injury. He also speculated that “the entire attack process [mediated by lymphocytes] may require only two or three components, compared to the fourteen proteins that make up the complement system” sirice “the direct and intimate contact between killer and target cells may suffice to focus the attack on the membrane of the target so as to spare
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bystander cells” (Mayer, 1982). And, “conceivably, appropriate cell-cell contact could cause a conformational change in a single species of protein on the killer cell surface, leading to exposure of hydrophobic peptides that can then become inserted in the lipid bilayer of the target cell.” It is now apparent that the accuracy of these statements has withstood the past few years of intensive investigation in several laboratories. Since it is generally accepted that C lyses cells by a colloid osmotic mechanism, any analogies between C and lymphocyte-mediated killing require understanding of the term colloid osmotic. When a channel is inserted into target membrane, it produces abruptly a semipermeable membrane; that is, a diffusion-limited passageway for small ions is formed in the plasma membrane. Small ions, mainly cations, are then driven through the membrane down their respective electrochemical gradients which are otherwise normally maintained across a plasma membrane. Since the intracellular polyelectrolytes (like proteins and other large-molecular-weight macromolecules) are either retarded or impeded from crossing the membrane, a transient Donnan effect ensues, with water flowing passively into the TC from the outside in response to osmotic pressure differences that are transiently built across the plasma membrane. The inflow of water leads to cell swelling and eventually to cell bursting. The so-called colloid osmotic mechanism predicts that small ions should leak across the membrane before cell lysis occurs, hence, the early observations of 86Rb+ efflux from cells damaged by lymphocytes. The colloid osmotic mechanism also predicts that exogenously added macromolecules, with effective Stokes diameters that exceed that of the channel size, should balance out the differences in transmembrane osmotic pressure and therefore block cell rupture. Ability of a given molecule to block cell lysis should provide an estimate of the upper limit of the channel diameter. As outlined above, all these aspects of colloid osmotic lysis have been observed for TC lysis mediated by lymphocytes. The first structural evidence for pores on TC membranes was provided by Dourmashkin and his collaborators (1980). Their examination by negative staining of erythrocyte ghost membranes after attack by lymphocytes in an ADCC reaction revealed circular lesions with an internal diameter of 15 nm. As noted by the authors, the dimensions of this type of lesion were larger than the lesions mediated by C, which generally assume an internal diameter of 10 nm. Podack and Dennert (1983) and Dennert and Podack (1983) were able to confirm and extend these findings to cloned NK cells and H2specific T killer cells. These investigators described two types of tubular lesions, one with an internal diameter of 16 nm, named polyperforin 1, and a second smaller lesion, with an internal diameter of 5 nm, or polyperforin 2. The putative monomer that assembles these lesions was named perforin, for its ability to perforate membranes. Interestingly, the tubular lesions ob-
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served by these investigators contained each a torus and membrane-embedded domain, which were reminiscent of C lesions. Of relevance, they also observed that lymphocyte granules are often found near the site of contact with TC, suggesting an active participation for cytoplasmic granules in cytolysis. In the schematic model proposed by Dennert and Podack (1983), tubular lesions previously assembled in the killer cell in the form of vesicular material are seen to be delivered directly to TC membranes by a putative process of membrane fusion. The components responsible for the assembly of perforin lesions have now been purified and their properties characterized in some detail. Rather than being preassembled in the effector cell, a protein monomer is now thought to be released into the extracellular medium which then assembles into the membrane lesions. These findings will be discussed in a later section. D. GRANULE EXOCYTOSIS MODELFOR CELL KILLING A transfer mechanism whereby the lymphocyte transfers cytotoxic substances to the TC has been speculated for a long time. Tight junctions have been postulated to occur between effector and target cell that allow the transfer of lymphocyte material (Sura et al., 1967; Sellin et al., 1971). This model was based on assays for transfer of cytoplasmic contents or fluorescent probes (fluorescein) from lymphocytes to TCs. However, the occurrence of cytoplasmic connections was not confirmed in subsequent studies (Kalina and Berke, 1976; Sanderson and Thomas, 1977). The notion of secretion as the cytotoxic mechanism was nevertheless brought forth by several other investigators, including Henney (1975), who considered the role of soluble cytotoxic mediators which are tentatively secreted during cell killing. Morphological evidence was provided by others that showed the deposition on the target surface of acid phosphatase (Thiernesse et al., 1977) and osmophilic staining material (Bykovskaja et al., 1978b), both presumed to be derived from similarly staining granules. These observations have led Zagury (1982) to suggest that secretion of hydrolytic enzymes of lysosomal origin at the CTL-TC contact site represents the delivery of the lethal hit. Several other pieces of evidence were supportive of the notion of a secretion mechanism for lysis. The well-known requirements for calcium during the lethal hit stage of killing by CTL have lent support to a secretory process (Henney, 1973b; Plaut et al., 1976). Carpen et al. (1981)and Pederson et al. (1982) showed that an intact secretory apparatus is required for NK cell killing and ADCC. These investigators showed that monensin, a well-known blocker of secretory pathways involving the Golgi, is also an effective inhibitor of cell-mediated killing. Similar results were later obtained for clones of NK-like cells (Acha-Orbea et al., 1983) and H-2 restricted killer cells (Dennert and Podack, 1983). Chloroquine, which accumulates in lysosomes caus-
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ing extensive vacuolation of cells, also blocks cytolysis mediated by lymphocytes (Brondz et al., 1973; Roder et al., 1980). Cytolysis is also suppressed by cytochalasin B and by colchicine at levels which do not block conjugate formation, but which would presumably inhibit secretion (Plaut et al., 1973). Using exogenously added proteases as inhibitors of NK cell killing, Hiserodt et al. (1983b)have suggested that protease-sensitive structures are delivered from the effector to the target during cytolysis. More recent evidence from Gray and Russell (1986) shows that CTL killing depends on extracellular C1-, since replacement of external C1- with other anions and treatment of CTL with stilbene disulfonate derivatives, known blockers of chloride fluxes across membranes, result in loss of lytic activity. These authors argue that since C1- fluxes have previously been implicated in secretion and exocytosis of a variety of cell types, the dependence of CTL function on intact CLfluxes suggests that the delivery of the lethal hit by CTL may involve granule exocytosis. An involvement for granules in cell-mediated killing was also supported by morphological evidence. As outlined earlier, a major rearrangement of cytoskeletal elements occurs rapidly following killer-target cell conjugation, with reorientation and polarization of the Golgi, the MTOC, and the granules toward the site of cell-to-cell contact. Fusion of granules directly with plasma membranes has been monitored continuously by high-resolution cinemicrographic studies (Yannelli et al., 1986). Other recent electron microscopic studies of NK cell-TC conjugates have provided further evidence for degranulation and the deposition of osmopholic granular material at the site of cell contact (Frey et al., 1982). Based on these observations, these authors have suggested that degranulation appears to be involved in the cytotoxic function of NK cells. The chief proponents of the secretory model, tying together granule exocytosis and assembly of tubular lesions on TC membrane, were Henkart and Henkart (1982) (see also Henkart, 1985). These investigators have suggested that the lymphocyte-mediated killing resembles in many aspects a stimulus-secretion coupling phenomenon, commonly observed for other well-known secretory cell types. The evidence they presented was mainly morphological, with the demonstration of granule fusion and release of granule contents at the site of cell-to-cell contact. Recently, Neighbour et al. (1982) have reported that strontium-induced loss of granules in human and mouse N K cells is correlated with a loss of cytotoxicity. Granules from CTL and NK-like cells have since been isolated and characterized. There remains little doubt now that the lytic mediator of lymphocytes resides in the cytoplasmic granule populations of these cells.
E. INTRACELLULAR DAMAGE An alternative view on cell-mediated killing proposes that the damage of TC initiates from within the target. Time-lapse cinematographic analysis of
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CTL-mediated killing and ADCC led Sanderson (1976) and Sanderson and Thomas (1977) to propose the following course of events thought to occur after conjugate formation: The lethal hit initiates with a progressive series of cytoplasmic convulsive movements in the TC accompanied by nuclear and plasma membrane blebbing, termed zeiosis, which precede an increase of transmembrane fluxes and loss of cytoplasmic contents. Zeiosis is not apparent when TCs are damaged by antibodies and C, suggesting that complement-mediated killing is remarkably different from the injury mediated by CTL and killer cells involved in the ADCC reaction. Several other morphological studies have confirmed and extended the notion that the process of TC disintegration may be significantly different, depending on whether cytolysis is produced by CTL, NK cells, or by C activation (Matter, 1979; Wyllie et a l . , 1980; Russel et d . , 1982). Russell and his colleagues recently proposed a novel “internal disintegration model” to explain the mechanism of cell-mediated lysis (reviewed by Russell, 1983). In this model, lymphocytes trigger an autocatalytic cascade within the target which results in nuclear membrane damage and DNA fragmentation. In support of their model, Russell and colleagues have taken into consideration previous morphological observations which have shown that the earliest nuclear changes in TCs mediated by CTL are a condensation of chromatin followed by blebbing of nuclear membranes. Similar changes are not observed during C-mediated lysis. Moreover, the pattern of release of cytoplasmic (51Cr) and nuclear ([ 1251]uridine and [ 1251]UdR)labels from mouse TCs lysed by CTLs is remarkably different from the pattern generated during C-mediated lysis (Russell et al., 1980); that is, when TCs are lysed by CTLs, 51Cr release is thought to be preceded by release of the nuclear label, suggesting an early onset of TC DNA breakdown. In contrast, during TC lysis mediated by antibody and C, 51Cr release has been shown to occur immediately, whereas release of nuclear labels is extremely slow. The nuclear membranes of TCs lysed by CTLs, but not C, become sensitive to low levels of nonionic detergent, and the TC DNA is rapidly digested to a size that is capable of escaping from the detergent-treated nucleus (Russell and Dobos, 1980). Kinetic analysis of this process revealed that nuclear membrane damage and DNA breakdown are initiated early on during the calcium-programming lytic stage (Russell et al., 1982). The early digestion of TC DNA proceeds with the generation of fragments that are multiples of base pairs ranging from 150 to 180 (Duke et al., 1983; Russell, 1983), which suggests the involvement of an internal endonuclease activity within the target. These observations have recently been confirmed for cell killing mediated by mouse NK cells (Sears and Christiansen, 1985). These same authors reported that mouse target DNA fragmentation is triggered by either mouse or human effectors, whereas DNA fragmentation does not occur in human TCs exposed to human effector cells. More recent studies by
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Gromkowski and his colleagues (1986) have shown that DNA fragmentation is not specific to the species of effector cell used. However, the pattern and the extent of TC DNA damage appears to be characteristic of the TC species, irrespective of the effector cells used. These data appear to lend support to the notion proposed by Russell (1983) that the degradation of TC DNA results from the activation of a TC enzyme during cell-mediated killing and not from the active transfer of lymphocyte DNase into TC nucleus. However, more experiments are needed to resolve this important issue.
F. ROLEOF OTHERSOLUBLECYTOTOXIC MEDIATORS IN CELLKILLING 1 . Lymphotoxin-Like Molecules Several other mediators have been considered as potential cytotoxic substances in the past. The most prominent one, lymphotoxin (LT), was discovered simultaneously by two groups in 1968 as a soluble product of antigen or mitogen-stimulated lymphocytes that causes cytolytic changes in certain TCs growing in the same culture (Ruddle and Waksman, 1968; Granger and Kolb, 1968). LT has been shown to be cytotoxic for a variety of tumor cells (Rosenberg et al., 1973; Williams and Granger, 1973; Sawada et al., 1976). Studies from a number of laboratories indicate that LT is a direct-acting cytotoxic lymphokine with cytostatic and/or cytolytic activities, depending upon the TC being affected (reviewed by Evans, 1982; Granger et al., 1982). LT has since been purified to homogeneity (Agganval et al., 1984) and cloned by recombinant cDNA techniques (Gray et al., 1984). The purified LT has been shown to synergize with interferon-y to produce antiproliferative effects on certain tumor cell lines not sensitive to either one added alone (Lee et al., 1984). LT has been proposed in the past to have some involvement in CTL killing (Russell et al., 1972; Hessinger et al., 1973; Walker and Lucas, 1973; Henney, 1975; Kramer and Granger, 1976). However, evidence used to argue against its involvement in lymphocyte-mediated killing included its slow mode of action, taking generally several hours for the cytotoxic reaction to complete, its lack of antigen specificity, and its apparent lack of requirement for calcium (Plaut et al., 1976; Okamoto and Mayer, 1978; Cerrotini and Brunner, 1974). Moreover, anti-LT antibody blocking experiments have produced contradictory findings, some reports describing effective blocking of CTL (Hiserodt and Bonavida, 1981; Leopardi and Rosenau, 1982) and NK (Weitzen et al., 1983) activity, while others describing their inefficacy on cytotoxic reactions (Gately et al., 1976; Ware and Granger, 1981). There is also some controversy concerning the cell type that produces and secretes
LT, the initial reports suggesting that LT is produced chiefly by Lyt-l+ cells rather than by Lyt-2+ CTL (Eardley et al., 1980). More recent reports from the same group, however, have indicated that Lyt-2+ CTL lines are also capable of producing LT upon stimulation with mitogen or with TNP-coupled syngeneic splenocytes (Conta et al., 1985; Schmid et al., 1986). Another soluble cytotoxin closely related to LT, named tumor necrosis factor (TNF) for its ability to produce hemorrhagic necrosis of a variety of tumors (Carswell et al., 1975; reviewed by Old, 1985), is known to be produced by macrophages. TNF has also recently been cloned and its sequence deduced by cDNA cloning (Pennica et al., 1984; Wang et al., 1985). LT and TNF are closely related, with 36% amino acid sequence identity and at least 51% homology. A third factor, also closely related to LT and TNF, has been described as a secretory product of NK cells by Wright and Bonavida (1981, 1982) and has been termed N K cytotoxic factor (NKCF). One important difference between NKCF and LT, according to these investigators, is that in contrast to LT, NKCF displays target specificity that correlates with that of the N K cells (Wright and Bonavida, 1983). The exact molecular nature of NKCF becomes even more uncertain when one considers that LT is also reportedly synthesized by NK cells (Leopardi and Rosenau, 1984). Furthermore, recent experiments by Trinchieri, Perussia, and their collaborators have clearly established that NKCF is biochemically, antigenically, and functionally similar or identical to the TNF produced by human moriocytes and myeloid cell lines (Degliantoni et al., 1985). Recent reports from the laboratories of Ruddle and Granger (Schmid et al., 1986; Yamamoto et al., 1986; Kobayashi et al., 1986) have firmly established that LT-like forms are produced by CTL and NK cells. Granger’s group describes a novel LT and TNF-like form produced by these cell types that is functionally and immunologically related, but not identical to LT and TNF. Furthermore, Schmid et al. (1986) have shown that LT-containing supernatants derived from CTL lines mediate DNA fragmentation into repeat units of 200 base pairs. Based on these results, Schmid et al. (1986)have proposed that the LT secreted from CTLs probably represents the species responsible for DNA fragmentation that occurs during lymphocyte-mediated killing as observed earlier by Russell and his colleagues (Russell, 1983). It remains unclear, however, how LT would permeate through the TC membrane to produce this effect. Recent observations made by Konigsberg and Podack (1986) have shown that granules mediate DNA breakdown in LTsensitive cell lines. Moreover, monoclonal antibodies specific for LT block the LT-like activity of the granules. We have also recently purified an LT and TNF-like polypeptide from the granules of a CTL line (presented later in this review together with other granule constituents). More experiments
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are needed to confirm the important results presented by Schmid et a2. (1986) that suggest a direct role for LT in lymphocyte-mediated changes in the target.
2 . Reactive Oxygen Metabolism lnterrnediates Using chemiluminescence assays for investigating the presence of reactive oxygen intermediates, Roder, Helfand, and their colleagues reported the release of these metabolites during killing mediated by NK cells (Helfand et al., 1982; Roder et al., 1982; Werkmeister et al., 1983). However, later studies using several different assays were unable to confirm a role for such metabolites in NK cell-mediated killing (Donzig and Moly, 1985; Kay et al., 1983, 1985; Storkus and Dawson, 1986). Nathan and his colleagues (1982) have also found that for CTL lines, although 0, is required for killing, toxic oxygen intermediates are not directly involved in the killing process. It should be noted that the lack of detectable reactive oxygen intermediates at the effector-target interface does not rule out the use of such metabolites intracellularly by the killer cell at some point during cell lysis. Thus, Duwe et al. (1985) have recently shown that OH- radicals, presumably produced in the lipooxygenase pathway of fatty acid metabolism, are apparently involved in NK-related reactions. Scavengers of O H - radicals (Duwe et al., 1985) and inhibitors of the lipooxygenase pathway (Suthanthiran et al., 1984) are effective blockers of NK cell killing.
3. Leukoregulin Leukoregulin is the name given by Evans and his collaborators to a lymphokine secreted by NK cells that predominantly plays a cytostatic function on a variety of tumor cell lines (Ransom et al., 1985; Barnett and Evans, 1986). The major form of human leukoregulin has a p l of 5.3 and a molecular mass of 32 kDa. It rapidly increases the plasma membrane permeability of tumor cells as detected by the loss of intracellular fluorescein and the uptake of extracellular propidium iodide. Further biochemical analysis will be required to elucidate the function of this novel mediator in cell-mediated killing. IV. Granule Proteins in Cell-Mediated Killing
A. CELLLINESOF CTL AND NK CELLS The biochemical analysis of effector mediators produced by CTL and NK cell has become possible largely due to the advent of new cloning and isolation procedures that have helped establish in uitro cell lines of these cell types (Gillis and Smith, 1977). Such cell lines are now routinely derived in
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the presence of lymphocyte-conditioned medium containing interleukin 2 (IL-2) and other growth factors. The presence of monolayers of mononuclear feeder cells is also known to enhance the growth of these cell types. With use of this approach, numerous cell lines have been derived and characterized in terms of their surface phenotype and target specificity (Dennert, 1980; Kornbluth and Dupont, 1980; Nabel et al., 1981; Bracilae et al., 1981; Dennert et al., 1981; Kedar et al., 1982; Brooks et al., 1982; Sugamura et al., 1982; Acha-Orbea et al., 1983; Dvorak et al., 1983; reviewed by Nabholz and MacDonald, 1982; von Boehmer and Haas, 1985). It is noteworthy to point out that the target specificity of cell lines maintained in long-term cultures may change. Some CTL lines, for example, are known to acquire NK-like function (Acha-Orbea et al., 1983; Brooks et al., 1983), whereas others may show both CTL and NK-like activity (see von Boehmer and Haas, 1985, for review). Among the more prominent morphological features of the killer cell lines established in culture is the presence of numerous, large, electron-dense cytoplasmic granules. The granules are occasionally scattered through the cytoplasm or, more commonly, appear concentrated in the perinuclear region. The granules show a distinct internum and externum. The internum, made up of a fine amorphous matrix, is bounded by a unit membrane surrounded by electron-dense membrane-bound vesicular material. The granules have been shown to contain certain hydrolytic enzymes and are known to display nonspecific esterase reactivity (Bozdech and Bainton, 1981; Grossi et al., 1982, Dvorak et al., 1983; Petty et al., 1984; Young et al., 1986a). The cell surface of large granular lymphocytes (LGL) (Grossi et al., 1982), cloned NK cells (Dvorak et al., 1983), and a cytolytic T cell line (Young et al., 1986a) also show nonspecific esterase activity. The cytoplasm of these cell lines contains abundant rough endoplasmic reticulum, which often shows dilated cisternae. A particulate material in the cytoplasm of some cell lines has also been reported (Dvorak et al., 1983; Young et al., 1986a) which appears to correspond to deposits of glycogen. B. CYTOPLASMIC GRANULES 1 . Isolation
If, in fact, the lytic apparatus of CTLs and NK cells resides in their cytoplasmic granules, as suggested by previous studies, then only subcellular fractionation studies and isolation of a purified population of granules would allow one to submit this hypothesis to a rigorous test. With the feasibility now of growing homogeneous populations of cytolytic cells to at least lo9 cells, several laboratories have succeeded in isolating granule populations from LGL, CTLL, and NK-like cells (Henkart et al., 1984; Millard et
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al., 1984; Podack and Konigsberg, 1984; Young and Cohn, 1985; Masson et al., 1985; Criado et al., 1985; Young et al., 1986b,c; see also reviews by Martz, 1984; Podack, 1985; Henkart, 1985; Young and Cohn, 1986a,b). The isolation procedure usually involves rupturing cells, followed by centrifugation of the nucleus-free lysate through continuous or discontinuous Percoll gradients. Because of their higher density, granules are usually separated from other organelles by velocity sedimentation. The different fractions collected from the Percoll gradient are tested for enzymatic and hemolytic activities. Granules of lymphocytes are lysosomal in nature (Henkart et al., 1984; Young et al., 1986a) and can be distinguished from the following cytoplasmic organelles based on differential distribution of enzyme markers: mitochondria (enriched for succinate dehydrogenase), lysosome (larger peak of acid phosphatase and p-glucuronidase), and plasma membrane (alkaline phosphodiesterase and 5'-nucleotidase). Isolated granules are highly enriched for hemolytic and serine esterase activities. This potent esterase activity has recently been described as a convenient marker for lymphocyte granules (Young et al., 1986a). It is not clear why serine esterase activities were reportedly negative in the granules in previous studies (Henkart et al., 1984; Millard et al., 1984). Because the routine and preparative separation of cell granules requires screening of a large number of subcellular fractions for hemolytic and enzymatic activities, several automated microassays for these activities have recently been developed that make use of microtiter plates and spectrophotometric reading of the reaction products by automated readers (Young et al., 1986d,e). Several cell lines initially characterized as CTLL, but that have lost their cytolytic capability in long-term cultures, do not contain hemolytic granules (unpublished observations). The expression of cytolytic granule contents appears to be related to the action of interleukins. A T cell hybrid (PC60) that can grow independently of IL-2 becomes cytolytic and acquires cytoplasmic granules following induction with a combination of IL-1 and IL-2 (Erard et al., 1984; Masson et al., 1985). Cell lines of this type that show selective deficiencies in the expression of granules or lytic activity may become powerful tools in dissecting the lytic mechanisms of lymphocytes.
2 . Structural and Functional Lesions Produced by Granules Work from several laboratories (reviewed by Henkart, 1985; Podack, 1985; Young and Cohn, 1986b) has established that the isolated granules are capable of mediating the assembly of tubular lesions on target membranes (Fig. 1).Tubular lesions with an internal diameter ranging from 150 to 170 A are observed only at temperatures exceeding 30°C and in the presence of submillimolar amounts of calcium. Henkart and his collaborators (1984) showed that calcium can be effectively replaced by strontium, in accord with
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FIG. 1. Morphology of isolated granules and the tubular lesions associated with granules. (A) Granules were isolated by centrifugation through Percoll gradients. (B) Selected images of circular lesions by granules on erythrocyte membranes. Arrows point to top views of circular lesions. Arrowheads correspond to longitudinal sections of the tubular lesions. Scale bars, 270 nm (A); 57 nm (B); 38 nm (C); 87 nm (D). From Young et al. (1986c).
observations made in the past for many other calcium-binding proteins. The tubular lesions formed by the granules resemble closely the previously described polyperforin 1 lesions on target membranes (Fig. 1). As mentioned earlier, granules isolated from LGL, CTLL, and NK-like cells contain a potent hemolytic activity, which is only expressed in the presence of calcium. Kinetic studies performed on the hemolytic reaction mediated by the granules, as measured by continuous monitoring of turbidity of the erythrocyte suspension at 700 nm, reveal that hemolysis is virtually complete within 10 minutes at 37°C (Young et al., 1986b,c). The hemolysis is completely blocked by chelating calcium in the erythrocyte medium with EGTA, but can be restored readily with subsequent addition of calcium. The hemolytic activity is markedly reduced when the hemolytic reaction is performed at room temperature. At temperatures below 4"C, hemolysis is completely abolished. The hemolytic activity is unstable and can be inactivated by exposure of granules to 37°C and/or to submillimolar amounts of calcium (Henkart et al., 1984; Young et al., 1986~). Granules depolarize rapidly the membrane potential of nucleated cells
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(Young et al., 1986b,c) and induce marker release from lipid vesicles. The membrane of lipid vesicles becomes leaky to carboxyfluorescein (Blumenthal et al., 1984), sucrose, Lucifer Yellow, and monovalent and divalent ions (Young et al., 1986b,c). These experiments reveal that granules are active on lipid bilayers without showing any target specificity. Sizing experiments using resealed erythrocyte ghosts as target membranes revealed that granules mediate release of polypeptides smaller than a-bungarotoxin (8000 Da) (Criado et al., 1985). Furthermore, the lytic activity expressed by the granules is optimal at neutral pH. Intact granules contain all the lytic activity. The rupture of granules and the release of soluble granule contents into the supernatant do not seem to be required for the lytic activity. This inference is suggested by experiments in which granules are washed several times by high-speed centrifugation. The final resuspended granules still show potent hemolytic activity, implying that possibly fusion of granules with target membranes may precede lysis (Young et al., 1986c, 1987a). Moreover, granules and solubilized granule contents have different requirements for calcium and pH for the optimal expression of hemolytic activities. Granule proteins can be readily solubilized with high amounts of salts (NaCl, phosphate, ammonium acetate, etc.), and the solubilized proteins can be separated from granule membranes by sedimentation of membranes by high-speed centrifugation. The solubilized granule proteins retain all the lytic activity originally contained in the intact granules. When tested against high-resistance voltage-clamped planar lipid bilayers which are capable of resolving the transmembrane flow attributed to individual channel molecules, granule proteins have been shown to induce a rapid change in membrane resistance (Young et al., 1986b,c). The change in membrane resistance occurs as a progressive incorporation of discrete ion channels into the lipid bilayer, measured as a stepwise increment of current steps, which usually proceeds until the membrane breaks down. The sizes of the channel steps are heterogenous, ranging 0.4 to 6 nS per channel in 0.1 M NaCl (1 S defined as ampere/volt). This range of unitary conductances reflects a flow rate of at least lo9 ions/second/channel. By analogy, constitutive channels normally found in biological membranes usually conduct at flow rates of 3-4 log magnitudes lower than the granule-derived channels. The larger scatter obtained for the unit conductances may be due to multiple sizes associated with the membrane lesions, which would include partially polymerized complexes, half-rings, complete rings, and double rings. The channels formed by the granule proteins remain permanently open and are highly resistant to closing by an increase of the transmembrane potential. This behavior indicates that large, stable, and voltage-resistance channels
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are formed by granule proteins, which are attributes that would favor an active role for these channels in mediating cytolysis. C. PORE-FORMING PROTEINISOLATED FROM GRANULES
1 . Purification Prior to its isolation, the putative pore-forming protein (PFP) from lymphocyte granules has been given different names, which followed mainly the morphological observations of tubular lesions produced on target membranes by lymphocytes. Henkart and his collaborators have named the putative pore former of rat LGL tumor as cytolysin (Henkart et al., 1984). Podack and Dennert (1983) have named the mouse CTL and NK cell pore formers as perforin 1 and 2. Since PFPs have now been isolated from a number of cell types, including human eosinophils (Young et al., 19860, E . histolytica (Lynch et al., 1982; Young et al., 1982), mouse lymphocytes with NK-like activity (Young et al., 1986c), and human NK cells (Liu et al., 1986), we will refer generically to these proteins here simply as lymphocyte poreforming proteins (PFP). The PFP from mouse CTLL and from an NK-like lymphocyte clone has recently been purified by a combination of molecular sieving and ion-exchange chromatography (Podack et al., 1985; Young et al., 1986c,g). PFP from CTLL has also been enriched only by molecular sieving chromatography (Masson and Tschopp, 1985).The fractions eluted from the columns are assayed for hemolytic activity using the microassay described earlier (Young et al., 1986d) and also directly for pore-forming activity in planar lipid bilayers (Young et al., 1986~).More recently, PFP from CTLL and human NK cells has also been purified by affinity chromatography using specific immunoglobulins attached to agarose as the immunoadsorbent (Young et al., 1986h; Zalman et al., 1986a; Liu et al., 1986). The purified monomeric protein migrates under reducing conditions with a molecular mass of 66 to 68 kDa, according to Masson and Tschopp (1985), and 70 to 75 kDa (Podack et al., 1985; Young et al., 1986c,d,g; Liu et al., 1986) when analyzed by SDS-polyacrylamide gel electrophoresis. The nonreduced form of this protein has an apparent molecular mass of 60 to 66 kDa as observed by gel electrophoresis and by molecular sieving chromatography (Young et al., 1986~). Recently, human peripheral blood LGLs isolated by density centrifugation (Zalman et al., 1986a)and NK cells isolated by indirect rosetting using a panel of monoclonal antibodies directed against different NK and T cell markers (Liu et al., 1986)were shown to contain a C9-related polypeptide in their granules. Antibodies prepared against C9 were used as immunoadsor-
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bents in the purification of a polypeptide of 70 kDa closely related to the PFP discussed here. This protein shows a number of biochemical and functional properties which are similar to the granule PFP isolated from murine CTLL and NK-like cells.
2 . Biochemical Properties of the Purified Pore-Forming Protein The purified protein polymerizes in the presence of calcium to form large tubular complexes of molecular mass exceeding 1 million Da (Fig. 2). This polymeric species resists at least partially dissociation by boiling and by treatments with SDS and reducing agents (Fig. 2; Young et al., 1986c,g). The polymerized material elutes in the void volume of Sephacryl S-200 columns. Under electron microscopy, the polymerized material assumes the shape of tubular lesions similar to the previously described lesions found in association with cells and their granules (Fig. 2). Ring-like structures with an internal diameter of 150-170 are typically observed. In the absence of
FIG. 2. Selected images of membrane lesions on erythrocyte membranes produced by isolated lymphocyte PFP/perforin. Ring structures of 16 nm internal diameter (arrows) and some incompletely polymerized tubules (arrowheads) are seen. Scale bar: Upper panels, 250 nm; lower panels, 85 nm. From Young et al. (1986g).
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Bee Venom Melfttfn --
A.
mellffera Gly-I1e-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala+
+
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Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH 2
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Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala+
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Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-CONH 2
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Gly-Ile-Gly-Ala-Ile-Leu-Lys-Val-Leu-A1a-Thr-Gly-Leu-Pro-Thrt
+
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Leu-110-Ser-Trp-Ile-Lys-Asn-Lys-Arg-Lys-Gln-CONH 2
Synthetic Melfttin-like Peptfde Lmu-Leu-Gln-Ser-Leu-Leu-Ser-Leu-Leu-Gln-Ser-Leu-Leu-Leu-Ser-Leu+
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Leu-Leu-Gln-Trp-Leu-Lys-Arg-Lys-Arg-Gln-Gln-CONH 2
Staphylococcal & - T o m Het-Ala-Gln-Asp-Ile-Ile-Ser-Thr-Ile-Gly-Asp-Leu-Val-Lys-Trpt
t
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-
Ile-Ile-Asp-Thr-Val-Asn-Lys-Phe-Thr-Lys-Lys-COO FIG.3. Primary structure of several surface-active peptide toxins. The sequences of melittin from several species of honeybee are shown. The melittin-like peptide is a synthetic peptide designed by IkGrado et al. (1981) that has cytolytic properties. Compiled from Fitton et al. (1980), Kreil (1973), and DeGrado et al. (1981).
calcium, polymerization is not observed. The isolated protein is sensitive to the effects of calcium and zinc, both of which rapidly inactivate its lytic activity (Podack et al., 1985; Young et al., 1 9 8 6 ~ ) . The purified protein has potent hemolytic activity. One nanogram of protein lyses completely 108 sheep red blood cells (Young et al., 1986d). Calcium is required for the expression of this potent lytic activity. The purified protein also lyses a variety of tumor cells, including EL-4, J774 macrophages, S49.1 lymphoma cells, K562, S194, YAC-1, and 3T3 cells (Young et al., 1986c,g). The amount of protein required to lyse nucleated cells is always severalfold higher than that required to lyse an equivalent number of erythrocytes, an observation consistent with the well-known mechanism of membrane repair that makes nucleated cells more resistant to lysis by poreforming substances. The lytic activity expressed by PFP on nucleated cells can be measured as a rapid depolarization of the resting membrane potential of target cells
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(Young et al., 1986c,g). Several different target cell types have been used to measure membrane potential changes. Because of their large size and consequently easy accessibility to impalement with microelectrodes, chicken embryo myocytes are convenient target cells for PFP. PFP has to be introduced close to the myocyte (within 100 pm) in order to produce any surface-active effect. This is due to the rapid inactivation of this protein at 37°C and in the presence of calcium, as described in more detail later. Calcium is required for the depolarization activity. PFP that has previously been polymerized in solution in the presence of calcium is totally inactive on the cells. PFP also inserts spontaneously into cell membranes, as measured by patch clamp in the whole cell configuration (Podack et al., 1985). PFP mediates increase of membrane current that can be resolved into discrete current steps which are usually indicative of incorporation of discrete ion channels into the cell membrane. Results obtained with excised membrane patches confirm the insertion of discrete channels into target cell membranes (unpublished observations). Lipid vesicles made of several types of lipid also become leaky to electrolytes and certain macromolecules, such as Lucifer Yellow (457 Da) and sucrose (342 Da) (Young et al., 1 9 8 6 ~ )The . membrane leakiness induced by PFP is nonspecific to several ions tested, including potassium, sodium, lithium, chloride, calcium, magnesium, zinc, and barium. Several biophysical properties of PFP in lipid bilayers have recently been elucidated (Young et al., 1986c,g). Like the granule extracts, the purified PFP forms large, voltage-insensitive, ion-nonselective channels in planar lipid bilayers. Bilayers treated with PFP become permeable to glucosamine+ (which has a Stokes diameter of 8 A), Tris-, and EGTA2-, implying a large functional diameter for the transmembrane tubules. It has recently been suggested that PFP forms functional lesions without having to polymerize completely into the tubular structures observed under electron microscopy (Young et al., 1986g). The isolated protein forms channels at room temperature under conditions in which ring-like lesions are not observed. Also, channels with a large scatter in sizes have been measured, indicating that various sizes are associated with the functional lesions. The tubules that have been polymerized prior to their incorporation into planar bilayers show higher unit conductances compared to the channels formed by the addition of monomeric protein to lipid bilayers. Thus, in our opinion, complete cireular polymerization is not an obligatory requisite for functional channel formation, although the circular lesions probably represent channels of the largest diameters that can be attained by polymerization. It should be noted that Berke and his collaborators have recently been cited by Marx (1986) to have provided arguments against the pore-formation model in view of their morphological studies of a large number of target membrane spec-
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imens of cells lysed by lymphocytes which have failed to reveal any circular lesions, in contrast to the early observations made by Dourmashkin et al. (1980) and Podack and Dennert (1983).There remains little doubt that the PFP represents a structural entity which can be isolated from lymphocyte granules. This protein, however, is thought to inflict multiple-sized lesions. in accord with the multiple conductance units measured in planar bilayers and patch-clamped membranes. Pore formation by lymphocyte PFP may involve a “barrel stave” model in which monomers would aggregate like barrel staves surrounding a nucleus pore that grows in diameter through the progressive recruitment of monomers. The monomers are thought to span the membrane and, through lateral movement in the bilayer, they oligomerize in such a way that the hydrophobic side of the molecule is exposed to the lipid while the hydrophilic sides line up the pore interior. This type of model would explain the multiple conductance states observed in association with single PFP channels. During cell killing, it is more likely that the formation of small channels (not observed by electron microscopy) is favored over the completely circularized lesions, based simply on statistical grounds. Membrane damage by the colloid osmotic mechanism would be operative as long as a channel large enough for the passage of small ions is formed. Only under optimal conditions (e.g., prolonged incubation, high concentration of PFP, millimolar amounts of Ca2+, neutral pH, low amounts of serum) would one expect a higher probability to find the circular lesions. Thus, the inability to demonstrate morphological lesions of large diameters cannot be used as an argument against the pore-formation model in cell-mediated killing. As noted earlier, the polymerized species is no longer active on bilayer and requires prior solubilization with detergent for its incorporation into the planar bilayer.
3. Evidence for a Membrane Binding and Pore-lnsertion Stage and lnactiuation of PFP Recent studies with isolated PFP have shown that at low temperatures (on ice), PFP binds to erythrocyte membranes without, however, producing hemolysis. Hemolysis occurs when the PFP-bound erythrocytes are subsequently warmed up to 37°C. This type of experimental evidence allows one to define a temperature-dependent pore insertion step which can be distinguished from the membrane binding event (Young et al., 1987a). Calcium and neutral pH are required for both membrane binding and pore insertion by PFP. The lack of calcium and reducing pH under 6. 2 produce a reversible inhibitory effect on the membrane binding and insertion activities of PFP. Serum, LDL, and HDL have recently been shown to block the lytic activity of PFP (Tschopp et al., 1986a). These reagents, and also heparin, inhibit PFP-mediated hemolysis by interfering only with the binding step (Young et
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al., 1987a). These reagents may compete with lipid bilayers for the lipidbinding domain of PFP. Our results also suggest that this lipophilic domain of PFP is only accessible when PFP is in solution and has not bound to lipid membranes. Once PFP has bound to erythrocyte membranes (on ice), the hemolytic activity is no longer susceptible to the inhibitory effects of neither one of these reagents. This mechanism may play an important protective function during cell-mediated killing whereby the extracellular serum could avoid the accidental injury of innocent bystander cells by secreted PFP. Due to the close apposition of effector-target cell membranes, it is expected that the released PFP would bind eficiently to the target bilayer. Any unbound PFP, however, would be rapidly inactivated by serum, preventing its further cytotoxic use. The lack of effect of serum and serum components on membrane-bound PFP suggests that once PFP has bound to bilayers, lysis would proceed to completion in the presence of a calcium and neutral pH environment, which approximates the conditions of the extracellular medium. These observations would also be consistent with previous reports establishing the presence of a killer cell-independent but calcium-dependent lytic stage in lymphocyte-mediated killing that occurs after contact and dissolution of the conjugates. Finally, the requirements for neutral pH and submillimolar amounts of calcium for expression of membranolytic activity would ensure that PFP is packaged in lymphocyte granules in an inactive form, since these cytoplasmic compartments are thought to be acidic in nature and low in free calcium (Steinman et al., 1983). 4 . PFP as a Secretory Protein CTLs stimulated with the calcium ionophore A23187 release PFP into the extracellular medium (Young et al., 1986h), suggesting that this protein is a secretory protein. Release of PFP is accompanied simultaneously by cell degranulation and functional pore formation, as measured in the planar lipid bilayer system. The protein released by mouse CTLs assembles into tubular lesions, binds to lipids, and has been identified as the lymphocyte PFP using specific antibodies as immunoadsorbents. Extracellular calcium is required for release. In the absence of calcium, 15% of the maximal release activity is observed. It is possible that during cell killing of target cells, the cytosolic levels of calcium may also increase to promote degranulation and secretion. In the case of NK cells, the binding of the NK cell surface Fc receptors may be actively involved in triggering secretion of granule contents by those cells. This possibility is currently being assessed in our laboratory. Recent studies by Bonavida and his colleagues (Graves et al., 1986) suggest the synergistic action of the calcium ionophore A23187 and phorbol esters on the release of NKCF by effector cells. These investigators have suggested a role for protein kinase C activation in the signaling of the release of NKCF.
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Analogously, a role for protein kinase C in degranulation of CTL and NK cells needs to be investigated. D. OTHERGRANULE CONSTITUENTS A N D THEIRROLE I N CELL-MEDIATED KILLING 1. Serine Esterase
Several laboratories have recently succeeded in isolating CTL-specific cDNA clones, all of which have turned out to encode for key sequences characteristic of serine proteinases (Lobe et al., 1985, 1986; Gershenfeld and Weissman, 1986; Brunet et al., 1986). That clones for serine esterases represent so far the only CTL-specific transcripts characterized in several laboratories suggests that these transcripts are truly CTL-specific transcripts, but also that they may represent abundant messages present in these cell types. A serine esterase activity characterized by using the ester compound N a benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) as the chromogenic substrate has previously been described as CTL specific (Pasternak and Eisen, 1985). This trypsin-like protease, which can be labeled with the affinity reagent for serine proteinases [3H]diisopropyl fluorophosphate (DFP), has a molecular mass of 28 kDa and is present at least 300 times higher in CTL than in other lymphocytes, including B cells, noncytotoxic T cells, and clones of helper T cells. Recently, three laboratories reported on the isolation of the trypsin-like serine esterase from granules of CTLL (Masson et a l . , 1986; Pasternak et al., 1986; Young et al., 1986a). Using lysine columns, Pasternak et al. (1986) purified a protein which is labeled on SDS-gels as a broad band with molecular mass of 28 kDa. Masson, Tschopp, and their collaborators (1986), and Young et al. (1986a) described the identification of two granule species which are labeled with ["HIDFP. The two proteins, referred to as serine esterases 1 and 2 (SE 1 and SE 2 ) by Young et al. and granzymes A and B by Masson et al., migrate with molecular masses of 3436 kDa and 28-30 kDa, respectively, under reducing conditions. The larger form contains all the trypsin-like activity, as measured with BLT, fibrin, and casein as substrates. Under nonreducing conditions, SE Ugranzyme A assumes a molecular mass of 60-66 kDa, suggesting that it may consist of two disulfide-linked subunits of 34-36 kDa each. It has a pI greater than 10 and optimal activity at pH 8. The substrate specificity of SE 2 is not known. The serine esterase activity is secreted by lymphocytes that have been stimulated with the cacium ionophore A23187 (Young et al., 1986a) and with target cells (Pasternak et al., 1986). None of these species has any plasminogen activator activity (Ossowski and Young, unpublished). A putative role of these enzymes in cell-mediated killing, perhaps in the processing of other lytic granule proteins which may be required for their activation, has been
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suggested. However, more experiments are required to elucidate their function in cell killing. It should be noted that recently, Kramer et al. (1986) questioned the specificity of this enzyme as a marker for CTL, as previously suggested by Pasternak and Eisen (1985). Kramer and his collaborators also identified two species of [3H]DFP-labeled polypeptides (40 and 32 kDa) in CTL as well as in other types of T cell lines. In addition to the trypsin-like activity mentioned above, they also identified proteases in CTL lysates with an amidolytic activity.
2 . Lymphotoxin and TNF-Related Polypeptides Previous studies have shown that an LT activity is present in the granules of CTLL (Konigsberg and Podack, 1986). Recent experiments in this laboratory have identified an LT and TNF-related polypeptide in the granules of CTLL (Young et al., 1987b). Isolated granules contain an LT-related polypeptide which can be affinity purified by using specific antibodies prepared against recombinant LT and TNF, suggesting that it is immunologically related to these two forms. The purified species has a molecular mass of 50 kDa under reducing conditions. Double-labeling immunofluorescence studies, using antibodies specific for PFP in one fluorescence channel and antibodies specific for TNF in another, point to its colocalization with PFP in the same cytoplasmic granules. It is conceivable that this LT-related polypeptide may be introduced into the TC via the transmembrane channel (16 nm) formed by fully polymerized PFP, and once in the TC, it may have direct cytotoxic function, resulting possibly in DNA fragmentation, as suggested by Schmid et al. (1986). Alternatively, the surface-active effect of PFP on the TC membrane might also produce a burst of endocytic uptake by the TC (see Young, 1985, for discussion), which could result in the enhanced uptake of LT or any other locally accumulated toxic mediator. 3. Proteoglycans Proteoglycans of the chondroitin sulfate A type have recently been identi-
fied in NK cell granules (MacDermott et al., 1985; Schmidt et al., 1985). These complex, highly negatively charged molecules are secreted into the extracellular medium during N K cell killing. Proteoglycans have also recently been identified in the granules of mouse CTLL (Young et al., 1987a). It is possible that these molecules provide a substratum to which other granule proteins are attached. Proteoglycans are not unique to lymphocytes and have also been observed in other cell types, particularly in mast cell granules. It has been suggested by MacDermott et al. (1985) and Schmidt et al. (1985) that proteoglycans may have a protective function in preventing self-inflicted injury by PFP or other toxic mediators. In our hands, however,
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chondroitin sulfate A is totally ineffective in blocking PFP-mediated hemolysis, and heparin is only partially effective at concentrations exceeding 0.5 mg/ml (Young et al., 1987a). Besides, PFP is reversibly inactivated by the absence of calcium and pH below 6, which presumably are the conditions found inside the granules, precluding in principle the need for any other inactivating mechanism. However, further studies addressing these issues are needed to assign a definitive role for the granule proteoglycans in cellmediated killing. V. Membrane Attack Complex of Complement
A. BACKGROUND: THE CHANNEL CONCEPTA N D NATUREOF C5b-9
THE
AMPHIPHILIC
Of the 15 proteins in the complement (C) system, 5 proteins (C5, C6, C7, C8, and C9) are intimately associated with the membrane attack complex (MAC) of C, which mediates the formation of lesions on target membranes. The proteins comprising the MAC are probably among the more widely studied surface-active proteins in nature. Several recent reviews have covered this topic in extensive detail (Muller-Eberhard, 1975; Mayer et al., 1981; Bhakdi and Tranum-Jensen, 1983). Here, we will only draw attention to certain aspects of this protein complex related to its membrane assembly and mechanism of action, which are probably applicable to studies of other soluble amphiphilic PFP, particularly the lymphocyte granule PFP. Initial studies by Shin and his collaborators (1968) demonstrated that C5 is cleaved into the fragments C5a and C5b during C activation. C5b that is generated in the fluid phase by enzymatic cleavage in the absence of a target membrane rapidly combines with C6, C7, C8, C9, and a serum protein known as S protein to form an inactive macromolecular complex in solution devoid of any cytolytic activity (Kolb et al., 1972, 1973). The S protein serves as an inactivator of the C complex by binding to the nascent hydrophobic surfaces of the C5b-7 complex, therefore inactivating its further cytotoxic attachment to lipid bilayers (Podack et al., 1977). On the other hand, C5b, which has been generated in the vicinity of a biological membrane, readily combines with C6 to form a stable complex that can bind to bilayers and initiate other terminal C lytic reactions (Shin et al., 1971; Goldlust et al., 1974). A major advance in our understanding of C-mediated lesions was provided by Thompson and Lachman (1970) and Lachman and Thompson (1970), who introduced the concept of “reactive lysis” in which isolated C5b6 and C7 were shown to form a stable intermediate on erythrocytes in the absence of any other C component, and upon subsequent addition of C8 and C9, hemolysis was observed. This monumental piece of work, later con-
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firmed with bacteria (Goldman and Austen, 1974) and lipid vesicles (Lachman et al., 1970) as target membranes, established that the sequential addition of the components of the reactive lysis system, i.e., C5b6, C7, C8, and C9, suffices to damage membranes in a nonspecific manner and in the absence of any other C component. As noted, much of the work on C-mediated lysis became possible only following the development of purification procedures which allowed the isolation of each C component in homogeneous form (reviewed by Muller-Eberhard, 1975; see also Podack et al., 1976; Kolb and Muller-Eberhard, 1975; Biesecker and Muller-Eberhard, 1980; Hammer et al., 1981). Early experiments on immune cytolysis involving C activation suggested that TC membranes are probably damaged, since small molecules are released from cells (Green et al., 1959a,b). Albumin added to the extracellular medium is capable of preventing cytolysis, suggesting to the authors that small discrete lesions must have formed in the membrane and that the colloid osmotic swelling is responsible for the terminal cell lysis. Numerous observations following that time have since confirmed these initial findings. An ultrastructural demonstration of membrane lesions produced by C was first presented by Borsos et al. (1964), who showed electron microscopy pictures of damaged erythrocyte membranes containing tubular structures. A positive correlation between the number of membrane ring-like lesions and the titer of C used was later provided by Humphrey and Dourmashin (1969). Typically, lesions with an internal diameter of 10 nm were observed. At about the same time, Lachman and his colleagues demonstrated that membranes damaged by the reactive lysis system, i.e., by association of C5C9, also presented the characteristic tubular lesions on their surface (Lachman et al., 1970; Hesketh et al., 1971). Further experiments at the time with liposomes made of phospholipid and cholesterol and containing trapped glucose showed that upon exposure to C activation, glucose is released from the liposomes (Haxby et al., 1968). Since a phospholipase activity of C had been excluded by the lack of measurable phospholipid degradation and by the ability of C to release trapped marker from liposomes prepared from phospholipid analogs that are not susceptible to phospholipases, an enzymatic attack of C on lipid bilayers was excluded (Kinsky, 1972; Lachman et al., 1973), but it was still considered possible that C could exert a detergentlike activity on membranes (Kinsky et al., 1971). In 1972, Mayer took into consideration the morphological evidence collected earlier by others and the previously established one-hit lysis theory of C action (Mayer, 1961) to propose the now famous “doughnut” hypothesis of membrane lesion (Mayer, 1972). This hypothesis describes the C lesion as a hollow transmembrane tubule lined up with a hydrophilic interior surface and a hydrophobic exterior surface facing membrane lipid molecules. Still as part of this hypothesis,
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Mayer suggested then that hydrophobic regions of C components become exposed during the interaction of the various C components. The hydrophobic regions then insert into the lipid bilayer to assemble the C channel. The bulk of experimental evidence that has been collected to this day appears to support fully the transmembrane channel concept introduced by Mayer and other earlier investigators. Each C5b6 complex binds one C7 molecule (Kolb et al., 1972, 1973). Each C8 molecule binds, in turn, one C5b-7 complex via its p chain (Kolb and Muller-Eberhard, 1975; Monahan et al., 1980; Monahan and Sodetz, 1981). The number of C9 that binds to C5b-8 has been a highly controversial issue and will be addressed separately later. Using radiolabeled C components, Hammer et al. (1975) in Mayer’s laboratory demonstrated that C5b can be readily eluted from erythrocytes exposed to Cl-C6 using high salts, but not from membranes containing Cl-C7. Similar experiments were obtained by Bhakdi and his colleagues (1975) by rocket immunoelectrophoresis of membranes and their salt extracts. These results have been taken as evidence that the C5b-7 complex inserts into bilayers. Enzymatic stripping experiments performed in several laboratories showed that during C activation, C5b, C7, C8, and C9 are protected from proteolytic digestion once they become associated with membranes, leading further to the notion that all the terminal components of C insert into lipid bilayers following activation (Hammer et al., 1975, 1977; Bhakdi and Tranum-Jensen, 1979; Bhakdi et al., 1980). These results, although suggestive of membrane insertion, are not definitive, since the protection against proteolysis afforded by C activation could also be due to protection afforded by other C components and/or by conformation changes in C components during activation, rendering them resistant to enzymatic digestion. Recently, using the membrane-restricted photoreacl-14C]glucosative glycolipid probe 12-(4-azido-2-nitrophenoxy)stearoyl[ mine, which presumably only labels integral membrane proteins, Hu and her collaborators (1981) established decisively that C5b, C6, C7, C8, and C9 all enter the hydrophobic interior of the membrane during C assembly. A parallel study using another photosensitive membrane-restricted probe, also pro[radiolabeled hexanoyldiiodo-N-(4-azido-2-nitrophenyl)tyramine], vided evidence that both C8a and C9 are the predominantly labeled species in the menibrane-bound complex C5b-9 (Steckel et al., 1983). The insertion of the various terminal C components into lipid bilayers to form functional channels was also elegantly demonstrated by using planar lipid bilayers as model membranes and high-resolution electrical measurements of transmembrane current observed in the presence and absence of C. Earlier experiments have already shown that the association of antigen, antibody, and complement results in increased membrane permeability (Barfort et al., 1968; Wobschall and McKeon, 1975). With use of defined C
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components, the permeability increase associated with the MAC was only observed when C8 and C9 were present (Michaels et al., 1976). Using even thinner membranes made of oxidized cholesterol, Michaels et al. were able to demonstrate an increment of membrane permeability with the addition of each of the C MAC components, including C5b6 and C7 (Michaels et al., 1978; Michaels and Mayer, 1978). These experiments clearly demonstrate that the process of membrane interaction initiates as early as the C5b6 stage. However, since these investigators did not clearly resolve single-channel fluctuations, i.e., the ionic current attributed to each individual channel, it cannot be concluded from these experiments that the interaction of the various C components resulted in channel formation, as argued earlier (Mayer et al., 1981). In more recent experiments using the high-resolution patchclamp recording system, Jackson and his colleagues (1981) were able to resolve single-channel currents obtained from cell membranes attacked by the complement reaction in the presence of antigen-antibody complexes. Since whole serum was used by these investigators as a source of complement, it is not clear to what stage of C aggregation the single channels could be attributed. Other experiments clearly demonstrating the C5b-9 expresses hydrophobic domains that interact directly with membranes included phospholipid and detergent-binding studies. C5b-9 is now viewed as an amphiphilic complex containing both hydrophilic and lipophilic regions. Wilson and Spitznagel (1968, 1971) showed that complement activation on the surface of Escherichia coli causes release of about 60-70% of the bacterial phospholipid into the medium. These studies were later confirmed and extended by Giavedoni and Dalmasso (1976), Inoue et al. (1977), and Kinoshita et al. (1977) with erythrocyte membranes and liposomes under C attack. A critical experiment that has helped to elucidate this mechanism of phospholipid removal from membranes under C attack was performed by Shin and her colleagues (1977). At high doses of C, labeled lipids from liposomes were seen to transfer from the membrane to C components, presumably C5b-7, C8, and C9. Another important piece of information provided in the same study was that functional channel formation, as monitored by ssRb release from liposomes, is produced with relatively low doses of complement, whereas removal of labeled lipids requires a much higher concentration of C. This study suggested that C can exert a detergent-like activity after all, as previously suggested by Kinsky et al. (1971), but this activity is measurable only when large quantities of C are present. The expression of nascent hydrophobic domains during the assembly of the C5b-9 complexes can also be measured by the detergent-binding capacity of these protein complexes. Thus, Bhakdi et al. (1978) and Podack and Muller-Eberhard (1978) presented evidence that the C5b6, C5b-7, C5b-8,
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and C5b-9 are amphiphilic complexes that bind to labeled detergents, in contrast to the hydrophilic behavior of the individual native proteins. Studies that gave further support to the notion of the amphiphilic nature of C5b-9 complexes came from reconstitution experiments performed by Bhakdi and Tranum-Jensen (1978). These investigators isolated C5b-9 complexes from membranes attacked by C with use of detergents. The isolated protein complexes were then reconstituted into proteoliposomes by dialysing the detergent away from the protein-lipid-detergent mixture. The reincorporated protein channels remained stably anchored in the bilayer and in the same original configuration observed in the membrane. These results suggested to the authors that lipid-binding regions of the C5b-9 complex enable the complex to penetrate into the bilayer. Other studies by Podack et al. (1979a)showed that the C complexes C5b6, C5b-7, C5b-8, and C5b-9 are all capable of binding stoichiometrically to labeled lipid. These same investigators introduced the notion of “protein-lipid micelles,” i. e., the formation of structures of protein packed with lipid in the lipid bilayer, which could destabilize the structural organization of lipid bilayers. In this model (Podack et al., 1979a; Esser et al., 1979; Biesecker et al., 1979), also known as the leaky patch or the mixed micelle model, the membrane leakage is viewed as a consequence of the detergent-like activity of C, and the transmembrane areas of leakage are delineated not by the interior linings of a protein tubule, but rather by the lipid disorganization surrounding the protein complex. One of the arguments used earlier by these investigators was based on the finding of lipid reorientation in the electron-spin resonance (ESR) experiments of Esser et al. (1979). However, to date, ESR studies with erythrocyte membranes have detected either a C-induced decrease, or increase, or no change in membrane lipid fluidity, depending only on the probe used in such experiments (Mason et al., 1977; Dahl and Levine, 1978; Nakamura et al., 1976). We agree with the view expressed by Mayer et al. (1981)that both channel formation and detergent action may play a role in C-mediated damage of membranes. Since channel formation is thought to require less C material than that required for phospholipid removal and lipid disorganization, it is likely that only the channel mechanism is physiologically relevant. In our opinion, channel-forming amphiphilic proteins, in general, are capable of sequestrating lipid from certain regions of the membrane to cause a lipid phase transition (micellar conversion) to occur. The reduced cohesion of lipid molecules and the overall perturbation of the lipid structure and organization that result from this micellar formation are expected to give rise to altered membrane permeability properties. However, as mentioned, this effect is expected to play a lytic role only when the amount of the lytic protein exceeds the amount of material required to produce discrete membrane leshns. Interestingly, the same type of controversy (i.e., pore forma-
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tion versus detergent effect) has accompanied studies of several other poreforming cytotoxins. B. SUBUNIT COMPOSITION OF OF THE LESIONS
THE
MAC
AND
FUNCTIONAL SIZE
The identity and the number of subunits of C that compose the pore has long been a controversial issue (see review of Bhakdi and Tranum-Jensen, 1983, for more extensive discussion). The controversy has been due, in part, to the multiplicity of aggregation states expected for an amphiphilic complex such as C5b-9. As described earlier, work from several laboratories has indicated that C5b-8 exists as a monomeric complex of each of the individual components C5b6, C7, and C8 (Kolb and Muller-Eberhard, 1975; Monahan and Sodetz, 1981). It is now well known that erythrocytes carrying only C5b-8 complexes undergo lysis, but the rate of lysis is much slower than that caused by the assembly of C5b-9 (Stolfi, 1968; Hadding and MullerEberhard, 1969; Tamura et al., 1972; Gee et al., 1980; Kitamura and Nagaki, 1981). The presence of small C5b-8 channels in the range of 1 nm has been confirmed by osmotic protection experiments (Kitamura and Nagaki, 1981). Other kinetics studies on the release of markers from resealed erythrocyte ghosts damaged with C5b-8 revealed that these channels allow membrane permeation of sucrose (0.9 nm molecular diameter), but not of inulin (3 nm) (Ramm et al., 1982). Formation of these sucrose-permeable channels by C5b-8 occurs with much slower kinetics and lower efficiency than C5b-9 lesions. On the controversial issue of number of C9 required to form the C5b-9 lesions, several initial studies have demonstrated that a multiple number of C9 is bound to each C5b-8 complex (Kolb et al., 1972; Kolb and MullerEberhard, 1974; Podack et al., 1982). Based on hydrodynamic properties of detergent-solubilized forms of C5b-9 complexes and following estimation of their molecular masses, Bhakdi and Tranum-Jensen (1981) have proposed that the unit structural lesion produced by C5b-9 is a monomer of C5b-6, C7, C8, and C9. This stands in contrast to the previously proposed formula of a dimer of (C5b-8) bound to multiple numbers of C9 [i.e., (C5b-8),C9,,] (Biesecker et al., 1979). Moreover, the reported number of C9 molecules bound per C5b-8 has varied between 1(Rommel and Mayer, 1973; Kitamura and Inai, 1974; Bhakdi and Tranum-Jensen, 1981), 6 (Kolb et al., 1972), and 12-16 (Podack et al., 1982). Recently, isolated monomeric C9 has been found to polymerize on prolonged incubation of 48-64 hours at 37°C (or on shorter incubations at temperatures exceeding 37°C) to form circular polymers (poly C9) resembling morphologically the MAC (Podack and Tschopp, 1982a,b; Tschopp et al., 1982, 1983). Polymerization of monomeric C9 (70-75 kDa) into a supramolecular tubule (molecular mass exceeding 1 million Da) appar-
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ently involves the self-association of 12-16 C9 molecules which, in the process, become resistant to dissociation by SDS, reducing agents, and proteolysis. In contrast, nontubular poly C9 containing less than 12 C9 molecules IS dissociable by SDS (Podack and Tschopp, 198213). According to Podack and Tschopp (1982a,b), the MAC tubule may be viewed as a hollow cylinder formed by poly C9, with the C5b-8 complex in the MAC assuming a peripheral morphology in the form of an “elongated structure” standing out next to the poly C9 tubule (Tschopp et al., 1982). In fact, the accessory function of C8 in the formation of C lesions and of poly C9 has recently been proposed by Tschopp et al. (1985), who have described the C5b-8 complex as an accelerator of C9 polymerization. These authors have shown that the tubular lesions associated with the MAC are generated by C9 only at high C9 multiplicity and that at low C9 : C8 ratios, C9 is mainly found in a nontubular form. Polymerization of C9 is accompanied by the formation of functionally large, voltage-resistant channels in planar lipid bilayers (Young et al., 1986i). Poly C9, which has been formed in liposomes, may be transferred to planar bilayers, and the current that flows through each poly C9 tubule has been measured, yielding estimates of channel sizes comparable to those measured for the lymphocyte PFP/perforin. The polymerization of C9 into structural and functional tubules may also be catalyzed by divalent metal ions (Tschopp, 1984; Young et d., 19863). Although poly C9 may form lesions on lipid vesicle membranes, poly C9 does not have any hemolytic activity even when polymerization is catalyzed by heavy metals. Hemolysis is observed only when the C9 is bound to C5b-8 complexes. Functional molecular sieving experiments have given variable estimates of the size of C-mediated lesions, with initial studies giving minimal diameter estimates of 4 nm (Giavedoni et al., 1979) and 5.5 nm (Ramm and Mayer, 1980). A study based on kinetics analysis of tracer exchange across the Cdamaged membranes has given estimates of average size ranging from 2 to 2.5 nm (Sims and Lauf, 1978). However, it should be noted that all these studies used complement at high doses. In fact, it is now generally agreed that C lesions are heterogeneous in size. This inference follows from osmotic protection studies, kinetic analysis of diffusion of various molecular size markers, ultrastructural examinations of C5b-9 complexes on membranes, and molecular weight determinations of C5b-9 complexes at various C9 input ratios (Boyle and Borsos, 1979; Boyle et al., 1979; Sims and Lauf, 1980; Dalmasso and Benson, 1981; Ramm et al., 1982, 1983; Bhakdi and TranumJensen, 1984). Boyle et al. (1979), and later several other investigators mentioned above, have presented the view that the number of C9 per C5b-9 complex accounts for the multiplicity of the sizes of C lesions. This view would be consistent with recent data showing that poly C9 also forms functional channels of multiple sizes, suggesting that the heterogeneity of sizes
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associated with the MAC is due to the multiplicity of C9 bound io C5b-8 (Young et al., 1986i). In fact, the formation of the poly C9-linked tubular structures appears not to be required for functional channel formation by C9. Thus, Dankert, Esser, and their collaborators have shown that the COOH-terminal fragment (C9b) obtained from C9 by enzymatic cleavage with thrombin is channel active on erythrocyte membranes and planar lipid bilayers (Dankert and Esser, 1985; Shiver et al., 1986). Cgb, however, does not form structural ring-like lesions. Dose-response analysis of functional C lesions has supported the view that the minimal C9 contribution required to produce a functional lesion is one molecule of C9 (Ramm et al., 1982). More impressively, Ramm et al. (1982, 1985) have been able to assign different sizes of C lesions to different numbers of C9. Thus, when one C9 is present, the formed channel allows passage of sucrose (0.9 nm in molecular diameter). Two C9 bound to C5b-8 result in a channel allowing transit of inulin (3 nm), and so forth, until a size presumed to correspond to 4 molecules of C9 is reached, which is large enough for the passage of ribonuclease A (3.8 nm). Bhakdi and TranumJensen (1984) more recently suggested that this functional heterogeneity matched with the subunit composition obtained from structural data. At low doses of serum, due to limiting concentrations of C9, these authors proposed that mean ratios of 2-3 to one C5b-8 complex are more common than the 68 molecules of C9 expected to bind to C5b-8 when C9 is in excess. These authors further suggest that only those complexes containing 6-8 molecules attached to one C5b-8 from the classical ultrastructural ring-like lesions attributed to C. The concept that low amounts of C9 per C5b-9 complex suffice to cause a stable functional lesion has recently been extended by Bhakdi and Tranum-Jensen (1986a). Previous studies by Boyle et al. (1978) have shown that C9 may bind to erythrocyte membranes containing C5b-8 complexes at 0" but without causing hemolysis. Warming up cells to 30"37°C results in immediate hemolytic activity, which helps to define distinct stages of membrane (C5b-8) binding and pore formation for C9. Bhakdi and Tranum-Jensen (1986a) performed a similar experiment to show that at 0", much lower amounts of C9 bind to each C5b-8 complex on erythrocyte membranes, in contrast to similar binding experiments performed at 37°C. After washing away the unbound C9 and warming up the erythrocyte suspensions from 0" to 37"C, hemolysis is observed, but without concomitant visualization of ring-like lesions on erythrocyte membranes. These results favor the view that oligomerization of C9 allows the formation of large circular lesions observed under electron microscopy. However, much smaller functional lesions may be formed at low C9 multiplicity and in the absence of any structurally demonstratable lesions. More importantly, these results would also tend to suggest that poly C9 may account for only part of the
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structural lesions attributed to the MAC. It follows that the C5b-8 complex may also form part of the hydrophilic channel associated with the MAC rather than being restricted to the “elongated structure” associated with the periphery of a MAC lesion, as previously suggested by others. More experiments are required, however, to define conclusively the subunit composition of C5b-9 lesions. C. ANALOGIESBETWEEN COMPLEMENT AND LYMPHOCYTE-MEDIATED KILLING The number of structural and functional similarities between C and lymphocyte-mediated killing have suggested the possibility that the individual effector proteins of these two systems are also structurally related (reviewed by Lachmann, 1983, 1986; Young and Cohn, 1986a). Antibodies prepared against target cell membranes damaged by lymphocytes have previously been shown to cross-react with neoantigens expressed on the MAC (Sundsmo and Muller-Eberhard, 1979; Ward and Lachmann, 1985). A monoclonal antibody has recently been raised that identifies neoantigens expressed on both C and lymphocyte-lysed membranes (Ward and Lachmann, 1985). This same monoclonal is capable of blocking ADCC, suggesting that the two systems must share common antigenic components. However, the identity of the cross-reactive proteins and the extent of the immunological crossreactivity were not determined in those studies. One obvious candidate of the C system which may resemble the lymphocyte PFP/perforin is C9. As noted earlier, C9 has a number of biochemical and functional features which resemble the lymphocyte PFP (summarized in Table I). The primary sequence of C9 has recently been deduced by cDNA cloning (DiScipio et al., 1984; Stanley et al., 1985; DiScipio and Hugli, 1985). Human C9 has 537 amino acids and the sequence is clearly amphipathic. The amino-terminal half contains predominantly hydrophilic residues and the carboxyl-terminal half contains more hydrophobic residues. The C9 sequence also contains cysteine-rich domains presumably engaged in disulfide bridge formation. These cysteine-rich domains show partial homology with the low-density lipoprotein (LDL) receptor. The function of these highly conserved domains remains unclear. In the case of LDL receptor, they are thought to be involved in ligand binding (Yamamoto et al., 1984). The homology between human C9 and mouse lymphocyte PFP/perforin was recently assessed using polyclonal antibodies raised against purified C9 and lymphocyte PFP (Young et al., 1986i; Table I). On immunoblots, immunological cross-reactivity between these two proteins has been verified. The cross-reactivity observed between C9 and lymphocyte PFP is only observed when the reactive antigens and the immunogens used to elicit antibodies are
TABLE I SIMILARITIES AND DIFFERENCES BETWEEN C9 AND LYMPHOCYTE PFP/PERFORIN~ Feature Molecular mass (SDS-PAGE) Polymerization into tubules catalyzed by Internal diameter of tubule by EM Unit conductance step in 0.1 M NaCl Electrical characteristics
c9 70-75 kDa (reduced) 62-66 kDa (nonreduced) C5b-C8, heavy metals (Zn2+ most effective), temperature >37T
70-75 kDa (reduced) 60-64 kDa (nonreduced) Ca2+, temperature >30"C
loo A
160 A 0.4-6 nSb Voltage-resistant, open state favored, slow channel kinetics Permeable to monovalent and divalent ions, Lucifer Yellow, sucrose, and glucosamine (+) Antiserum to reduced C9 (-) Antiserum to nonreduced C9 Hemolytic; cytolytic to a variety of tumor cell lines; cytolysis requires Ca2+
0.2-4 nS Voltage-resistant, open state favored, slow channel kinetics
Functional size
Permeable to monovalent and divalent ions, Lucifer Yellow, and sucrose
Antigenic cross-reactivity
(+) Antiserum to reduced perforin (-) Antiserum to nonreduced perforin Hemolytic activity requires activation of C5b-C8; cytolytic to a variety of tumor cell lines
Cytotoxicity
Lymphocyte PFP
Lymphocyte PFP/perforin refers to material purified from mouse cytotoxic T cell lines and NK-like lymphocytes, whereas C9 refers to human material. This range of channel sizes includes data obtained on channels formed by lymphocyte PFP/perforin added directly to the aqueous phase of planar bilayers at room temperature and also by polyperforin polymerized in lipid vesicles at 37°C prior to incorporation into planar bilayers. The range of channel sizes for C9 shown here pertains only to poly C9 complexes previously formed and transferred to planar bilayers. From Young et al. (19861). a
b
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reduced and alkylated (Table I). These results indicate that the cross-reactivity observed between the two species is restricted to cysteine-rich domains which are normally masked by disulfide bridges and become antigenically exposed only upon chemical reduction. Recent studies by Tschopp and his colleagues (1986b) showed that antibodies raised against a synthetic peptide prepared to mimic the region of homology (in the cysteine-rich area, beresidues 101-111: Asp-Asn-Asp-Cys-Gly-Asp-Phe-Ser-Asp-Glu-Asp) tween C9 and LDL receptor also react against mouse PFP. These authors also showed that the synthetic peptides inhibit the hemolytic activity of granule PFP/perforin. The immunological cross-reactivity is not limited to C9 and PFP. Recent studies have shown that the lymphocyte PFP is also immunologically related to C5b-6, C7, and C8 (Young et al., 19863’). The antigenic epitope(s) shared by these proteins is also restricted to the cysteine-rich domain(s). In parallel studies, antibodies raised against the synthetic peptide mentioned above also react against the other components of the MAC and the polymerized complexes of C5b-9 (Tschopp and Mollnes, 1986; Tschopp et al., 1986b). The cysteine-rich domain that appears to be conserved in these molecules may be exposed following major structural rearrangement of these proteins, which is thought to accompany membrane insertion and polymerization. This region of homology may play some function related to their attachment and/or in their subsequent pore formation in the membrane. Recently, rabbit polyclonal antisera directed against human C9 have been used in the affinity purification of a PFP localized in human large granular lymphocytes (Zalman et al., 1986a) and in human peripheral blood NK cells separated by using a panel of monoclonal antibodies directed against N K and non-NK surface antigens (Liu et a l . , 1986). Zalman and her colleagues (198613) showed, furthermore, that monoclonal antibodies directed against C9 which also react against the PFP of LGLs are capable of blocking killing of K562 cells by LGLs. Polymerization of the isolated polypeptide resulted in the formation of two different circular structures, with internal diameters of 6 and 12.5 nm. Polymerized PFP in liposomes gave rise to channels of two functional diameters of 5-9 and 10.2 nm, as determined by liposome marker-retention assays. Polyclonal antibodies raised against residues 101-111 of C9, mentioned above, also block killing by LGLs (Zalman et al., 1986a). In our studies with purified NK cell populations, a large scatter of channel sizes was observed by morphological analysis, but rings with 16-nm internal diameter were the most commonly observed lesions (Liu et al., 1986). In our hands, the functional channel sizes formed by the NK cell polypeptide in planar lipid bilayers are also quite heterogeneous, showing a large scatter which may correspond to the different aggregation or polymerization states. Functionally, the lymphocyte PFP and poly C9 form ion nonselective
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channels which remain permanently open (Table I). The pores formed by these two complexes resist to closing induced by changes in the membrane potential. This behavior indicates that stable and voltage-resistant channels are formed by these protein complexes, which are attributes that would favor an active role of these channels in cytolysis. Functional sieving experiments have suggested that the pores formed by these two entities are heterogeneous in size, with sizes attaining 6-8 nm in functional diameter. Both these complexes are thought to form pores by a barrel stave model, with the pores consisting of discrete membrane nuclei that enlarge in size through the uptake of monomers. The recent demonstration that lymphocyte granules contain potent serine esterase activities which may play some role related to cytotoxicity (Pasternak and Eisen, 1985; Pasternak et al., 1986; Masson et al., 1986; Young et al., 1986a)has further suggested the connection between lymphocyte-mediated killing and cytolysis mediated by C. This analogy has recently been discussed in more detail by Reid (1986). It is now well known that the assembly of C lesions also depends on the activity of the complex serine proteases C4b2a3b and C3bBb3b that are involved in the generation of C5b. Moreover, C6 has also recently been described as a serine protease, and its enzymatic activity appears to be linked with the formation of C5b-9 complexes (Kolb et al., 1982). Like the C serine proteases, the granule serine proteases may also play an intermediate processing function, perhaps in the conversion of other lytic granule proteins into their active forms. The collective results obtained in several laboratories further support and extend the notion of “complement supergenes” initially proposed for C6 and C7 (Lachmann and Hobart, 1978; Podack et al., 1979b). On the basis of structural and functional similarities, a genetic relationship has also been proposed for several other complement proteins, including Clr and Cls, C3, C4, and C5, and C2 and factor B (reviewed by Campbell et al., 1986). The observed immunological similarities between lymphocyte PFP and several C components of the MAC suggest the possibility that the lymphocyte PFP may be an additional member of the complement supergene family. It is possible that all these effector molecules may have emerged from the same ancestral protein during evolution, but diverged and became specialized later to carry out either humoral or cellular immune responses. Studies on C-mediated cytolysis have been highly controversial over the years until the recently unified views on the molecular nature of C lesions. It is also expected that future studies on lymphocyte-mediated killing will also generate controversy and disaccord. Thus, Berke and his colleagues (as cited in a review article by Marx, 1986) have claimed that lymphocyte-damaged membranes do not exhibit the ring-like lesions described by other workers. This observation was taken as a piece of evidence against a role for pore
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formation in lymphocyte-mediated killing. It might be noteworthy to point out, however, the differences between functional channel formation and ultrastructural observations of structural tubular lesions. The lymphocyte PFP is thought to damage membranes by forming functional channels on target membranes. Under certain favorable conditions (protein density, time of incubation, temperature), the protein may aggregate to form circular lesions of high PFP multiplicity. However, in our opinion, formation of the macromolecular tubular lesions is not a requisite for functional channel formation, and therefore, the morphological criteria may not be taken as synonymous of membrane damage by channel formation. Functional channels are, in faci, thought to be formed prior to complete circular polymerization. Similar notions have been put forth in the C field.
VI. Other Cytolytic Pore-Forming Proteins
A. OTHERPORE-FORMING PROTEINS I N CELL-MEDIATED KILLING 1 . Eosinophil Cationic Protein Eosinophils play an active role in allergic reactions and in the antibodydependent killing of a number of helminthic parasites (reviewed by Dessein and David, 1982; Venge et al., 1980; Spry, 1985; Gleich and Loegering, 1984). Eosinophil granule proteins have long been implicated in this type of cxtotoxicity. A number of cationic proteins have been isolated from eosinophi1 granules of several species and have been partially characterized. A major basic protein (MBP) of 9-11 kDa has been isolated from eosinophils of several species by Gleich and his colleagues (Gleich et al., 1973, 1974, 1976). A major eosinophil cationic protein (ECP) with a molecular mass of 21 kDa has also been isolated from human eosinophil granules (Olsson and Venge, 1974; Olsson et al., 1977). Other basic proteins that have been isolated from eosinophil granules include EP-X (Peterson and Venge, 1983), eosinophil peroxidase (Carlson et al., 1985), and the eosinophil-derived neurotoxin (EDN) (Durack et al., 1979, 1981). The strategy used by these investigators to isolate eosinophil proteins that are highly basic in nature has consisted of solubilization of granule contents in low pH and purification of proteins using cation-exchange chromatography. Since all these proteins represent abundant proteins within eosinophil granules (Ackerman et aZ., 1983), their role in parasite and microbial killing has been investigated in several laboratories. MBP has been shown to damage parasites (Buttenvorth et al., 1979; Wassom and Gleich, 1979) and mammalian cells (Gleich et al., 1979) at concentrations exceeding lop5 M . The human ECP, however, has been shown to damage schistosomula larvae
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of the intravenous parasite Schistosoma mansoni at concentrations as low as 10-7 M (McLaren et al., 1981). ECP also produces the classic paralytic syndrome known as the Gordon phenomenon after intrathecal injection into guinea pigs (Fredens et al., 1982). Furthermore, E C P has been detected by radioimmunoassays in the supernatants of human degranulated granulocytes stimulated via the Fc-linked mechanism (Venge et al., 1980), and a secretory form of ECP has recently been identified in the granules of eosinophils (Tai et al., 1984). Recent studies with purified ECP suggest that this protein may form functional channels in lipid bilayers (Young et al., 19863’). ECP depolarizes the membrane potential of cultured nucleated cells and induces ion flow through model lipid bilayers. Purified ECP forms channels which show characteristics similar to those produced by C9 and lymphocyte PFP. ECP channels are resistant to closing by high transmembrane voltages and appear to be stable transmembrane entities, remaining permanently open once inserted into the bilayer. Ion-selectivity experiments show that ECP channels are relatively nonselective to all the monovalent ions tested, being slightly more permeable to anions. The channel-forming activity is only observed when the acid form of ECP is diluted in the presence of target membranes in a neutral environment. Extensive dialysis of E C P against buffers of neutral pH drastically lowers its channel-forming activity, suggesting that this protein may form inactive aggregates that are no longer membrane active. ECP has recently been observed to form ring-like lesions on the surface of liposomes (Young, Peterson, and Venge, unpublished observations). The lesions formed by ECP are heterogeneous in size, varying between 2 and 5 nm. It is not known whether purified MBP which is also cytolytic is capable of assembling membrane channels like ECP. The functional similarities between ECP and lymphocyte PFP and C9-mediated lesions suggest that E C P may also damage cells by a colloid osmotic mechanism.
2 . PFP of Amoebas Entamoeba histolytica is the enteric human parasite responsible for dysenteric amebiasis (reviewed by Ravdin and Guerrant, 1982). This infection is characterized -by an invasive enteric illness that may spread to multiple organs. In culture, E . histolytica is cytolytic to a variety of cell types, including leukocyte. Several laboratories have shown that the cell killing mediated by the amoeba is surface contact dependent (reviewed by Ravdin and Guerrant, 1982; Gitler et al., 1984; Young and Cohn, 1985). Following contact, the amoeba may rapidly ingest the target cell. Eaton et al. (1969) have suggested that a surface triggering mechanism may be involved in killing in which lysosomal contents are released at the site of surface contact between the cells. This suggestion has been substantiated by more recent cin-
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emicroscopic and kinetic studies (Ravdin et al., 1980; Ravdin and Guerrant, 1981) indicating that the cytolysis mediated by the amoeba may precede the phagocytic event, therefore raising the possibility of an extracellular cytolytic event triggered upon surface contact. In many respects, the mode of killing mediated by amoebas resembles that produced by immune cells described earlier in this review. A PFP of amoebas has recently been isolated (Lynch et al., 1982; Young et al., 1982; Gitler et al., 1984; Young and Cohn, 1985). The isolated polypeptide assumes a molecular mass of 15 kDa under reducing conditions. Under nonreducing conditions in the presence of the nonionic detergent p-D-OCtylglucoside, it assumes an apparent molecular mass of 28-30 kDa as determined by molecular sieving chromatography (Young and Cohn, 1985). The amoeba PFP forms voltage-dependent ion channels in liposomes and in planar lipid bilayers, and several of its biophysical properties have been studied in some detail. One of the more remarkable properties of this PFP is its tendency to aggregate in the lipid bilayer to assume channels of multiple sizes that function synchronously as individual units. This behavior is reminiscent of the barrel stave model. Following stimulation of their surface with the calcium ionophor A23187, LPS, or concanavalin A, amoebas release PFP rapidly into the the extracellular medium (Young et al., 1982). The amoeba PFP lyses a variety of tumor cell lines as assayed by conventional 51Cr release assays (unpublished observations).
B. OTHERTOXINSAS POREFORMERS A number of other toxins are present in nature, and their modes of action have been extensively studied in several laboratories (for reviews, see Rogolsky, 1979; Alouf, 1980; Latorre and Alvarez, 1981; Bernheimer and Rudy, 1986; Bhakdi and Tranum-Jensen, 1986b). Here, we will only discuss toxins that mediate target membrane damage by means of pore formation and, in particular, those aspects related to C and lymphocyte pore formers. For the sake of brevity, the toxins will be subdivided into several broad arbitrary categories as follows. 1 . Small Peptides Included in this category are small peptides that produce cytolysis by forming either aqueous channels which span the membrane bilayer or other undefined structures which increase membrane permeability. a . Melittin. Melittin is the prototype of this group and is the most widely studied model membrane lytic peptide. Melittin is the main component of the venom of the honeybee Apis mellt$era and may comprise up to 50% of the bee venom by weight (Haberman, 1972). Melittin is a small basic peptide of 26 amino acids (Fig. 3). The amphipathic nature of this peptide is
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clear from its primary sequence. It contains a hexapeptide at the carboxylterminus carrying a cluster of positive charges, followed by a long stretch of hydrophobic amino acids (Fig. 3). Such distribution of amino acids and charges has been observed with a number of integral membrane proteins that span the bilayer. The peptides of two other species of honeybee have recently been sequenced, and they all show conservative changes in their structure, maintaining the above-mentioned segregation of charges (Kreil, 1973; Fig. 3). When the hexapeptide segment is removed from melittin, the remaining 20 amino acid segment, while capable of binding to erythrocytes, does not lyse them (Schroeder et al., 1971). It has recently been suggested that these peptides tend to form amphiphilic a helical structures, with the amino acid side chains segregated on either a hydrophobic or a hydrophilic side (Kaiser and Kezdy, 1983, 1984). Kaiser and Kezdy have proposed that the ability to form such amphiphilic secondary structures is vital to the biological function of a number of peptides, including hormones, apolipoprotein A-I, and melittin. Thus, it is thought that the amphiphilic a helix may be essential for the lytic activity of melittin,
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exists as a monomer, dimer, tetramer, or some larger oligomer (see Bernheimer and Rudy, 1986, for review of the relevant literature). Two models for membrane damage have been proposed for melittin, which take into account the channel and detergent-like activities described earlier. The first model suggests that melittin forms aqueous channels in lipid bilayers and lyses cells by a colloid osmotic mechanism (Tosteson et al., 1985). The second group of models proposes that melittin does not span completely the bilayers and that it disrupts the phospholipid structure of membranes through its detergent-like activity (Dawson et al., 1978; DeGrado et al., 1982; Tenvilliger et al., 1982). These two models are reminiscent of similar controversies that have accompanied the C field. In either model, the activation of phospholipase A, activity by melittin is thought to result from melittin making membrane phospholipids more susceptible to enzymatic activity rather than a direct modification of the enzyme by melittin (Yunes et al., 1977). b. Alamethicin. Alamethicin and its analogs suzukacillin A and trichotoxin A-40 are peptides produced by different strains of the fungus Trichoderma uiride. Alamethicin is one of the best known channel-forming peptides, and many of its gating and other biophysical properties have been studied in detail (reviewed by Latorre and Alvarez, 1981). Alamethicin is the prototype of the barrel stave model proposed by Bauman and Mueller (1974) and Boheim (1974)and discussed earlier in this review. This model assumes that the alamethicin pore consists of a nucleus that grows in diameter through the uptake of monomers, which behave as barrel staves, with an internal hydrophilic and external hydrophobic surface. Since each monomer is long enough to span the lipid bilayer, the barrel stave channel would be formed by simple lateral oligomerization of various molecules in such a way that the hydrophobic side of the molecule is exposed to the hydrocarbon while the hydrophilic sides line up the pore. c. Staphylococcal 6-Toxin. Like melittin, staphylococcal 6-toxin is a peptide of 26 amino acids (Fig. 3) secreted by certain pathogenic strains of Staphylococcus aureus (Fitton et al., 1980; reviewed by McCartney and Arbuthnott, 1978). It shares a number of structural features with melittin, including a positively charged carboxyl-terminal end, but which is shorter in the &toxin. Like melittin, it interacts with phospholipid monolayers and increases the permeability of phospholipid vesicles (Bhakoo et al., 1982; Freer et al., 1984). Freeze-fractured membranes that have been exposed to &-toxin exhibit transmembrane particles suggestive of formation of transmembrane pores (Freer et al., 1984). These same authors have suggested that 6-toxin is capable of forming alamethicin-like barrel stave pores by aggregation of several monomers in the membrane to form pores of in-
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creasingly larger diameters. Staphylococcal &toxin has also been shown to activate the host membrane phospholipase A, activity and, like melittin, to cause cell release of arachidonic acid metabolites (Durkin and Shier, 1980). d . Gramicidin A . This linear pentadecapeptide with alternating D and L amino acids is produced by several strains of Bacillus brevis. Its structure and mode of action have been extensively studied by Andersen and his colleagues (Andersen, 1984). A transmembrane pore of 2.5 nm is formed by the head-to-head interaction of two gramicidin molecules, each of which spans only half the bilayer. In the membrane, this peptide is arranged in a phelical form, with the hydrophobic residues exposed to the outside. Thus, there are examples of both a-and p-helical structures with channel-forming function. e. Other Peptide Toxins. Several other toxins with modes of action similar to those mentioned above have been described. Barbatolysin, found in the venom of the red harvester ant, Pogonomyrmex barbatus, consists of 34 amino acid residues and appears to resemble melittin and &toxin (Bernheimer et al., 1980). Other toxins isolated from hymenopteran insects include mastoparan (14 amino acids) from the wasp Vespula lewisii (Hirai et al., 1979), cabrolin (13 amino acids) from the European hornet Vespa cabro (Argiolas and Pisano, 1984), and bombolitins (17 amino acids) from the bumblebee Megabombus pennsylvanicus (Argiolas and Pisano, 1985). These peptides are all amphiphilic in nature and may have melittin-like activity on lipid bilayers. Recently, pardaxins (33 amino acids) from the marine sole Pardachirus pavonius have been isolated which presumably are secreted by the sole and have strong shark-repelling activity (Thompson et al., 1986). Like melittin, pardaxins, also contain terminal carboxyl-termini, which are hydrophilic, followed by a hydrophobic remainder. These peptides show marked physical and pharmacological similarities to melittin, including the observation that they are strongly surfactant and lyse erythrocytes, and yet they lack sequence homology (Thompson et al., 1986). These results suggest that numerous toxins, widely distributed throughout nature, may have predicted membrane lytic activity by assuming amphipathic secondary structures favoring their insertion and orientation in the bilayer. It will also be exciting to see whether melittin-like domains (i.e., clusters of charges followed by hydrophobic segments) are also present in the structure of C9 and lymphocyte PFP which may be responsible for their attachment to lipid bilayers and/or membrane pore formation.
2. Cytolytic Proteins In addition to the small peptides mentioned above, a number of wellknown polypeptide toxins have been described over the past few years. We
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will cite only a few, particularly those appearing to have a mode of action closely related to C proteins and lymphocyte PFP. a . Staphylococcal a-Toxin. This toxin is produced and secreted by virulent strains of S. aweus (reviewed by Harshman, 1979; Freer and Arbuthnott, 1982; Bhakdi and Tranum-Jensen, 198613). It is secreted as a water-soluble monomer of 34 kDa, and on contact with a target cell, the monomers attach to and oligomerize on the membrane to form circular lesions with an internal diameter of 2-3 nm (Fussle et al., 1981; Bhakdi et al., 1984b). The circular lesion is formed from an annular hexamer of molecular mass of 200 kDa composed of six identical molecules of the native toxin (Freer et al., 1968; Fussle et al., 1981). The amino acid sequence of atoxin has been deduced by cDNA cloning (Gray and Kehoe, 1984), and at least three short runs of highly hydrophobic sequences have been identified which are thought to interact with lipophilic domains of target bilayers. aToxin binds to target membranes (Cooper et d., 1964a,b; Cassidy and Harshman, 1976; Bhakdi and Tranum-Jensen, 1986b) and to the detergent deoxycholate (Bhakdi et al., 1981), causing toxin monomers to self-associate into the hexameric tubules. A positive correlation between the amount of cell-bound toxin and the extent of hemolytic activity has also been reported (Bhakdi et al., 1984a). Interestingly, low levels of calcium inhibit lysis of erythrocytes by this toxin, and a suggestion has been made that calcium may impede the lateral movement of a-toxin in the lipid bilayer necessary to form the oligomeric pores (Harshman and Sugg, 1985). Planar lipid bilayer experiments have also shown that calcium closes the ionic channels associated with a-toxin (Menestrina, 1985). b. Thiol-ActivatedLysins. There are at the present time at least 15 members of this group which comprise polypeptides produced by gram-positive bacteria inactivated by mild oxidation and reactivated by reduction with thiols (reviewed by Smyth and Duncan, 1978; Alouf and Geoffroy, 1984; Bernheimer and Rudy, 1986). They all bind to cholesterol and closely related sterols and are antigenically related. Among the better known members of this group are streptolysin 0, pneumolysin, tetanolysin, perfringolysin 0, cereolysin, thuringiolysin 0, and alveolysin, which have recently been purified and comprise polypeptides with molecular masses ranging from 48,000 to 68,000 (Smyth and Duncan, 1978; Alouf and Geoffroy, 1984). Early morphological studies have indicated that streptolysin 0 produces membrane “holes” (Dourmashkin and Rosse, 1966; Pendleton et al., 1972). These membrane hole:
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al., 1984b, 1985). It is suggested by these investigators that the initial binding of streptolysin 0 to cholesterol triggers conformational changes accompanied by a hydrophilic-amphiphilic conversion of the monomers, which then self-aggregate to form oligomeric structures with different internal diameters. Bhakdi and his colleagues have proposed that the ring structures are themselves the functional lesions. Others have proposed that due to sequestration of cholesterol by streptolysin 0, the lytic effect is produced by its perturbation of the lipid structure of bilayers, leading to permeability changes and membrane rupture (reviewed by Smyth and Duncan, 1978; Bernheimer and Rudy, 1986). In this context, the circular lesions are regarded as incidental by-products of lysis. From studies of other well-known pore-forming proteins, it is likely that both the pore-forming and lipidsequestration effects are operative during cell lysis, the relative contribution of each effect being dependent on the toxin concentration. It is unlikely, however, that only the large oligomeric structures represent functional channels. It should be expected that functional streptolysin 0 channels are probably generated even when ultrastructural lesions are not visualized and that the 30-nm-diameter lesions represent only the largest channel structures within a broad spectrum of channel sizes. Further experiments are required to address this issues. c. Diamphotoxin. A potent cytolytic basic protein with a molecular mass of 60 kDa, named diamphotoxin, has been purified from the pupae of the South African chrysomelid beetle (de la Harpe et al., 1983; Woolard et al., 1984). Like the lymphocyte PFP and C proteins, the hemolytic activity of this protein can be resolved into two events: binding to membranes and insertion into lipid bilayers producing discrete channels allowing potassium leakage and colloid osmotic lysis. Unlike the lymphocyte PFP, diamphotoxin binds to membranes in the absence of calcium. However, calcium is required to cause a conformational change in the membrane-bound toxin molecule which is thought to be involved in its insertion and subsequent formation of ion channels (de la Harpe et al., 1983). d. Other Toxins. Colicins are bacterial toxins produced by certain strains of E . coli containing the plasmids that carry their structural genes (Jacob et al., 1952; Holland, 1975). For the E l family of colicins, these toxins are known to depolarize the resting membrane potential of sensitive cells (Gould and Cramer, 1977; Weiss and Luria, 1978)and to form ion channels in planar phospholipid bilayers (Schein et al., 1978). Recently, several investigators have attempted to define the channel-forming domain of colicin by limited enzymatic cleavage followed by assay for channel activity (Dankert et al., 1982; Cleveland et al., 1983). Thus, Cleveland and his colleagues (1983)have defined an upper limit of 152 amino acid residues from the carboxyl-terminus which is required for channel formation. This type of experimental
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approach coupled with the possibility of site-directed mutagenesis of domains thought to be involved in channel formation should help to elucidate the molecular basis for toxin insertion into membranes and its subsequent formation of ion channels. Of relevance to studies of C and lymphocyte-derived channels is another type of toxin, the so-called yeast killer toxin, secreted by Saccharomyces cereuisiae, which is lethal to sensitive yeasts (Palfree and Bussey, 1979). It consists of an ap-subunit protein, each with apparent size of 9 kDa. This toxin has recently been cloned (Bostian et al., 1984; Boone et al., 1986). An immunity mechanism has been described for both colicins and yeast killer toxin whereby the cells secreting the toxin are immune to its action. For bacteriocins, a plasmid-encoded protein has been identified which presumably confers immunity by forming an inactive complex with the toxin (Weaver et al., 1981; Bishop et al., 1985). i n the case of yeast killer toxin, an immunity-coding region has been identified in the precursor of the native toxin by site-directed mutagenesis (Boone et al., 1986). It is thought that the precursor confers immunity by competing with mature toxin for binding to a membrane receptor. Similar proteins or precursors may also play an immunity role in C and lymphocyte-mediated lysis. Recently, a human erythrocyte membrane protein of 65 kDa has been purified using C9-Sepharose columns that, when incorporated into liposome membranes, are capable of blocking C5b-8- and CH-mediated channel formation (Zalman et al., 1986b). It will be exciting to verify whether analogous “immunity” proteins are also present in lymphocyte granules or plasma membranes which would prevent the suicidal killing of the lymphocyte that is actively secreting PFP during TC killing. VII. Conclusion
How killer cells kill their targets will remain an enigmatic research problem for a number of years, but there already is an emerging consensus about the role of certain polypeptides in these cytotoxic reactions. A general mechanism of killing involves inflicting target membrane damage by pore formation. Pore formation is not restricted to cell-mediated killing and other immune responses, but rather it is a powerful membrane injury mechanism that is widely spread in nature. i n our future studies of immune cell killing mediated through the action of PFPs, it should be exciting to verify what aspects are common to all these cytotoxins. It will also be useful to examine the biochernical and physiological properties of other well-known pore formers, since much of the methodology used to this day to examine these properties should also be applicable to the dissection of PFPs of lymphocytes or other immune cells. The advent of cDNA cloning and site-directed muta-
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genesis will make it possible to define the functional domain(s) of these proteins in regard to membrane binding, membrane insertion, and pore formation. Once the primary sequence of these proteins has been deduced by cDNA cloning, it should also be possible to synthesize peptides with predicted functions and test these peptides in their ability to mimic given functions of the native protein. It is also emerging from current studies that cell-mediated killing is a complex phenomenon, and pore formation may answer for only one of the many mechanisms available to the cell in its delivery of target cell injury. Nevertheless, it is apparent that there seems to exist a certain unity in humoral and cellular immunological mechanisms which must not have occurred by accident during evolution.
ACKNOWLEDGMENTS We wish to acknowledge several people who made important contributions to the studies described in this review: Drs. E. R. Podack, C. G. B. Peterson, P. Venge, H. Hengartner, C. F. Nathan, M. A. Palladino, and B. Perussia for collaborative studies; and L. G. Leong, A. Damiano, M. A. DiNome, and S . S . KO for excellent technical assistance. Special thanks are also due to Dr. C.-C. Lin for her contribution to more recent studies. This work was supported in part by grants from the Cancer Research InstituteIFrances L. & Edwin L. Cummings Memorial Fund Investigator Award and the Lucile P. Markey Charitable Trust to J.D.-EY.and by grants CA30198 and A107012 from NIH to Z.A.C. J.D.-EY.is a Lucille P. Markey Scholar.
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ADVANCES IN IMMUNOLOGY, VOL 41
Biology and Genetics of Hybrid Resistance’ MICHAEL BENNEll Department of Pathology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235
1. Introduction
When I began my research career with Gustavo Cudkowicz at Oak Ridge National Laboratory in 1963, he was in “big trouble.” He had recently discovered the phenomenon of hybrid resistance to bone marrow cell and nonparenchymal liver cell grafts by irradiated F, hybrid mice (Cudkowicz and Cosgrove, 1960, 1961). Few believed him and investigators were trying to prove that he was wrong. The genetic laws of transplantation are based upon the assumption that histocompatibility antigens are inherited codominantly (Snell, 1953; Snell et al., 1953). Hence, F, hybrid progeny mice of a mating between two mice of inbred strains must express all of the antigens of each parent. It was therefore impossible for an F, hybrid to recognize a foreign antigen on parental strain cells. He was trying to break a genetic law! Almost as unusual (unlikely) as the genetics, Gustavo made the observation by grafting cells into lethally irradiated mice. Few immunologists in the early 1960s believed specific immune reactions could occur in animals so deprived of lymphoid cells. It was fortunate that Oak Ridge National Laboratory had several excellent geneticists to call upon for advice. I think the key to the success of the studies of hybrid resistance was based upon the decision by Gustavo to use a genetic as well as an immunological approach to the problem of hybrid resistance. If hybrid resistance really existed, sooner or later other investigators were bound to make similar observations. In fact, one of the first published observations that parental strain cells fail to succeed well in F, hybrid mice was made by Snell (1958). Two C57BL/10 tumors, C1498 and S913, were less able to grow and kill C57BL/10 x C3H hosts than C57BL/10 hosts. Pretreatment of the F, hybrid mice with C57BL/10 thymocytes or with a nonlethal inoculation of S913 cells did not enhance hybrid resistance. Hence, lack of imniunological “memory” was demonstrated and is the usual finding
1 This review is dedicated to the memory of my mentor, Gustavo Cudkowicz, who discovered hybrid resistance and who pioneered the field of “natural resistance.”
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in hybrid resistance. Hauschka and Furth (1957) predicted that the outcome of grafts would not always be based upon the expression of conventional histocompatibility antigens after they made similar observations (Hauschka et al., 1956). Parental strain normal spleen cells, especially of the C57BL strain (H-2b), often failed to survive in F, hybrid hosts, even when the cells were donated by mice immunized against the opposite strain partner in the cross (Boyse, 1959; Corer and Boyse, 1959). These observations led to the idea that the parental cells were dying due to an “allergic” reaction to alloantigens. This idea was expanded upon by Hellstrom following his observations that parental strain lymphoma cells often grow poorly in F, hybrid hosts (Hellstrom, 1963). He initially named the phenomenon “syngeneic preference” (Hellstrom et al., 1964), but later named it “allogeneic inhibition” when he concluded that exposure to alloantigens was the mechanism of poor growth (Hellstrom and Moller, 1965). Lengerova et al. (1973a)made similar, but not identical, observations in studies with normal bone marrow cells. Meanwhile, to demonstrate the intense interest or concern about hybrid resistance at Oak Ridge, Popp and colleagues (Popp, 1961, 1964; Popp et al., 1964) reported that C57BL marrow grafts regressed in irradiated F, hybrid mice after temporary engraftment, Celada and Welshons (1962) noted that parental strain immune cells faired poorly in F, hybrid hosts, and Goodman (1965) searched in vain for parental antigens not expressed on cells of F, hybrid mice. Popp and Cudkowicz (1965) observed that acute and chronic rejection of (C57BL x 101)F, marrow grafts by (C57BL x 101)F, hosts differed genetically, suggesting that two mechanism may be operative. Gene Shearer, Gustavo’s first graduate student, and I accompanied Gustavo when he moved to Roswell Park Memorial Institute in 1965. The analysis of the effector cells involved in hybrid resistance was initiated by Cudkowicz and colleagues in Buffalo. This work helped to pioneer a new field in immunology, that of “natural resistance” (Cudkowicz and Bennett, 1971a,b), which was later greatly stimulated by the discovery of natural killer (NK) cells (Kiessling et al., 1977a,b; Herberman et al., 1975a,b). I recommend early reviews on this subject by Cudkowicz (1965a,b, 1968a,b, 1970, 1975b, 1978), Cudkowicz and Lotzova (1973), Hellstrom and Hellstrom (1965, 1967, 1968), and Lengerova et al. (1973a), and recent reviews by h e l l (1976a,b, 1979), Trentin et al. (1976), Dicke et al. (1978); Cudkowicz and Hochman (1979), Clark and Harmon (1980), Lotzova (1977b,c, 1982), Cudkowicz and Nakamura (1983), and Miller (1984). Fox (1958) wrote a review on the genetics of antigen expression and stimulated the idea of interaction between two or more genes to “realize” the appearance of an antigen.
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II. Hybrid Resistance to Normal Hemopoietic Cells
A. BONEMARROWCELLS An impetus for experimental bone marrow transplantation studies in the early 1950s during the Cold War was the knowledge that a major complication of exposure to ionizing radiation was hematological failure. Lorenz et al. (1952) observed that irradiated mice and guinea pigs could be protected by injections of bone marrow. Initially, it was thought that “humoral factors” of the marrow were responsible for protection (Jacobsen, 1952; Congdon and Lorenz, 1934). Later studies indicated clearly that donor cells repopulated recipient mice (Lindsey et al., 1955; Ford et al., 1956; Nowell et al., 1956). The first complication of bone marrow transplantation noted was “homologous or secondary disease,” which usually occurs after early recovery of hemopoietic cells (Trentin, 1956) and is due to a graft-versus-host reaction (Schwartz et al., 1957; Kaplan and Rosston, 1959; Cole and Garver, 1960; van Bekkurn et al., 1959; Congdon and Urso, 1959; Billingham, 1958;Barnes and Loutit, 1956; DeVries and Vas, 1959). In that disease, there is extensive proliferation of histiocytes and epithelioid cells, infiltration of various organs by lymphoid cells, and eventually depletion of lymphoid and hemopoietic elements. In light of what we know about NK cell ontogeny, it is interesting to note that F, mice (unirradiated) become resistant to graft-versus-host disease at 3 weeks of age (Fiscus et al., 1962). The first mention of “a possible immune response by the F, hybrid against the parent” was made by Cudkowicz and Cosgrove (1960) when they were assessing the ability of C57BL (H-2b) or 101 (H-2k)liver nonparenchymal cells to induce fatal graft-versushost reactions in irradiated F, hybrid mice infused with syngeneic F, bone marrow cells. The improved survival of mice infused with syngeneic marrow cells suggested to Cudkowicz that F, marrow-derived cells were exerting an immune response against the liver cells of parental strain origin. In the succeeding studies (Cudkowicz and Cosgrove, 1961; Cudkowicz, 1961b), they noted that parental strain C57BL marrow cells failed to protect lethally irradiated F, hybrid mice if preincubated with B10 x 101 F, (H-2 heterozygous) liver cells prior to infusion. In two succeeding papers, evidence was presented that F, hybrids could be immunized against parental antigens. In the first, Cudkowicz (1961a) observed that prior injection of large numbers of parental spleen cells 30 and 15 days or placement of parental skin grafts 100 days prior to irradiation and infusion of lo7 marrow cells resulted in a greater incidence of mortality than if the hosts were not “preimmunized.” In the second study of this sort, Cudkowicz and Stimpfling (1964a) showed that multiple injections of parental strain spleen cells weak-
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ened hybrid resistance, but that injections of parental strain marrow cells or grafting of parental skin strengthened hybrid resistance. Alternative explanations for these results are now possible. The parental spleen cells infused prior to irradiation could have survived in the F, hybrid mice and could have caused death (graft-versus-host reaction) in the survival study and could have inhibited hybrid resistance in the second study (Hakim and Shearer, 1986). The H-2b marrow cell injections and skin grafts could have stimulated a host antibody response to H-2b alloantigens. This would allow host N K cells to lyse grafted stem cells by antibody-dependent cellular cytotoxicity if IgG antibodies were elicited and any IgM antibodies could lyse the cells in the presence of complement. The injections of H-2b erythroleukemia cells into H-2b heterozygous F, hybrid mice did lead to the production of anti-H-2h antibodies (Risser and Grunwald, 1981). A different interpretation of the failure of parental stem cells to grow well in F, hybrids was called “CFU [colony-forming unit] repression” (McCulloch and Till, 1963). The development of the spleen colony technique to evaluate hemopoietic stem cell function (Till and McCulloch, 1961) stimulated research in this field to a great extent. Evidence was presented to favor the notion that stem cells ‘died’ (entered irreversible differentiation) or selfreplicated at random (Till et al., 1964); a stochastic model of stem cell proliferation was developed. However, a few years later, Wolf and Trentin (1968) provided evidence that the spleen and marrow organ stroma dictated the patterns of differentiation of stem cells. Thus, in the spleen the “hemopoietic inductive microenvironment” favored erythropoiesis, and the ratio of erythropoietic to granulocytic (E : G) colonies was 2.5 to 3 : 1. In contrast, the E : G ratio in the bone marrow was 0.5 : 1. They even showed discrete anatomical areas where single spleen colonies abruptly changed from erythropoiesis to granulopoiesis. Whichever model for stem cell development was favored, one could view the failure of C57BL stem cells to proliferate in spleens of F, hybrid mice as an inefficient interaction between the stem cell and the stromal cell, i. e., inappropriate “seed-soil” relationship. This concept preceded the development of H-%restricted interactions involved in immune cell interactions. Gregory et al. (1972) developed antibodies in mice against non-H-2 antigens which were able to abrogate CFU repression or hybrid resistance, and the sera were named “derepressive” sera. The best strain combinations to elicit such antibodies were C3H antiBlO.BR, LP anti-B6, and 129 anti-B6. These strains are mentioned now because C3H and LP have “poor responder” genetic status against grafts of H-2b marrow cells (Cudkowicz and Bennett, 1971a; Cudkowicz, 1971), and 129 mice have recently been used to generate monoclonal antibodies to NK-1.1 and NK-2.1 antigens following immunization with enriched sources of H-%identical C57BL/6 NK cells (Ouellet-Talbot et al.,1986). NK-1.1 and
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NK-2.1 are two markers so far specific for murine NK cells (Burton and Winn, 198 1). Till et al. (1970) noted that horse anti-mouse thymocyte serum also reversed CFU repression by F, hybrid mice. Another alternate mechanism to explain poor growth of parental strain cells was a lack of stimulation of stem cells by the host environment. This poor growth of C57BL marrow cells in F, hybrid hosts could be reversed if large numbers of C57BL/6 thymocytes were infused along with the marrow cells (Goodman and Wheeler, 1966, 1968; Goodman and Matthews, 1966; Goodman and Shinpock, 1968, 1972). The idea to study the thymus was based upon observations that thymomas are associated with erythropoietic failure (Roland, 1964); erythropoiesis was inhibited in the absence of the thymus (Miller et al., 1965) and later sustained by the finding that T cells stimulated stem cells (Wisktor-Jerdrezejezak et al., 1977; Cerny et al., 1975). Goodman and colleagues irradiated F, hybrid mice and infused inocula of lo6 C57BL marrow cells. They observed that C57BL, but not F, hybrid or opposite parental strain thymocytes were effective in restoring erythropoiesis as judged in terms of "Fe uptake in spleens or erythrocytes 6 days after cell transfer. The thymocytes were effective if given between 2 days before or 1 day after marrow cell transfer. The thymus cells had to be viable, and exposure to 100 Gy, but not to 10 Gy (Goodman, 1971), abrogated the effect. The ability to induce a graft-versus-host reaction by the thymocytes was not related to the stimulation of erythropoiesis (Goodman et al., 1972), since thymocytes from donors tolerant to host alloantigens were three times more effective than cells from nonimmune donors. Moreover, lymph node cells were unable to stimulate erythropoiesis. The failure to immunize F, hybrids against parental strain grafts (Goodman and Bosma, 1967) and the failure to detect histological evidence of an immune response to explain the poor growth of parental strain marrow cells in F, hybrid mice (Goodman et al., 1970) were taken as strong evidence against the hostversus-graft hypothesis of Cudkowicz. In support of the concept that thymocytes have a nutritive or supportive role in erythropoiesis, Lord and Schofield I 1973) observed that infusion of syngeneic thymocytes enhanced endogenous spleen colony formation by sublethally irradiated mice and enhanced the function of transplantable/exogenouscolony-forming cells following an exposure of mice to 1.8 Gy. On balance, Harrison et al. (1979) observed that thymectomy did not prevent successful cures of W/Wv anemia by histocompatible bone marrow cells, and Metcalf (1960) noted that thymectomy had no effect on red blood cell (RBC) counts or hematocrit values. In a series of experiments, Lengerova and colleagues investigated the mechanism of deficient spleen colony formation in irradiated mice which differ at H - 2 only, as they used B10 congenic mice (Lengerova and Zeleny, 1968, 1969; Lengerova and Matousek, 1969; Lengerova et al., 1970, 1971,
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1972, 1973a-c). In their experiments, inocula containing 7 to 25 x 104 marrow cells were infused into irradiated hosts, and spleen colonies were enumerated 9 or 10 days later. Growth was quantitated by calculating the slopes of the lines relating colony numbers to cell inoculum size. They observed that parental strain marrow cells grew less well in F, hybrids than in syngeneic hosts and that F, hybrid cells were also inhibited in parental strain hosts. Using nonimmunized recipients, they observed that H-2D differences were more important than H-2K differences. If marrow cells were preincubated with lymph node cells from donors immunized against H-2 alloantigens prior to inoculation into hosts syngeneic to the marrow donor, they observed that H-2K differences were more important, i.e., produced greater impairment of colony formation, than H-2D differences. Cortisone pretreatment of the hosts abolished the impaired growth of incompatible marrow cells. They also observed that exposure of marrow cells to allogeneic, semiallogeneic, or parental strain lymphoid cells or nonliving homogenates of lymphoid cells resulted in relatively poor growth in syngeneic host mice. F, hybrid mice were splenectomized and neonatal spleens from both parental strains were transplanted into opposite poles of the kidney. Later, the mice were irradiated and infused with one or the other parental strain marrow cells; inhibition of colony formation was detected in the allogeneic as compared to the syngeneic spleen graft. The stem cells, under these conditions, would be exposed to F, hemopoietic cells in the two spleens as well as any residual parental strain stromal or hemopoietic cells that had survived the 1 month after grafting prior to irradiation. Their data are remarkably similar to those of Hellstrom in his fascinating studies of allogeneic inhibition to tumor cells (Hellstrom and Hellstrom, 1965, 1967, 1968). However, these investigators went further in showing that it was matching of H-2 antigens rather than exposure to H-2 antigens that was critical. F, hybrid mice were irradiated and repopulated with a 1: 1 mixture of the two parental strain marrow cells. The mice were subsequently irradiated a second time and infused with F, hybrid marrow; no inhibition of colony formation was observed. These investigators “modulated” the expression of H-2 antigens on marrow cells by treatment with specific anti-H-2 serum followed by rabbit anti-mouse Ig serum. If B10 x BIO.A cells were treated with B10 anti-BIO.A serum, the cells subsequently grew without impairment in B10 hosts. In contrast, similarly treated F, cells grew less well in syngeneic hosts, as if the marrow cells were phenotypically H-2b only. Any diminution of growth of antibody-treated marrow cells could be attributed to antibody-dependent cellular cytotoxicity mediated by radioresistant host cells, probably NK cells. Such an explanation fails to account for enhanced growth of marrow stem cells so treated. The experiments just cited were interpreted to indicate that H-2 antigen matching between donor
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and host is optimal for stem cell function, and a mismatch of any type can lead to relatively poor growth of stem cells. A histological analysis of colony formation revealed that parental strain cells would sometimes proliferate in the white pulp of the spleen, as if the host alloantigens were stimulating lymphopoiesis. Thus, they considered the possiblity that stem cells may be diverted from erythropoiesis and would therefore generate less numbers of spleen colonies. This observation was not made when F, cells were transplanted into parental strain host. Finally, exposure of bone marrow cells in uitro to whole cells or homogenates of lymphoid cells which either had an “excess” (a marrow cells exposed to a X b) or a “deficit” (a X b marrow cells exposed to b) of antigens inhibited subsequent colony formation in syngeneic hosts. If a x b marrow cells were exposed to mixtures of a and b cells or extracts, no inhibition was observed. As mentioned earlier, Cudkowicz embarked upon genetic and immunological approaches to understand hybrid resistance to bone marrow grafts. The determinants for hybrid resistance to C57BL marrow cells were mapped to the H-2b region (Cudkowicz and Stimpfling 1964a-c), specifically at the D end of H - 2 . Another genetic influence on the outcome of bone marrow grafts was detected when the immunobiology was studied (Cudkowicz and Bennett, 1971a). The ability of irradiated mice to reject allogeneic H-2b and H-2d marrow grafts was shown to be a determinant-specific and strain-dependent trait, with the “good responder” trait dominant over the “poor responder” trait. For example, mice of three H-2k strains, namely, C3H, AKR, and 101, did not resist grafts of B10 marrow cells, whereas C57BR and B10. BR (also H-2k) strongly resisted grafts of B10 cells; C3H x B10.BR also were good responders. Similarly, 129 mice were poor responders to H-2d marrow grafts while B10, B10 x 129, and 129 x B10 mice were good responders. An analysis of F, progeny mice between matings of C3H x B10. BR F, hybrids and of 129 x B10 F, hybrids indicated that two or more genes, not linked to the H-2, agouti, chinchilla, pink eye dilute, or dilute coat color loci, regulated the ability of mice to reject marrow cell grafts in a determinant-specific manner (Cudkowicz, 1971). The ability of mice to resist H - 2 allogeneic or parental strain marrow cell grafts matures at about 3 weeks of age (Cudkowicz and Bennett, 1971a,b). Resistance is relatively radioresistant, but progressive loss of resistance can be detected in whole-body exposures between 6 and 10 Gy of irradiation. Exposure of good responder mice to 3 to 5 Gy 7 to 14 days before lethal irradiation weakens resistance to parental and allogeneic strain marrow grafts, indicating that precursors of the effector cells were radiosensitive. Thymectomized mice, which were irradiated and reconstituted with syngeneic marrow cells, were able to reject allogeneic marrow cells. This indicated that T cells were not necessary; eventually. nude athymic mice were
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observed to reject marrow cells much better than euthymic littermates (Cudkowicz, 1975a). To determine how fast parental or allogeneic strain marrow stem cells were rejected, two-step experiments were performed. Thus, primary recipient B10.D2 or B10 x BlO.D2 F, mice were irradiated and infused with 2.5 x lo6 B10 marrow cells. At intervals between 3 and 120 hours, the spleen cells from the primary hosts were reinfused into irradiated B10 secondary hosts previously immunized against B10. D2 antigens. Survival and/or proliferation of grafted stem cells was reflected by the degree of splenic uptake of 541251]iodo-2‘-deoxyuridine(IUdR) 5 days after the second cell transfer. The data indicated that 12-24 hours elapsed before significant rejection or stem cell death occurred. The ability to reject marrow cell grafts was shown to be a property of hemopoietic cells, since both good responder and poor responder status against grafts of H-2d marrow cells could be transferred by marrow cells infused from B10 to 129 and from 129 to B10 sublethally irradiated mice, respectively. We assessed the potential role of macrophages by injecting mice with heat-killed Corynebacterium parvum (now Proprionobacter acnes) organisms 4 to 21 days before irradiation and marrow cell transfer. Corynebacterium parvum organisms are a powerful adjuvant and greatly stimulate macrophage phagocytic function (Halpern et al., 1963; Neveu et al., 1964). Both allogeneic and hybrid resistance to marrow cell grafts were abrogated 7-18 days after injection of C. parvum (Cudkowicz and Bennett, 1971a,b). In light of knowledge regarding NK cells obtained since 1971, one can explain the inability of neonatal mice to resist incompatible marrow grafts to the functional immaturity of NK cells (Koo et al., 1982). The explanation for the effect of C. parvum organisms could be that suppressor cells are induced that inhibit NK cell function (Savary and Lotzova, 1978; Hackett et al., 1986a; Milisauskas et al., 1986a). Immunological memory against parental strain antigens has not been convincingly demonstrated, but evidence for specific tolerance to parental strain marrow graft antigens, now called hybrid histocompatibility-1 (Hh-1) antigens, had been obtained. Thus, several weekly injections of C57BL spleen cells weakened hybrid resistance, but not the ability to reject “third-party” marrow cell grafts (Cudkowicz and Stimpfling, 1964a; Cudkowicz and Bennett, 1971b). As mentioned, the recent data of Hakim and Shearer (1986) indicate that injections of C57BL spleen cells induce a graft-versus-host mediated immunosuppression. However, Cudkowicz (1965a) observed that if B10 x A marrow cells were used to repopulate the hemopoietic system of irradiated B10 host mice, such chimeras were not able to resist, after irradiation a second time, grafts of B10 marrow cells. The mechanism of “tolerance” to marrow cell grafts has not been determined. Whereas tolerance to host-type solid tissue grafts and unresponsiveness to host-type alloantigens have been demonstrated in H-2 allogeneic radiation bone marrow chimeras
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(Zinkernagel et al., 1980; Rayfield and Brent, 1983; Auchincloss and Sachs, 1983; Maestroni et al., 1982, 1983; Krown et al., 1981; Maki et al., 1981), tolerance to allogeneic marrow grafts has not been assessed. Lotzova and Cudkowicz (1971, 1972, 1973) discovered three new instances of hybrid resistance to murine bone marrow cells. Marrow grafts from NZW, WB/Re, and NZB donor mice were resisted by irradiated H-2 heterozygous F, hybrid mice as well as H-2 allogeneic recipients. NZW mice are H-2”; from the data presented it would appear that recipients of the good responder type to grafts of H-2h marrow grafts and not homozygous for H-2Db were also good responders to NZW marrow cell grafts. Genes of the C57BL background endow mice with this responder status. Since B10 x NZW F, mice did not resist grafts from NZW donors (Lotzova and Cudkowicn:, 1971), it is conceivable that H-2” and H-26 share a common Hh-1 determinant. WB/Re mice are H-2, although the designation was H-2“ at the time. The j haplotype shares with the b haplotype the D” determinant, as well as Qa-2., Tla”, and Qa-lb. Therefore, it could be a “natural recombinant” between the b a n d j haplotypes (Klein et al., 1983). Nonetheless, WB x B10 F, hybrids strongly resist grafts of WB marrow cells. As a matter of fact, the only H-2 allogeneic mice which did not resist WB marrow grafts (probably due to a poor responder status) were A/He, SJL, DBA/2Ha, and 129/Rr mice. WB x SJL mice weakly resisted, while WB x A.SW mice strongly resisted grafts of WB marrow cells. Since both of those F,s are H-%/H-29, it follows that the WB strain and the SJL strain mice have poor responder status against WB marrow grafts. An analysis of WB x (WB X BlO.tf,T)F, and (B10.tf,T x WB)F, x WB backcross progeny mice used as recipient of WB marrow grafts was made. The animals were typed for the W/ + dominant spotting gene (white spot), the expression of H-2.5 public antigen, and for short tail ( t l f ) . The results indicated that the Hh determinant mapped 11.6 units away from T and 3.8 units away from H-2 and therefore was linked to both. The results did not permit the conclusion that the WB Hh determinant was located at H-2D or at Hh-1 (Lotzova and Cudkowicz, 1972). If the determinants for Hh antigen expression for both H-2” and H - 2 map in the same (Hh-1) locus, Hh-1 must not be in the region from H-2D to Qa-1. WB and B10 differ in expression of H-2-associated Hh antigen expression and are identical in this portion of H-2. NZB mice are H-2”. Lotzova and Cudkowicz (1973) noted extremely strong hybrid resistance by NZB x NZW F, hybrids in that 4 x lo7 marrow cells were resisted! This fact will be recalled when I discuss the potential role of antibodies in hybrid resistance. In this study, allogeneic and F, hybrid hosts were challenged with NZB marrow grafts. Those hosts, which were H-2Dd/H-2Dd, accepted the grafts irrespective of their responder status to
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H-2d marrow cell grafts. Mice that were not homozygous for H-2Dd did or did not resist NZB marrow cells based upon their responder status to H-2d marrow grafts. The results of grafts of NZB marrow cells into backcross progeny mice of matings between NZB and (NZB x NZW)F, or (BlO.tf,T x NZB)F, indicated that probably two genes determined resistance, since 26 of 75 or 35 of 115 of the respective mice were resistant (closer to 25% than to 50% resistant). Since 11135 resistant animals in the second cross were typed negative for the H-2.5 antigen of H-2b, they concluded that one of the two genes was loosely linked (31.4 units away) to H-2. In a later communication, Cudkowicz (1978) reported that B10.D2 ( H - 2 9 marrow grafts were rejected by H-2 heterozygous F, hybrid host mice and that the locus, called Hh-2, mapped 16 crossover units to the right of H-2. Warner (1978) and Cudkowicz (1978) reported that there was hybrid resistance to SJL (H-2") marrow grafts by hosts heterozygous for H-2D". Exceptions were SJL x C57BL, SJL X BlO.A(SR), SJL x BlO.A(4R) and SJL X B10. HTG F1 mice, suggesting that H-2" and H-2b share a common determinant at the H-2DIHh-1 locus. All of the genetic analyses by Cudkowicz and colleagues involved backcrossing F, hybrid mice to the parental strain of interest and testing the ability of the progeny mice to reject parental strain grafts after determining other genetic traits. I tried a different approach and tested progeny mice of various crosses as bone marrow donors (Bennett, 1972). Irradiated (C3H x BlO)F, (H-2klH-2b) mice reject parental strain B10 (H-2b) and allogeneic DBA/2 ( H - 2 9 marrow cells. However, under the conditions used (grafts sizes of 2 X lo6 cells; measurement of IUdR uptake in the spleen 4 days later), grafts of (C57BL x DBAI2)F, (H-2blH-2d) marrow cells grew without impairment. The F, mice were backcrossed to C57BL or DBA12 strain mice, and each progeny mouse was typed for H-2, using RBC agglutination by anit-H-2b serum that did not agglutinate DBAI2 RBC and anti-H-2d serum that did not agglutinate C57BL RBC. Marrow cells from each progeny mouse were infused into groups of (C3H x BlO)F, irradiated hosts. Without exception, all H-2 homozygous donors contained marrow cells that were rejected, and all H-2 heterozygous donors had marrow cells that proliferated extensively. C3H x B10 F, hosts were challenged with marrow grafts from inbred strain and F, hybrid from a variety of donors. H-2 types of the following were rejected: blb, jlj, dld, and j l d , and H-2 types of the following were accepted: kld, j l b , bld, jlk, and klk. The exceptional heterozygote, jld, was analyzed further by graftingjlj, dld, j l d , and jlk marrow cells into recipients capable of rejection of both j / j and dld marrow cell based upon responder status and into recipients capable of rejecting jlj, but not capable of rejecting dld grafts. The former type of hosts resisted j l j , dld, and jld grafts, but not jlk grafts, while the latter type of host resistedj/j grafts, but
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not j l d , dld, or j l k marrow grafts. These results suggested to me that there were two Hh determinants in the j haplotype, one of which is shared with the d haplotype. These data indicated that homozygosity for Hh (probably Hh-1) determinants is required for optimal immunogenicity of marrow cells for irradiated recipient mice. The data helped to resolve one question, but raised another. If C57BL x DBAI2 F, marrow cells fail to express any detectable Hh-1 antigens, why do such mice reject C57BL marrow cells, but accept DBA/2 marrow cells? I made the suggestion that a gene in a cis position near the Hh-1 gene regulated expression of the gene product so as to allow or not to allow enough expression of Hh-1 antigens in the F, to produce “self-tolerance. In a more recent study, advantage was taken of two mutant haplotypes to assess the genetics of marrow transplantation (Bennett et al., 1980). The D region contains at least two class I genes, D and L (Demant et al., 1975; Lemmonier et d . , 1975; McKenzie et d . , 1977; Hansen et d., 1978, 1983, 1984; Kohn et al., 1978; Demant and Neuport-Sautes, 1978). The H-2dm2 mutation involves the “loss” of L”, and this mutation occurred in a BALB/c mouse (Melvold and Kohn, 1976; McKenzie et al., 1977). The H-2dm1mutation occurred in a B10. D2 mouse and is a “gain-plus-loss” mutation (Egorov, 1967; Egorov and Blandova, 1974; Morgan et al., 1978), which is due to a deletion of DNA between the 3’ end of D and the 5‘ end of L with the formation of a hybrid DIL gene (Sun et al., 1985). The loss of Ld in the dm2 mutation did not affect the immunogenicity of BALBIcKh (C) marrow cells for irradiated allogeneic host mice. Both B6 x C and B6 x C-H-2dm2 F, hybrids rejected B6 marrow cells and accepted C or C-H-2dm2 marrow grafts. The dml mutation appeared to result in a change in the immunogenicity of marrow cell grafts. B10. D2 and B10. D2-H-2dm1marrow grafts were equally resisted by H-2 allogeneic hosts, but B10. D2-H-2dm1 marrow cells were much less immunogenic than B10. D2 cells for B6 x B10. D2 or B6 x B10. D2-B-2dm1 F, hybrid recipients. These results have two potential implications. First, the original mapping of the B10.D2 Hh locus (Hh-2) 16 crossover units to the right of H-2 may be incorrect, or the D-L region regulates the degree of expression of an Hh antigen whose determinant maps 16 units away. Second, it is conceivable that separate genes code for Hh antigens recognized by allogenic and F, hybrid recipient mice. Contrasting evidence (Morgan and McKenzie, 1981) suggested that H-2L antigens may be recognized as targets for rejection. In a recent analysis of the immunogenics of murine marrow graft rejection, Drizlikh et al. (1984) observed that the I and K regions could be involved. They grafted lo4 to lo7 marrow cells and enumerated spleen colonies on day 8. They observed a weak resistance by B6 x BALBIc F, hybrids to BALB/c marrow cells, which had not been detected before. Using ”
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a series of H-2Db donors that differed in genotype between K and S, they observed quantitative differences in the ability of B6 x BALB/c and B6 x C3H recipients to reject the grafts. However, there was resistance to all of the grafts, which included D2.GD, B10. HTG, BlO.A(2R) and BlO.A(4R). Relatively speaking, they observed that resistance to 2R and 4R was much better in the B6 x BALB/c than in the B6 x C3H hosts, which they interpreted to be caused by sharing of H-2k genes between K and I E . In contrast, the HTG cells were more strongly resisted by B6 x C3H than by B6 x BALB/c hosts, presumably due to sharing of H-2d genes between K and S . A series of donors which are Dd, but different between K and S with BALB/c, were sources of marrow cells infused into B6 x BALB/c F, hybrids in numbers between lo4 and 105. These donors included 5R, A.TL, A.AL, A.TFR.5, BALB/c, and C3H.OH. The BALB/c and A.TFR.5 cells were resisted, but the other cells were not. The inability to express the Ia.22 combinatorial specificity was associated with poor growth. This led the investigators to suggest that class 11 antigens may be involved in marrow rejection. However, they did not test for the expression of Ia.22 on bone marrow stem cells. C3H.OH marrow cells, which are d from K to S and k at D, were resisted modestly by B6 x C3H hosts. This was taken as evidence that genes to the left of D were responsible for expression of an antigen recognized by the host mice. C3H.OH marrow cells were not tested in other recipient mice which are able or not able to reject H-2d (B6 or C3H, respectively) or H-2k (129 or B6, respectively) marrow cells. Using inocula of 5 or 10 x lo4 marrow cells from BlO donors, colony formation in H-2Db allogeneic hosts was tested. These hosts differed from B10 between H-2K and H-2s. There was modest allogeneic resistance manifested by B10. HTG, BlO.A(2R), and BlO.A(4R) hosts. Their results are not unlike those of Lengerova et al. (1973a). These investigators compared the ability of B10. HTG x BALB/c and B6 x BALB/c F, hybrid mice to resist grafts from BALB/c donors. Note that B10.HTG is d from K to S and is H-2Db. BALB/c marrow cells between numbers of lo4 to lo5 grew much better in the B10.HTG F, hybrid, strengthening their argument that lack of unique Ia determinants can regulate graft rejection. Finally, three different H-2Db homozygous marrow cells grew to different extents in irradiated B6 x BALB/c hosts; B6 grafts were strongly rejected, B10.HTG cells were rejected less well, and B10.HTG x B6 cells were only weakly resisted. Using B6 x C3H hosts, the same three types of marrow grafts were resisted well and to about the same extent. The interpretation is that unique transcomplementation for Ia antigens in b x d crosses are antigens recognized by b x k B10 x C3H F, hybrid mice. Their results obviously complicate matters, but Ia and K differences must be considered when analyzing the immunogene-
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tics of marrow transplantation, especially when small cell inoculum sizes are used. Early studies had indicated that Hh determinants mapped at or near the D end of H-2. However, Cudkowicz and Warner (1979) observed that there was a reactivity toward H-2Kk marrow cells. In this case, 129 mice were good responders and C57BL/6 and D1.LP mice were poor responders, even though all three strains are H-2b. In contrast to previous studies of resistance to grafts of H-2b or H-2d marrow cells (Cudkowicz, 1971), susceptibility was dominant over resistance to grafts of H-2Kk marrow cells in B10 x 129 or 129 x B10 crosses. This locus was named Hh-3. No hybrid resistance to H-2Kk marrow cells was detected; however, the F, hybrids tested would not have been expected to reject based upon the dominance of susceptibility over resistance. For example, B10.BR X 129 F, mice are H-2k/H-2b, but inherit the poor responder status from the B10 background (non-H-2) genes and fail to reject B10.BR marrow grafts. Before one can assume that hybrid resistance cannot occur against K k marrow cells, F, hybrid mice need to be stimulated with interferon or interferon inducers. This treatment augments marrow allograft reactivity and endows poor responder mice with the ability to reject marrow grafts (Afifi et al., 1985). Such treated mice can then be challenged by mice with Kk marrow grafts. Alternatively, the H-2k haplotype could be transferred to the 129 strain to produce 129.H-2k congenic resistant mice. 129 x 129.H-2k F, hybrids could then be challenged with H-2Kk marrow cells. If hybrid resistance to Kk marrow cells still does not occur, this is a situation like most Dd marrow grafts. Whereas homozygosity for Dd is necessary for immunogenicity of the stem cells, most Dd heterozygous F, hybrids accept Dd homozygous transplants (Bennett, 1972). The exceptions are grafts of NZB and B10.D2 H-2Dd marrow cells which are rejected by their F, hybrids. Rossi et al. (1970, 1971, 1973) discovered a new Hh locus which is not linked to H - 2 . It was discovered by transplanting bone marrow cells of DBAI2 mice shortly after infection with Friend leukemia virus. This Hh locus was not linked to H-2, and resistance to normal (uninfected) DBAI2 marrow cells was “weak” in that inocula greater than 3 X 105 cells were not resisted, as detected by splenic IUdR uptake (9%) values 5 days after cell transfer (Chdkowicz and Rossi, 1972). F, hybrid mice were resistant to 10 times that many Friend virus-induced leukemia cells or marrow cells soon after infection with Friend virus. DBA/2 marrow cells grew well in DBA/2 hosts after Friend virus infection, but C57BL/6 x DBA/2 F, host mice were strongly resistant and BALB/c x DBA/2 F, mice were moderately resistant. The ability to reject was not obviously related to two resistant genes to Friend virus, namely, Fu-1 and Fu-2 (Pincus et al., 1971; Lilly, 1970). This
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rapid change in expression of Hh antigens after infection with Friend virus invites the speculation that the effector cells involved in hybrid resistance may function normally to eliminate recently “transformed” leukemic cells. One can perform bone marrow transplantation experiments in unirradiated W/W” dominant spotting anemic mice (Russell and Bernstein, 1967; Harrison and Cherry, 1975; Harrison and Doubleday, 1976). W/W” marrow stem cells are greatly deficient in function, and the mice suffer from a macroscopic anemia and are very radiosensitive with respect to the hemopoietic system. Nevertheless, these mice are immunocompetent (Mekori and Phillips, 1969; Bennett, 1971; Harrison and Cherry, 1975). Recipient WB x B6 F, W/W” mice were infused with 5 to 20 x lo6 marrow cells from B6 or B10 congenic resistant mice which differed at different minor H antigens. Skin grafts were performed in the same strain combinations. Cures of the anemia occurred in those strain combinations where skin graft rejection was weak or absent; the genetic loci so defined were H-37, Thy-1, H - 1 6 , Ea-2, H - 3 0 , H-18, H - 7 , H-20, H-34, H - 1 9 , H - 1 , and Ly-1. Antigens specified by H - 2 , H - 3 , H-4, H - 2 5 , and H - 2 8 were strongly immunogenic for marrow and skin grafts, H-17 and H-24 antigens were immunogenic for skin but not marrow grafts, and H - 1 2 antigens were more strongly immunogenic for marrow than for skin grafts. Therefore minor H antigens can be expressed on marrow stem cells and can be rejected by nonirradiated recipient mice. It should be pointed out that in all cases except for the B6.H-2d donor, the donor marrow cells were homozygous for H-2b, while the recipients were heterozygous. Presumably the cell numbers used “overrode” hybrid resistance. Using normal recipient mice, Snell et al. (1967) demonstrated that one can successfully immunize mice such that rejection of marrow cells can occur across a weak (H-13) histocompatibility barrier following exposure to lethal doses of irradiation. After lethal doses of irradiation, mice can be used as recipients of xenogeneic rat bone marrow cells. However, there is a genetically regulated ability of mice to resist such marrow cell grafts (Rauchwerger et al., 1973a,b, 1976, 1977). A, BALB/c, DBA/2, C3H, and CBA mice developed countable spleen colonies 8 days after infusion of lo5 to lo6 LEW strain rat marrow cells, whereas no colonies were detected in C57BL or C57BL x A hosts unless more than 3 X lo7 cells were grafted. Yet, as in mouse-to-mouse transfers, the genetic resistance to xenogeneic marrow cells can be overridden by large cell numbers. Lotzova et al. (1975b)performed a genetic analysis of this ability to reject LEW marrow cells by testing the backcross progeny of matings between (C57BL/6 x A) F, and A mice. Grafts of lo7 rat marrow cells were infused and splenic IUdR uptake 5 days later was the measure of growth of the cells. The mice fell into clearly discernible groups, resistant (0.01-0.11% uptake) and susceptible (0.2-1.5% uptake). The ratio
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of resistant to susceptible offspring was 441108, or 28.9%. The data were indicative of a 2-gene regulation of resistance. Immunomodulating influences, e. g., age, cyclophosphamide, split-dose irradiation, fractionated irradiation (225 cGy each week 4 times), massive doses of irradiation, and C . parvum organisms, had similar effects on resistance to rat marrow grafts as have been observed for mouse marrow grafts (Rauchberger et al., 1976, 1977; Trentin et al., 1973; Miller, 1981; Miller et al., 1981; Fohlmeister and Hohentanner, 1985). Survival of recipient mice for a 30-day period in rat to mouse marrow radiation chimeras depended upon colonization of the bone marrow, which occurs at a much smaller cell inoculum size than colonization of the spleens of recipient animals (Rauchwerger et al., 1973). This latter point is important, since many experimental hematologists confuse splenic repopulation with hemopoietic recovery. One should think of the spleen as a convenient test organ, since the effectors of hybrid resistance are concentrated there following lethal irradiation. Survival depends upon marrow repopulation under most conditions. Carlson t?t al. (1986) recently observed that irradiated mice reject IUdR labeled normal BMC or cells derived from long-term bone marrow cultures. Rejection did not occur during the first 4 hours, but was detected at 24 hours. The immunogenetics of the response matched that using the proliferative assay for marrow growth. B6 BMC were accepted by B6 and allogeneic mice, which were H-2DblHh-lb.There was no defect in the ability of beige B(3 x DBAI2 F, mice to reject B6 BMC. Harrison and Carlson (1983) had previously noted that beige mice were deficient in inhibiting CFU-S development by allogeneic BMC. Anti-asialo GM1 serum prevented the rejection of IUdR-labeled BMC. B6 X DBA/2 F, BMC were not rejected by 136 or DBAIB host mice. This set of results suggests that many BMC express Hh-1 antigens, since the percentage of reduction in counts retained in the spleens was 50-80%. Several short reviews list the strains of donor mouse and rat that have been used as donors, mouse strains that have been used as donors, and mouse strains that are or are not good responders to grafts from those donors (Cudkowicz, 1968a,b, 1970, 197513; Cudkowicz and Lotzova, 1973; Lotzova, 1977b; Lotzova et al., 1977; Cudkowicz and Nakamura, 1983). As expected, the last review by Cudkowicz and Nakamura (1983) has the most complete summary of the Hh types known. To briefly summarize the information dealing with hybrid resistance only, the Hh types associated with the b or bc haplotypes were termed Hh-lb, as were the haplotypes g, h, h2, and h4 which are H-2Db. The Hh types associated with H-2Sand H-2" are termed Hh-1"and Hh-l", respectively. The Hh type associated with H-2ia of the WB strain was termed Hh-WB, because of the experiment which appeared to map the locus 3.8 units away from H-2. The Hh type associated with the
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BlO.D2 strain was called Hh-2-D2, since this locus appeared to map 15.8 units away from H - 2 . The NZB strain's H h type was termed H h - N Z B , since it mapped 31.4 units away from H - 2 . Finally, H h - D B A I 2 is the H h type which maps completely away from H - 2 on another chromosome. Hh-3 is a locus that contains an H h type so far associated only with allogeneic resistance to H-2Kk marrow grafts. For reasons mentioned above, I do not think that the appropriate experiments have been done yet to rule out the possibility that hybrid resistance to H-2Kk could be detected. Rats have not been used very much to create knowledge of the genetics or biology of hybrid resistance to bone marrow grafts. However, Santos and Owens (1968) first demonstrated the ability of cyclophosphamide to inhibit rejection of marrow allografts in rats. I became involved in rat marrow transplant experiments in Boston for two reasons. John Dittmer and I wanted to devise a method to successfully transplant organs across MHC barriers (Dittmer and Bennett, 1975), and mice were too small for heart or kidney transplant surgery. A second reason was our inability to observe rapid erythropoiesis in the spleens of irradiated rats infused with syngeneic marrow cell grafts (Rodday et al., 1976). We had to resort to the use of cytotoxic drugs, iron deficiency anemia, or erythropoietin preparations to stimulate growth of syngeneic rat marrow cells. Under conditions when syngeneic grafts succeeded, we observed that irradiated rats, like mice, could reject MHC-incompatible grafts. Thus, LEW or F344 (RTIz)hosts pretreated with dimethylmyelran to stimulate erythropoiesis were able to reject W F (RTI") marrow grafts of 20-50 x lo6 cells, and iron-deficient (LEW x BUF)F, (RTIzIRTIb)-irradiatedrats resisted grafts of W F marrow cells. We eventually used nandrolone decanoate, the anabolic steroid, to stimulate erythropoiesis in rats. We observed that BN marrow cells were strongly resisted by irradiated W F x BN and LEW x BN, but not by BN x DA F, hybrid rats. A parallel hybrid resistance pattern to BNML promyelocytic leukemia cells of BN strain origin was observed in similar unirradiated F, hybrid rats (Williams et al., 1980) or in radiation chimeras (Singer et al., 1980). The data suggested that immune response-like genes regulating responder status exist in rats as they do in mice. We performed experiments to determine if homozygosity for determinants at the RTI locus was necessary for optimal immunogenicity of rat marrow cell grafts (Rodday et al., 1981; Bennett et al., 1982). (WF X DA)F, (RTIUIRTIa)X W F and (WF X LEW)F, (RTIUIRTIq x W F backcross progeny rats were typed for RTI by performing one-way and two-way mixed lymphocyte reactions with peripheral blood and spleen lymphocytes. Recipient LEW rats were stimulated with nandrolone decanoate and irradiated before inocula of 30 x 106 marrow cells were infused. Splenic IUdR uptake was determined 5 days after cell transfer. In the first cross, 13/14 grafts from donors which typed
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ulu were rejected, with uptake values ranging from 0.003 to 0.03%. One graft grew well (0.92%). All 18 of the ula marrow grafts proliferated significantly. In the second cross, 617 of the ulu grafts were rejected, while 0/7 of the ull grafts were rejected. Thus, it would appear that the immunogenetics of hybrid and allogeneic resistance to marrow cell grafts is similar in rats and mice. The rat is actually a good species to study hybrid resistance. The reaction is strong and reproducible, rats survive irradiation well for short period of time, and their large size allows one to harvest many cells for transfer or for in vitro studies. Rolstad and Benestad (1984) recently reported that normal and nude rats could reject radiolabeled allogeneic BMC. They separated BMC on a density gradient which removed RBC and their precursors. The cells were labeled with 51Cr and infused. Tissues were removed and the 51Cr radioactivity was measured 21 hours later. A 0 (Rtl") BMC were destroyed especially well by nude PVG (RT1") recipient rats. The deficit in counts was greatest in circulating leukocytes. The nude PVG spleen cells were able to lyse 51Cr-labeled A 0 BMC to a modest extant in vitro in a 4- to 6-hour incubation period. Serum from the nude rat did not enhance the lysis, suggesting that antibodies were not sensitizing the BMC for antibody-dependent cellular cytotoxicity. Another animal used to investigate the immunobiology of bone marrow transplantation is the dog. These large animals are usually irradiated at a much lower rate than mice or rats, i.e., 7-16 rather than 80-150 cGy/min, with total doses of 900-1200 cGy. Animals larger than rabbits are about twice as radiosensitive with respect to hemopoiesis as small animals (Leong et al., 1964). Transplants between dog leukocyte antigen (DLA) identical littermates at inocula of 4 x lo8 marrow cellslkg body weight are usually successful (Storb et al., 1973, 1979; Rapaport et al., 1972, 1973), while DLA incompatible grafts fail (Epstein et a l . , 1968; Storb et al., 1979; Deeg et al., 1979). Antigens specified by serological (DLA-A, B, and C) or mixed lymphocyte response tests (DLA-D) could not always predict rejection, suggesting that additional determinants were important (Vriesendorp et al., 1976; Storb et al , 1979). The diagnosis of rejection was usually failure of white blood cell (WBC) counts to rise or to fall after a brief rise and marrow aplasia at autopsy. In one study, donor-host pairs were chosen such that the donor and hosts were DLA identical or the donor had no DLA antigens not shared by the host. In 16 transplants, 8 were rejected. Mutual cell-mediated lympholysis responses were negative in that study. There were three instances of rejection and two cases of "takes" when the donor was homozygous and the host was heterozygous for one or more DLA antigens (Deeg et al., 1982). Since DLA identical littermate transplants are much more successful than DLA identical unrelated animals, determinants not detected and associated
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or not with DLA are probably involved. Vriesendorp et al. (1976, 1981a,b) used agents known to weaken marrow allograft rejection in mice and were only partially successful in dogs. Silica particles worked to some extent, but preirradiation of dogs with 6 Gy 3 days before a second dose of 6 Gy was successful. Storb et al. (1978b) also had partial success with silica. In 1979, Deeg et al. performed an experiment which succeeded in abrogating resistance to DLA-incompatible unrelated marrow grafts. Large numbers (7.7 X lo8) of thoracic duct lymphocytes of donor origin infused with the marrow cells prevented rejection; however, all 8 dogs died rather rapidly of rampant graft-versus-host disease. Irradiated donor lymphocytes had no effect on graft takes. The studies of canine marrow grafts were usually performed by investigators involved in clinical bone marrow transplantation. A major problem in clinical cases is the incidence of rejection in HLA identical matches. Since many patients have had blood transfusions, much effort was expended in canine studies to determine if previous blood transfusions sensitized dogs to DLA identical grafts (they did) and if one could prevent marrow rejection under these conditions. One injection of blood could sensitize for rejection, and methotrexate, procarbazine, and antithymocyte serum alone did not prevent rejection, but the combined use of procarbazine and antithymocyte serum was successful (Storb et al., 1973, 1974). Storb and Deeg (1986) have recently reviewed this subject very well, and provided evidence that non-T cells can reject BMC in dogs, since cyclosporin failed to prevent rejection. Recently, Deeg et al. (1986) observed that exposure of dog leukocytes to 1.35 J/cm2 of ultraviolet light prevented the cells from sensitizing against subsequent marrow grafts and from stimulating in a mixed lymphocyte reaction or serving as accessory cells in a mitogen response, even though cell viability was not affected. Whereas the cellular mechanisms of rejection of marrow grafts are probably very similar in dogs as in rats or mice, the immunogenetics of dog marrow rejection will be difficult to resolve. Dogs are expensive and are slow to breed compared to rodents. There is a similarity in radiosensitivity to humans, which can recommend dogs as models for human bone marrow transplantation. Studies of human bone marrow transplantation have been extensive, and I will not attempt to cover that vast literature. I do recommend the following references: Pillow et al. (1966), Shank et al. (1981), Ramsay et al. (1980), Thomas et al. (1977), Rapaport et al. (1981), Sontos (1983), Hansen et al. (1981), Opelz et al. (1978), Braine et al. (1982), Storb et al. (1977, 1978a), Gale et al. (1982), Reisner et al. (1983).Briefly, bone marrow rejection does occur in patients even after total body irradiation with or without chemotherapy. No hemopoietic histocompatibility antigens have been demonstrated in humans to date. Storb et al. (1978a) could not correlate pretransplant antibodies in aplastic anemia patients with subsequent rejection
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of marrow grafts. Attempts to cure patients with severe combined immune deficiency, some of which have near-normal NK cell function (Buckley et al., 1983; Peter et al., 1983; Pierce et al., 1986), with bone marrow grafts have been met with rejection in some instances, particularly when the patients have detectable NK cell function (Peter et al., 1986). NK cell lines can be derived from patients with severe combined immunodeficiency disease (SCID) using interleukin 2 (IL-2) (Flomenberg et al., 1983). The ability to prevent graft-versus-host disease by selectively removing T cells from bone marrow cell suspensions (Reisner et al.,1983) leaves marrow graft rejection as a major impediment to successful bone marrow transplants. B. LYMPHOID CELLS If the preceding section on the immunogenetics of bone marrow transplantation appears complicated, imagine the nuances introduced when the “target” lymphoid cells have the ability to react against the host! The original observation of hybrid resistance to lymphoid cell is an excellent example (Boyse, 1959). Boyse hyperimmunized C57BL or A strain mice against sheep or human RBC. In some instances, the donors were immunized against the opposite parent, e . g . , A anti-C57BL. Spleen cells and spleen fragments were injected intraperitoneally into A, C57BL, or A x C57BL F, hybrid hosts. The mice were immunized with a small number of sheep or human RBC such that only already immune cells could mount an antibody response. Rising antibody titers were produced quite rapidly in syngeneic hosts, and titers were present only transiently in allogeneic hosts. A strain spleen cells continued to produce antibody in F, hosts, even if the donors were immunized against C57BL antigens, whereas C57BL spleen cells failed to generate high titers of antibody in F, mice, and preimmunization of C57BL against A antigens resulted in almost no antibody formation. The failure of antibody formation could have been caused by rejection of the C57BL antibody-forming cells by the F, mice or by a graft-versus-host reaction. A pathological study of the graft-versus-host reaction in F, hybrids injected with C57BL spleen cells led Gorer and Boyse (1959) to the conclusion that the donor cells were dying of an “allergic reaction.” This idea may have been the seed for the allogeneic inhibition hypothesis which soon followed. It is somewhat ironic that Cosgrove et al. (1959) interpreted one of their early experiments as “inhibition of foreign spleen reaction by inactivation of donor cells with recipient antigen. C57BL spleen cells preincubated with F, hybrid liver cells lost the ability to induce graft-versus-host reactions. In the seminal observation of hybrid resistance by Cudkowicz and Cosgrove (1960), marrow-derived cells of the F, hybrid protected irradiated F, mice against the graft-versus-host reaction mediated by liver nonparenchymal cells. Perhaps the “active” function of F, marrow cells stimu”
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lated the “hybrid resistance” interpretation. In 1962, Celada and Welshons “immunized’ F, hybrids with 3 x lo7 parental spleen cells 10 days before transferring 24 x lo6 spleen cells from donors immune to rat RBC. C57BL spleen cells failed to produce antibodies in irradiated (C57BL x C3H)F, hybrid mice, while C3H spleen cells were able to produce antibodies in similar F, mice. These results extended those of Boyse to irradiated host mice. Claman et al. (1969) tested the function of C57L versus A strain marrow, thymus, and spleen cells in irradiated F, hybrid mice. They observed hybrid resistance to C57L marrow cells, as judged in terms of spleen weight 8 days after cell transfer. C57L marrow + thymus cells generated more anti-sheep red blood cell (SRBC) plaque-forming cells (PFC) in C57L than in C57L x A F, hybrid mice, and F, marrow + thymus cells responded better in F, than in C57L hosts. In contrast, A versus F, marrow + thymus cells grafted to F, hosts or to A hosts functioned equally well. Thus, there was hybrid resistance to C57L cells and resistance to F, cells by C57L hosts. These results agree with those of Lengerova et al. (1973a) using marrow grafts. When donor spleen cells were used, in every combination more PFC were generated in syngeneic hosts than in F, or parental strain hosts. When C57L spleen cells were immunized in diffusion chambers implanted into the peritoneal cavities of C57L or F, hosts, no hybrid resistance was observed, i.e., PFC production was not inhibited. Again, the failure of function of parental spleen cells in F, hosts could be explained by a graft-versus-host reaction; the failure of F, to C57L grafts to function could have been due to a host-versus-graft reaction or could have been interpreted as an instance of allogeneic inhibition. The lack of inhibition of antibody synthesis when cells were sealed inside diffusion chambers indicated that donor : host cell contact was required for suppression or rejection to occur. Using the splenic IUdR incorporation method developed for marrow grafts, I observed that lymph node cells (LNC) proliferated extensively in spleens and lymph nodes of lethally irradiated allogeneic mice (Bennett, 1971). LNC from H-2Db homozygous donors failed to proliferate in H-2Db heterozygous recipients. Thus, hybrid resistance was detected in that the donor cells were prevented from proliferating. Allogeneic bone marrow cells and lymph node cells were grafted into irradiated allogeneic hosts which were good responders or poor responders to marrow cell grafts. A parallel was seen with the outcomes of LNC grafts, e.g., DBA/2 H-2d marrow and node cells were rejected by irradiated C57BL/6 H-2b hosts, but were accepted by 129 recipients. Elkins and Quant (1981) also observed hybrid resistance to parental alloreactive T cells. While the immunogenetics of marrow stem cell and lymph node graftversus-host cell grafts of Hh-lb is similar, we did discover an interesting difference when analyzing grafts of C3H LNC (Eastcott et al., 1981).We had developed the IUdR assay for measuring LNC proliferation grafting C3H
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LNC into C3H x BlO F, hosts, since H-2k was thought to have a “null” gene at Hh-1 when bone marrow cell grafts were analyzed (Cudkowicz and Bennett, 1971a). That was a fortuitous choice. C3H LNC grafted into H-2‘! NZB irradiated hosts failed to proliferate, while H-2d BALB/c hosts accepted the cells. In contrast, C3H marrow cells grew well in both NZB and BALB/c hosts. This result indicated that C3H LNC, but not marrow cells, expressed an Hh antigen recognized by NZB effector cells due to good responder genes. C57BL/6 x DBA/2 and C57BL/6 X NZB F, recipients also rejected C3H LNC, but not C3H bone marrow cells (BMC). Not surprisingly, C57BL/6 BMC and LNC were rejected by allogeneic NZB and C57BL/6 x DBAI2 F, hybrid hosts. What did surprise us was that C3H x C57BL/6 F, LNC, but not BMC, were also rejected by NZB and C57BL/6 x DBAI2 F, recipients. This result suggested that H-2k and H-2b shared a determinant for an Hh antigen expressed on LNC, but not on BMC. Since C3H x C57BL/6 F, mice reject CS7BL/6 BMC, that shared determinant would not be expected to be Hh-lb. An analysis involving a number of inbred-strain mice indicated that this Hh antigen was associated with the H-2k genetic region. Whereas the Hh-3 locus which specifies a BMC Hh antigen maps to H - 2 K (Cudkowicz and Warner, 1979), the LNC Hh determinant that we detected maps to N - 2 D . We should have performed the appropriate genetic crosses to prove that one genetic locus, i.e., H-2D, was the source of that Hh antigen. Whereas one can deliberately immunize A x B F, hybrid animals against A anti-B receptors expressed on parental strain lymphocytes (Bellgrau and Wilson, 1978, 1979), Polison and Shearer (1980; Shearer and Polison, 1980, 1981; Shearer et al., 1980) observed hybrid resistance by nonimmunized F, hybrid mice to H-2b parental lymphoid cells capable of inducing a graftversus-host immunodepressive condition in unirradiated mice. Shearer and Polison mapped the determinant on the donor cell to H-2D of the b haplotype. These studies led Shearer (1983) to the interesting hypothesis that week natural resistance of humans to allogeneic T lymphocytes capable of exerting graft-versus-host immunosuppression may contribute to the pathogenesis of acquired immune deficiency syndrome (AIDS), since many victims are frequently exposed to allogeneic lymphocytes. Ishikawa et al. (1982, 1984) and Kubota et al. (1983) performed various genetic studies to map determinants for antigens on lymphocytes capable of inducing graft-versus-host-induced immunosuppression which were recognized by F, hybrid mice. They detected determinants in the DBAI2 and DBA/1 strains not linked to H-2 which specified antigens recognized by F, host mice. DBA/2 X B10.D2 F, and B10.D2 spleen cells were not “resisted” by B10 x DBA/2 F, hosts, indicating a requirement for homozygosity for optimal immunogenicity. The assay for take of the spleen cells (inocula of 4 x lo7 cells) was the inability of host spleen cells to mount anti-
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H-2 allogeneic cell-mediated lympholysis assays 2 weeks after cell transfer. When (B10.D2 x DBA/2)F1 x DBA/2 individual backcross progeny mice were tested for the ability to induce immunosuppression in B10 x DBA/2 F, recipient mice, 41 of 48 failed to do so. This result was consistent with the interpretation that three genes were involved; i.e., homozygosity for any one of the three genes results in failure of the spleen cells to function. These data with spleen cells are similar to those using normal DBA/2 marrow cells or marrow or spleen cells following infection with Friend leukemia virus (Rossi et al., 1970, 1971, 1973; Cudkowicz et al., 1972) except that three and not one genetic regions were implicated. They also observed hybrid resistance to H-2k homozygous spleen cells from C3H, AKR, or B10.BR donors. The ability of BIO.A and the inability of B10.BR spleen cells to induce immunosuppression in B10.D2 x C3H F, hosts mapped the determinants to the S,D region of H-2. This confirms and extends previous studies of H-2k lymph node cell grafts assessed by splenic IUdR uptake in irradiated mice (Eastcott et al., 1981). C3H X C3H.SW F, mice resisted spleen cell grafts from H-2k donors better than B10.BR x B10 F, hosts (both H-2k/H-2b), suggesting that the non-H-2 background genes of C3H contributed good responder status, and vice versa for B10 background genes. They determined how many genes regulated the ability to resist H-2k spleen cell grafts. B10 x (B10.BR x AKR)F, and B10 x (B10.BR x C3H)F, backcross mice were challenged with B10.BR spleen cells and later tested for immunosuppression. There were 23/33 and 50/58 resistant mice, respectively, suggesting that 3 C3H and 2 AKR genes dominant for ability to resist engraftment were involved. These data resemble the genetic studies of Cudkowicz and colleagues testing the ability of mice to reject bone marrow grafts. Hybrid resistance to lymphochoriomeningitis virus (LCMV) immune T cells responsible for the inflammatory response induced in the meninges of infected mice was observed (Doherty and Allan, 1986). Donor cells from LCMV-infected mice were infused into recipient mice pretreated with large doses of cyclophosphamide and infected with LCMV (the suppressive effect of the drug was counterbalanced by the stimulatory effect of the virus on the NK cell function of the host). The numbers of inflammatory cells obtained from the cisterna magna were the measure of success of the grafts. Spleen cells from donor mice homozygous, but not from mice heterozygous, at H-2Db were relatively unable to mount an inflammatory response in F, hybrid mice heterozygous at H-2Db. This hybrid resistance could be overridden by inocula of 20 x lo6 cells. One can argue that inhibition offunction of lymphoid cells exposed to alloantigens is a consequence of a graft-versus-host reaction and not a host-versus-graft reaction. However, no inhibition was seen when allogeneic, but H-2Dbheterozygous spleen cells were infused. This finding is in agreement with previous studies of marrow grafts (Bennett, 1972).
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Athymic nude mice can reject allogeneic and xenogeneic lymphoid cells (Piguet and Vassal1 1978, 1980, 1981, 1983). A second graft 40 days later is rejected even faster! The mechanism of immunological memory in nude mice was the induction of antibodies by nude B cells using “help” from the transferred T cells. Failure of the first transplant could also be related to alloantibody production, since it occurred within 3-5 days after cell transfer. F, to parent nude host transfers did not result in antibody production or graft-versus-host reaction (GVHR). These observations are possible in unirradiated host mice. The resistance to GVHR induced by parental cells is very radiosensitive in that 5 X lo7 parental spleen cells are usually not fatal in unirradiated mice, while 105 spleen cells can kill irradiated F, host mice. The authors concluded that a peculiar form of “allogeneic suicide” is possible when unirradiated F, hosts are infused with parental strain T cells, and that mechanism is a donor T cell : host B cell collaboration to produce IgG antibodies cytotoxic for the donor T cells. A more direct method of detecting destruction of allogeneic or parental strain lymphocytes could be studies of the fate of radiolabeled lymphocytes infused into recipient animals. In a number of studies in mice and rats, allogeneic cells survived less well than syngeneic cells, and the differences in survival could be detected within 24 hours (Bainbridge and Gowland, 1966; Zatz et al., 1972; Viklicky and Mickova, 1979; Viklicky and Pavlik, 1981; Degos et al., 1979; Pincott and Bainbridge, 1980; Viklicky et al., 1976; Heslop and Hardy, 1971; Rolstad, 1979; McNeilage and Heslop, 1980; Heslop and McNeilage, 1983; Bainbridge, 1983). The major difference detected is the survival of cells in the lymph nodes of recipient mice or rats. Natural resistance to allogeneic cells in the rats was absent until after 3 weeks of age and became maximal at 7 weeks of age (Heslop and McNeilage, 1983). There is a genetically regulated degree of natural resistance to the transfer of allogeneic lymphocytes in rats, reminiscent of the responder status of mice to bone marrow allografts (Cudkowicz, 1971). A study of (AS x DA)F, x AS backcross progeny rats individually challenged with DA lymphocytes indicated that a minimum of 2 genes appeared to determine the ability to reject the cells (McNeilage et al., 1982). One gene was linked to RTI, as should be expected when using two strains differing at RTI in the study. McNeilage et al. were analyzing simultaneously the donor determinants recognized and the host responder status. Between the numbers 20 and 150 x 106 cells, a constant fraction of the cells was recovered in allogeneic versus syngeneic lymph nodes. This result suggests that a subpopulation of lymphocytes was the target for rejection. The target antigens appeared to have determinants near or in the RTl locus. Rejection did not require host T cells (Tonnessen and Rolstad, 1983). The spleen appeared to be critical for the rejection of allogeneic lympho-
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cytes in rats (McNeilage and Heslop, 1980), but not in mice (Viklicky and Pavlik, 1981). Not surprisingly, preimmunization of hosts against donor alloantigens greatly strengthens resistance, and alloantibodies have been implicated as the mechanism of rapid lysis of incoming lymphocytes (Heslop and Hardy, 1971). The injection of neonatal rats with allogeneic bone marrow cells abrogated the ability of these rats later to lyse allogeneic lymphocytes of the strain used to induce tolerance. However, injection of the tolerant rats with normal syngeneic lymphocytes could restore the resistance to allogeneic lymphocytes (Heslop and McNeilage, 1983). In the donor : host strain combinations which result in strong resistance, F, to parent and parent to F, combinations greatly reduced the resistance. This result would suggest that neither hybrid resistance nor allogeneic inhibition is the mechanism of resistance to lymphocyte transfer. In contrast to the rat data, F, donor lymphocytes were resisted by parental strain recipient mice (Bainbridge and Gowland, 1966; Pincott and Bainbridge, 1980). Irradiation of rats or mice greatly reduced the resistance to or rejection of allogeneic lymphocytes (Heslop and McNeilage, 1983; Zatz et al., 1972). Since irradiation of rats 1 or 7 days before cell transfer had similar effects, the role of circulating “natural antibodies” was discounted as a likely mechanism of natural resistance to lymphocyte transfer. With use of athymic nude PVG strain rats, strong “allogeneic lymphocyte cytotoxicity” (ALC) to Wr-labeled thoracic duct lymphocytes (TDL) was detected by measuring a deficit of counts in lymph node, lung, and WBC, and an increase in counts in the kidney and blood plasma (Rolstad and Ford, 1983). Of interest, there was a slight increase in counts in the spleen and much more in the liver and bone marrow. The higher than expected counts in those organs were due to reutilization of the released isotope by macrophages, according to a comparison of results with W r and [3H]uridine. Localization to the nodes was the greatest indication of survival of the transferred cells. Examination of autoradiographs of sections of tissues indicated that very few lymphocytes survived longer than 24 hours, since the label was detected in host macrophages and/or “interdigitating cells.” These investigators noted that nude rats, like nude mice, generated antibodies to donortype lymphocytes, sometimes as early as 3 days. Nude rats are even resistant to the graft-versus-host reaction detected in popliteal lymph nodes after injection of allogeneic lymphocytes into their footpads. These authors discount the role of the antibody made by nudes as the mechanism of ALC, since not all strain combinations resulted in detectable antibody formation and no antibodies were detected during the first 24 hours. These arguments do not rule out the possibility that the antibodies were generated rapidly within the lymph nodes themselves and participated in the lysis of allogeneic lymphocytes. They ruled out the need for donor T cells, since nude lympho-
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cytes were quite sensitive to ALC. Moreover, “filtered” (Ford and Atkins, 1971) DA strain cells infused into irradiated DA x PVG F, or syngeneic hosts and recovered 24 hours later to remove alloreactive cells (for PVG) were equally susceptible to ALC by nude PVG rats. F, hybrid to parent transfers also experienced ALC in nude rats, and removal of T cells with antibodies and C also failed to render the remaining lymphocyte population resistant to ALC. In some cases, F, cells are almost totally resistant to ALC, but usually there is detectable ALC to RTI heterozygous cells. With use of a strong donor : host combination and a relatively small inoculum size (lo7 cells), only 7% of F, cells remained in the blood of nude rats 24 hours later. These results can be compared to the consistent finding of Lengerova et al. (1973~)that small numbers of F, marrow cells were resisted by irradiated parental strain hosts. Rolstad and Ford (1983) favored the idea that “gene dosage” of RT1 antigens was the best explanation for the weaker ALC against F, lymphocytes. Rolstad (1979)observed that ALC was as strong 1week after exposure to 6 Gy irradiation as if the rats had not been irradiated. This result differs from that of McNeilage and Heslop (1983). An ingenious experiment designed by Rolstad and Ford (1983)demonstrated that PVG nude thoracic duct lymphocytes were capable of the adoptive transfer of ALC to PVG x DA F, hosts of DA, but not of PVG radiolabeled lymphocytes. To detect this ability, small numbers of test target cells (6 x 106 cells) and large numbers of nude lymphocytes (250 x lo6 cells) were employed. Thoracic duct lymphocytes (TDL) of nude rats are 93% Ig+ cells (Fossum et al., 1980). Unlike TDL of euthymic normal rats, TDL of nude rats have NK cell activity (Zoller et al., 1982b). Their results led to the speculations that ALC could be mediated by N K cells via antibody-dependent cellular cytotoxicity (ADCC), and that the preexisting antibody was elicited by infection with bacteria capable of eliciting antibodies that cross-react with MHC antigens (Chase and Rapaport, 1965). Nude rats have at least normal levels of IgG isotypes other than IgG,, (Bazin et al , 1980). However, Rolshd et al. (1985)recently treated nude rats from birth with anti-p chain antibody and induced a state almost free of immunoglobulins and B cells. ALC was stronger in nude rats treated with anti-p chain serum than in those treated with normal rabbit serum. Moreover, sera of nude rats were unable to sensitize allogeneic TDL for ADCC and failed to confer ALC to neonatal rats, whereas alloantiserum was capable of both activities. In contrast, anti-asialo GM1 serum did abolish ALC, supporting the idea that N K cells are necessary for that response (Ford et al., 1984). Morphological evidence was presented to support the idea that N K cells lysed the allogeneic lymphocytes and that Ia antigen-negative interdigitating cells phagocytosed the dead lymphocytes (Fossum and Rolstad, 1986a). Veiy recently, Fossum and Rolstad (1986b) observed that enriched
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populations of rat NK cells, especially from anti+ chain-treated nude rats, could lyse allogeneic TDL in uitro. F, TDL were also lysed, but to a much lesser extent. 111. Hybrid Resistance to Leukemia/Lymphoma Cells
The observation that F, hybrid mice resist the growth of parental strain tumor cells has been a source of fascination to a number of immunologists and oncologists for many years. The first observation published may have been the significantly greater resistance of C3H F, hybrid mice than of parental strain mice to the growth of transplantable C3H mammary tumors following irradiation of the tumor in situ (Cohen and Cohen, 1950). Hauschka et al. (1956) inoculated small numbers of a C3H/St 6C3HED lymphosarcoma cells into syngeneic and C3H/St X Swiss F, hybrid mice and noted that the F, mice were refractory to the tumor cells. They speculated that the F, mice reacted immunologically against the parental strain cells. As mentioned earlier, Snell (1958) analyzed a similar “exception” to the genetic laws of transplantation that he helped to establish (Snell, 1953). Even before the first reported observations of hybrid resistance to tumors, Barrett and Deringer (1950) noted that passage of C3H mammary tumors in DBAI2 or BALB/c X C3H F, hybrid mice for only one transplant generation resulted in selection for tumors relatively resistant to rejection by F, or by F, x C3H strain backcross mice. The “Barrett-Deringer phenomenon” may have diverse mechanisms, but the data suggested that some type of resistance system was encountered by the tumor in the F, hybrid mice. A series of experiments of the “opposite kind’ to those of Barrett and Deringer eventually led the Hellstroms to the discovery of the phenomenon of allogeneic inhibition. If one transplants H-2 heterozygous F, hybrid tumors into parental strain mice, one can select for cells expressing only those H-2 antigens of the parental strain, presumably due to immunoselection (Klein et al., 1960; Hellstrom, 1960; Dhaliwal, 1964; Ozer et al., 1965). Such F, tumors now grow progressively in the parental strain used for selection and are “parental” with respect to expression of H-2 antigens. The changes are usually permanent. In many of their studies, A x A.SW F, lymphomas, LNSF, which had been selected in A or A.SW mice, were used as “parental” tumors subjected to allogeneic inhibition in A x A.SW F, hosts (Hellstrom, 1961). Hellstrom (1963, 1964) detected the differential growth of lymphomas, sarcomas, and carcinomas in syngeneic versus F, hybrid recipients, and that phenomenon was termed syngeneic preference (Hellstrom and Moller, 1965). Using a method developed by Haughton (1964) to prepare extracts of cells enriched in H-2 antigens, Hellstrom et al. (1964) exposed fibrosarcomas of A x A. CA or C57BL origin to spleen cell extracts from A or
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C57BL donor mice. The extracts were added to cells growing in culture for 2 days, and recovery of viable cells on day 4 was the measure of survival of the tumor cells. C57BL extracts were inhibitory to A x A.CA tumor cells, while A extracts were inhibitory to C57BL sarcoma cells. Even extracts of tumor cells were inhibitory to the growth of H-2 disparate tumor cells. These observations were the basis for the hypothesis that exposure to alloantigens can be toxic or inhibitory to the growth of tumor cells. Moller (1965)made a similar observation using lymph node cells of A.CA, A.CA x C57BL F,, or C57BL mice which were aggregated with C57BL MC57S sarcoma cells by phytohemagglutinin (PHA). Both A.CA and the F, cells inhibited the growth and/or survival of the MC57S cells. Holm et al. (1964) had recently observed that PHA enabled nonimmune cells to lyse allogeneic, but not syngeneic cells in uitro. The Mollers (1965a,b) demonstrated that mouse embryo fibroblasts could be lysed, with the aid of PHA, by allogeneic, parental strain or F, hybrid lymph node cells. Since mouse RBC contain H-2 antigens, they tested for the ability of RBC to lyse embryo cells. The RBC did not exert a cytotoxic effect. Exposure of F, mice to 4.5 Gy or F, hybrid cells to 10 or 100 Gy of irradiation failed to abolish allogeneic inhibition in uiuo or in uitro (Hellstrom and I~ellstrom,1965; Hellstrom, 1967). That was not surprising, since sonicated, freeze-thawed, or mechanically homogenized cells were effective. They performed “Winn assays” of neutralization (Winn, 1959). Syngeneic, allogeneic, or F, hybrid lymphoid cells and tumor cells at 10 to lo5: 1 F, cell : tumor cell ratios were incubated in uitro in the presence of PHA for only 45 minutes. The cells were seen to agglutinate together even in the syngeneic combination. Inocula of cells were injected subcutaneously into hosts syngeneic with the tumor and exposed to 3 or 4 Gy of irradiation. Both allogeneic and F, hybrid cells “neutralized” the subsequent growth of tumor cells (Bergheden and Hellstrom, 1966; Hellstrom et al., 1965). Cell-free homogenates were also effective in the Winn assay type of experiment, and PHA was not necessary for the phenomenon to be detected. Antibodies specific for the H-2 antigens of the extracts or cells interfered with allogeneic inhibition (Hellstrom and Hellstrom, 1965). Moller (1967), using similar tumors and tissue culture methods, demonstrated that the destruction of allogeneic, parental strain or F, hybrid tumors by nonimmune lymphocytes in the presence of PHA could be inhibited by antibodies directed against the effector cells. Subsequent growth of incubated cells in a colony-forming assay in uitro gave similar results (Hellstrom, 1967). Treatment of F, mice with cortisone 2 days before injection of tumor cells abrogated allogeneic inhibition in uivo, and the addition of cortisone to the mixture of antigeneic preparations with tumor cells in vitro prevented allogeneic inhibition (Hellstrom et al., 1965). Cortisone did not prevent lym-
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phocytes from aggregating around tumor cells in vitro. They justifiably concluded that cortisone was acting in a nonimmunological manner. One could imagine that allogeneic inhibition involved the secretion of arachidonic acid metabolites, e. g., prostaglandins or leukotrienes, and that cortisone acted by inhibiting that biochemical pathway. To return to the Barrett-Deringer phenomenon, Hellstrom (1966a,b) sought to develop tumor lines with decreased sensitivity to allogeneic inhibition. The passage of A strain YAA lymphoma cells or strain A compatible variant LNSFO of A x A.CA F, lymphoma LNSF (expressing only H-2” antigens) through A, A x A.SW, or A X A.CA mice serially for up to 5 generations, resulted in a decrease, but not in the loss of allogeneic inhibition. There was some specificity in that cells passed through A x A.CA F, mice grew better in A X A.CA F, than in A x A.SW F, mice. Cudkowicz (1965) observed hybrid resistance to two B10 H-2b lymphomas, S1033 and S1043. A genetic analysis indicated that homozygosity and heterozygosity at H-2D of recipient mice were associated with susceptibility and resistance, respectively. F, mice less than 3 weeks of age were not resistant to these lymphomas, a finding similar to the age of maturation of hybrid resistance to marrow grafts. Other investigators also presented evidence to support the notion that a host-versus-graft reaction was involved in the failure of parental cells to grow well in F, hybrid recipients (Glynn et al., 1964; 0 t h and Burg, 1967a,b; Sandford, 1967). There likely are three reasons for F, hybrids to resist the growth of parental strain tumors, according to Burg and 0 t h (1967). These are hybrid resistance, allogeneic inhibition, and hybrid hyperreactivity. Hybrid hyperreactivity meant a more vigorous response by F, hybrids than by parental strain mice to tumor-specific antigens ( 0 t h and Burg, 1967a,b). Heumer (1965) observed that the C57BL lymphoma 21B, after passages in F, hybrid mice, grew as well in that type of F, as in C57BL mice; this was a “slow” type of Barrett-Deringer phenomenon. However, such an adapted tumor passed through B6 x DBA/2 F, mice was still resisted by B6 x A F, hosts (Heumer, 1969). By changing F,s in subsequent passages, the ability to grow in the B6 x DBAI2 F, host was lost. This result argued against the idea that allogeneic inhibition was the mechanism of poor growth in F, hybrid mice in the first place. Similarly, if loss of Hh-lb was the mechanism of adaption to hybrid resistance, the 21B lymphoma cells adapted to B6 x DBA/2 F, hosts should also have been adapted to B6 x A F, hosts. Therefore, adaptation to hybrid hyperreactivity was considered the mechanism in this instance. On balance, Hewitt et al. (1976) analyzed 27 spontaneous tumors of mice and concluded that immune responses were not important; i.e., the tumors were not immunogeneic. Allogeneic inhibition, as judged in terms of proliferation of lo4 A strain
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lymphoma ([YAC)cells injected intraperitoneally into A X CBA F, or A X DBAI2 F, host mice, was not affected by prior thymectomy followed by exposure to 6 Gy irradiation and infusion of syngeneic marrow cells (Moller et al., 1969 Lindholm et al., 1970). Moreover, the induction of tolerance to A x CBA F , alloantigens by the neonatal injection of F, spleen cells did not prevent lymphocytes from such mice from lysing A X CBA or CBA mouse embryo fibroblasts in the presence of PHA. These data strengthened the concept that allogeneic inhibition was not an immunological event, but rather was due to exposure to alloantigens. Note, however, that YAC cells were the tumor cells tested and the NK cells are not thymus-dependent cells. Thus, in light of knowledge concerning NK cells, these investigators may have made the first observation of NK(YAC) cell activity in vivo! The discoveries that immune responses were strongly regulated by genes at the MHC (McDevitt and Benacerraf, 1969; Ellman et al., 1970; Benacerraf and Dorf, 1974; Klein et al., 1974) and that resistance to viral leukemogenesis was determined by genes at H-2 (Lilly, 1966; Lilly and Pincus, 1973) greatly changed the approach to analyzing the immunogenetics of tumor transplantation. The finding that parental tumors were resisted by F, hybrids had to take into account the existence of “immune response” genes. Indeed, in an analysis of H-2-linked genetic control of resistance to transplants of parental strain fibrosarcoma or mast cell tumors, the precise control of resistance could not be mapped to the I or the D or K regions (Williams et al., 1975), although the D region appeared to contribute strongly to the hybrid resistance to a B10 fibrosarcoma. Harmon et al. (1977) utilized B10 congenic resistant mice and their F, hybrids to map the resistance gene to transplants of C57BL EL-4 lymphoma cells. Heterozygosity at H-2D was required for hybrid resistance. They tested for the ability of spleen cells from nonimmunized mice of the same strains and F, hybrids to lyse 51Cr-labeled EL-4 cells over a period of4-6 hours. They observed that donors resistant to EL-4 cells in vivo had spleen cells more cytotoxic for EL-4 cells than spleen cells from susceptible mice. These investigators extended their observations to 3 B10.A(4R) fibrosarcomas and to more intra-H-2 recombinant inbred strains and F, hybrid mice (Clark et al., 1977). Hybrid resistance, as measured by mean survival times, was observed if EL-4 lymphoma cells or 4RMF1 or 4RMF2 fibrosarcoma cells were injected intraperitoneally, but not if they were injected subcutaneously. Hybrid resistance was observed in F, host mice, e.g., 5R x 4R or BIO.A x B10, which are H-2Db heterozygotes, but 2R x 4R or 2R X B10 H-2Db homozygous F, mice were susceptible. Identical results were obtained with EL-4 and the two 4R fibrosarcoma cell lines. Therefore, unless one considers fibroblasts as members of the hemopoietic system, the data indicate that Hh antigens are not limited to hemopoietic tissue. One exception to the general pattern was observed;
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B10. S(7R) x 2R or 4R did not exhibit hybrid resistance. 7R is s from K to S and d at D , 2R is k from K to IA, d from ZE to S and b at D , and 4 R is k from K to IA and b from IE to D . Therefore, 7R x 2R or 4R are dlb at D , and yet accept the parental tumor cells. 7R x 2R are sld at S and 7R x 4R are slb at S. If one recalls the data with bone marrow cell grafts, the Hh-1 determinants of H-2b and H-2" appear to be identical (Cudkowicz, 1978; Warner, 1978). Since 7R X 2R are sld heterozygous at S , it would appear from these data that Hh-1 maps between S and D . Thus, Clark et al. (1977) suggested that the 7R recombinant, H-2t2, resulted from a crossing over between H-2D and Hh-1. In a later study, however, Clark and Harmon (1980) observed that male B10 X 7R F, mice and male and female B10 X B1O.S F, mice were resistant to EL-4 cells, as defined by mean survival times. B1O.S (21R) is a recombinant between 7R and B10 and is b from K to S and d at D. B10 x 21R F, mice rejected EL-4 cells, suggesting that a resistance gene was inherited from the 7R, perhaps Hh-ld or Hh-1". An alternative explanation offered was that a second locus at H-2 affected whether heterozygosity at H-2DIHh-1 resulted in hybrid resistance. There is no simple explanation why B10 x B10. S F, mice reject EL-4 cells, since 7R is derived from B10. S and 21R is derived from 7R. The Hh-1 antigens expressed on marrow stem cells and on malignant lymphoid cells (EL-4) may be slightly different. In a similar study, Harmon et al. (1979) compared the growth of 100 EL-4 cells injected into 23 different F, hybrid mice, all B10 congenic resistant. All 8 H-2Db homozygous F, hybrids with B10, i.e., BlO.HTG, BlO.Rl01, B10.Rl02, BlO.BDR1, BlO.A(2R), BlO.A(15R), BlO.A(lR), and BlO.AM, were susceptible. Fourteen H-2Db heterozygous F, hybrids were resistant; these included mice which were d, du,J k, q, and s at D . The 7R F, was the fifteenth studied; females are susceptible and males are resistant. Genes in mice regulate the ability of their spleen cells to lyse to YAC-1 A strain lymphoma (Kiessling et al., 1975c; Petranyi et al., 1975), and there is a correlation between the ability of spleen cells to lyse YAC-1 cells and the in vivo resistance to growth of YAC-1 tumor cells (Petranyi et al., 1976; Haller et al. 1977a). There was a similar correlation between the ability of spleen cells to lyse Rauscher virus-induced RBL-5 lymphoma cells in vitro and the resistance to the same cells in vivo. The resistance gene is linked to H-2. C57BL mice are susceptible, C57BL x DBAl2 F, mice are resistant, H-2blH-2b C57BL x F, mice were susceptible and had low lytic activity, and H-2blH-2d backcross progeny mice were resistant in vivo and ,their spleen cells lysed R B L J cells well. With use of the same type of backcross mice, a similar pattern of spleen cell cytotoxicity for YAC-1 and human MOLT-4 tumor cells was observed. They discounted the probability that Hh-lb antigens were expressed on the H-2a YAC-1 cells and on the human
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MOLT-4 cells. Klein et al. (1978) compared the immunogenetics of resistance to outgrowth of inoculated leukemias, lymphomas, sarcomas, and carcinomas. H-2 heterozygosity was the most important genetic influence regulating hybrid resistance to the leukemias and lymphomas. However, resistance to the A strain carcinoma, S3A, and the C57BL fibrosarcoma, MC57X, was not associated with H - 2 heterozygosity in the backcross progeny mice. The one exception was hybrid resistance by A x A.CA F, mice to S3A cells. To pursue the idea that NK cell activity toward a tumor regulated the host’s resistance to that tumor, AKR x CBA F, mice were thymectomized or not, irradiated, and reconstituted with marrow cells from AKR (low NK cell activity) or CBA (high N K cell activity). The CBA radiation marrow chimeras had greater splenic NK(YAC-1) activity than AKR chimeras (Riesenfeld et al., 1980). Two different AKR T lymphomas were compared for growth in uiuo in these chimeras; 1-522 and 1-51 are sensitive and insensitive target cells for NK effectors in uitro. The CBA marrow chimeras were much more resistant than the AKR marrow chimeras to the 1-522 cells and somewhat more resistant to the 1-51 cells. The thymectomized mice were somewhat more resistant than the intact chimeras. In a similar study (Klein et al., 1982), thymectomized or sham-operated, irradiated C57BL or F, hybrid mice were reconstituted with syngeneic fetal liver cells. When challenged with a variety of C57BL lymphoma cells, the T cell-deprived mice were not significantly more resistant than the control chimeras, although hybrid resistance was detected. However, a thymic effect was noted when C57BL/6 mice were irradiated, following similar surgeries, and reconstituted with anti-Thy-1.2 + C-treated congenic bg/+ or bglbg (beige) marrow cells. The thymectomized chimera given bg/ + cells was significantly more resistant than the sham control, while both types of hosts were very susceptible if given beige marrow cells. The fact that removal of thymic influences enhances resistance of syngeneic C57BL/6 mice as much as that of resistance of F, hybrids emphasizes that resistance to tumor cells may be more complex than resistance to normal hemopoietic cells. If recognition of H h - l b antigens by NK cells was the only important mechanism of resistance to EL-4 cells in thymectomized mice, why did C57BL/6 mice become so resistant? The difficulty in using tumor cells to assess the immunogenetics of hemopoietic hisf ocompatibility was brought to our attention when studying the lysis of EL-4 tumor cells in uitro by NK-like cells in an 18-hour incubation period (Kumar et al., 1979a; Luevano et a l . , 1981). In that system, hybrid resistance was detected, as originally described by Harmon et al. (1977), and we noted that cells taken from good responder mice (to H - 2 b l H h - l b marrow grafts) lysed EL-4 cells better than spleen cells from poor responder mice. However, using “cold target competition” assays and adsorption of spleen
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cells onto monolayers of tumor cells, we observed that EL-4 and YAC-1 cells shared a common target structure or antigen, i.e., unlabeled EL-4 and YAC-1 cells could inhibit the lysis of either Wr-labeled YAC-1 or EL-4 cells. These types of experiments could be interpreted to suggest that YAC-1 cells express Hh-lb antigens even though YAC-1 cells are H-2a; alternatively, EL-4 cells could express “NK target structures” in addition to H h - l b antigens. In an extensive analysis of hybrid resistance to a number of H-2b tumors, Klein and Klein (1981) observed that H - 2 heterozygosity for the haplotypes d, a, f, or k in F, or backcross mice was associated with resistance to implantation of virally or chemically induced lymphomas. However, C57BL x A. SW F,, b x s, mice were susceptible. This result would be expected if Hh-lb and Hh-ls antigens are identical (Cudkowicz, 1978; Warner, 1978). The differential resistance of C57BL x A.SW and B10 X BIO.S F, hybrids, both b x s, to grafts of EL-4 cells (Harmon et al., 1979; Klein and Klein, 1981) indicates that genes outside H - 2 must regulate hybrid resistance. Ahrlund-Richter et al. (1983, 1985) analyzed these background genes and transferred them to the A/Sn strain. A clear case of T cell-dependent hybrid resistance to tumor cells was described by Gronberg et al. (1982). YWA is Moloney murine leukemia virus-induced lymphoma of A.SW (H-2”) origin. There is strong hybrid resistance to YWA in H - 2 heterozygous F, hybrids, including b x s hosts. Thymectomized, irradiated mice reconstituted with fetal liver cells lose their genetic resistance to YWA cells. Moreover, F, hybrid and syngeneic mice can be actively immunized against YWA cells (Merino et al., 1984). A.SW x A.BY F, hybrid spleen cells, s x b, can be used to generate cytolytic T lymphocytes in cell-mediated lympholysis assays. Finally, YWA cells are not sensitive to NK cell-mediated lysis. In light of these data, the acceptance or rejection of H-2b tumor cells by b x s hosts could be explained by the absence or presence, respectively, of a T cell response to the tumor. It should be pointed out that Harmon et al. (1979) and Klein and Klein (1981) performed experiments in unirradiated mice, and therefore both NK and T cells could have functioned quite well. Klein and Klein (1984) went further to demonstrate that immunization of syngeneic and F, hybrid mice to 5 different lymphomas almost abolished the difference in resistance between syngeneic and F, hybrid hosts. Carlson and Wegmann (1977) performed a rapid in vivo study of hybrid resistance to tumor cells. Tumor cells growing as an ascites were labeled with IUdR and infused into syngeneic or F, hybrid mice. Whole-body counting over the next few days and counting individual organs at different time points allowed them to follow the survival of the infused cells. They observed hybrid resistance to DBA/2 L1210 leukemia cells in H - 2 hetero-
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zygous F, hybrid mice. Localization of tumor cells in the spleen was the best indication of hybrid resistance. Exposure of mice to 8 Gy of irradiation almost abolished the hybrid resistance, as measured by whole-body counting. Interestingly, with use of spleen retention as the measure of hybrid resistance, C3H x DBA/2 F, mice lost hybrid resistance after irradiation, but C57BIJ6 x DBAI2 F, mice were still resistant. With use of C3H congenic resistant mice, disparity at K and ZA or D only allowed unirradiated allogeneic hosts to reject L1210 cells, but irradiation abolished resistance. Genes of the C57BL/6 background appeared to confer radioresistance of the hybrid resistance. Carlson et al. (1980) extended the observation to other tumor cells and noted that athymic BALB/c nulnu mice were quite resistant to EL-4/G cells. Treatment of mice with silica particles or sgSr, which weakens hybrid resistance to bone marrow grafts, also weakened the ability of mice to eliminate IUdR-labeled tumor cells. With use of both C-dependent lysis and a radioimmunoassay with lZ5I-labeled goat anti-mouse Ig, natural antibodies were found which could bind to tumor cells. However, H - 2 disparities were not related to the presence of the antibodies, which were easier to detect in older mice. Carlson et al. incubated L1210 cells with as much as 1 ml of C3H or C3H x B10 F, serum prior to infusion into genetically susceptible C3H x DBAI2 F, hosts and did not observe any rapid elimination of the radiolabeled cells. Using antibodies to L121O cells generated in CBA mice, they observed that destruction of L1210 cells in vivo was radioresistant even in those strains whose “natural” or hybrid resistance was radiosensitive. Beige mice were shown to be defective in eliminating IUtlR-labeled tumor cells, with a delay of about 24 hours (Carlson et al., 1984a). While this delay might be due to defective NK cells, they also observed somewhat defective cytolytic T cell and humoral antibody responses to allogeneic tumor cells in beige mice. If preformed antibody was injected into beige mice, they eliminated tumor cells as well as b g / + controls. In a genetic study comparing NK cell activity against YAC-1 tumor cells and the ability to eliminate IUdR tumor cells (Carlson et al., 1984b), differences between donor and host at H - 2 was much more important than NK(YAC-1) activity. For example, 23 BXD (C57BL/6 x DBAI2) recombinant inbred-strain mice were challenged with H-2d tumor cells, and only mice of the 8 H-2h strains were resistant. NK cell function among the various BXD strains was not linked to H - 2 or to the ability to eliminate tumor cells. Therefore, even though N K cells are probably responsible for the elimination of IUdR-labeled tumor cells rapidly after infusion, one cannot predict the ability to eliminate such cells with the routine NK cell assay. The results suggest that separate receptors exist for the N K target structures and Hh-1 or other Hh antigens. To emphasize the role of NK cells, Carlson and Marshall (1985) recently observed that severe combined immunodeficient
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(SCID) C. B-17 scidlscid mice were able to eliminate H-2 allogeneic tumor cells and that the ability to reject the tumor cells was weakened by injection of rabbit anti-asioalo GM1 serum known to eliminate NK cells. SCID mice have no T or B cell functions and little or no circulating immunoglobulins (Bosma, 1983), yet NK cell function is normal (Dorshkind et al., 1984, 1985) and marrow progenitor cells for NK cells are intact (Hackett et al., 198613). Bonmassar and colleagues (Bonmassar and Cudkowicz, 1976; Bonmassar et al., 1971) studied hybrid resistance to lymphoma and leukemia cells in lethally irradiated mice and observed that H-2DIHh-1 -specified antigens were recognized during rejection. Extending those findings, Iorio et al. (1978) observed that in some tumor : host combinations recipients could reject H-2 identical grafts and that “responder status” to a given Hh-1 type of marrow graft did not always predict rejection of the tumor cell graft. If the host was a good responder to marrow grafts, tumor grafts were rejected, but sometimes poor responders, e.g., SJL mice, rejected lymphoma cells. H-2k lymphoma cells, 6C3HED, were particularly immunogenic, unlike normal marrow cells. C3H mice were treated with lethal doses of irradiation or a combination of two cytotoxic drugs and were challenged with allogeneic BALB/c or C57BL/6 marrow cells or with BALB/c LSTRA virus-induced lymphoma cells. IUdR uptake was measured 4 or 5 days later. The C3H host mice accepted the bone marrow allografts, indicating their poor responder status to H-2b and H-2d marrow cells. However, the mice were quite resistant to growth of LSTRA cells in the spleens, livers, and lungs (Campenile and Bonmassar, 1980).This indicates that tumor cells can be recognized by more than one system, even if NK cells are the major effector cells mediating rejection. Athymic nude mice rejected normal marrow allografts quite well (Cudkowicz, 1975a) and also rejected lymphoma cells well after lethal irradiation (Bonmassar et al., 1975; Campenile et al., 1977). Nude mice rejected H-2 allogeneic tumor cells, but not H-2 identical cells. Rejection by nudes and by controls was much better in the spleen than in the liver, with one exception. The same two drugs mentioned above, 5(3,3’-dimethyl-l-triazeno)imidazole-4-carboxamide(DTIC) and cyclophosphamide (CY), were used to treat nude and euthymic mice prior to challenge with incompatible marrow or lymphoma cell grafts (Bonmassar et al., 1980a,b). DTIC CY suppressed the T-dependent allograft responses of unirradiated mice to grafts of allogeneic lymphoma cells. Rejection of parental strain marrow and lymphoma cells by euthymic mice, and rejection of H-2 allogeneic lymphoma cells by nude mice was observed. NK cell function was suppressed markedly 24 hours after the last drug (CY) injection, but activity was moderately well preserved at 5 hours. The macrophage “poison” carrageenan abrogated rejection of lymphoma cells in the drug-treated mice. An ingenious method to determine if two different parental strain lympho-
+
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mas express a common Hh-1 antigen was developed (Iorio et al., 1981). Lethally irradiated C57BL/6 x DBA/2 F, mice were infused with 5 x 105 viable B10.129 (5M) L5MF-22 (H-2b) lymphoma cells. Three hours prior to the infusion of viable lymphoma cells, the mice were infused with 1-4 x 107 heavily irradiated (100 Gy) H-2b EL-4, LSMF-22, R B L J lymphoma or normal C57BLJ6 spleen cells, or H-2d lymphoma cells. Splenic IUdR uptake 4 days after cell transfer was measured. The H-2b lymphoma cells partially, but significantly, inhibited rejection of LSMF-22 cells. The H-2d lymphoma cells and the normal C57BL/6 spleen cells did not affect rejection. The results support the idea that the various lymphoma cells shared common antigens, probably Hh-lb antigens. Although hybrid resistance by C57BL/6 x DBA/2 F, mice to normal DBA/2 marrow or spleen cells is weak, DBA/2 P388 lymphoma cells are strongly rejected. Irradiated DBA/2 P388 and L1210 cells and, to a lesser extent, BALBIc LSTRA H-2d lymphoma cells inhibited rejection, whereas H-2b LSMF-22, H-2" BIO.A LAF-17, and normal H-2d DBAI2 or BALB/c x DBAI2 F, spleen cells did not. Even though the LAF-17 cells expressed H - 2 D d , they failed to inhibit rejection. This poses a problem in interpretation; which, if any, Hh antigen was recognized in the first place? It could have been the one minor DBA/Hh antigen detected by 13ossi et al. (1970) on marrow stem cells or one of the three minor Hh antigens detected by Kubota et al. (1983) on lymphocytes responsible for graft-versus-host reactions. Alternatively, the relevant antigen could have been Hh-Id, but LAF-17 may not express Hh-ld. A variety of other instances of hybrid resistance to tumor cells have been described. AKR x DBA/2 F, are much more resistant than AKR mice to transplants; of spontaneous lymphoma cells derived from the thymuses or spleens of AKR mice (Schmitt-Verhulst and Zatz, 1977). This was correlated with a successful response by AKR x DBA/2 F,, but not AKR spleen cells to generate cytolytic T lymphocytes against AKR lymphoma cells in vitro. Hybrid resistance to the guinea pig L,C leukemia was observed in irradiated strain 2 x 13 F, animals (Bhan et al., 1979). There is no polymorphism for class I antigens in guinea pigs, and there was no allogeneic or hybrid resistance to strain 2 or 13 normal bone marrow cell grafts. Both Ia+ and IaL,C leukemia cells were rejected. Therefore, the nature of the antigen recognized by the irradiated guinea pigs was not determined. Since 13-dayold pigs failed to reject the L,C cells, it was suggested that NK cells may have been responsible for the hybrid and allogeneic resistance observed. Hybrid resistance to the BALBIc plasmacytoma, MPC-11, has been investigated in great detail (Walker and Phillips-Quagliata, 1979a,b). A genetic study indicated that F, hybrids between BALB/c and C57BL/10 and all of the B10 congenic resistant strains, C57BL/6, C57L, C57BL/Ks, AKR, and DBA/1, were resistant to MPC-11 cells, while F, hybrids between BALB/c
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and A or its congenic resistant strains, SJL, DBA/2, or BALB/c, were susceptible. The major gene for resistance was not linked to H-2, although expression of the GI, antigen by F, hybrid mice was associated with a relative lack of hybrid resistance (Walker et al., 1985).This hybrid resistance was radiosensitive and was not abrogated by silica particles. When resistant BALB/c x B10 F, mice were compared with susceptible BALB/c x BALB.B F, mice, both types of mice had similar NK cell function toward YAC-1 lymphoma cells and no activity against MPC-11 cells. The ability of their spleen cells to generate cytolytic T lymphocytes (CTL) against MPC-11 cells in uitro before and after tumor inoculation was also similar until 28 days after cell transfer when the susceptible BALB/c x BALB. B mice developed adherent, cyclophosphamide-sensitive suppressor cells (Phillips-Quagliata et al., 1985; Marsili et al., 1986). A very similar genetic control of hybrid resistance to another BALB/c plasmacytoma, LPC-11, was observed (Walker and Phillips-Quagliata, 1985). With these plasmacytomas, delivery of effector T cells to the tumor may be the basis of genetic resistance. Antibody responses to the tumor cells did not occur, and no role of antibody-dependent cellular cytotoxicity could be found. This is a type of hybrid resistance where immune responses to tumor-specific antigens (hybrid hyperreactivity) is the basis of hybrid resistance. In a recent series of experiments, Klas Karre and colleagues presented evidence suggesting that lack of class I antigens on tumor cells can increase their sensitivity to NK cell-mediated lysis in vitro and protection in uiuo (Ljunggren and Karre, 1985; Pointek et al., 1985; Karre et al., 1986a,b). EL-4, RBL-5, and YAC-1 tumor cells were mutagenized and selected for H-2 loss variants. The variants so selected were permanently changed. The variant cell lines were exquisitely sensitive to NK cell-mediated lysis in uitro, and this sensitivity was not altered after in uiuo passage. The variant cell lines were strongly resisted by syngeneic and F, hybrid recipients, including athymic nude mice. Interferon treatment of normal YAC-1 cells render them less sensitive to NK cell-mediated lysis, but the variant cells were not affected by interferon. Survial of IUdR-labeled H-2 variant cells at 24 hours after infusion was much less than control tumor cells. These investigators concluded that the NK cell system functions in immune surveillance against tumor cells by eliminating cells which fail to express self class I antigens, especially H-2D antigens. Since gain of H-2K class I antigens has been associated with an increase in immunogenicity of tumors and loss of metastatic properties (Eisenbach et al., 1983; Gooding, 1982; Hui et al., 1984; Wallich et aZ., 1985), it may well be that T cell responses to H-2Kassociated tumor-specific transplantation antigens make up one arm of the surveillance system, while NK responses to the lack of H-2D antigens make up another arm of the system. The highly oncogenic adenovirus decreases
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H-2K expression on cells to perhaps increase their tumorigenicity (Schrier et al., 1983). The earlier studies of Lengerova et al. (1973a) with normal bone marrow cells also indicated that allogeneic inhibition based upon H-2 differences was stronger at H-2D using nonimmune lymphoid cells and at H-2K using immune lymphoid cells. How would an NK cell recognize a lack of H-2 class I antigen? Does the NK cell have a receptor for a cell surface target structure somehow “masked” or “covered” by class I antigens? Alternatively, do the tumor cells themselves bind to the class I antigens on NK cells which they lack, for some reason, to initiate the response? Previously, Stern et al. (1980) had shown that embryonal carcinoma cells lacking H-2 antigens were lysed by NK cells. The prototypic human NK target cell, K562, does not express HLA antigens. Induction of differentiation of K362 or other N K target cells by various agents, e.g., 12-0tetradecanoyl phorbol acetate, into more mature forms expressing MHC antigens was associated with the loss of sensitivity of lysis by N K cells (Gidlund at al., 1981). Moreover, fetal human thymus and bone marrow cells were more sensitive to lysis by N K cells than adult cells (Hansson et al., 1981). This subject has been thoughtfully reviewed by Kiessling and Wigzell (1981). With these thoughts in mind, one might expect to observe hybrid resistance to certain embryonal tumors which fail to express H-2 antigens. A limitation of such a study is the finding that transplantation of teratocarcinomas into genetically resistant mice results in an expression of H-2 antigens on their surface (Artz and Jacob, 1974; Stern et al., 1975; Nicolas et al., 1976). IV. Effector Mechanisms of Hybrid Resistance
When Gustavo Cudkowicz first entertained the idea that hybrid resistance was an immunological host response to parental strain hemopoietic cells, what were the effector mechanisms to consider in 1961? Obviously, he considered that “antibody forming cells of the F l hybrid mice may have reacted immunologically against parental marrow cell antigens” (Cudkowicz and Cosgrove, 1961). His and others’ (Goodman, 1965) attempts to detect F, anti-parental antibodies to antigens not shared by the F, were not successful. While in preliminary experiments, parental strain skin grafts and marrow cell injections appeared to enhance hybrid resistance (Cudkowicz, 1961; Cudkowicz and Stimpfling, 1964a), this has not been a consistent finding. Therefore he realized that he not only had discovered a peculiar immunogenetic system, but was also dealing with unusual effector cells. It was therefore important to determine if the effector cells were part of the hemopoietic system. Therefore, mice were exposed to sublethal doses of irradiation to deplete the precursors of most hemopoietic elements. Later at
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different intervals of time, the mice were irradiated and challenged with parental or allogeneic marrow grafts. This procedure weakened the ability to reject incompatible marrow grafts (Cudkowicz and Bennett, 1971a,b). We took advantage of the fact that non-H-2 genes determined the ability to reject marrow grafts (Cudkowicz, 1971) and sublethally irradiated good responder C57BL/10 or C57BL/6 and poor responder 129 mice (to H-2d marrow) and infused marrow cells from syngeneic or H-2 identical allogeneic donors. These experiments indicated that marrow could transfer responder status and confirmed that the effector cells were part of the hemopoietic system. Cudkowicz considered the possibility that the effector were part of the natural immune system that defends against infectious agents prior to the acquisition of specific humoral or cell-mediated immunity. Therefore, to determine if rejection occurs quickly after cell transfer, resistant allogeneic or F, hybrid mice were irradiated and infused with incompatible C57BL/lO marrow cells. At different intervals, later, the spleen cells of these “primary hosts” were removed and infused into irradiated C57BL/ 10 “secondary recipient” mice in a two-step assay described earlier. Splenic IUdR uptake 5 days after spleen cell transfer measures stem cell activity. The data indicated that rejection began no sooner than 12 hours and was completed between 24 and 36 hours (Cudkowicz and Bennett, 1971a,b). That procedure made the exact timing difficult, since the frequency of stem cells in the spleens of syngeneic recipients decreases about 2-fold between 8 and 24 hours. The data did indicate that rejection was relatively fast and much more rapid than tissue graft rejection or peak antibody response. Santos and Owens (1968, 1969) introduced the drug cyclophosphamide as an effective agent to inhibit marrow allograft reactivity. This drug affects so many hemopoietic cell types that it may not be useful in identifying the effector cells for hybrid resistance. The ability to maintain mice in excellent health in specific pathogen-free conditions allowed Lotzova et al. (1976) and Rauchwerger et al. (1977) to determine the life span of the effectors. By delaying the infusion of parental strain or xenogeneic rat BMC by 5 days after irradiation, they observed that resistance to incompatible cells was lost. Thus, effectors are relatively shortlived.
A. MACROPHAGES Phagocytic cells, granulocytes, and macrophages are part of the natural immune system which can interact rapidly without any need for immunological “priming.” Therefore, macrophages were seriously considered. We obtained from Dr. G. Biozzi heat-killed C.parvum organisms, which are the most powerful of macrophage stimulants in the mouse (Halpern et al., 1963;
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Neveu et al., 1964). The height of the response of macrophages to this agent is between 7 and 14 days after injection. We assessed the ability of mice to reject incompatible marrow grafts. To our surprise, marrow allograft reactivity was weakened (Cudkowicz and Bennett, 1971a,b). This result suggested that activated macrophages may have inhibited the cells reponsible for rejection or may have created an environment so conducive to stem cell function that the effector cells were “overridden” by an effective high cell inoculum. Recently, mouse macrophages have been shown to be a good source of factors capable of stimulating hemopoietic progenitor cells (Rich et al., 1981; Rich, 1986). Nevertheless, the importance of macrophages as effector cells was demonstrated in a very convincing manner by Lotzova and Cudkowicz (1974). Silica particles cytotoxic for macrophages (Allison et al., 1966)were obtained from Dr. K. Robock and were quite effective in inhibiting rejection of marrow allografts. Not all preparations of silica particles are effective, and many workers in the field are indebted to Dr. Robock. Moreover, the macrophage “stabilizer,” poly-2-vinylpyridine-N-oxide (PVNO), which reverses several effects of silica on macrophages, also reversed the suppressive effects of silica on marrow allograft reactivity. Various carrageenans, which are sulfated ~-galactansextracted from seaweed, also inhibited the ability of mice to reject incompatbile marrow cell grafts (Cudkowicz and Yung, 1977; Yung and Cudkowicz, 1977, 1978). Since carrageenans have anticomplementary, anticoagulant, and Hageman-factoractivating properties, control experiments were performed. Cobra venom factor, which depletes complement, heparin, and ellagic acid, which activates Hageman factor, did not inhibit marrow graft rejection. PVNO did not reverse the suppressive effect of carrageenans on the ability to reject marrow grafts. Lotzova et al. (1975a) and Buurman and van Braggen (1975a,b, 1976) made very similar observations. Lohmann-Mathes et al. (1979) claimed that promonocytes were responsible for NK cell activity. How might macrophages function during rejection of bone marrow cell grafts? They could directly lyse donor stem cells after being activated by interferon-y (Spitalny and Havell, 1984). Since athymic nude mice can reject marrow cells, presumably a cell other than a T cell would have to secrete the interferon-y; that cell might be an NK cell. The macrophages could lyse stem cells by antibody-dependent cellular cytotoxicity, since they have Fc receptors; antibodies to the stem cells would have to be present. Another potential mechanism is the secretion of interferon-a/@by macrophages so as to activate NK cells. Evidence for this possibility was presented (Mifi et al., 1985). Treatment of mice with polyinosinic : polycytidylic acid [poly(I-C)] results in the augmentation of NK cell activity due to interferon secretion (Gidlung el al., 1978). A similar treatment of mice with poly(1-C)augmented
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hybrid resistance to marrow allografts and even “converted” poor responder mice into good rejectors of marrow allografts. However, if the mice were treated with silica, poly(1-C) failed to boost marrow allograft reactivity. If mice were injected with silica and then treated with interferon-a/p, marrow allograft reactivity was augmented. We interpreted the data as follows: Macrophages secrete interferon-a/P during marrow allograft rejection so as to stimulate NK cell function. Macrophages are the source of interferon that stimulates NK cell function when poly(1-C) is administered (Djeu et al., 1979). What we have failed to determine is the mechanism of stimulation of macrophages to secrete interferon. Klimpel et al. (1982) demonstrated that C57BL/6 marrow cells secreted interferon-a/p in response to allogeneic or semiallogeneic spleen cells. Therefore it is at least conceivable that alloreactive cells in the donor inoculum, at least when normal hemopoietic cells are transplanted, could stimulate host macrophages to secrete interferon. Macrophages may have functions other than secreting interferon. We observed that silica particles and carrageenan inhibited the ability of irradiated mice to reject BALB/c FLD-3 erythroleukemia cells (Afifi et al., 1986). However, the ability to reject FLD-3 cells was not inhibited by antibodies to interferons-a/P or -y and was not stimulated by the administration of i n t e r f e r o n 4f3. Hybrid resistance was not exerted by macrophage monolayers on cellulose acetate membranes in the peritoneal cavities of irradiated mice injected with parental marrow cells (Kitamura et al., 1973). Thus, macrophages may be heterogeneous in function, or macrophages alone cannot reject marrow cell grafts.
B. NATURAL KILLER CELLS Miller (Miller and Mitchell, 1969) and Cooper et al. (1965)discovered that the thymus and the bursa of Fabricius were the central lymphoid organs necessary for the differentiation of T and B cells, respectively. Since the effector cells responsible for marrow allograft rejection did not appear to be T or B cells, they did not seem to fit into the scheme of differentiation of immunologically competent cells with specificities. Eric Mayhew and I attempted to develop an in vitro model for hybrid resistance, using embryonic fibroblasts as target cells (1971, 1974). We observed that spleen, lymph node, and bone marrow cells were cytotoxic in that assay. Since marrow cells were thought to be more a source of precursors of cytotoxic effector cells than a source of effector cells themselves, these results were unexpected. I decided therefore to test the hypothesis that the marrow tissue itself was the central lymphoid organ for effector cells responsible for hybrid resistance. Fried et al. (1966) had shown that the administration of the bone-seeking isotope, sQSr, chronically irradiates the bone marrow to induce aplasia. The
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spleen takes over stem cell functions and the animals can survive. C3H x B10 F, hybrid mice were treated with 89Sr one or two times at 28-day intervals and tested for the ability to reject parental or allogeneic marrow cell grafts :I-4 weeks after the last injection. In a dose-related manner, 89Sr abrogated the ability to reject incompatible marrow cell grafts (Bennett, 1973). The treated mice had competent B, T, and even accessory (macrophage/aritigen-presenting) cell functions (Bennett et al., 1976). Kincade et al. (1975) observed that fetal liver cells could differentiate into B cells when grafted into Y3r-treated mice, indicating that mammalian B cells were bone marrow derived but not bone marrow dependent. Stutman (1976) observed that yolk sac cells failed, while bone marrow cells succeeded in repopulating the thymus of irradiated mice previously treated with Y3r. Thus, T cell development from primitive stem cells may need the marrow microenvironment. Moreover, Van Zant (1984) observed that the ability to form 14-day spleen colonies by bone marrow cells was impeded in irradiated mice also previously treated with s9Sr. It is conceivable that primitive stem cells normally pass through the bone marrow before migrating to the spleen to undergo hemopoiesis. Thus, marrow-dependent cells were necessary for hybrid resistance, but what were they? Natural killer (NK) cells were simultaneously discovered by Kiessling et al. (1975a,b) and Herberman et al. (1975a,b). YAC-1 lymphoma cells grown in tissue culture became the prototypic NK target cells, and the effector cells capable of lysing YAC-1 in a short-term assay appeared to have characteristics very similar to the effector cells responsible for rejection of marrow allografts (Kiessling et al., 1977; Datta et al., 1979). These similarities included (1)age of maturation of function at 3-4 weeks, (2) relative resistance to acute total-body irradiation at doses of 11 Gy or less, but not to doses of 22 Gy or greater, and (3) susceptibility to suppression by silica particles and carrageenan. Haller and Wigzell (1977) observed that 89Sr treatment also abrogated NK cell function, indicating that NK cells, like the cells responsible for rejection of marrow allografts, were marrow dependent. Emmanuel et al. (19811) observed that 90Sr also depleted NK cell function. Haller et al. (1977b) reported that bone marrow cells could adoptively transfer NK cell function to irradiated mice. Hackett et al. (1985, 1986b) noted that N K progenitors were different from myeloid or T or B progenitors because there were no defects in the ability of W/Wv progenitors or C.B-17 scid progenitors to generate NK cells upon infusion into irradiated histocompatible mice. In those experiments, the in vivo assay for NK cell function, namely, “lung clearance” of YAC-1 cells, was utilized (Riccardi et al., 1979). Marrow cultures give rise to diffuse colonies of cells which function like NK cells (Claesson et al., 1982). However, other marrow-derived cells function in a manner similar to NK cells; these are referred to as natural cytotoxic
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(NC) cells (Stutman et al., 1978). The cells that lyse EL-4 in vitro were thought to be NC cells (Kumar et al., 1979a; Lust et al., 1981). Recently, Bykowsky and Stutman (1986) observed that almost any hemopoietic cell type can perform NC cell functions! Therefore, NC function rather than cell type should be considered; this function is potentially important immunobiologically as tumor necrosis factor mediates NC function (Ortaldo et aZ., 1986). Strong support for the concept that NK cells and cells responsible for hybrid resistance were marrow dependent was the observation (Seaman et al., 1978, 1979a,b; Seaman and Gindhart, 1979) that congenital or estradiolinduced osteopetrosis depleted mice of both functions. Even the very strong hybrid resistance exerted by NZB x NZW F, hybrid mice against NZB marrow cells was inhibited by induction of osteopetrosis using estradiol. There was a correlation between the development of osteopetrosis with loss of marrow tissue and the loss of NK cell function and hybrid resistance. The development of specific antisera to NK cells was important in the field. Glimcher et al. (1977) immunized BALB/c x C3H F, mice with CE lymphoid cells to produce an antibody specific for NK cells, called antiNK-1.1 serum. Pollack et al. (1979) produced a similar antiserum. While the anti-NK-1.1 sera C eliminated NK cell function, a variety of T cell functions were unaffected. A second specific marker for NK cells is anti-NK-2.1, which was generated in CE x NZB F, mice against CBA lymphoid cells (Tai and Warner, 1980), in CE mice against CBA cells (Burton and Winn, 1981), and in NZB mice against BALB/c cells (Pollack and Emmons, 1982). Koo and Peppard (1984) developed a monoclonal antibody (PK136) after immunizing C3H x BALB/c F, mice against CE lymphoid cells. Koo et al. (1980) reported the phenotype of N K cells. The ability of C57BL/6 mice to reject BALB/c marrow cells was weakened after the administration of polyclonal anti-NK-1.1 antiserum (Lotzova et al., 1983), which also suppressed N K cell function for 24 hours. Further evidence that N K cells function during marrow allograft rejection was presented. Roder and Duwe (1979) reported that the beige mutation impairs NK cell function in mice. Harrison and Carlson (1983) observed that C57BL/6 b&/b& mice had low NK cell function and an impaired ability to reject incompatible marrow grafts. Kaminsky et al. (1983) analyzed the low NK cell function of SJL mice which also cannot reject marrow allografts. Gallagher et al. (1976) observed that treatment of mice with fractioned irradiation (1.75 Gy four times at weekly intervals), which induces thymic lymphomas in C57BL mice, also abrogates marrow allograft reactivity and NK cell function. Rabbit anti-asialo GM1 eliminates NK cell function in vitro and in vivo (Habu et al., 1979, 1981; Kasai et al., 1979, 1980, 1981). Although mac-
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rophages and activated T cells also express asialo GMl, the administration of anti-asialo GM1 serum in uivo primarily affects N K cells (Saijo et al., 1984). Anti-asialo GM 1 serum inhibits marrow allograft reactivity very efficiently (Okumura et al., 1982; Afifi et al., 1985). Miller (1983) characterized the cells that could adoptively transfer the ability of irradiated mice to reject marrow cells. The cells so studied appeared to be similar to or identical with NK cells. The demonstration that cloned lymphoid cells with NK activity (Dennert, 1980) could adoptively transfer the ability to reject bone marrow allografts (Warner and Dennert, 1982) strengthened the concept that N K cells are intimately involved in resistance to marrow grafts. These investigators stimulated C57BLJ6 +/ + or nulnu spleen cells with concanavalin A-conditioned medium and developed clones of cells that had surface markers similar to normal NK cells. A clone called NKB61A2 was studied; these cells were infused into C57BL/6 mice which were unable to resist BALB/c marrow allografts for two different reasons. One group had been exposed to 1.75 Gy of gamma rays four times at weekly intervals and another group was beige (bglbg)C57BL/6 mice. The cells were infused 6 days before the mice were irradiated and challenged with the H-2 allogeneic marrow cells. The NKB61A2 cells restored the ability of the mice to reject allogeneic BMC, but failed to restore hybrid resistance to C57BL/6 x C3H F, bglbg mice challenged with C57BL/6 BMC. Whereas marrow allograft reactivity was restored by the cloned cells, NK cell function in the spleens of these mice was not restored. Therefore the precise mechanism of how these cloned cells restored marrow allograft reactivity was not ascertained. The cloned cells did not attack .H-2b identical BALB.B marrow cells infused into C57BL/6 beige hosts. In any event, there is little doubt that NK cells function during the process of marrow graft rejection. There remains the problem of how socalled “H-2 unrestricted” killer cells can mediate the specificity seen in bone marrow transplantation experiments. Recently (Sonada et al., 1986), the rejection of parental WB and B6 marrow mast cell precursors by unirradiated WB x C57BL/6 F,-W/W” mice in the skin was observed. This is of interest because the epidermis contains a Thy-l+ cell with NK-like activity (Nixon-Fulton et d., 1985). In addition to their ability to reject marrow grafts and to lyse certain tumor cells, NK cells are also capable of lysing virally infected cells (Trinchieri and Sontoli, 1978; Lee and Keller, 1982; Minato et al., 1979). This observation has provoked speculation that N K cells may play a role in early resistance to viral infections. Evidence supporting such a possibility includes the following: (1)Mice depleted of NK activity in uivo by infusion by anti-asialo GM1 are significantly more susceptible to influenza virus, murine cytomegalovirus, murine hepatitis virus, and herpes simples virus (Stein-Streilein and
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Guffee, 1986; Bukowski et al., 1983, 1985; Habu et al., 1984); and (2) resistance of mice to Friend leukemia virus, herpes simplex virus, and murine hepatitis virus appears to be mediated by cells which require an intact bone marrow microenvironment (similar to NK cells) for functional maturation (Kumar et al., 1974; Kumar and Bennett, 1975, 1976; Levy-Leblond and Dupuy, 1978; Lopez et al., 1978). Welsh (1978, 1981, 1984) has reviewed this subject in detail. Natural killer cells also appear to be capable of interacting with some normal cell types, such as immature thymocytes (Hansson et al., 1979a,b, 1981), bone marrow cells (Hansson et al., 1982; Riccardi et al., 1981), and dendritic cells which have been pulsed with antigen (Shah et al., 1985, 1986). A potential role for NK cells in the regulation of hemopoiesis was first suggested by Cudkowicz and Hochman (1979). Cells with NK characteristics appear to be involved in inhibition of in uitro myeloid and erythroid colony formation (Hansson et al., 1982; Mangan et al., 1984; Degliantoni et al., 1985) and may be responsible for the clinical symptoms of neutropenia seen in patients with large granular lymphocytosis (Rambaldi et al., 1985). Recent studies also suggest that NK cells may be capable of regulating both T and B cell responses (Abruzzo and Rowley, 1983, 1986; Shah et al., 1985, 1986; Gilbertson et al., 1986). Clark and Holly (1981) observed that challenges of mice with H-2 allogeneic cells can activate NK cells. C. ANTIBODIES If mice are immunized against H-2 alloantigens by injection of lymphoid cells or grafting skin, antibodies are made which can passively transfer the ability to reject bone marrow cell grafts in irradiated mice (Santos et al., 1959; Garver and Cole, 1961; Loutit and Micklem, 1961). Thus, radioresistant effector cells and/or complement can utilize preformed antibodies to eliminate incompatible bone marrow cells. N K cells are endowed with Fc receptors for IgG and are perfectly capable of antibody-dependent cellular cytotoxicity (ADCC) (Pollack and Kraft, 1977; Herberman et al., 1977; Ojo and Wigzell, 1978). Indeed, some evidence was presented to suggest that the serum used in NK cell assays might contribute the antibodies utilized by the NK cells to lyse the target cells by ADCC (Takasuchi et al., 1977; Troye et al., 1977; Akira and Takasugi, 1977; Kall and Koren, 1979; Kay et al., 1979). This led to the hypothesis that N K cells were killer (K) cells armed with antibodies (Koide and Takasugi, 1977; Pope et al., 1979). Haller provided evidence that NK function was independent of the serum source used (1978). With use of serum from patients with pure red cell aplasia, effector cells from peripheral blood could lyse allogeneic but not autologous marrow erythroblasts (Krantz and Dessypris, 1982). This latter observation suggests that ADCC may be more complicated than previously thought; perhaps
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ADCC is inhibited when autologous target cells are employed or the effector cell must utilize its Fc receptor and some other receptor for the target cells. Warner and Dennert (1984) recently observed that serum from good responder mice could transfer the ability to reject bone marrow allografts to poor responder mice. Moreover, serum from these good responder mice were able to sensitize appropriate target cells for ADCC mediated by NK cells. For example, 129 mouse serum endowed C57BL/6 mice with the ability to reject H-2k allogeneic C3H marrow allografts. They could even sensitize the marrow cells in uitro with serum from the good responder mice. Their efforts were less successful in attempting to detect natural antibodies in the serum of F, hybrid mice capable of transferring the ability to reject parental strain marrow grafts. In a second study, they preincubated marrow cells with monoclonal anti-H-2 antibodies and demonstrated that antibodies of the IgG,, or IgG,,,, but not IgM, isotypes could sensitize the cells for rejection by irradiated mice not normally capable of rejecting the cells (Dennert et al., 1986). Using recipient mice treated with fractionated irradiation to eliminate N K cell function, they demonstrated that administration of cloned NKB61BlO NK cells 4 days before irradiation and challenge with C3H marrow cells preincubated with anti-H-2k antibodies resulted in rejection. Depletion of C3 by cobra venom factor had no effect. However, C57BL/6 X C3H F, beige mice reconstituted with cloned NKB61A2 N K cells of C57BL/6 origin were unable to reject C57BL/6 marrow cells, but were able to reject BALB/c marrow grafts. Presumably, if natural antibodies mediate hybrid resistance by ADCC, the beige mice should have had those antibodies to allow the injected NK cells to lyse the parental stem cells. The NKB61B10 cells were capable of ADCC. C57BL/6 X C3H F, mice normally reject C57HL/6, but not C3H marrow cells. However, C3H BMC preincubated with anti-H-2k antibodies were rejected by C57BL/6 x C3H F, mice. When BMC were sensitized with antibodies and infused into irradiated syngeneic recipient mice, rejection was observed in only a fraction of the cases. This suggested to the investigators that dual receptors are involved, i.e., Fc receptors can use the appropriate antibody to lyse stem cells by ADCC, but other N K cell receptors directly recognize target cell antigens to participate in the response. Since the cloned NKB61BlO and NKB61A2 cells express T cell p chain rearrangements in the DNA and RNA (Yanagi et al., 1985), they concluded that N K cells can recognize allogeneic cells much like T cells. If natural antibodies mediate marrow allograft rejection by ADCC using NK cells, one would expect to find such antibodies in athymic nude mice which reject marrow allografts quite well. Chow et al. (1981) did find C fixing antibodies in the serum of nude mice capable of lysing SL2 tumor cells. The antigens recognized by such anti-tumor natural antibodies are not neces-
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sarily expressed on stem cells. Miller et al. (1983)observed that treatment of tumor cells with interferon increased their sensitivity to lysis by natural antibodies + C. Since interferon enhances the expression of H-2 antigens (Lindhal et al., 1974), it is conceivable that some natural antibodies are directed against H-2 antigens. F, hybrid mice immunized with parental H-2b Friend virus induced-erythroleukemia cells generated antibodies to H-2Kb (self) antigens (Risser and Grunwald, 1981). Chow (1984) selected tumors for the ability to grow in uiuo in mice and selected for cells resistant to natural antibodies. Natural antibodies detected in mice are often directed against leukemia retroviral antigens (Martin and Martin, 1975; Nowinski and Klein, 1975; Kende et al., 1981). Wolosin and Greenberg (1981) detected natural antiH-2 antibodies, and antibodies to fetal antigens were detected (Colnaghi et al., 1982). The ability of natural antibodies in A x C57BL F, mice to bind to A strain tumor cells could be inhibited by C-type virus particles, bacterial sonicates, and various glycoproteins (Gronberg et al., 1985). Murine natural antibodies are often broadly reacting autoantibodies (Avrameas et al., 1983). Lymberi et al. (1985) generated IgM monoclonal antibodies by fusing normal spleen cells of newborn or adult BALB/c mice with myeloma cells. Most of the monoclonals shared common idiotypic determinants, which were also detected on IgGZb natural antibodies. A given antibody might react with several antigens, including actin, tubulin, myosin, trinitrophenylated bovine albumin, and DNA, most of which are intracellular antigens. Nonetheless, it is entirely conceivable that natural antibodies could exist in mice capable of binding to transplanted stem cells and allowing rejection by NK cells via ADCC. Anderson et al. (1981) summarized a number of experiments demonstrating that IgM as well as IgG antibodies can sensitize target cells for lysis by effector cells with receptors for IgM and IgG. Murine T and B cells have Fc receptors for IgM and can lyse Moloney sarcoma virus-induced tumor cells (Lamon et al., 1975a,b). Mice (Ivanyi et al., 1982; Cerny-Provaznik et al., 1985), rats (Gunther et al., 1983), chickens (Longnecker and Mosmann, 1980; Neu et al., 1984), and humans (Tongio et al., 1985) develop with increasing age IgM antibodies specific for allogeneic MHC components. Some antiviral antibodies are directed against virus self MHC components (Wylie et al., 1982). Perhaps very low levels of these antibodies exist in young animals, and such antibodies conceivably could function during BMC graft rejection. However Asjo et al. (1977) performed genetic studies which indicated that the ability to make antibodies against YAC cells was not related to NK activity against YAC cells. Mice were treated from birth with rabbit anti-p chain serum to deplete B cells and immunoglobulins. Natural resistance was stimulated in such mice, including the ability to reject incompatible BMC grafts (Brodt et al., 1981;
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Brodt and Gordon, 1982). The IgG levels were low, but not absent. Nonetheless, the increased ability to reject with poor ability to make antibodies does argue against the natural antibody hypothesis. Similar B cell-deprived mice were shown to lack certain T suppressor cell subsets, particularly those suppressor cells whose functions are Igh restricted (Flood et al., 1984). Given that NK or other effector cells can lyse antibody-sensitized stem cells, is this the usual manner in which marrow allografts are rejected? Until the putative antibodies are detected and analyzed, that question will be unanswered. What one can do is ask if antibodies are absolutely necessary for marrow allograft rejection. That experiment is now possible due to the discovery and careful analysis of severe combined immunodeficient (SCID) mice (Bosnia et al., 1983; Custer et al., 1985). A SCID mouse was discovered in a population of C.B-17 strain mice when a routine Ig typing test was performed and no Ig was detected. The mice were subsequently inbred in a specific pathogen-free environment to produce an independent strain called C.B-17 scid. The SCID mouse is similar to SCID humans in that there is a stem cell defect in the production of functional T and B cells. SCID mice can be immunologically reconstituted by bone marrow grafts from C.B-17 donor mice, and their otherwise extremely small thymus is fully repopulated. The marrow of SCID mice can reconstitute the myeloid system of irradiated C.B-17 mice, but fails to produce T or B cells. Dorshkind et al. (1984, 1985) detected no functional B or T cells, but did note that NK cell function was normal or near normal. Hackett et al. (1986b) discovered that the progenitor cells in bone marrow of SCID mice were as capable as progenitors from C. B-17 mice to generate functional N K cells upon transplantation into compatible irradiated mice. If SCID mice are capable of rejecting allogeneic bone marrow cells, natural antibodies must not be necessary for this function. Therefore, H-2d C. B-17-scid mice were irradiated and challenged with grafts of 5 x lo6 bone marrow cells from H-2b/Hh-lb C57BL/6 mice, H-2d identical DBA/2 mice which differ at the “minor” Hh-DBA locus, C57BL/6 x DBA/2 F, H-2-semiallogeneic/Hh-l mice, and from syngeneic SCID mice. The C57BL/6 cells were strongly rejected, the DBA/2 cells were moderately well rejected, and the C57BL/6 x DBA/2 F, and SCID cells grew without impairment (Murphy et al., 1987a). Serum Ig levels in the mice used were absent or 1% of normal values. We therefore concluded that natural antibodies are not required for marrow allograft rejection, with specificity, to occur. The observation that SCID mice were able to resist large numbers of DBAIB marrow cells suggests the possibility that some T and/or B cell function(s) of normal mice may actually regulate either the recognition or rejection or foreign stem cells by NK cells. Nonetheless, the observation that NZB x NZW F, mice can resist grafts of 4 X lo7 NZB marrow cells (Lotzova and Cudkowicz, 1973) suggests that antibodies conceivably function in hybrid resistance in certain instances.
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If NK cells do not use antibodies to reject stem cells, what is the mechanism of recognition? NK cells were purified using the anti-NK-1.1 monoclonal reagent and were propagated in vitro by stimulation with human recombinant interleukin 2. No functional RNA transcripts of the T cell receptor a,p, or y chains could be detected (Tutt et al., 1986). Similar findings were made with purified human NK cells (Lanier et al., 1986) and rat large granular lymphocytic leukemia cells with NK cell activity (Reynolds et all, 1986). Therefore NK cells must have their own perhaps unique receptors which can be used to recognize Hh antigens on stem, lymphoid, and tumor cells. D. T CELLS Even though athymic nude mice and rats and SCID mice without T cell functions can reject marrow, normal lymphoid, and tumor cell grafts, T cells may still function to reject hemopoietic grafts. Bau and Theirfelder (1973) observed that rabbit anti-mouse lymphocytic serum treatment would allow the survival of otherwise incompatible rat marrow cells in lethally irradiated mice. Similarly, rabbit anti-rat serum allowed mouse cells to repopulate irradiated rats. They monitored the granulocytes of the chimeric animals, since rat granulocytes are alkaline phosphatase positive and mouse cells are negative for that enzyme. The inoculum sizes used were large, 250 x lo6 mouse BMC and 100 x lo6 rat BMC. Recently, Thierfelder et al. (1986) used a rat IgGa anti-Thy-1 monoclonal reagent to prevent both graft-versushost disease and graft rejection. CBA or CBA X C57BL/6 F, mice were irradiated and were infused with 50 X 106spleen cells and 20 x 106marrow cells from C57BL/6 donors. Administration of the antibody allowed 100% chimerism without any graft-versus-host disease. One can argue that the inoculum size was too large to determine if the antibody actually had any effect on the host response to the graft. A better case for the potential importance of host T cells was made by observations of Aizawa et al. (1980). C3H mice were irradiated and infused with C57BL x C3H F, bone marrow cells. With use of the short-term IUdR method to assess growth, no rejection was detected, as expected. However, spleen cellularity after 5-7 days dropped for a few days before subsequent repopulation of the spleen. Aizawa et al. detected cells in the spleen of these recipient mice which were capable of lysing C57BL EL-4 tumor cells in vitro. The effector cells could be eliminated by anti-Thy-1 antibody C. In a subsequent study, these investigators (Alzawa et al., 1981, 1984; Sad0 et al., 1985; Hirokawa et al., 1985) noted that functional T cells, including alloreactive cytolytic T lymphocytes, could be recovered from irradiated mice repopulated with allogeneic or semiallogeneic donor bone marrow cells. The dose of irradiation required to “suppress” the emergence of these
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radioresistant T cells varied with the strain studies. For C57BL mice, the dose was 8.5 Gy, and for C3H mice the dose was 11 Gy. The detection of host radioresistant T cell function is quite apparent when one transfers SCID marrow cells into irradiated mice (Hackett et al., 198613). In a series of experiments (von Melchner et al., 1980; von Melchner and Lieschke, 1981; von Melchner and Nicola, 1981; von Melchner, 1983; von Melchner and Bartlett, 1983a,b; von Melchner and Hoffken, 1984), evidence was produced to support the idea that radioresistant host T cells were able to mediate early marrow graft rejection. The unique test system used was the organ culture of spleen slices removed within a few days after irradiation and transfer of 5 X lo6 syngeneic or H - 2 allogeneic BMC. This organ culture system allowed the continued growth of CFC in uitro colonyforming cells. The basic protocol was to irradiate CBA H-2k mice and to infuse or not allogeneic BALB/c H-2d marrow cells. Six days later, BALB/c, C57BL/6 II-2b, or CBA BMC were infused into the mice. The spleens were removed the following day and the spleen slices were incubated in uitro for 6 days. At the end of the incubation, cell suspensions were made and assays for CFC were performed. In parallel groups, spleen cells were removed from irradiated CBA mice at different intervals of time after infusion of BALB/c BMC. No CFC were detected in the spleens of the latter mice when spleen cells were tested directly from the spleen. In CBA mice infused with BALB/c BMC at day -7, neither BALB/C nor CBA BMC infused on day - 1 generated CFC well in the organ cultures. There was a greater inhibition of donor than of host-type BMC. With use of the organ culture system and testing at intervals after transfer of BALB/c BMC, donor-derived CFC were obtained 1 to 5 days after cell transfer, with a peak at day 3. If lymph node cells were mixed with the BALB/c BMC injected 1day before setting up the organ cultures, these investigators observed that either donor or host-type nodal cells (BALB/c or CBA) prevented the expansion of CFC numbers in vitro. This suggested to them that alloreactive T cells could be activated to nonspecifically inhibited differentiation of CFU-S into CFC. To test that notion, spleen cells were removed from days 1-7 BALB/c + CBA BMC chimeras and tested for the ability to inhibit CFC growth of either BALB/c or CBA normal BMC. The spleen cells removed on days 5 or 7 were inhibitory, whereas cells removed on days 1 or 3 were not suppressive. If irradiated BMC were injected on day -7, no inhibition of day - 1 BALB/c or CBA BMC occurred. Presumably the irradiated BMC were incapable of sensitizing host T cells. In a test for specificity, no BMC, BALB/c BMC, or C57BL/6 BMC were infused on day -7. On day -1, CBA, BALB/c, or C57BL/6 BMC were infused and the spleens were cultured one day later. The injection of BMC on day -7 resulted in some inhibition of CFC generation no matter the
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source of BMC on day -1. However, suppression was much greater if the allogeneic BMC infused on days -7 and -1 were of the same type, e.g., C57BL/6. They noted that serum from the recipient mice taken 1-6 days after cell transfer was not suppressive of CFC generation in the organ cultures, even when used at a 30% concentration. Irradiated host-type marrow or spleen cells, a potential source of effector cells, did not inhibit the ability of BALB/c BMC from generating CFC upon transfer into CBA hosts. Mixed lymphocyte cultures between CBA and BALB/c spleen cells (oneway or two-way) generated supernatant factors(s) capable of inhibiting CFC generation in the organ cultures. Control experiments indicated that both interleukin 2 and colony-stimulating factor were produced in the supernatant fluids. By using donors and hosts which differed at Thy-l, namely, BALB/c Thy-1.2 and-AKR Thy-1.1, they observed that only host-type AKR Thy-1.1 cells were generated in vivo in spleen cell suspensions or in vitro in the organ cultures. Spleen cells were removed from BALB/c -+ AKR 7day chimeras and were treated with either anti-Thy-1.2 + C or with antiThy-1.1 c . The cells were then tested for the ability to inhibit CFC function of normal BALB/c or AKR in agar cultures. Only anti-Thy-1.1 antibodies prevented the suppression of CFC function. Thus, host-type Thy-1 cells appeared to be responsible. Finally, BALB/c + AKR BMC chimeras were injected daily with anti-Thy-1.1 or with anti-Thy-1.2 antibodies. CFC numbers in recipient spleens and femurs were enumerated. In mice treated with anti-Thy-1.2, spleen CFC rapidly declined after day 4, which is observed if no antibody is administered. However, CFC in the spleens and femurs increased in numbers after day 4 in mice injected with anti-Thy-1.1 antibodies. The data indicated to these investigators that hosttype T cell can respond to H-2 allogeneic BMC and generate nonspecific inhibitory factor(s) capable of preventing CFU-S from differentiating into CFC. Since only Thy-1 antibodies were used, the proof that the effector cells were T cells is lacking. About 50% of NK cells express Thy-1 antigens (Hackett et al., 1986a). These very interesting experiments should be repeated to phenotype the host effector cells more accurately. The use of antiNK-1.1 antibodies to inhibit NK cell function and anti-L3T4 and anti-Lyt-2 antibodies to inhibit T cell functions could conclusively demonstrate whether host T cells mediated this inhibition of CFU-S differentiation. Dennert et al. (1985) provided evidence that host T cells can reject H-2 allogeneic, but not parental strain BMC. They irradiated C57BL/6 +/ or bg/bg mice (7 Gy) and infused C3H BMC. The first graft was capable of sensitizing either + / + or bglbg mice against C3H BMC. The beige mice immunized with C3H BMC did not reject BALB/c BMC. Genetically poor responder +/+ or bglbg mice could be immunized against C3H BMC by the previous injection of H-2k tumor cells, whereas C57BL/6 nulnu mice
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could not be so immunized, and accepted the C3H BMC. The transfer of T killer cell clones into bglbg host mice or into poor responder 129 mice could transfer specific ability to reject H-2 allogeneic BMC. However, C57BL/6 x C3H F, + / + or bg/bg mice could not be immunized in uiuo to reject parental C3H or C57BL/6 (in the case of bglbg) BMC. C57BL/6 x DBA/2 F, antiparental BALB/c T cells generated in uitro failed to cause rejection of BALB/c BMC in irradiated C57BL/6 x DBA/2 F, mice. The data are certainly consistent with their interpretation that host T cells were sensitized and therefore able to lyse incoming stem cells of the second grafts. They did not rule out an antibody response, even though nude mice failed to be sensitized and the conditions of the experiment may have precluded ADCC function by hosts at the time of the second cell transfer. A more interesting test of the 17, antiparental T killer cells would have been the use of such cells generated against C57BL/6 target cells and infused into C57BL/6 x C3H F, bglbg hosts. A very interesting study of the potential of T cells to function after large doses of irradiation was recently reported (Gassman et al., 1986). LEW (RTI? rats were exposed to 10.5 Gy (50% lethal) or 12 Gy (100% lethal) of 6oCo gamma rays and were challenged with CAP (RTI") skin grafts. Some of the mice were simultaneously immunized with lo8 irradiated CAP BMC. Even the rats exposed to 12 Gy were able to reject skin grafts by 7 days if they were immunized with the BMC. When LEW rats exposed to 10.5 Gy were immunized with CAP BMC at different intervals of time after application of the CAP skin graft, progressively slower rejection times were recorded as the increase in delay of immunization occurred. This is the first instance reported that rejection of an organ graft can be so vigorous after such large doses of irradiation. The data suggest that effector cells capable of such a reaction can survive at least 1 or 2 days. Since skin grafts were rejected, it is conceivable that marrow allografts might also be attacked by a classical tissue allograft-type response in irradiated mice. Two earlier studies indicated that delayed hypersensitivity responses were relatively radioresistant (Uhr and ScharE, 1960; Volkman and Collins, 1968). Cobbold et al. (1986) observed that treatment of irradiated mice with a mixture of monoclonal reagents directed against L3T4 and Lyt-2 antigens prevented rejection of H-2 allogeneic marrow + spleen cell grafts. They used adult thymectomized mice as donors and depleted marrow and spleen of T cells with antibodies to L3T4 and Lyt-2 complement. Engrdtment was tested by determining the percentage of donor versus host lymphoid cells. This regimen of treatment was designed to attack radioresistant host T cells, and the results support the concept that rejection indeed can be mediated by T cells. The apparent need to use both anti-L3T4 and anti-Lyt-2 reagents suggests that both major types of T cells are involved in the rejection process.
+
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Finally, Kosmatopoulos et al. (1987) have recently described a Thy-1 Lyt-l+2- nylon wool nonadherent spleen effector cell which can transfer hybrid resistance to syngeneic mice treated with 5-fluorouracil or with splitdose irradiation (Kosmatopoulos and Orbach-Arbouys, 1987). Before accepting the conclusion that this cell is a T cell, it would have to express L3T4, an antigen more restricted to the T cell lineage. Its presence in SCID and in nude mice need to be determined, i.e., is it truly a thymus-dependent (T) cell? +
E. CONDITIONS CONDUCIVE TO MARROWENGRAFTMENT
1 . Specific Unresponsiveness The “specific” loss of hybrid resistance can be induced in F, hybrid mice by the multiple injections of parental strain spleen cells prior to irradiation and marrow cell transfer (Cudkowicz and Stimpfling, 1964a; Cudkowicz, 1965; Cudkowicz and Barnett, 1971; Lotzova, 1977a). Usually 4 or 5 injections of 1 or 2 x 107 spleen cells at weekly intervals were made, the last one 7-18 days prior to the test grafts. If C3H x C57BL/10 F, mice were injected with C3H spleen cells multiple times, hybrid resistance to C57BL/10 BMC was not suppressed. Moreover, multiple injections of C57BL/6 spleen cells into C57BL/6 x DBA/2 F, mice induced loss of hybrid resistance to C57BL/6 marrow cells, but not loss of allogeneic resistance to WB strain BMC (Lotzova, 1977a; Eastcott et al., 1981). In a detailed study, Lotzova (1977a) observed that the stronger the hybrid resistance to a given parental strain BMC donor, the more times 2 x lo7 spleen cells had to be injected to achieve unresponsiveness. For example, 5 injections of NZB spleen cells were required to inhibit hybrid resistance of NZB X NZW F, mice. The duration of unresponsiveness was related to the number of injections and spleen cells injected. Unresponsiveness could last 120 days after the last spleen cell injection. Both adherent and nonadherent spleen cells and lymph node cells were capable of inducing unresponsiveness, but thymocytes were very weakly effective and liver cells were not effective. Whereas this procedure reproducibly inhibits hybrid resistance to parental strain marrow grafts, we observed that C3H x C57BL/6 F, mice so treated did not allow C57BL/6 lymph node cells to proliferate after irradiation (Eastcott et al., 1981). Kitamura et al. (1977)observed that spleen colonies could be developed in unirradiated C57BL X CBA-T6T6 F, mice if 75 x lo6 C57BL/6 lymph node cells were infused 9 days before C57BL/6 BMC were transferred. The colonies developed 9 days after BMC injection. No detailed immunological investigation was performed to determine the mechanism of suppression of hybrid resistance.
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Hakin and Shearer (1986) recently performed experiments which cast doubt on the previous interpretations of induction of unresponsiveness to parental strain marrow cells by injection of spleen cells. They examined immune functions of unirradiated F, hybrid mice injected with large numbers of parental spleen cells and detected a state of immunodepression, with loss of cell-mediated T cell functions. They considered the possibility that the loss of hybrid resistance was due to graft-versus-host induced immunodepression. To test this hypothesis, they injected B10 x BIO.A F, hybrid mice with various B10 congenic resistant spleen cells and observed that exposure of the spleen cells to class I or class I1 antigens was the key to induction of unresponsiveness. An instructive case was the abrogation of hybrid resistance in B6.bm12 x BlO.A(5R) F, mice (H-2Db/H-2Dd) by the injection of BlO X BlO.A(5R)F, (also H-2Db/H-2Dd) spleen cells. Only class I1 antigenic differences was required to induce unresponsiveness. The ability of the mice injected with BlO parental strain spleen cells to reject grafts of the opposite parent was interpreted to be due to the generation of cytotoxic T cells. On balance, Kosmatopoulos and Orbach-Arbouys (1987) detected specific suppressor cells in F, hybrid spleens of mice injected with parental B6 spleen cells. Moreover, Lemeneva and Chertkov (1976) induced loss of hybrid resistance with parental cells not expected to induce a graft-versushost reaction. Cudkowicz (1965)also observed the loss of hybrid resistance if 810 x A F, marrow cells were used to repopulate the hemopoietic tissues of BlO-irradiated mice. If, however, marrow cells from the F, 3 B10 chimera were used to repopulate irradiated A or B10 x A secondary irradiated hosts, hybrid resistance was “restored.” This result was interpreted to mean that the F, effector cells were “paralyzed” by exposure to B10 antigens. It would follow that transplantable suppressor cells were not generated in the F, 3 BlO primary recipient mice. This model may be the proper one to study the mechanism of tolerance to Hh-1 antigens, since it is not complicated by a graft-versus-host reaction. Waterfall et al. (1984) induced specific unresponsiveness to H-2b parental marrow ct4s by exposure of neonatal CBA x C57BL F, mice to H-2b homozygoiis cells. The F, mice were injected with 15 X lo6 C57BL BMC intraperitoneally within 24 hours of birth. When the mice were young adults, they were irradiated and challenged with marrow grafts from F,, C57BL, and SJL (H-2”)donors. C57BL BMC were now accepted by the F, mice injected neonatally with C57BL BMC. However, the SJL BMC were rejected. Since H-2“ and H-2b were thought to have similar Hh-1 determinants (Cucikowicz, 1978; Warner, 1978), this latter finding was somewhat surprising. In a second experiment, F, hybrid mice were injected with 40 x 106 C57BL marrow cells neonatally. When these animals were adults, their
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spleen cells were tested for the presence of suppressor cells by transferring inocula of lo8 cells into normal CBA x C57BL F, mice 6 days prior to irradiation and challenge with C57BL or SJL BMC. The cells successfully adoptively transferred tolerance to C57BL, but not SJL BMC, and this ability was abolished by prior treatment of the spleen cells with anti-Thy-1 antibody + C. Their interpretation was that H-2-specific T suppressor cells were infused in the neonatally injected mice and were capable of interacting with semiallogeneic F, NK cells so as to suppress hybrid resistance. The lack of unresponsiveness to H-2" BMC was not easily explained. It is possible that H-2S has two Hh-1 determinants, one of which is shared with H-2b, and tolerance therefore was only induced to one of two Hh-1" antigens. Wegmann et al. (1980)observed that the serum of DBA/2 mice parabiosed to DBA/2 x C3H F, mice generated anti-H-2k antibodies strongly reactive with C3H hemopoietic cells, including stem cells. The transfer of large numbers o i DBA/2 spleen cells along with serum from the DBA/2 parabiont produced chimerism in unirradiated C3H mice without graft-versus-host disease. Later, the administration of monoclonal anti-host antibody was shown to enable incompatible spleen cells to engraft and not to cause a graftversus-host reaction (Wegmann et al., 1981). T suppressor cells have been detected in radiation marrow chimeras, which could explain tolerance to hemopoietic or other grafts (Tutschka et al., 1981). 2. Other Conditions Associated with Lack of Rejection of Hemopoietic Cells One can take advantage of the immunological immaturity of fetal animals to transplant otherwise incompatible hemopoietic stem cells, provided that the recipient animal has a defective stem cell system (Fleischman and Mintz, 1979, 1984; Fleischman et al., 1982; Blanchet et al., 1982). Mice with dominant spotting anemia, W/W, Wf/Wf, etc. have severely defective stem cells. If fetal liver cells from normal donors are injected into the fetal circulation on day 11 of gestation, long-term chimerism of erythrocytes and lymphoid cells is possible. Adult bone marrow cells are less successful, but H-2-compatible allogeneic BMC are often successful. H-2-incompatible BMC repopulate the recipients until 3-5 months of age, when the grafts apparently fail and the mice often die. The immune systems of these stem cell-deficient mice are intact (Harrison and Cherry, 1975; Mekori and Phillips, 1969; Bennett, 1971). It is therefore conceivable that immune rejection eventually occurs. Successful engraftment of sheep was accomplished by injecting allogeneic fetal liver cells into fetal animals (Flake et al., 1986). Total lymphoid irradiation, TLI, is a method for treatment of Hodgkin's disease and results in long-term immunosuppression (Fuks et al., 1976). Mice, rats, and dogs received a comparable treatment by exposing their
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lymphoid organs, while shielding most of the bones, to 2 Gy on 17 occasions during a period of 3-4 weeks. These animals now accept bone marrow allografts without developing graft-versus-host disease and accept a variety of tissue allografts (Slavin et al., 1977, 1978a,b, 1979; Gottlieb et al., 1979). Okada and Strober (1982) observed that mice exposed to TLI and neonatal mice had similar types of suppressor cells capable of inhibiting mixed lymphocyte reactions. The suppressor cells were not T or B cells (Oseroff and Strober, 1984; May et al., 1983). Eventually natural suppressor cells from these mice were cloned and shown to have surface markers identical with NK cells, but were not lytic for YAC-1 target cells (Hertel-Wuff et al., 1984; Schwadron et al., 1985). The natural suppressor cells have not been tested for their ability to inhibit hybrid resistance to marrow cells directly. Adrenocortical steroids, e. g., hydrocortisone, suppress NK cell function, but did not inhibit marrow allograft reactivity in one study (Hochman and Cudkowicz, 1977), although Lengerova et al. (1973a)observed that cortisone did inhibit rejection of BMC. Lotzova and Savary (1981) may have resolved the discrepancy by observing that rejection during the first 5 days was not prevented by steroid administration, but by 7 or 8 days rejection ability was weakened. Dexamethasone can suppress murine NK cells directly in uitro (Cox et al., 1982). Animals bearing transplantable tumor cells and patients with advanced stages of malignancy are often immunosuppressed (Roth, 1983; Naor, 1979). Suppressor cells capable of inhibiting NK cell function have been described in animals a.nd in patients with tumors (Gerson, 1980; Gerson et al., 1981; Uchida et al., 1984; Tarkkanen et al., 1983; Johnson and Pope, 1986). The suppressor cells described have included adherent, macrophage-like cells, and such cells may function by secretion of prostaglandins of the E series, which inhibit NK cells (Goto et al., 1983; Koren et al., 1981). Mice bearing syngeneic transplanted tumors lose the ability to reject allogeneic or parental strain BMC grafts (Kumar and Bennett, 1975; Gardner et al., 1980). In the latter study, there was a correlation between host spleen size, lack of NK cell function, and loss of hybrid resistance. The ability to reject BMC allografts is not lost in mice infected with Friend leukemia virus, even at a time when the spleen is large and humoral antibody formation is greatly depressed (Bennett and Steeves, 1970). Pierpaoli et aZ. (1980, 1981, 1985) discovered factor(s) in bone marrow tissues that prevented rejection of H-2 allogeneic marrow cells and also prevented graft-versus-host disease. Aging of humans and animals is associated with a decline and dysregulation of immune functions and an increased susceptibility to neoplasia (Wigzell and Stjernward, 1966; Walford, 1969, 1974; Burnet, 1970; Enstrom, 1979). When “old” mice of ages 15-18 months were compared with
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“young” mice 2-6 months of age, Harrison (1981) observed a decrease in the ability to reject parental or allogeneic BMC grafts. This was observed in the relatively short-term spleen colony assay system as well as in the long-term system which requires the replacement of erythrocytes of W/Wu anemic mice. When mice between the ages of 2 and 26 months were compared, a severe loss of NK cell function was observed (Fitzgerald and Bennett, 1983a). Stimulation of interferon secretion by the administration of poly(I-C) only slightly boosted NK activity of mice greater than 18 months of age. The ability of mice 17 months of age or older to reject BMC grafts after irradiation was tested. The very old mice could reject H-2 allogeneic BMC grafts, but old C3H x C57BL/6 F, or C57BL/6 x DBA/2 F, mice failed to reject C57BL/6 parental strain BMC. Stimulation of old F, hybrid mice with poly(1-C) restored hybrid resistance to BMC grafts. In a parallel study, cellmediated lympholysis assays involving stimulating spleen cells with H-2 allogeneic or parental strain-irradiated spleen cells were performed with mice of different ages. Whereas both of these responses are mediated by T cells, the F, anti-parent reaction declined in old mice, while the anti-H-2 allogeneic response persisted (Fitzgerald and Bennett, 1983b). Chertov and Guretvitch (1981) also observed weakened genetic resistance to marrow grafts in old mice. The anti-tumor and immunosuppressive drug cyclophosphamide is particularly effective in preventing rejection of allogeneic marrow grafts (Santos and Owens, 1968, 1969; Santos and Haghshenass, 1968). This drug affects so many cell types that its precise mode of action has not been determined.
F. REGULATIONOF SYNGENEIC STEM CELL FUNCTIONS Since hemopoietic stem cells are required for protection against radiationinduced marrow aplasia, a large body of literature and knowledge exists concerning the nature of stem cells. I will mention here only those properties that may be relevant to understanding the mechanism of hybrid and allogeneic resistance to BMC grafts. One of the most important questions to be answered is whether NK cells normally down-regulate the self-replication or differentiation of syngeneic stem cells. This is important, since the argument that NK cells recognize Hh determinants on incompatible stem cells and mediate hybrid resistance has been mentioned several times in this review. Cerrottini et al. (1973) reported that interferon preparations and inducers of interferon inhibited the development of syngeneic spleen colonies in irradiated mice. This observation was made prior to the discovery of NK cells in 1975. Even earlier, Jullien and d e Maeyer-Guignard (1971) observed that stem cells of the marrow and the spleen, as measured by spleen colony-forming units (CFU-S), were markedly depleted, as were all nucleated cells, by injections of poly(1-C). Reticulocyte counts dropped from
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3.3 to 0.1% and leukopenia was observed. An important control experiment was performed which indicated that the “f” fraction was not changed, i.e., the fraction of stem cells infused that can be recovered in the host spleen a few hours after cell transfer. The f fraction is an important variable when one tries to quantitate stem cell functions when either donor or host has been manipulated. The lack of additivity when poly(1-C) and the cycle-active agent vinblastine (Bruce et al., 1969) were given together suggested that poly(1-C) also acted upon these stem cells in active cell cycle. These investigators did not think that interferon was the mechanism of suppression of stem cells, since the injection of Newcastle disease virus had no effect on stem cells, yet induced high levels of circulating interferon. If several daily injections of poly(1-C) were made, marrow CFU-S were almost totally depleted, but splenic CFU-S were maintained at a level able to prevent death due to hematological failure (Martelly and Jullien, 1974). No tests for N K cell function were done, but one would predict that N K cell function would have been high after the injection of poly(1-C), Newcastle disease virus, or interferon preparations. Eckner and Hettrick (1979) observed that mice infected with the Friend spleen focus-forming virus developed stem cells which were resisted by syngeneic irradiated mice. Later, Eckner et al. (1987) provided evidence that NK cells prevented the development of Friend erythroleukemia by inhibiting the progression of CFU-S into preleukemic cells in mice infected with Friend virus. There are several conditions in which there is an association between lack of marrow allograft reactivity, low N K cell function, and intense hemopoiesis in the spleens of mice. These include the neonatal mouse (Savary and Lotzova, 1978), mice treated with 8QSror estradiol (Bennett, 1973; Seaman et al., 1979a,b), sublethally irradiated mice after a number of days (Hochman et al., 1978:1,mice treated with killed C. paruum organisms (Cudkowicz and Bennett, 1!371a,b), and in aged mice (Fitzgerald and Bennett, 1983a,b). Normal bone marrow has low NK cell function, but NK cells sorted away from other marrow elements with anti-NK-1.1 antibodies are quite active (Hackett et al., 1986a). There seems to be, therefore, an inverse relationship between NK cell activity and hemopoiesis. Kalland (1986a) recently observed that interleukin 3, a stimulator of hematopoiesis, inhibits the differentiation of N K cells from marrow precursors. We observed that mice infected with murine cytomegalovirus (MCMV) had very high levels of N K cell function and greatly diminished splenic hemopoiesis, although marrow cellularity was not affected (Masuda and Bennett, 1981). Mice infected with Listeria monocytogenes develop high NK cell function, and those NK cells sorted with the anti-MAC-1 reagent were able to inhibit syngeneic CFU-S function (Holmberg et al., 1984). They incu-
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bated the purified NK cells with syngeneic BMC overnight and tested for spleen colony formation by the surviving BMC. A decrease of 50-60% of function was observed. Welsh (1978) reviewed those factors which activate NK cells. Lymphochoriomeningitis virus (LCMV) is a powerful stimulus of NK cell function (Welsh 1981, 1984). Recently, Thomson et al. (1986)observed that infection of mice with LCMV 3-5 days before irradiation resulted in the “rejection” of syngeneic marrow cell grafts. Rabbit anti-asialo GM1 serum reversed the ability of infected mice to resist syngeneic BMC grafts. O’Brien et al. (1983)noted that normal bone marrow cells inhibited NK cell activity by normal spleen cells and suggested that NK cells may normally regulate hemopoiesis. Lord et al. (1977) and Wright and Lord (1977) described factors within marrow which can control the cycle status of syngeneic stem cells. Finally, Lichter et al. (1980) observed that a 3-day delay in marrow infusion after total-body irradiation hastened repopulation of syngeneic recipient thymus and other tissues. This suggests that a short-lived radioresistant element normally can retard the growth of syngeneic stem cells. The stimulation of NK cell function and marrow allograft rejection by poly(I-C) or by interferon-a/P was not associated with a diminution of proliferation of syngeneic stem cells in lethally irradiated mice (Afifi et d . , 1985). Even H-2 allogeneic F, hybrid (Hh antigen-negative) marrow cells proliferated well in spleens of irradiated mice. The anti-stem cell effect of viral infections may be mediated by effector cells/factors other than NK cells. Biron et al. (1984)observed that lysis of radiolabeled L-929 cells in uiuo was greatly stimulated in LCMV-infected mice. Moreover, elimination of NK cells by treating mice with anti-asialo GM1 serum did not abrogate this function significantly. This function was sensitive to treatment with cyclophosphamide or hydrocortisone. Therefore, effector cells other than NK may be activated by viruses which may act upon syngeneic stem cells. ORGAN? G. MARROWMICROENVIRONMENT:A CENTRALLYMPHOID The observations that chronic irradiation of marrow tissue with the boneseeking isotope 89Sr weakens hybrid resistance to parental strain marrow grafts (Bennett, 1973) and inhibits NK cell function (Haller and Wigzell, 1977) suggest that the marrow microenvironment is necessary for an essential stage in differentiation of effector cells responsible for these two activities. Strong support for this concept was presented by Seaman et al. (1979a,b), who observed that congenitally osteopetrotic milmi mice and adult mice treated with pharmacological doses of estradiol to induce osteopetrosis also had poor NK cell function and ability to reject incompatible marrow grafts. These data are consistent with the hypothesis that NK cells
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responsible for hybrid resistance are a “third” type of lymphocyte whose central lymphoid organ is the marrow. However, that concept can be challenged along the following lines of arguments: (1)Treatment of mice with 8gSr destroys so many stem cells that effector functions could be lacking due to the inability of the spleen stem cells to provide enough NK cells to provide normal functions; (2) the spleen, after taking over the stem cell function of the mice, is filled with proliferating hemopoietic cells which could intedere with N K cell functions by simply occupying the functions of the N K cells in the spleen; (3)suppressor cells may emerge in the spleen of treated mice, which inhibit the function of otherwise normal NK cells. Evidence for the presence of suppressor cells capable of inhibiting NK cells in the spleens of mice treated with 89Sr or with estradiol has been presented (Cudkowicz and Hochman, 1979; Milisauskas et al., 1983). The presence or absence of suppressor cells capable of inhibiting N K cell functions in spleens of mice with low NK cell function has been a controversial subject The method of analysis may have had something to do with the results. When suppressors were detected, a constant number of suppressor cell candidates were added to each well that had various numbers of competent spleen cells. Suppressor cells were consistently detected using this method. U‘hen the number of cells/well was held constant and differing numbers of normal or experimental (cells from mice treated with 89Sr) spleen cells were added, no suppression was observed (Kumar et al., 1979b; Mellen et al., 1982). Using the technique of Hochman and Cudkowicz, Seaman and colleagues failed to detect suppressor cells in the spleens of mice treated with estradiol (Seaman et al., 1978, 197913; Seaman and Ginhart, 1979). The strains of mice were different; Hochman and Cudkowicz (1979) used C57BL/6 x C3H F, mice, while Seaman used NZBZ x NZW F, and BALB/c mice. Savary and Lotzova (1978) were able to detect suppressor c& for NK cell function in spleens of infant mice, F, mice injected with parental spleen cells to induce “tolerance” and mice treated with heat-killed C . paruum organisms. The normal mouse thymus, particularly in neonatal mice, contains target cells for normal NK cells of the spleen (Hansson et al., 1979). Mice of the A strain, which have low NK cell function, had the most sensitive cells. Thymus glands of mice also contain N K cells which can only be “revealed” by isolating the light density fraction (Zoller et al., 1982a). Therefore, it is conceivable that spleens of mice treated with 8QSr or estradiol do have functional NK cells, but “natural target cells” coexist in such high numbers that their ability to function cannot be easily assessed. This could explain the discrepancies in studies concerning the presence or absence of suppressor cells capable of inhibiting NK cell function of marrow allograft reactivity. The ability to sort for NK cells based upon their expression of NK-1.1
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antigens allowed one to test if NK cells are normal in mice treated with estradiol (Hackett et al., 1986a). NK cells purified from spleens of normal mice were extremely competent, with significant ability to lyse YAC-1 target cells at a 1 : 1 E : T ratio. However, when NK-1.1+ cells were sorted from mice treated with estradiol or from infant mice, the cells could bind to YAC-1 cells, but their lytic ability was very deficient and that poor ability improved only marginally after exposure of the cells to interferon. In contrast, NK-1.1+ cells sorted from normal bone marrow cell suspensions or spleen cell suspensions from mice treated with C. parvum organisms were quite active. These data indicate that lack of a functioning marrow prevents the complete differentiation of N K cells to the level of competence. Moreover, the results suggest that either suppressor cells or natural target cells in marrow or spleen cell suspensions can indeed inhibit NK cell function. Even if the marrow is the “central lymphoid organ” for NK cells, i.e., NK cells are marrow-dependent effector cells, differences still exist between NK cell and B or T cell differentiation in their central lymphoid organs. In athymic nude mice, the actual numbers of T cells are grossly deficient. In bursectomized birds, the numbers of B cells and plasma cells are also very low (Miller and Mitchell, 1969; Cooper et al., 1964). It would appear that NK cell numbers are not low (in the spleen) in mice deprived of marrow tissue, since only the later stage(s) of differentiation requires the marrow microenvironment. The overall numbers of NK cells are obviously greatly deficient, since no marrow exists. What microenvironment cell is responsible for inducing differentiation of NK cells? Volkman et al. (1983) observed that one injection ofsgSr induced a severe monocytopenia for over a month, even though splenic hemopoiesis was vigorous. The macrophage content of the peritoneal cavity was maintained. Thus, a defect in “delivery” of monocytes to the blood was apparent. It is conceivable that a defect in cells of the monocyte/macrophage lineage is associated with the lack of terminal differentiation of NK cells. If the infusion of normal marrow cells into mice treated with 8gSr fails to correct the monocytopenia, then this isotope may induce a defect in monocyte growth, development, and function (Bennett and Kumar, 1983). Osteopetrotic oplop rats have defects in both macrophage and T cell function (Hochman et al., 1982). The thymus atrophies after weanling and the rats die of “runting disease”; this can be prevented by infusion of syngeneic bone marrow cells (Milhaud and Labat, 1978). Labat et al. (1983) observed that a subpopulation of peritoneal macrophages were depleted in 8-week-old mice treated from birth with (dich1oromethylene)diphosphonate(C1,MDP) to induce osteopetrosis by 4 weeks of age. Peritoneal exudate cells obtained from mice injected with thioglycollate were centrifuged over bovine serum albumin gradients; four bands were detected in normal mice. The treated mice lacked the low-
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density band. Phase contrast microscopy indicated that the “granular” type
of macrophage was missing in the treated mice. A light-scattering analysis by a fluorescence-activated cell sorter indicated that macrophages of the mice treated with C1,MDP were small and nongranular. C1,MDP also reduces NK cell function in mice (Labat et al., 1984). In that study, cessation of therapy was associated with recovery of N K cell function in 2 weeks when osteopetrosis was still evident. The data with osteopetrotic and s’Sr-treated mice do not pinpoint the precise microenvironmental cell, but a subpopulation of cells of the monocyte/macrophage series, e.g., osteoclast, deserves investigation. Tavassoli (1986) did report that marrow tissue can exist separately from bone or cartilage.
H. In Vttro ASSAYSFOR HYBRID RESISTANCE Several attempts have been made to develop an in vitro method which reflects the immunogenetics and immunobiology of hybrid resistance to bone marrow or lymphoid cell grafts in uivo. Eric Mayhew and I (1971, 1974) utilized adherent target cells, either L929 cells or embryonic fibroblastic cells. The target cells were plated in wells of Terasaki microtiter plates and effector cells were added at E : T ratios of 1000 to 8 : 1one day later. After a 24-hour incubation, nonadherent cells were removed and the viable adherent cells were stained and counted. This technique detected cytotoxic lymphoid cells from nonimmune mice which were present in the spleen, bone marrow, and lymph nodes, but not in the thymus. In fact, thymocytes inhibited the ability of lymph node cells (LNC) to lyse target cells. The effector cells were relatively radioresistant, did not require mitogens for activity, did require protein synthesis for the first 2 hours, were not sensitive to elimination by rabbit antilymphocytic serum, but were sensitive to cyclophosphamide. When C57BL/10 x C3H F, LNC were tested against C57BL/ 10 and C3H early-passaged embryonic fibroblasts, the C57BL/ 10 cells were much better lysed than the C3H fibroblasts, and C57BL/10 LNC did not lyse syngeneic fibroblasts well. Although this method appeared to reflect hybrid resistance well, continued growth of the embryonic fibroblasts resulted in cells sensitive to lysis by syngeneic LNC. Therefore, the technique was not considered reliable. The correlation between the in uitro lysis of tumor cells by F, hybrid effector cells and the resistance to growth of the cells in uivo has already been discussed (Petranyi et al., 1976). Using the EL-4 system worked out hy Harmon et al. (1977), Kumar et al. (1979) were able to correlate good responder versus poor responder status to C57BL/6 (H-2b) marrow grafts with the ability to lyse EL-4 cells in an 18-hour 51Cr-labeled release assay. This relatively simple assay for hybrid resistance detected effector cells which had properties similar to those detected in the assay with embryonic fibroblastic
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cells. The EL-4 system had its drawbacks; EL-4 cells from various sources differed greatly in their sensitivity to lysis by F, hybrid effector cells. Even EL-4 cells propagated in our own lab lost their sensitivity to lysis unpredictably. Williams et al. (1977) detected hybrid resistance to SV40-transformed BN strain rat fibroblasts by W F x BN F, and by DA x BN F, spleen cells. Four different clones of SV4O-transformed cells were found to be more sensitive to lysis by F, hybrid than by BN strain spleen cells. In this system, target cells were labeled with and spleen cells at 200 to 5 : 1 E : T ratios were incubated for 18 hours. The effector cells detected in this system were not characterized. The use of unfractionated lymphoid cell suspensions may impair the ability to detect effector cells that recognize antigens on target cells in a specific manner. Osband and Parkman (1978) detected cells in spleens or normal mice which could lyse syngeneic, but not allogeneic fetal fibroblasts in an overnight assay utilizing 1%-labeled amino acid target cells. To detect these “autocytotoxic” cells, spleen cells were separated by centrifuging the cells through a bovine serum albumin gradient. One fraction, 2B, was not only directly cytotoxic, but caused a local graft-versus-host reaction when injected into the footpads of syngeneic mice. The cells even caused wasting and splenomegaly in syngeneic newborn C57BL/6 mice. The autocytotoxicity was inhibited by a Thy-1 cell in a lighter density band of the gradient. The cytotoxic cell was inhibited by aggregated IgG, suggesting that it expressed an Fc receptor. A similar cell was detected in human peripheral blood (Parkman and Rosen, 1976). The precise nature of that cell has not been determined, but such a cell could complicate the detection of specific recognition of Hh antigens by normal spleen cells. Shearer and Cudkowicz (1975) developed an in vitro assay that appeared to reflect the immunogenetics of hybrid resistance very well. B6 x C3H F, hybrid spleen cells were incubated with irradiated parental strain B6 (H-zb) spleen cells at a 2 : 1responder : stimulator ratio for 5 days. After 5 days, the ability of the cultured cells to lyse H-2b EL-4 cells or other tumor cells was measured in a 4-hour assay. Cytotoxic cells were generated successfully against EL-4 cells, and third-party H-2d tumor cells were not lysed. The immunogenetic analysis indicated that the region of H - 2 recognized by these cytotoxic effectors was H-2DIHh-1. The instructive case was the ability of BIO.A X BlO.A(SR) F, spleen cells to respond to BlO.A(2R)stimulator cells to generate effector cells that lysed EL-4 cells. If B6 X C3H F, mice were injected with lo7 B6 spleen cells three times at weekly intervals to induce unresponsiveness to B6 BMC grafts, the spleen cells of these mice were unable to generate effector cells capable of lysing EL-4 cells. However, the spleen cells were able to repond to H-2 allogeneic spleen cells. These results
+
HYBRID RESISTANCE
395
seemed to confirm their concept that this cell-mediated lympholysis (CML) assay detected hybrid resistance to Hh-lb antigens, and that it detected the induction of specific tolerance. Infant F, hybrid cells failed to respond in this assay, as would be expected if the assay reflected hybrid resistance in uiuo. In a more detailed study (Shearer et al., 1976a,b), this F, anti-parent CML system was compared to an anti-H-2 allogenic CML. The response by B6 x DBA/2 F, splenocytes to parental B6 cells was not quite as rapid or as intense as the response to H-2k C3H cells. Very weak but detectable F, antiparent responses were detected against DBA/2 cells, suggesting that this in uitro system may be more sensitive than the in uiuo assay for hybrid resistance. Mitogen-stimulated normal cells could be used as target cells, and they observed that this CML response was quite dependent upon the batch of fetal calf’ serum used, supporting the idea that it is a relatively weak reaction (or one that is easily inhibited). Carrageenan impaired this F, antiparent CM L response if added before the fourth day of a 5-day assay (Yung and Cudkowicz, 1978). Carrageenan treatment in uiuo suppressed the response to parental cells more than it did to H-2 allogeneic cells. Silica particles had a similar effect (Shearer et aZ., 1976a,b); in that study the mature cytdytic cells themselves were not sensitive to silica. A detailed analysis by Shearer et al. (1977) appeared to indicate that this CML reaction was very similar to hybrid resistance in uiuo at the genetic and biological levels, with the exception that the responding and effector cells were T cells. Cudkowicz (1978) reported that 89Sralso inhibited this CML reaction. There was a parallel loss of hybrid resistance in uiuo and F, anti-parent reactivity in uitro in aging mice (Fitzgerald and Bennett,. 1983a,b). Nakamura and Cudkowicz (1979) observed that parental T cells were required as stimulator cells, but not as target cells in this system. This conclusion was based upon the inability of B10 nude spleen or peritoneal cells to stimulate the response even though similar cells could be lysed by F, antiparent CTI,. F, hybrid anti-parental CML responses to H-2k cells were observed (lshikawa and Dutton, 1979; Warner and Cudkowicz, 1979). A genetic analysis indicated that the genes for the determinants recognized by these F, anti-parental T cells mapped to the H-2KIHh-3 region. These are differences in the immunogenetics of in viuo rejection of H-2k BMC grafts and this F, anti-parent H-2k CML response. In the in uiuo studies, no hybrid resistance was observed, only allogeneic resistance (Cudkowicz and Warner, 1979). The responder status, however, to BMC grafts and to H - 2 k cells in uitm was quite similar, with the following exception: In uiuo the poor responder status was genetically dominant over the good responder status, while the opposite was true in the CML response. In addition to F, anti-parental CML responses to H-2b (Nakano et aZ. 1981a) and H-2k, responses to parental H-2d (Nakamura et al., 1983), H - 2 f
396
MICHAEL B E N N E l T
(Nakano et al., 198lb), H-2dm1, and H-2dm2 (Nakamura et al., 1979) were observed. In the H-2d response, both the H - 2 L and H - 2 D loci had genes for determinants recognized by F, anti-parent cytolytic T lymphocytes (CTL). Another difference, however, was noted between the in vivo and in vitro systems. Cells syngeneic with the F, hybrid responder cells, although resistant to lysis by F, anti-parent CTL, were capable of inhibiting lysis of parental cells, i.e., could act as “cold target cell competitors” (Nakamura et al., 1983; Nakano et al., 1981a). In a recent method for detecting cold target inhibitors of hybrid resistance in vivo (Daley and Nakamura, 1984), F, hybrid H-2 heterozygous tumor cells were not able to inhibit rejection of parental BMC. Two other differences between the in vivo and in uitro systems are notable (Ichiro Nakamura, personal communications): (1)H-2” x H-2” F, mice do not reject homozygous H-2b or H-2” BMC, suggesting that there is a sharing of common Hh-1 determinants; however, spleen cells from similar F, hybrid mice can generate anti-parent CML response to H-2b or H-2“ cells; and (2) H-2b x H-2i are both H-2Db, and yet, H-2b x H-2i F, hybrid mice reject H-2b or H-2i BMC grafts (Bennett, 1972); spleen cells from similar mice do not generate CML responses to parental cells, as if there is a sharing of determinants. Despite the differences between the two systems, the in vitro CML response has correlated better with hybrid resistance in vivo than any other method. Gale and Moran (1979) performed a similar study with human effector cells. Peripheral blood mononuclear cells were incubated with irradiated allogeneic BMC for 5-7 days. The effector cells generated were able to inhibit, in a specific manner, CFU-C function by BMC during a 16-hour incubation period. The F, anti-parent murine effector CTL have not been tested against normal hemopoietic progenitor cells. Bordignon et al. (1985) may have finally developed an in vitro model which reflects hybrid resistance in vivo in every respect. They enriched for NK cells by percoll density centrifugation and incubation of these cells, after irradiation, with parental strain normal BMC for 6-8 hours in a liquid culture. The cell mixture was then assessed for the ability to form colonies in vitro. They were able to detect immunogenetically “correct” hybrid resistance to burst forming units-erythroid (BFU-E), which are primitive erythropoietic precursor cells, and colony-forming units-granulocytic, monocytic (CFU-GM). There was no hybrid resistance detected against CFU-monocytic (CFU-M) or CFU-erythroid (CFU-E). This system allows the analysis of hybrid resistance in vitro, using normal hemopoietic cells. Another in vitro assay for hybrid resistance utilizes purified N K cells which are sorted with the anti-NK-1.1 reagent and propagated in uitro with human recombinant interleukin 2 (rIL-2). This assay has the advantage of using only NK cells as the effectors, but so far only tumor cells have been used as target cells (Murphy et al., 1986).
HYBHIV RESISTANCE
397
V. Genetics of Antigen Expression
One potential explanation of the apparent “recessive inheritance” of Hh antigen expression is the existence of codominant trans-acting regulatory genes. In other words, the detection of Hh-lb antigens on EL-4 tumor cells or C57BL/6 marrow cells reflects the absence of a gene capable of downregulating the expression of Hh-lb antigens. This could explain the requirement for H-2 homozygosity for optimal immunogenicity of BMC grafts (Bennett, 19721. The development of the method to detect Hh-lb antigen expression 011 tumor cells by the ability to inhibit hybrid resistance to BMC grafts (Daley and Nakamura, 1984)allowed us to test this “trans gene hypothesis” (Rembecki et al., 1987a). Potter et al. (1983) were able to select for H-2 antigen loss mutants in a population of Ableson virus-transformed pre-B cell lymphoma cells derived from a BALB/c x BALB. B F, hybrid (H-2“/H-Zb) mouse, called ACCb cells. This was accomplished by treating the cells with monoclonal anti-H-2 reagents and C and plating the cells in agar culture. The surviving cells were stable variants that had lost all or just one or two H-2 molecules. We reasoned that the loss of genes at H-2 of one of the two haplotypes of the heterozygous cells might remove the down-regulation of the Hh-1 antigen expression coded for by genes of the opposite haplotype. The experimental protocol was the following: B6 x DBAI2 F, mice were irradiated and infused with inocula of 5 x lo7 ACCb, ACCb H-2d-, ACCb H-2b-, ACCb Dd-, ACCb Dd-Ld-, EL-4 (H-2b), or P815 (H-2d) cells, 3 hours before inoculating 5 x lo6 B6 BMC. Splenic IUdR uptake 5 days after cell transfer was the measure of growth of BMC. The tumor cells were exposed to 80 Gy gamma rays or to mitomycin C to prevent their proliferation in vivo. The control EL-4 cells inhibited hybrid resistance, P815 cells did not inhibit, and the parental ACCb cells failed to prevent hybrid resistance. However, the H-2d-, Dd-, and Dd- Ld- variant ACCb cells did inhibit rejection of B6 BMC. The H-2b- cells had no effect. Control experiments indicated that all cell types infused reached the spleens ofrecipient mice to the same extent and no large increases in expression of H-2Db antigens were detected on the variant cell lines which lacked Dd, Dd + Ld, or all of H-2d antigens. A summary of the data is presented in Table I. Kaminsky et al. (1986) made similar observations with somatic cell hybrids between H-2b EL-4 cells and H-2k R1 tumor cells. In two of three lines studied, the introduction of H-2k resulted in the down-regulation of expression of Hh-1” antigens. While other explanations of the data are possible, the results are consistent with the idea tested, i.e., codominant genes at or near H - 2 D down-regulate the expression of Hh-1 antigens. If the trans gene hypclthesis proves to be correct, it is conceivable that the mapping of genes responsible for hybrid resistance reflects the positions of these regulatory genes. The structural genes for Hh-1 antigens could be at H-2 or
398
MICHAEL B E N N E l T
TABLE I GROWTHOF BMC DETERMINED BY MEASURING SPLENIC UPTAKE OF IUdR 5 DAYSAFTER IRRADIATION A N D BMC TRANSFER BMC donor B6 B6 B6 B6 B6 B6 B6 B6 B6
Recipient mice
B6 B6 B6 B6 B6 B6 B6 B6 B6
X
X X X X
X X X
DBA/P DBA/2 DBA/2 DBA/2 DBA/2 DBA/2 DBA/P DBA/2
Tumor cells infused None None EL-4 ACCb ACCb ACCb ACCb ACCb P815
Growth of BMC
+++ -
++ -
H-2bH-2dDdDd-Ld-
-
++ ++ ++ -
could be elsewhere. It is possible that the structural genes for all of the Hh antigens detected are present in all members of the species. There are transacting genes which control the temporal development of realization of acid hydrolases; these can be linked or unlinked to the structural genes (Paigen, 1979; Berger et al., 1979; Lusis et al., 1983; Paigen and Jakubowski, 1985). Bl0.RQDB is a new intra-H-2 recombinant inbred strain derived from BlO.A(ZR)H-2h2and BlO.T(GR)H-2y2 parents (Levy et al., 1985). BlO.A(2R) mice are k from K to Ea, are d at S, and are b at D , while BlO.T(GR)mice are q from K to S and are d at D and L. The B1O.RQDB mice are q from K to S, are d at D , but also b at D! The mice do not express Ld antigens. Therefore, the crossover must have occurred between D and L, which is the first instance of such a crossover, and Db is really Lb. There is both serological and biochemical evidence supporting the idea that Db is very much like Ld and that the b haplotype has only one gene in the DIL region (Steinmeftz et al., 1982; Sher et al., 1984; Sun et al., 1985; Hunt et al., 1985). In a collaborative effort with Chella David, we have analyzed the expression of Hh-1 antigens on BMC of the RQDB mice and on BMC ofF, hybrids between RQDB and B6 or BALB/c mice (Rembecki et al., 1987b). If the Hh-1 locus mapped to the right of H-2D, one would expect that RQDB BMC express Hh-lb, but not Hh-ld antigens; the opposite would be expected if Hh-1 mapped to the left of H-2L. RQDB BMC were rejected by B6 hosts, accepted by B6 x DBA/2 F, hosts, and rejected by NZB x B6 F, hosts. This is the pattern expected of BMC, which expressed Hh-ld, but not Hh-lb antigens. BMC grafts from RQDB x B6 F, donors, although homozygous for Db (now Lb) antigens, were not rejected by any of the above host mice. Moreover, RQDB X B6 F, mice
399
HYBRID RESISTANCE
TABLE II ~~
S
C57BL (B6) BALB/c (C) DBA/2 (D2) NZB (NZ) BlO.A(2R) BIO.T(6R) RQDB RQDB X B6
b d d d d 9 s s
RQDB
b s
X
C
Growth in
H-2
Strain/F hybrid donor of BhlC
D
L
B6
B6D2
B6NZ
h d d d
+
-
-
d d d d d d
b d h b
d
b b
d
B6DZU
d b
d b
d
B6NZa
d b
d b
d
d
d
B6D2 = B6
X
DBA/2 F1; BGNZ = B6
-
b d d d b d d
+
+
Null
-
+
-
d
+
+
+
Null
+
+
+
Null
-
-
+
+ + + -
-
+ +
+
-
X
Deduced Hh-l
-
-
NZB FI.
rejected grafts ofB6 BMC. RQDB x BALB/c F, BMC grafts were rejected by B6 and NZB X B6 F, hosts, and were accepted by B6 x DBA/2 recipients, i.e., behaved exactly as RQDB or BALB/c BMC grafts. RQDB X BALB/c F, mice rejected B6 BMC and accepted BALB/c BMC. These results indicate that the crossover occurred between H - 2 L and S. The two parental strain mice, B10 A(2R) and BlO.T(6R), have BMC which express Hh-lb and Hh-ld antigens, respectively. A summary of those results is given in Table 11. These data clearly indicate that Hh-1 maps to the left of L. Since the b haplotype only has one class I antigen coded for by the gene(s) in the D region, it follows that Hh-lb is not a class I antigen. An analysis of many recently derived intra-H-2 recombinant strains of mice has revealed several instances which map Hh-1 determinants to the region between S and D (Bennett et al., 1987). There is a high incidence of crossing over in the region between S and D. Usually the Hh-1 type correlates with the “D side” of the crossover, but has correlated with the “S side” on a number of occasions. If Hh-1 maps between Ea and probably between S and D, what other genes could it be associated with? In a recent review of genes associated with H-2 (Klein et al., 1983), a few candidate genes are mentioned. The G locus
400
MICHAEL B E N N E l T
specified the H-2.7 antigen, which is a C4d fragment detected on erythrocytes. Therefore, this locus no longer exists as such, and the H-2.7 antigen is a product of the S region which codes for class 111 products related to complement components. A locus between S and D was proposed to control the ability of lymphocytes to form rosettes with erythrocytes (Primi et a l . , 1979). O'Neill and Parish (1981) detected antibodies to class I1 antigens by immunizing mice with cells differing only at H - 2 D . The Rfv-l locus controls the recovery from Friend leukemia virus infection and maps at H - 2 D (Chesebro and Wherly, 1974). One of the three resistance genes to Moloney leukemia virus maps between Eb and D (Debre et al., 1979). The association between resistance to viral infection and H - 2 class I loci could be ascribed to H-2-restricted T killer cell lysis of infected target cells (Zinkernagel and Doherty, 1974). The association of murine leukemia virus sequences with the H-2 region (Meruelo et al, 1977, 1984), as well as with minor H loci and genes for lymphocyte differentiation antigens (Meruelo et al., 1983; Rossomando and Meruelo, 1986), is consistent with the idea that retroviruses may either affect differentiation of hemopoietic cells or affect expression of nearby H antigens. The S region of H - 2 contains a number of complement genes (Shreffler and David, 1972; Hansen et al., 1984) over a distance of some 100 kilobases (Campbell and Bentley, 1985; Chaplin, 1985; Ogata, 1985; Tosi et al., 1985). From left to right are the genes for C2 and factor B; after a gap of 40 kb are Slp and 21 hydroxylase A. This is followed after 60 kb by C4 and 21-hydroxylase B. An examination of the C4 and Slp levels of mice of various H-2 types does not suggest any relationship between complement components and Hh-1 antigens. The activity of neuraminidase and other enzymes is regulated by the Neu-1 locus, which maps to the H - 2 region (Womack et al., 1981; Womack and David, 1982; Figueroa et al., 1982; Peters et al., 1981; Samollow et a l . , 1986; Klein et al., 1986). If the Hh-1 antigens are downregulated by codominant genes, as suggested above, enzymes are candidate gene products. B10.RVB mice are o from K through S and are Db. Their Neu-1 phenotype is similar to B10. SM ( H - 2 9 , but their BMC express Hh-1" antigens (R. M . Rembecki, personal communications). Thus, Neu-1 probably is centromeric to Hh-1 in the S-D interval. The murine tumor necrosis factor (TNF) genes a and p, which code for cachectin and lymphotoxin, respectively, map just 70 kilobases centromeric or 5' to H-2D (Muller et al., 1987). Polymorphic restriction sites were detected in the TNF-a gene, using two different enzymes, and H-2d differed from H-2k and H-2b. Human cachectin augments the expression of HLA class I antigens at the cell surface of fibroblasts and endothelial cells (Collins et al., 1986). It is possible that cachectin or lymphotoxin has the ability to inhibit the expression of Hh-1 antigens. In order to regulate the expression
401
HYBRID RESISTANCE
of specific Hh-1 determinants the TNF molecules would have to be polymorphic themselves. VI. Proposed Mechanisms of Antigen Expression and Mechanisms of Rejection
A. A
"TuNS
GENE"MODEL FOR Hh-1 ANTIGEN EXPRESSION
The model described here is based upon the information already summarized in this review and includes some preliminary observations by Rembecki, Bennett, Kumar, and David. The assumptions used to construct the model are (1)the regulatory genes are dominant, trans acting, and map to the Hh-1 locus; (2) the structural genes are present in all strains of inice and may or may not be linked to H-2; (3) the Hh-1 phenotype is directly governed by the presence or absence of the down-regulatory genes; (4)bone marrow, lymphoid, or leukemia/lymphoma cells expressing determinants not present on host cells will be recognized as foreign; (5) rejection will occur or not, depending upon cell inoculum size, responder status of the host for the Hh-1 determined, and presence or absence of conditions conducive to engraftment of Hh-1 incompatible cells (Table 111). Operationally, we determine the Hh-1 phenotype of a new strain using the following criteria: (1)strains A and B with identical Hh-1 antigens will accept grafts from each other and will reject BMC from mice C, which express a different Hh-1 antigen; (2) BMC grafts from mice of strains A and B with identical Hh-1 antigens will exhibit the same pattern of acceptancelrejection when transplanted into a panel of hosts; (3) F, hybrids between strains A and B with identical Hh-1 phenotypes will express that same Hh-1
TABLE 111 Hh-l type"
H-2 types
o (null)
4.r
k I>
k b (z?)
S
s
d
dLp j
.i
Determinants expressed
Regulatory genes ,
1' 1'
(Z?)
1' 1'
2' 2' 2'
3' 3' 3' 3' 3'
None
4'
4' 4' (4'?)
1 1
4 4
2 2 2
3
(4')
H-2Lhas not been tested for determinants 2 or 4 but appears to share determinant 1 with H-2', and H-2s. H-2J has not been tested for determinant 4. Determinant 4 was detected only when using (NZB X B6)FL mice as hosts, which reject H-2k and H-2k/H-2", but riot H-2k X H-2s, H-2q, H-2r, or H-2k X H-2d marrow cells. C3H lymphoid cells may express a similar determinant (Eastcott et al., 1981).
402
MICHAEL BENNETT
antigen on the BMC and will accept parental A or B BMC grafts; (4) F, hybrids between strains A and C with nonidentical Hh-1 antigens will not express the Hh-1 antigens of A or C (exception is determinant 2 i n j x d F,s) and will usually reject parental A or C grafts; (5) rejection of marrow grafts from strains A and B with identical Hh-1 antigens will be inhibited by the infusion of strain A or B Hh-1 antigen + tumor cells (in uiuo cold target cell competition). This model appears to conflict somewhat with the idea that H h - W B , Hh-20 2 , and Hh-NZB loci map 4, 16, or 32 centimorgans to the right of H - 2 (Cudkowicz and Nakamura, 1983). Perhaps genes in those positions affect hybrid resistance, and could even be the structural genes discussed above. Those mapping studies were used to locate the genes regulating hybrid resistance to BMC grafts, whereas the model presented above functions for both allogeneic and hybrid resistance. On balance, Nakamura et al. (1986) did detect Hh-lb antigens on WB x B6 F, cells. If the Hh-1 genes are regulatory genes, what gene products might they “control” or “modify” on the cell surface of stem cells or other progenitor cells? The nearest (genetically speaking) gene products could be class I or class I1 antigens themselves. Singh and and David (1983) suggested that enzymes encoded by genes in the S / D region of H - 2 might alter Ia antigens so as to affect immune functions. Class I, but not class 11, antigens are expressed very strongly on stem cells such that anti-H-2kb antibodies have been used to purify mouse marrow stem cells (Visser and Eliason, 1983; Visser et al., 1984). Therefore, Hh-1 genes could modify class I antigens, perhaps enzymatically (Warner, 1978), so as to prevent their recognition by host effector cells, probably NK cells. O’Neill and Blanden (1979) measured the ability of spleen cells of parental BlO.A(BA) and BlO.A(SR) or F, hybrid mice to absorb anti-H-2 antibodies and the ability of their macrophages to serve as targets for cytolytic T lymphocytes. There was a disproportional loss of expression of H-2Kb and H-2Dd antigens on the F, hybrid cells. Such data are in keeping with the possibility that Hh-1 can d e c t expression of class I antigens. However, preformed anti-class I antibodies and presensitized T cells can lead to rejection of BMC, indicating that class I antigen recognition would have to be different by N K cells. Milisauskas et al. (1986) recently observed that loss of Db antigens from RBL-S tumor cells was not associated with loss of Hh-lb antigens. Other candidates include drug receptors; Byron (1972, 1973) detected P-adrenergic and cholinergic receptors on CFU-S, which regulate the cell cycle status of these cells. Insulin receptors have been associated with class I H-2 and HLA antigens (Edidin, 1986; Due et al., 1986). Therefore, it is conceivable that Hh-1 genes function to affect hormone or drug receptors which may or may not be associated with H-2 antigens. An appealing possibility is that viral gene products constitute the
HYBRID RESISTANCE
403
antigen recognized and that the Hh-1 genes determine the expression of the viral products on stem cells. Pampeno and Meruelo (1986) detected retroviral sequences in the T l a region of H-2; it is conceivable that similar gene products are present throughout the MHC region of mice. These could be acted upon by the Hh-1 regulatory genes. The H - 2 D region can determine the rate of synthesis of Friend leukemia virus by cultured leukemic cells (Bubbers et al., 1977; Freedman et al., 1978). Hyman and Cunningham (1986) described a trans-acting gene which regulates cell surface expression of Thy-1 by controlling transcription. Another possible target for the putative Hh-1 regulatory genes could be the large gl ycoproteins that are thought to constitute the target structures on NK-sensitive cells (Roder et al., 1979; Obexer et al., 1983; Henkart et al., 1986). The ability of laminin to block NK cell function, including binding of NK cells to their targets, suggested that laminin receptors could be target structures on cells recognized by N K cells (Hiserodt et al., 1985). If these surface molecules are modified by Hh-1 gene products, they could be involved in hybrid resistance. The extreme sensitivity of H-2-negative YAC-1, RBL-5, and EL-4 tumor cells to N K cell-mediated rejection (Ljunggren and Karre, 1985; Pointek et al., 1985; Karre et al., 1986) could be explained by a loss of function of the down-regulating trans genes at Hh-1. Perhaps such cells express all H-2controlled Hh-1 determinants (and perhaps other Hh antigens) and are thus much better targets for NK cell-mediated lysis. This could explain why a B6 (H-2b/Hh-lb) mouse might recognize H-2-negative EL-4 cells almost as well as an F, hybrid; those cells would express determinants 2 and 3 as well as determinant 1. In the human system, loss of HLA was associated with an increased sensitivity to N K cell lysis (Hard-Bellan et al., 1986). Bach (1978) maintained that hybrid resistance was governed by density of class I antigens expressed on homozygous versus heterozygous hemopoietic cells.
B. A MISMATCHINGOF H-2 ANTIGEN BETWEEN DONORAND HOST PREVENTS OPTIMALGROWTHOF TRANSPLANTED CELLS When dealing with transplants of hemopoietic cells, even into irradiated recipients, it is hard to ignore the potential for host-versus-graft reactions. Preformed natural antibodies, N K cells, and presensitized T cells can function under those conditions. Nonetheless, it is equally difficult to disregard the data of Hellstrom and Lengerova and their colleagues, which suggest that mismatching of H-2 antigens can lead to poor growth of transplanted bone marrow or leukemia/lymphoma cells. Snell(1976a, b, 1979) envisioned that a mismatch of H-2 antigens between donor stem cell and host effector cell could trigger a destructive reaction by the effector cell. Carlson et al. (1984b) suggested that “cell positioning” of doncr stem cells with respect to
404
MICHAEL BENNETT
host organ stroma or microenvironment could be the basis of poor growth. Karre et al. (1986a,b) conceived the idea that loss of “self” H-2 antigens on tumor cells or a lack of self H-2 antigens on stem cells would “unmask’ cell surface target structures that could be recognized and attacked by NK cells. One way to test Karre’s hypothesis would be to transplant (A x B)F, marrow cells into irradiated C hosts, where A, B, and C are different H-2/Hh-1 types. Several years ago in Boston, Michael Williams and I observed that B10.BR (H-2k) hosts rejected B10 (H-2b) or B10.D2 (H-2d) but accepted (B10 x BlO.D2)F, marrow grafts, using a 4-day 1251UdRassay. This results conflicts with the “non-self’ hypothesis but Lengerova et al. (1973a) have observed rejection of small numbers of H-2 heterozygous marrow cells, using the spleen colony assay after day 8. While writing this review I became more aware of the possibility that T cells might be able to function in marrow allograft rejection, even during the first week after cell transfer. Therefore H-2k/H-213(C3H x B6)F, marrow cells were infused into irradiated H-2d BALB/c or SCID mice, and growth of marrow was assessed by measuring splenic 1251UdRuptake on days 4 or 8. In BALB/c mice, (C3H x B6)F, mice grew without impairment on day 4 but were rejected by day 8. In contrast, the F, cells grew well in SCID mice on both days 4 and 8. These preliminary experiments performed by Bill Murphy do not support the idea that lack of “self’ H-2 antigens is the trigger for rejection of marrow stem cells. Curtis and Rooney (1979) observed that epithelial cells from adult kidneys of mice exhibit contact inhibition when grown in vitro. By mixing cells or different H-2 types, they noted an enhanced degree of contact inhibition, especially if the two cell types differed for class I antigens, i.e., H-2D or H-2K antigens. If a similar phenomenon occurs in spleens or other hemopoietic organs of mice after transfer of stem cells, a deficient cell interaction could lead to “CFU-S repression” (McCulloch and Till, 1963). C. A MODELTO EXPLAINH o w HEMOPOIETIC CELL GRAFTSARE REJECTED The existence of natural antibodies or the induction of antibodies by deliberate or accidental immunization can lead to rejection by targeting stem cells for lysis by NK cells or other cells capable of antibody-dependent cellular cytotoxicity (ADCC). The ability to sensitize T cells in irradiated mice can lead to direct lysis of stem cells by CTL. The mechanism of rejection by these two effective immune systems is straightforward and requires no new model to explain how marrow grafts might be rejected. One should not underestimate the importance of T cell-mediated responses to BMC allografts or the existence of preformed antibodies. The mechanism of rejection of BMC grafts by NK cells without the aid of antibodies has not been determined. Let us propose the idea that NK cells
HYBRID RESISTANCE
405
have specific receptors for Hh-1 and other Hh antigens that are expressed on donor stem cells. There is a limited degree of polymorphism so far detected for Hh-1 antigens. If the model described above is correct, no more than 4 different Hh-1 determinants exist. However, there are Hh-3 and Hh-DBA “minor” antigens and potentially different Hh antigens on normal lymphoid cells. Therefore, N K cells may have clonally distributed anti-Hh-1 receptors. The other alternative is that each NK cell expresses receptors for all Hh antigens of the species. Since boina fide N K cells do not express T cell receptors, N K cells must utilize their own type of receptor for Hh antigens. What triggers the destruction of incoirnpatible stem cells by host NK cells? The findings that antibodies to interferon-a/p inhibit hybrid resistance and that macrophages are necessary for the secretion of interferon (Afifi et al., 1985) indicate that NK cells by themselves are unable to mediate graft rejection. Macrophages or products of macrophages are apparently required to stimulate NK cells in some way that endows them with the ability to reject stem cells. Mice from poor responder strains for a given Hh-1 antigen fail to reject normally, but will reject if their NK cells are stimulated by interferon or interferon inducers. Therefore, an early recognition event must lead, in the good responder mice, to the elaboration of at least interferon by macrophages. The finding that athymic nude mice and SCID mice can reject BMC allografts well reduces the cell types necessary for the response to N K cells and macrophages. ‘The process of rejection does not appear to be a simple, rapid destruction of stem cells by NK cells similar to the lysis of YAC-1 cells. Using the antiasialo GM1 serum which inactivates NK cells immediately in uiuo, Sentman et al. (1987) observed that 24 hours were required for rejection of parental marrow cells, even in mice stimulated with polyinosinic:polycytidylic acid. Hybrid resistance was inhibited even when the antiserum was injected 24 hours after marrow cell infusion. Therefore, the series of events that may occur during hybrid resistance to H-2”IHh-lb BMC by B6 x I>BA/2 F, hybrid mice could be the following:
1. NK cells with anti-Hh-lh receptors recognize BMC expressing Hh-lh and are stimulated in some way, but cannot yet mediate rejection. 2. NK cells so stimulated by recognizing Hh-ll’ antigens interact with syngeneic macrophages, stimulating them to secrete interferon-a/@. This event is obviously not understood, but may well be the critical one that makes a mouse a good responder or a poor responder to a given Hh-1 -incompatible inarrow graft. 3. Macrophages can be stimulated in other ways to secrete interferon, e.g., by the administration of agents that induce interferon secretion by macrophages.
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MICHAEL BENNETT
TABLE IV CELLSURFACE ANTIGENS EXPRESSED BY NK CELLSA N D OTHERPOTENTIALLY RELATED CELL TYPES
NK cells Murine Ly-1 Ly-2
L3T4 Mac-I (CR3) Asido GM1 Thy-1 NK-1.1 NK-2.1 Qa-2 Qa-5 Human CD2 CD3 CD4 CD7 CD8 C D l l (CR3) CD16 Leu 7 Leu 19 Leu M3
-
+ + +I+ + + + +
-
T cells
Monocytes
Granulocytes
+ + + + + + -a -a
+ + + + + + + + -b + +
a Has been identified on some Ly-2+ cell lines with or without non-MHC-restricted cytotoxicity (Brooks et al., 1982a,b). b A rare subset of T cells has been reported to express low-density CD16 antigen (Lanier et a!., 1985).
4. The interferon augments the lytic function by NK cells with specific anti-Hh-lb receptors. Interferon recruits pre-NK cells and enhances the kinetics of lysis (Silva et al., 1980). 5 . The Hh-lb-specific NK cells lyse stem cells which express Hh-lb antigens.
Even though interferon can definitely stimulate NK cells to reject BMC allografts, there are other agents which could have similar activities. Thymosin a-1 synergizes with interferon to stimulate NK cell maturation (Favalli et al., 1985) and function (Henney et al., 1981; Umeda et al., 1983). Interleukin 2 augments NK cell function (Kalland, 198613; Koo and Manyack, 1986), and tumor necrosis serum (Chun et al., 1979) has a distinctive stimulating effect on NK cells. Perhaps tumor necrosis factor can also activate
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TABLE V STRAINDISTRIBUTION OF N K ALLOANTIGENS~ Strains
NK-l.lb
NK-2.1c
C58/J C57BL/6J B1O.A CE/J C57Brl J SJL Ma/My NZB/J SM/J A/J BALB/c C3H/J Ba1b.H-26 CBA/J RF AKR AU LP/J 129/J DBA/2 DBA/1
+ +d + + + + + + +
+ + -
-
+ + + + + + + + + -e + +e
As determined by complement elimination. (C3H X BALB/c)FI anti-CE; C3H anti-CE; CBA anti-CE; (C3H X DBA/2)FI anti-CE CE anti-DBA; (NZB X CE)F1 anti-CBA; (CE X CBA)F1 X CE anti-CBA; NZB anti-BALB/c. Also on B6.H-2k; B6.Lyt-1.1, 2.1, 3.1; B6.PC+; B6.Gix+; B6.Tla; B6.Kl. By FACS analysis, 129/J lacks detectable NK-2.1 (NZB anti-BALB/c) and DBA/1 is positive for both NK-1.1 and NK-2.1 (Pollack and Emmons, 1982. f n t . Not tested. a
N K cells. Wakowiak et al. (1986) described a Thy-l+ Lyt-l+ cell destroyed by 5-flourouracil that appears to participate in hybrid resistance to BMC grafts. The role of this latter cell type and other cytokines/lymphokines in the rejection process have yet to be tested in any detail. The lytic molecules of N K cell:<(Podack and Dennert, 1983; Millard et al., 1984; Zalman et al., 1986; Young and Cohn, 1986) have not been tested for the ability to inactivate stem cells. Lymphotoxin (Williams and Granger, 1969) enhances N K cell-mediated lysis (Ransom and Evans, 1982) and therefore could be involved in !hybrid resistance.
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MICHAEL BENNETT
TABLE VI OF VARIOUS MOUSESTHAINS REJECTIONCAPABILITIES
Strains as hosts B6, B10, B1O.A (ZR), BIO.A (4R) BIO.S B10.D2, BIO.A (5R), BIO.A (3R), B1O.A NZB B10. WB C3H B10.BR B6 X DBA/2, B6 X BIO.A (5R), B6 X BIO.A NZB X B6, NZB x NZW C3H X B6 129 129 X C3H SJL
Their BMC express determinant 4
1 1
2 2 2 2
2
3
1
3
1 1
3
None
1 1
None
1 4
1
4b
1
3 3
4 4
46 1
They can reject BMC expressing determinantsa
2
1
3 3 3
2
2
3 2
4b
4
6
3 NoneC 3 None
a Determinant 1 is common to Hh-1 b, H h - l s , and Hh-12;determinant 2 is common to Hh-l d , H h - l j , and H-2s; determinant 3 is associated with Hh-Y, which also has determinant 2; determinant 4 is common to H - 2 k and H-26. Only NZB X B6 tested for ability to reject BMC expressing determinant 4 only; certain donors not yet tested. 129 mice do resist grafts of H-2Kk/Hh-3k BMC.
D. POSSIBLE MECHANISMS OF REGULATION OF MARROWALLOGRAFTREACTIVITY The observations that athymic nude mice and SCID mice have augmented abilities to reject BMC allografts suggest that in normal mice, T and/or B cells function to regulate that ability. One simple hypothesis is that NK cell function, in general, is augmented in these mice. That is a possibility, but the C.B-17 scid and C. B-17 mice have relatively low NK cell function, even though the frequency of NK cells is increased in spleen cell suspensions of SCID mice (M. Tutt, personal communications). The first indication that T and/or B cells might normally regulate marrow allograft reactivity was our observations that SCID mice rejected large inocula of H-%identical DBAIB BMC (Murphy et al., 1987a). Radiation bone marrow cell chimeras were generated by grafting SCID BMC into irradiated H-2 semiallogeneic B6 x DBAI2 F, mice. Both normal B6 x DBA/2 F, mice and the SCID : B6 x
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TABLE VII STRAINDISTRIBUTION OF “MINOR” Hh ~
Hh antigen(s) Hh-3 (H-2Kk) Hh-DBA Unnamed Unnamed, may be part of H h - l
(H-2Dk)
ANTIGENSO
_____________
Strains
Cell types
C3H, B10.BH CBA DBA/2
Marrow stem cells
DBA/2, DBA/l C3H, CBA C58, C57BR C3H X C57BL
Marrow stem cells, leukemia cells Graft-vs-host cells Graft-vs-host cells
a Cudkowicz and Nakamura (1983) also list H h - W B , H h - 2 , and H h - N Z B as separate loci for antigens recognized by the FI hybrids (strictly hybrid resistance). These are located 4, 16, and 32 units to the right of H - 2 , respectively.
DBA/2 F, chimeras “accepted” grafts of 5 x lo6 DBAI2 BMC (Kumar et al., 1987). An interpretation of that data could be that H-2-restricted T cells of host origin inhibited the ability of SCID marrow-derived NK cells to reject large numbers of DBA/2 BMC. A second observation was even more remarkable. H-2” SCID BMC were used to repopulate irradiated H-2b B6 mice. These chimeras were irradiated and challenged with B6 BMC grafts; the grafts were rejected. The chimeric mice contained radioresistant T cells in that host-type T cells responsive to concanavalin A were present. However, the N K cells were of SCID donor origin. This suggests that T cells must share H-2 antigens with N K cells in order to regulate their function. To test that notion, BALB/c x B6 F, thymocytes were infused into irradiated B6 mice recently injected with SCID BMC. These “mixed” SCID BMC, F, hybrid thymocyte radiation chimeras, when tested, now accepted test grafts of B6 BMC (W. J. Murphy et al., 1987b). This result suggests that F, hybrid T cells can be “tolerized” to Hh-lh antigens and can transfer a “tolerization signal” to SCID NK cells which share the H-2” haplotype. The data can explain the mechanism of tolerizatiori to Hh-lb antigens observed by Cudkowicz (1965) when B10 x BIO.A F, marrow cells were used to repopulate irradiated B10 mice. The observatioiis by Waterfall et al. (1984) that neonatal exposure of F, hybrid mice to parental strain BMC can induce tolerance to Hh-1 antigens and that the tolerance can be transferred by Thy-1+, presumably T cells fit nicely with this idea. If the idea is correct, it suggests that some T cells, probably T
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MICHAEL BENNETT
suppressor cells, have receptors for Hh-1 antigens. The sequence of events could be as follows:
1. B6 X DBA/2 F, T cells are exposed to large amounts of Hh-lb antigens on bone marrow or other hemopoietic cells so as to receive a tolerization stimulus. This can occur by inoculating neonatal mice with parental strain BMC or by using F, BMC to repopulate irradiated B6 recipient mice. 2. The B6 X DBA/2 F, tolerized T suppressor cells can transmit a signal to NK cells with which they share H-2 antigens; i.e., they act in an H-2restricted manner. Which H-2 antigens are the restricting elements has not been determined. 3. The transmission of this signal probably requires the presence of Hh-lb antigens on the BMC used in the test graft. 4. NK cells so affected by the T suppressor cells do not recognize and reject Hh-lb BMC. These observations are very recent and need extensive repetition and further analysis. They at least suggest that NK cells may be under regulatory control by T cells. Any regulatory role of antibodies on NK cells during marrow graft reactions has not yet been tested. Tables IV-VII are intended to summarize data discussed in the review.
ACKNOWLEDGMENTS I want to first thank Ms. Deborah Scott for her heroic task in typing the manuscript and utilizing a computer to assemble all the references in alphabetical order. Robert Strickland guided us through the use of the computer programs. I am in greatest debt to Gustavo Cudkowicz, who was more than an excellent mentor and friend. He was a role model and I have been only able to try to live up to his standards. I with to acknowledge the collaborative help of the following people in studies of hybrid resistance along the way: Richard Steeves, Robert Eckner, Eric Mayhew, Edwin Mirand, and Tin Han when I was in Buffalo; Elinor Levy, John Dittmer, Donna Yonkowski, Pamela Rodday, Joe Vitale, John Lust, Vincent J. Merluzzi, Edgar Cathcart, Morton Scheinberg, Jean Eastcott, Robert Burton, Tom Moran, Eduardo Luevano, Gerald Sonnenfeld, Atul Bhan, Roger Melvold, Henry Kohn, Michael Williams, Pat Fitzgerald, Aoi Masuda, and the late Sidney Cooperband when I was in Boston; and Joan Stein-Streilein, Donald Mann, Bill Kuziel, Phil Tucker, Michael Charley, Anwar Mikhael, Aly El-Hag, Robert Clark, Peter Lipsky, Mary Lipscomb, Gloria Koo, Dorothy Yuan, and Chella David since I have been in Dallas. I wish to especially thank the Graduate Program in Immunology here and graduate students John Hackett, Richard Rembecki, Michelle Tutt, Bill Murphy, and Charles Sentman who have worked in this lab. Discussions of hybrid resistance with Jim Forman have been very helpful. Finally, I wish to pay tribute to my long-term collaborator and friend, Vinay Kumar, whose intelligence, analytic skills, and creativity have been absolutely essential to the progress made in this laboratory since 1973. I am also in debt to my Chairman, Vernie Stembridge, who has created an atmosphere conducive to successful research and scholarly endeavors. My work cited in this article was supported by Grants CA36921 and CA36922 from the National Institute of Health.
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Index
Agretope, T cell subsets and, 53, 54 Alamethicin, cytolysis and, 315 Albumin hybrid resistance and, 392, 394 membrane attack complex of complement and, 300 Alkaline phosphatase B lymphocyte formation and, 186, 213 hybrid resistance and, 380 Allergic reaction, hybrid resistance and, 351 Alloantibodies, hybrid resistance and, 356 Alloantigen B lymphocyte formation and, 190, 193, 194, 197, 198 cytotoxic T lymphocytes and, 135, 136, 169, 170 amino acid, 159, 163 Pz-microglobulin, 156 hybrid resistance and, 333 antibodies, 376 bone marrow cells, 336-340 leukemia/lymphoma cells, 359, 361 lymphoid cells, 354, 356 T cell subsets and cell surface molecules, 44 H-2 molecules in thymus, 99, 107-109 H-%restricting antigen recognition, 63, 70, 74 T cell specificity, 111 Allogeneic inhibition, hybrid resistance and, 334, 358-361, 369 Allogeneic lymphocyte cytotoxicity, hybrid resistance and, 356, 357 Allografts cytotoxic T lymphocytes and, 135 cytotoxicity and, 270 hybrid resistance and antibodies, 379 antigen expression, 404, 405, 408-410 hone marrow cells, 348, 350 effector mechanisms, 370 leukemia/lymphoma cells, 366 lymphoid cells, 355 macrophages, 371, 372 marrow engraftment, 387
A Abelson virus B lymphocyte formation and, 195, 209, 217, 219, 225 hybrid resistance and, 397 Accessory cells B lymphocyte formation and, 220 hybrid resistance and, 350, 373 T cell activation and, 7-14, 21 T cell subs.ets and, 63-66, 69 Accessory molecules, cytotoxic T lymphocytes and, 150-152, 158, 168-170 Acid hydrolysis, hybrid resistance and, 398 Acid phosphatase B lymphocyte formation and, 212, 213 cytolysis and, 281 Acquired imimune deficiency syndrome, hybrid resistance and, 353 Actin cytolysis and, 275 hybrid resistance and, 378 Activated T cells, T cell subsets and, 52-59 Adenovirus, hybrid resistance and, 368 Adhesion B lymphocyte formation and, 185, 235, 237, 239 B cell precursors, 194, 195, 198, 200, 201 bone marrow cultures, 209, 212, 213, 219, 220 genetically determined defects, 228, 231 lymphohempoietic tissue organization. 186-188 population dynamics, 208 soluble mediators, 234 stromal cells, 214-216 cytotoxicity and, 271, 272 hybrid resistance and, 368, 393 Adipocytes, B lymphocyte formation and, 210 Adsorption, hybrid resistance and, 363 Agglutination, hybrid resistance and, 342, 359 447
448
INDEX
marrow microenvironment, 391 N K cells, 372-375, 377 syngeneic stem cells, 389, 390 T cells, 383 T cell subsets and, 43, 92-95 Alloreactivity B lymphocyte formation and, 190 cytotoxic T lymphocytes and, 135-137, 169, 170 amino acid, 159, 162, 163, 165 carbohydrate moieties, 153, 153 exon shuffling, 141, 142, 144-148 monoclonal antibodies, 156-158 Pz-microglobulin, 156 somatic cell class I variants, 167 hybrid resistance and, 357, 372, 380, 381 T cell subsets and, 40 cell surface molecules, 44 H-2 alloantigen recognition, 78-83 H-2 molecules in thymus, 100-102 H-%restricted antigen recognition, 68 Amino acids B lymphocyte formation and, 197, 198 cytolysis and, 285 cytototoxic T lymphocytes and, 135, 170 exon shuffling, 142, 147, 148 H-2 mutant strains, 158-163 HLA class I subtypes, 163-165 HLA subtyptypes, 163-165 mononoclonal antibodies, 157 Pz-microglobulin, 154 somatic cell class I variants, 166, 167 human T lymphocyte activation and, 3, 5, 10, 13 hybrid resistance and, 394 membrane attack complex of complement and, 307 pore formers and, 313-318 T cell subsets and, 85 activated T cells and hybridomas, 54, 56 cell surface molecules, 46, 47 H-2 alloantigen recognition, 83 Amoebas, pore-forming proteins of, 312, 313 Amphiphilic nature of C5b-9, membrane attack complex of complement and, 299304 Anemic cells, hybrid resistance and, 346, 348. 388
Anemic mice, B lymphocyte formation and, 224 Anions, cytolysis and, 282, 312 Antibody B lymphocyte formation and, 182, 183 B cell precursors, 189, 191, 192, 194, 201, 202 bone marrow cultures, 213 Ig genes, 203, 204 lymphohemopoietic tissue organization, 187 cell mediation, 271, 272 cytolysis, 275, 278, 283 cytotoxic T lymphocytes and exon shuffling, 141, 144-146 HLA class I antigens, 150, 151 Pz-microglobulin, 156 monoclonal antibodies, 156-158 hybrid resistance and antigen expression, 400, 402-405 bone marrow cells, 336, 338, 341, 349, 350 effector mechanisms, 370, 376-380 leukemia/lymphoma cells, 359, 365, 368 lymphoid cells, 352, 356, 357 macrophages, 371, 372 marrow engraftment, 386, 387 NK cells, 374 T cells, 383 lymphotoxin and, 298 membrane attack complex of complement and, 301, 302, 307, 309 pore-forming protein, 291, 296 T cell activation and, 6, 7, 9, 11, 13 T cell subsets and, 39, 40 activated T cells and hybridomas, 54,56 cell surface molecules, 49 effector phase, 76 H-2 alloantigen recognition, 83-86, 8993 H-2 molecules in thymus, 97, 98, 105 H-2 restricted recognition of antigen, 50, 52 T accessory molecules, 61, 62 T cell receptor, 40 unprimed and resting T cells, 64,65, 70, 72 Antibody-dependent cell-mediated cytotoxicity (ADCC), 269, 273
449
INDEX
cytolysis and, 275, 277-281, 283 hybrid resistance and bone marrow cells, 336, 338 effector mechanisms, 376-378 lymphoid cells, 351 macrophages, 371 T cells, 383 membrane attack complex of complement, 307 Antigen expression, hybrid resistance and genetics of, 397-401 hemopoietic cell grafts, 404-407 Hh-I, 401-403 marrow allograft reactivity, 408-410 transplanted cells, 403, 404 Antigen-presenting cells (APC) B lymphocyte formation and, 225 human T lymphocyte activation and, 1, 30 accessoty molecules, 14 cell surface molecules, 2 T cell antigen receptor, 2, 5, 7 T cell subsets and H-2 alloantigen recognition, 80, 83, 8892 H-2 molecules in thymus, 100-102, 105, 10;' H-2-restricted antigen recognition, 5356, 58, 60-62, 68,70, 74, 75 T cell specificity, 110, 111 T cell triggering, 111, 112 Antithymocyte serum, hybrid resistance and, 350 Aplasia, hybrid resistance and, 349, 372, 376, 388 Aplastic anemia, 236, 350 Arachidonic acid, hybrid resistance and, 360 Asparagine, cytotoxic T lymphocytes and, 152, 153 ATP cytolysis and, 277 T cell activation and, 16, 18 Autoantibodies B lymphocyte formation and, 227, 228, 231 hybrid resistance and, 379 Autocytotoxic cells, hybrid resistance and, 394 Autoradiography B lymphocyte formation and, 187 hybrid resistance and, 356
B B cell growth factor 11 B lymphocyte formation and, 235, 236 T cell subsets and, 76 B cell precursors, B lymphocyte formation and, 188, 189 cell size changes, 202, 203 cell surface markers, 189 distinctions, 198, 199 functional assays, 199-202 Ly-5 family of glycoproteins, 189-193 markers, 193-195 phosphatidylinositol-linked lymphocyte antigens, 197, 198 technical considerations, 196, 197 tumor cell lines, 195, 196 B cell stimulating factor-1 (BSF-1) B lymphocyte formation and, 236 B cell precursors, 193, 194, 197 genetically determined defects, 231 inducible cell line, 222 soluble mediators, 234, 235 T cell subsets and, 66, 76, 77, 95 B cells cell-mediated killing and, 297 cytolysis and, 273 human T lymphocyte activation and, 29 hybrid resistance and antibodies, 378, 379 antigen expression, 397, 408 leukemia/lymphoma cells, 366 lymphoid cells, 355, 357 marrow engraftment, 387 marrow microenvironment, 392 N K cells, 366 T cell subsets and, 39, 40 cell surface molecules, 42, 45, 46 H-2 alloantigen recognition, 89-91 H-2 molecules in thymus, 99, 100, 102 H-2-restricted recognition of antigen, 51, 54, 68-71, 76, 77 T cell specificity,llZ B lymphocyte formation, 181-185, 235-239 B cell precursors and, 188, 189 cell size changes, 202, 203 cell surface markers, 189 distinctions, 198, 199 functional assays, 199-202
450
INDEX
Ly-5 family of glycoproteins, 189-193 markers, 193-195 phosphatidylinositol-linkedlymphocyte antigens, 197, 198 technical considerations, 196, 197 tumor cell lines, 195, 196 bone marrow cultures and, 208, 209 CBA/N mice, 226 characteristics, 216-218 inducible cell line, 223 lymphocyte adhesion to stromal cells, 214-216 recent innovations, 219, 220 structural organization, 209-214 W/W anemic mice, 224 genetically determined defects, 223, 224 CBA/N mice, 226, 227 C3H/HeJ mice, 230, 231 cyclic neutropenia, 228, 229 moth-eaten mice, 231, 232 NZB mice, 227-229 SCID mice, 224-226 W/W anemic mice, 224 immunoglobulin genes, 203-205 inducible cell line, 220-225 lymphohemopoietic tissue organization, 185-188 population dynamics, 205-208 soluble mediators, 232-235 B lymphocytes, T cell activation and, 22 B lymphoma cells, cytotoxic T lymphocytes and, 149 Bacteria cytotoxicity and, 269, 270 cytolytic proteins, 317, 318 membrane attack complex of complement, 300 hybrid resistance and, 357, 378 Basement membrane, B lymphocyte formation and, 186 Bone marrow B lymphocyte formation and, 183, 184, 235-238 B cell precursors, 191, 193-199, 201, 202 C3HeJ mice, 231 cyclic neutropenia, 229, 230 Ig genes, 204, 205 lymphohemopoietic tissue organization, 186, 187
moth-eaten mice, 231 NZB mice, 227-229 population dynamics, 205-208 SCID mice, 225, 226 soluble mediators, 233, 234 cytotoxicity and, 270 hybrid resistance and, 333, 334 antibodies, 376-378 antigen expression, 397-399, 401-410 effector mechanisms, 370 in oitro assays, 393, 395, 396 leukemia/lymphoma cells, 362, 365, 367, 369 lymphoid cells, 353, 355, 356 marrow engraftment, 384-388 marrow microenvironment, 391 NK cells, 372, 373, 375 normal hemopoetic cells, 335-351 syngeneic stem cell functions, 388, 390 T cells, 380-383 Bone marrow cultures, B lymphocyte formation and, 208, 209 CBA/N mice, 226 characteristics, 216-218 inducible cell line, 223 lymphocyte adhesion to stromal cells, 214-216 recent innovations, 219, 220 structural organization, 209-214 BP-1 marker, B lymphocyte formation and, 193, 217, 225 Bromodeoxyuridine, T cell subsets and, 101, 102 Bursa of Fabricius B lymphocyte formation and, 181, 237 hybrid resistance and, 372 Burst-forming units-erythroid, hybrid resistance and, 396 Bystander cells, cytotoxicity and, 280, 296 Bystander help, T cell subsets and, 76 77
C Cachetin, hybrid resistance and, 400 Calcium cell mediated killing and, 288-290, 292297, 299 cell mediation, nature of, 271-273 cytolysis, 274, 278, 281, 284 cytolytic proteins, 317, 318
INDEX
human T lymphocyte activation and, 31 cell surfatce molecules, 2,12 gene regulation, 27-29 receptor-mediated signal transduction, 19-26 synergy, phobol esters and, 15-19 pore-f0rmin.g proteins and, 317, 318 T cell subsets and, 65 Calf serum B lymphocyte formation and, 209, 216, 218, 21.9, 222, 223 hybrid resistance and, 395 Carbohydrate P B lymphocyte formation and, 197 cytotoxic T lymphocytes and, 138, 152154, 165, 170 Carboxfluorescein, cell-mediated killing and, 290 Carcinoma, hybrid resistance and, 358, 363, 369 Carrageenan, hybrid resistance and, 366, 371-373, 395 Casein, cell-mediated killing and, 297 Cations B lymphocyte formation and, 214, 215 cytolysis and, 280, 311 cDNA cytotoxic T lymphocytes and, 138, 148 cytotoxicity and, 319, 320 cell-mediated killing, 297 hydrolytic enzymes, 276 lymphotcixin-like molecules, 284, 285 pore formers, 317 human T I>rmphocyteantigen and, 3, 10, 13 T cell subsets and, 41, 50 Cell-mediated immunity, hybrid resistance and, 370 Cell-mediated killing, 319, 320 granule prciteins and cell lines, 286, 287 cytoplasnnic granules, 287-291 lymphotoxin, 298 membrane attack complex, 310 proteoglycans, 298, 299 serine esterases, 297, 298 TNF-related polypeptides, 298 pore-forming proteins and amoebas., 312, 313 biochemical properties, 292-295
451
eosinophil cationic protein, 311, 312 membrane binding, 295, 296 purification, 291, 292 Cell-mediated lympholysis hybrid resistance and, 355, 364, 394-396 T cell subsets and, 43, 45, 51, 57, 108 Cell-mediated lysis, hybrid resistance and, 407 Cell surface molecules T cell activation and, 1, 2 accessory molecules, 14, 15 IL-1 receptor, 13, 14 T cell antigen receptor, 2-8 T1, 13 T11, 8-10 Thy-I, 10, 11 Tp44, 11-13 T cell subsets and, 40, 43-48 accessory molecules, 49, 50 T cell receptor, 40-43 Cellulose acetate, hybrid resistance and, 372 C-fos, T cell activation and, 27, 28 Channel formation cytolytic protein and, 318, 319 membrane attack complex of complement, 301-303, 306, 307, 311 Chemotherapy, hybrid resistance and, 350 Chicken ovalbumin, T cell subsets and, 53 Chloroquine, 56, 57, 281 Cholesterol, cytotoxicity and, 279, 300, 302, 317, 318 Chondroitin sulfate A, cell-mediated killing and, 298, 299 Chromatin B lymphocyte formation and, 204, 221 cytolysis and, 283 Chronic myeloid leukemia, B lymphocyte formation and, 188 Cleavage cytolytic protein and, 318 membrane attack complex of complement and, 299, 306 Clones B lymphocyte formation and, 182, 184, 185, 238 B cell precursors, 191, 196, 200, 201 bone marrow cultures, 213, 216, 217, 219, 220 C3H/HeJ mice, 231 Ig genes, 203, 205
452
INDEX
inducible cell line, 220-222 lymphohemopoietic tissue organization. 187 NZB mice, 226, 229 population dynamics, 207 SCID mice, 225, 226 WIW anemic mice, 224 cytolysis and, 274, 276, 280, 281, 285 cytotoxic T lymphocytes and, 167 amino acid changes, 162 carbohydrate moieties, 154 exon shuffling, 138, 142, 144-146, 149 HLA class I antigens, 149-151 Pz-microglobulin, 156 monoclonal antibodies and, 162, 166, 167 cytotoxicity and, 319, 320 granule proteins, 286, 287, 291, 297 nature of mediation, 271, 273 pore formers, 317, 319 hybrid resistance and antibodies, 377 antigen expression, 405 in uitro assays, 394 marrow engraftment, 387 NK cells, 375 T cells, 383 T cell activation and, 1 gene regulation, 27 IL-1 receptor, 13 receptor-mediated signal transduction, 20-22, 24 synergy, 17 T cell antigen receptor, 2, 3, 6, 7 T11, 9 Thy-1, 10 T cell subsets and cell surface molecules, 40, 49. 50 H-2 alloantigen recognition, 79, 87, 94 H-%restricted antigen recognition, 52, 53, 57, 59-61, 64,66, 71, 72, 74, 75 C-myc, T cell activation and, 27, 28 Cognate help, T cell subsets and, 77 Coimmunoprecipitation, T cell activation and, 4, 5 Colchicine, cytolysis and, 282 Cold target cell competitors, hybrid resistance and, 396, 402
Colicins, cytolysis and, 318, 319 Colloid osmotic killing membrane attack complex of complement, 300 membrane damage, 280 pore formers, 315, 318 pore-forming protein, 295, 312 Colony-forming cells B lymphocytes and, 182, 191, 200, 230, 237 hybrid resistance and bone marrow cells, 336, 344 leukemia/lymphoma cells, 359 NK cells, 376 T cells, 381, 382 Colony-forming unit, hybrid resistance and, 336, 396 Colony-stimulating factor B lymphocyte formation and, 219, 228230, 234, 236, 237 hybrid resistance and, 382 Complement-mediated cytotoxicity, see Cytotoxicity, lymphocyte and complement-mediated Complementarity-determining regions, T cell subsets and, 52 Complete Freund’s adjuvant, T cell subsets and, 70, 71 Concanavalin A B lymphocyte formation and, 198, 229 hybrid resistance and, 375, 409 pore-forming protein and, 313 T cell activation and, 8, 28 T cell subsets and, 63-65 Concanavalin A-activated spleen cells, T cell subsets and, 64,65, 70 Conjugate formation cell mediation and, 272 cytolysis and, 274-276, 278, 282, 283 Corticosteroids, B lymphocyte formation and, 197 Cortisone, hybrid resistance and, 338, 359, 360 Corynebacterium parvum B lymphocyte formation and, 208 hybrid resistance and bone marrows, 340, 347 macrophages, 370 marrow microenvironment, 391, 392 syngeneic stem cell functions, 389
INDEX
Cross-linkage cell mediation and, 272 T cell activation and, 4, 5, 9, 11, 12, 25 T cell subsets and, 62, 111 Cross-priming, T cell subsets and, 74 Cross-reactivity lymphocytes and, 144, 147, cytotoxic ’I 162, 170 hybrid resistance and, 357 membrane attack complex of complement and, 307, 309 T cell subsets and, 79, 88,112 Cyclic neutropenia, B lymphocyte formation and, 222, 228-230, 234, 237 Cyclophosphamide, hybrid resistance and bone mamow cells, 347, 348 effector mechanisms, 370, 388, 390, 393 leukemia/lymphoma cells, 366, 368 lymphoid cells, 354 Cyclosporin, 60, 350 Cyclosporin .4, T cell activation and, 29, 30 Cysteine cytotoxic I’lymphocytes and, 148, 163 membrane attack complex of complement and, 307, 309 Cytochalasin B, cytolysis and, 282 Cytochrome c, T cell subsets and, 53 Cytolysin, cell-mediated killing and, 291 Cytolysis af27 polarity, 273-275 cell mediation, 272 colloid osmotic killing, 280 cytoplasmic granules, 287, 288, 291 granule exocytosis, 281, 282 hydrolytic enzymes, 273-275 intracellular damage, 282-284 leukoregulin, 286 lymphotoxin-like molecules, 284-286 membrane attack complex of complement, 299, 300, 310 membrane damage, 277-281 polypeptide toxins, 316-319 pore-forming proteins amoebas, 312, 313 cytolytic proteins, 316-319 eosinopliil cationic protein, 311, 312 polypeptide toxins, 316-319 small peptides, 313-316 reactive oxygen metabolism intermediates, !286
453
Cytolytic T cell clones, 9, 12, 14 Cytolytic T cells, 365, 367, 368 Cytolytic T lymphocytes, hybrid resistance and, 364, 380, 395, 396, 402, 404 Cytoplasmic granules, cytotoxicity and, 281, 287-291 Cytosol, T cell activation and, 17, 22 Cytotoxic T cells, hybrid resistance and, 385 Cytotoxic T lymphocyte cell lines cell-mediated killing and, 287-289, 291, 297 cytolysis and, 273 Cytotoxic T lymphocyte differentiation factor, T cell subsets and, 64, 76 Cytotoxic T lymphocytes, 269, 270 granule exocytosis, 281 granule proteins, 286, 287 cytoplasmic granules, 287 pore-forming protein, 291, 296, 297 serine esterase, 297, 298 hydrolytic enzymes, 275-277 intracellular damage, 283 lymphotoxin-like molecules, 284, 285 mediation, 270-273 polarity, 273-275 recognition by MHC molecules, 135-138 carbohydrate moieties, 152-154 exon shuffling, 138-149 H-2 mutant strains, 158-163 HLA class I antigens, 149-152 HLA subtypes, 163-165 Pz-Microglobulin, 154-156 monoclonal antibodies, 156-158 somatic cell class I variants, 165-167 T cell subsets and, 39, 51 activated T cells and hybridomas, 54, 56, 57 alloreactivity, 78, 81, 82 effector phase, 75, 92, 94, 95 H-2 molecules in thymus, 97, 99-103, 108 resting T cell subsets, 83-88 T accessory molecules, 60 unprimed and resting T cells, 65, 71-75 Cytotoxicity hybrid resistance and, 359, 361, 362, 366, 372, 393, 394 lymphocyte and complement-mediated, 269, 270, 319, 320 at27 polarity, 273-275
454
INDEX
colloid osmotic killing, 280 CTL cells, 270-273 granule exocytosis, 281, 282 hydrolytic enzymes, 275-277 intracellular damage, 282-284 leukoregulin, 286 lymphotoxin-like molecules, 284-286 membrane attack complex of complement, 299-307 membrane damage, 277-281 NK cells, 270-273 pore-forming proteins, 311-319 reactive oxygen metabolism intermediates, 286 Cytotoxins, 269, 270
D Degranulation, cytotoxicity and, 282, 296, 297, 312 Delayed hypersensitivity, hybrid resistance and, 383 Delayed-type hypersensitivity, T cell subsets and, 76, 77, 94 Deletion B lymphocyte formation and, 203 cytotoxic T lymphocytes and, 139, 167 Dendritic cells, T cell subsets and H-2 alloantigen recognition, 89-92 H-2 molecules in thymus, 103-106, 108110 H-2-restricted antigen recognition, 74, 75, 77 Deoxyguanosine (dGuo), T cell subsets and, 104, 106, 108 Deoxynucleotidyltransferase, B lymphocyte formation and, 194, 203, 204, 220, 231 Depolarization cell-mediated killing and, 273 pore-forming protein and, 293, 294 Desotope, T cell subsets and, 53, 58 Dexamethasone, hybrid resistance and, 387 Dexter’s culture system, B lymphocyte formation and . bone marrow, 208-210, 213, 215-217, 219, 220 W/W anemic mice, 224 Diacylglycerol, T cell activation and, 15-19, 30 Diamphotoxin, cytolysis and, 318
Differentiation B lymphocyte formation and, 181, 235237, 239 B cell precursors, 188, 189, 191-192, 194, 195, 197, 199-201 bone marrow cultures, 210, 212, 214218, 220 genetically determined defects, 223226, 230, 231 inducible cell line, 220, 221, 223 lymphohemopoietic tissues, 185, 187 population dynamics, 206, 207 soluble mediators, 232, 235 cytolysis and, 270 hybrid resistance and antigen expression, 400 bone marrow cells, 336 leukemia/lymphoma cells, 369 marrow microenvironment, 390, 392 NK cells, 372, 373 syngeneic stem cells, 388 T cells, 381 T cell activation and, 22 T cell subsets and H-2 molecules in thymus, 98-102, 104, 105, 107, 108 H-2-restricted antigen recognition, 52, 64,66, 76, 77 DNA B lymphocyte formation and, 183, 184, 203, 217, 235 cell-mediated killing and, 298 cytolysis and, 277, 283-285 hybrid resistance and, 343, 377, 378 T cell activation and, 29 T cell subsets and, 41, 44, 46-48, 78 Dog leukocyte antigen, hybrid resistance and, 349, 350
E Effector cells cell-mediated killing and, 296 cytolysis and, 274, 275 granule exocytosis, 281 hydrolytic enzymes, 276 intracellular damage, 283, 284 membrane damage, 277 cytotoxic T lymphocytes and, 148, 150, 170
INDEX
cytotoxicit) and, 269, 270, 272 hybrid resistance and, 334, 370 antibodies, 376, 378, 379 antigen expression, 402, 403 bone marrow cells, 346 in uitro :assays, 393-396 leukemia/lymphoma cells, 359, 366, 368 lymphoid cells, 353 macrophages, 371 marrow imicroenvironment, 390 NK cells, 372, 373 syngeneic stem cells, 390 T cells, 382-384 Effector mechanisms of hybrid resistance, 369, 370 antibodies, 376-380 in oitro assays, 393-396 macrophages, 370-372 marrow engraftment, 384-388 marrow microenvironment, 390-393 NK cells, 2872-376 syngeneic stem cell functons, 388-390 T cells, 380-384 Effector phase, T cell subsets and, 75-77, 92-95 Electron microscopy B lymphocyte formation and, 212 cell-mediated killing and, 292, 294, 295 cytolysis arid, 282 membrane attack complex of complement and, 300, 306 T cell subsrets and, 106 Electron-spin resonance, membrane attack complex of complement and, 303 Embryo, B lymphocyte formation and, 181, 193, 199, 201, 224, 227 Endocytosis, cell-mediated killing and, 298 Endothelid cells B lymphocyte formation and, 186, 222225, 235, 236 hybrid resistance and, 400 T cell subsets and, 46, 90 Endotoxin, B lymphocyte formation and, 224, 230 Eosinophil cationic protein, 311, 312 Eosinophil-derived neurotoxin, 311 Eosinophils, Icytotoxicity and, 269, 270, 291, 312 Epithelial cells hybrid resistance and, 335
455
T cell activation and, 1, 10 T cell subsets and cell surface molecules, 46 restricted T cells, 103-106 T cell development, 96-98 T cell specificity, 113 tolerance induction, 107-100 Epitopes B lymphocyte formation and, 191, 192 cytotoxic T lymphocytes and, 138, 169, 170 amino acid changes, 159, 162, 163 carbohydrate moieties, 152, 154 exon shuffling, 139-142, 144, 145, 147 HLA class I antigens, 150, 151 Pz-Microglobulin, 155 monoclonal antibodies, 156-158, 165167 T cell activation and, 1, 9, 11, 20 T cell subsets and cell surface molecules, 44, 47 H-2 alloantigen recognition, 79, 80, 83 H-%restricted antigen recognition, 53, 54, 56, 62 T cell specificity, 111, 112 Epstein-Barr virus, cytotoxic T lymphocytes and, 144, 165, 167 Erythroblasts, hybrid resistance and, 376 Erythrocytes B lymphocyte formation and, 189, 208 cell-mediated killing and, 289, 290, 293, 295 cytolysis and, 277, 278, 280 hybrid resistance and, 337, 376, 386, 388, 400 membrane attack complex of complement and, 299-301, 303, 304, 306 pore formers and, 314, 316, 317, 319 Erythroid cells, B lymphocyte formation and, 186, 213 Erythroleukemia, hybrid resistance and, 336, 372, 378 Erythropoiesis, hybrid resistance and, 336, 337, 339, 348 Erythropoietin, hybrid resistance and, 348 Escherichia coli, 302, 318 Esterase B lymphocyte formation and, 212, 213 cell-mediated killing and, 287 Estradiol, hybrid resistance and, 374, 389392
456
INDEX
Exogenous help, T cell subsets and, 84, 85, 87, 112 Exon shuffling, CTL and, 144-148, 169 altered cytoplasmic regions, 148, 149 class I gene transfection, 138, 139 CTL recognition, 141-144 monoclonal antibodies, 156, 165 serology, 139-141
marrow engraftment, 387 NK cells, 376 Friend spleen focus-forming virus, hybrid resistance and, 389 Friend virus, 168, 378, 389 Fungi, cytotoxicity and, 269, 270
F
G protein, CTL and, 154 Gene dosage, hybrid resistance and, 357 Glucose, membrane attack complex of complement and, 300 Glutaraldehyde, T cell subsets and, 53 Glycans, CTL and, 152, 153 Glycogen, cell-mediated killing and, 287 Glycolipid, membrane attack complex of complement and, 301 Glycoprotein B lymphocyte formation and, 189-193, 227 CTL and, 152 human T lymphocyte activation and, 2, 4, 8 hybrid resistance and, 378, 403 T cell subsets and, 43, 44, 46, 49 Glycosylation B lymphocytes and, 190, 192, 215 CTLand, 135, 138, 152, 153 cytotoxicity and, 272 T cell subsets and, 41, 50 Golgi apparatus cytolysis and, 275, 281, 282 T cell subsets and, 57 Graft, hybrid resistance and antigen expression, 397, 399, 401, 402, 404-407, 410 bone marrow cells, 336-351 effector mechanisms antibodies, 376, 377 in oitro assays, 393, 395 macrophages, 371 marrow, 384-388, 390 NK cells, 373-375 syngeneic stem cell functions, 388, 390 leukemia/lymphoma cells, 362, 364-367 lymphoid cells, 352-355 Graft rejection, hybrid resistance and antigen expression, 404-407 bone marrow cells, 344, 350
Fc receptors B lymphocyte formation and, 194 cytotoxicity and, 269, 271, 279, 296 hybrid resistance and, 371, 376-378, 394 T cell activation and, 9 Fibroblasts B lymphocyte formation and, 235, 236, 238 genetically determined defects, 230 Ig genes, 205 lymphohemopoietic tissue organization, 186, 188 CTL and, 145, 150-153 hybrid resistance and antigen expression, 400 in vitro assays, 393, 394 leukemia/lymphoma cells, 359, 361 NK cells, 372 T cell activation and, 10 T cell subsets and, 46, 89-91, 94 Fibronectin, B lymphocyte formation and, 215 Fibrosarcoma CTL and, 45 hybrid resistance and, 359, 361, 363 Fluorescein isothiocyanate, CTL and, 166, 167 Fluorescence B lymphocyte formation and, 196 cell-mediated killing and, 298 Fluorescene-activated cell sorter, T cell subsets and, 84 Friend erythroleukemia, hybrid resistance and, 389 Friend leukemia virus, hybrid resistance and antigen expression, 400, 403 hone marrow cells, 345 lymphoid cells, 354
G
INDEX
effector mechanisms, 370, 371 antibodies, 376, 378 marrow engraftment, 385, 388 NK cells, 375 syngeneic stem cells, 390 Graft-versus-host disease (GVHD), T cell subsets and cell surface molecules, 43 H-2 alloantigen recognition, 83, 84, 86, 88, 92, 95 H-&-restricted antigen recognition, 71, 72 Graft-versus-lhost reaction, hybrid resistance and antigen expression, 403 bone marrow cells, 335, 336, 340, 350 in vitro assays, 394 leukemia/lymphoma cells, 360, 367 lymphoid cells, 351-356 marrow engraftment, 385-387 T cells, 380 Gramicidin A , cytolysis and, 316 G r a d e exocytosis, cytotoxicity and, 273, 281, 282 Granule proteins, cell-mediated killing and cell lines, 286, 287 cytoplasmic granules, 287-291 lyrnphotoxin, 298 membrane attack complex of complement, 310 pore-forming protein, 291-297 proteoglycans, 298, 299 serine esterases, 297, 298 TNF-related polypeptides, 298 Granulocytes B lymphocytes and, 238 B cell piocursors, 191, 195 bone marrow cultures, 209, 210, 219 geneticallly determined defects, 229, 230 cell-mediated killing and, 312 hybrid resistance and, 336, 370, 380 Granulopoiesis, hybrid resistance and, 336
H H-2 moleculm alloantigen recognition and, 78 alloreact ivity, 78-83 antigen-presenting cells, 88-92 effector phase, 92-95 resting T cell subsets, 83-88
457
antigen recognition and, 51, 52 effector phase, 75-77 T accessory molecule function, 59-62 triggering of activated T cells and hybridomas, 52-59 triggering of unprimed and resting T cells, 62-75 B lymphocyte formation and, 213 CTL and, 136, 170 carbohydrate moieties, 152-154 exon shuffling, 138-148 HLA class I antigens, 149, 152 Pz-Microglobulin, 155, 156 monoclonal antibodies, 156, 157, 166, 167 mutant strains, 158-163, 165 cytotoxicity and, 269, 279-281 hybrid resistance and antibodies, 376-378 antigen expression, 397-404 bone marrow cells, 336-348 in oitro assays, 395, 396 leukemia/lymphoma cells, 358-369 lymphoid cells, 352-354 marrow engraftment, 386-388 NK cells, 375, 376 syngeneic stem cells, 390 T cells, 381-383 T cell subsets and, 40, 43-48 thymus and, 95, 96 development, 96-99 restricted T cells, 99-107 tolerance induction, 107-110 Haplotype CTL and, 159 hybrid resistance and antigen expression, 397-399, 409 bone marrow cells, 341, 343 leukemia/lymphoma cells, 364 lymphoid cells, 353 T cell subsets and, 47, 48, 78, 79, 102 Helper T cells CTL and, 136, 137 cytolysis and, 276 Hemagglutinin, T cell subsets and, 57 Hemolysis, cell-mediated cytolytic proteins and, 317, 318 cytoplasmic granules, 288-290 membrane attack complex of complement and, 299, 305, 306, 309
458
INDEX
membrane damage and, 279 pore-forming proteins and, 291, 293, 295, 296 proteoglycans and, 299 Hemopoiesis, B lymphocyte formation and, 181, 182, 235-238 B cell precursors, 188, 191, 196, 197, 200, 201 bone marrow cultures, 208-210, 212, 214, 215, 217, 219, 220 genetically determined defects, 224, 225, 228, 229, 231, 232 inducible cell line, 220, 221, 223 lymphohemopoietic tissues, 185-188 soluble mediators, 233 Hemopoietic cells, hybrid resistance and antigen expression, 400, 403, 405-407, 410 bone marrow cells, 335-351 effector mechanisms, 369, 370 in witso assays, 396 macropbages, 371, 372 marrow engraftment, 385-388 marrow microenvironment, 391, 392 NK cells, 373, 374, 376 syngeneic stem cell functions, 388-390 T cells, 380 leukemia/lymphoma cells, 361, 363 Hemopoietin 1, B lymphocyte formation and, 234 Heparin cell-mediated killing and, 295, 299 hybrid resistance and, 371 Hepatocytes, T cell activation and, 16 Herpes simplex virus, 153, 375, 376 Heterogeneity B lymphocyte formation and, 196, 216, 217 CTL and, 138, 139, 152 cytoxicity and cell-mediated killing, 312 mediation, 271 membrane attack complex of complement, 305, 306, 309, 310 hybrid resistance and, 372 T cell activation and, 27 T cell subsets and, 43, 79, 98 Histocompatibility antigens (HA) H-2 alloantigen recognition and, 82, 87 H-2 molecules in thymus and, 99, 100
H-%restricted antigen recognition and, 51, 57, 71-75 hybrid resistance and, 333, 334, 350 Histiocytes, hybrid resistance and, 335 Histotope, T cell subsets and, 53, 58 Homeostasis, B lymphocyte formation and, 185, 208, 236 Homogeneity B lymphocyte formation and, 221, 228 cytotoxicity and, 270, 284, 287, 300 hybrid resistance and, 338, 339 T lymphocyte activation and, 13 Homology CTL and, 136 exon shuffling, 140-142, 144-146 &-Microglobulin, 154, 155 monoclonal antibodies, 156, 158 cytotoxicity and, 285, 307, 309, 314, 316 human T lymphocyte activation and, 3 accessory molecules, 14 gene regulation, 29 synergy, 17 T cell antigen receptor, 4 thy-ll,lO hybrid resistance and, 335 T cell subsets and, 41, 46, 49, 50 Hormones B lymphocyte formation and, 196, 229 hybrid resistance and, 402 pore formation and, 314 Horse red blood cells, T cell subsets and, 68 Horse serum, B lymphocyte formation and, 209, 219 Human leukocyte antigen (HLA) CTL and, 168, 170 amino acid, 163-165 carbohydrate moieties, 153 exon shuffling, 138, 139, 141, 142, 144 Pz-Microglobulin, 155, 156 monoclonal antibodies, 158, 165, 167 transfected cells, 149-152 cytotoxicity and, 270 hybrid resistance and, 350, 369, 400403 Hybrid histocompatibility antigens antigen expression, 397-399, 401, 405407, 409, 410 trans gene model, 401-403 bone marrow cells, 340-343, 345, 347
459
INDEX
genetics of expression, 397 in oitro assays, 394-396
leukemia/lymphoma cells, 360-367 lymphoid cells, 352, 353 marrow engraftment, 385, 386 Hybrid hyperreactivity, 360, 379 Hybrid resistance, 333, 334 antigen expression genetics, 397-401 hemopoietic cell grafts, 404-407 Hh-1, 401-403 marrow allograft reactivity, 408-410 transplated cells, 403, 404 effector mechanisms, 369, 370 antibodies, 376-380 in oitro assays, 393-396 macrophages, 370-372 marrow engraftment, 384-388 marrow microenvironment, 390-393 N K cells, 372-376 syngentic stem cell functions, 388-390 T cells, 380-384 hemopoietic cells hone m.arrow cells, 335-351 lymphord cells, 351-358 leukemia/ lymphoma cells, 358-369 Hybridization cytotoxicity and, 276, 288 T cell subsets and, 41, 100, 101 Hybridomas B lymphocyte formation and, 223, 225, 237 CTL and, 150, 168, 169 T cell activation and, 1, 23, 25 T cell subsets and H-2 alkiantigen recognition, 79, 82, 91 H-2-restricted antigen recognition, 5260, 62, 70 T cell receptor, 41, 42 T cell triggering, 111 Hybrids B lymphocyte formation and, 199, 226 CTL and, 169 exon shuffling, 139, 142, 144, 145, 147 HLA class I antigens, 150, 151 &-Microglobulin, 156 monoclonal antibodies, 158 Hydrocortisone, hybrid resistance and, 387, 390 Hydrolysis, T cell activation and, 15, 16, 23, 25, 30
Hydrolytic enzymes, cytolysis and, 275-277, 281 Hydrophobic domains human T lymphocyte activation and, 5, 13, 25 T cell subsets and, 45, 54 Hydrophobicity B lymphocyte formation and, 197, 198 cytotoxicity and cell-mediated killing, 295 cytolysis, 279, 280 membrane attack complex of complement, 299-302 pore formers, 314-317 Hypersensitivity, B lymphocyte formation and, 183
1 Immune response genes hybrid resistance and, 361 T cell subsets and, 44, 51, 54, 55 Immunodeficiency, B lymphocyte formation and, 199, 200, 207, 216 genetically determined defects, 224-226, 231 Immunodepression, hybrid resistance and, 385 Immunofluorescence B lymphocyte formation and, 188, 221 cytotoxicity and, 275, 298 Immunoglobulin B lymphocyte formation and, 182, 183, 237, 238 B cell precursors, 188, 189, 191, 193195, 199-202 bone marrow cultures, 209, 217, 218 CBA/N mice, 226 inducible cell line, 221, 223 lymphohemopoietic tissue organization, 187 population dynamics, 206, 207 SCID mice, 225 cell-mediated killing and, 291 CTL and, 168 hybrid resistance and, 357, 365, 366, 378, 379 Immunoglobulin G B lymphocyte formation and, 223, 237 cytotoxicity and, 271
460
INDEX
hybrid resistance and, 336, 355, 357 effector mechanisms, 376-378, 394 Immunoglobulin genes human T lymphocyte antigen and, 3, 10 T cell subsets and, 40, 41, 46, 52 Immunoglobulin H, B lymphocyte formation and, 198 Immunoglobulin M B lymphocyte formation and, 183 B cell presursors, 194, 200 inducible cell line, 221, 222 population dynamics, 206 soluble mediators, 234 hybrid resistance and, 336, 377, 378 Immunoglobulin receptors, T cell subsets and, 77 Immunoprecipitation B lymphocyte formation and, 221 T cell activation and, 19 Immunosuppression, hybrid resistance and, 340, 353, 354, 386-388 Inflammatory response B lymphocyte formation and, 208, 237 lymphoid cells and, 354 Influenza virus CTL and, 136, 168 amino acid, 165 exon shuffling, 144, 146 HLA class I antigens, 151, 152 monoclonal antibodies, 151, 166, 167 hybrid resistance and, 375 Inhibition, see also Allogeneic inhibition B lymphocyte formation and, 236 B cell precursors, 91, 92 bone marrow cultures, 216 inducible cell line, 222, 223 CTL and, 168, 169 amino acid, 162 carbohydrate moieties, 152-154 exon shuffling, 139 HLA class I antigens, 149-151 monoclonal antibodies, 157, 158 cytotoxicity and cell-mediated killing, 295, 296 cytolysis, 276, 281, 282 cytolytic proteins, 317 membrane attack complex of complement, 309 hybrid resistance and antibodies, 378
antigen expression, 397, 402, 404, 409 bone marrow cells, 337-340, 347, 348 effector mechanisms, 370 in vitro assays, 394-396 leukemia/lymphoma cells, 358, 364, 367 lymphoid cells, 351, 352, 354, 356 macrophages, 371, 372 marrow engraftment, 387 marrow microenvironment, 390-392 N K cells, 374-376 syngeneic stem cells, 388, 390 T cells, 381, 382 T cell activation and, 9, 22, 26 T cell subsets and H-2 alloantigen recognition, 79, 82, 85, 86, 90,91 H-2 molecules in thymus, 109, 110 H-%restricted antigen recognition, 60, 61, 74, 76 Inositol triphosphate, T cell activation and, 15, 16, 21, 24, 26, 30 Insulin B lymphocyte formation and, 197 hybrid resistance and, 402 Interdigitating cells, hybrid resistance and, 356, 357 Interferon B lymphocyte formation and, 228, 234, 265 hybrid resistance and, 368, 377 antigen expression, 405, 406 bone marrow cells, 345 macrophages, 371, 372 marrow microenvironment, 392 syngeneic stem cells, 388-390 Interferon-? B lymphocyte formation and, 197, 222, 230 cytotoxicity and, 272, 284 hyman T lymphocyte antigen and, 6, 11, 27-29 hybrid resistance and, 371 T cell subsets and H-2 alloantigen recognition, 89, 90, 94, 95 H-2 antigen recognition, 64, 66, 69, 76, 77 H-2 molecules in thymus, 106 Interleukin- 1 B lymphocyte formation and, 192, 208, 222, 236, 237
461
INDEX
genetically determined defects, 228, 230 soluble mediators, 234, 235 cell-mediated killing and, 288 T cell activation and cell surface molecules, 2, 8, 13, 14 receptor-mediated signal transduction, 20 22, 23, 26 T cell subsets and, 63-66, 84, 85, 107, 111 In terleukin-2 B lymphocyte formation and, 193, 228, 237 cell-mediated killing and, 287, 288 human T lymphocyte activation and, 1, 6, 11-14 gene regulation, 27-300 receptor-mediated signal transduction, 22 25 synergy, 15, 19 T11, 9, 10 hybrid resistance and, 351, 380, 382, 396 T cell subsets and H-2 alloantigen recognition, 87, 93-95 H-2 molecules in thymus, 98, 99, 107 H-2-restricted antigen recognition, 52, 53, 56, 62-66, 72-74, 76, 77 T cell triggering, 111, 112 Interleukin-3 B lymphocyte formation and, 228-230, 232-234, 236, 237 hybrid resistance and, 389 Inulin, cytotoxicity and, 304, 306 Iodo-2’-deouynridine (IUdR), hybrid resistance and antigen eupression, 397, 404 bone marrow cells, 340, 342, 345, 347 effector mechanisms, 370 leukernia/’lymphorna cells, 364-368 lymphoid cells, 352, 354 T cells, 380 Ionomycin, T cell subsets and, 65, 98 Irradiation, hybrid resistance and antigen eupression, 397, 403, 404, 408410 bone marrow cells, 339, 344, 346-348, 350 effector mechanisms, 369, 370 antibodies, 376, 377, 379 in uitro assays, 394, 396
marrow, 384-386, 388, 390 NK cells, 373-375 syngeneic stem cells, 389, 390 T cells, 380-384 leukemia/lymphoma cells, 358, 359, 361, 363-367 lymphoid cells, 352, 354-357
K Keratinocytes, B lymphocyte formation and, 234 Keyhole limpet hemocyanin, T cell subsets and, 70, 71 Kidney CTL and, 149 hybrid resistance and, 338, 348, 356, 404
1 L cells B lymphocyte formation and, 198 CTL and amino acid, 163 exon shuffling, 138-140, 144, 147, 148 HLA class I antigens, 149-152 Laminin, hybrid resistance and, 403 Langerhans cells, T cell subsets and, 69, 92, 94 Large granular lymphocytes cytotoxicity and, 271, 287, 289, 291, 309 hybrid resistance and, 376, 380 Lectin B lymphocyte formation and, 201, 210 CTL and, 150 human T lymphocyte activation and, 7-9, 12, 28 T cell subsets and, 60, 76 Lesions, cytotoxicity and, 288-291, 305307, 318 Leucine, CTL and, 166 Leukemia B lymphocyte formation and, 185, 191, 193, 205, 220, 233 hybrid resistance and, 358-369 antibodies, 378, 380 antigen expression, 401, 403 bone marrow cells, 346, 348 Leukocytes B lymphocyte formation and, 192, 194, 198, 229
462
INDEX
cytotoxicity and, 270, 312 hybrid resistance and, 349, 350 T cell subsets and, 108 Leukopenia, hybrid resistance and, 388 Leukoregulin, cytolysis and, 286 Leukotrienes, hybrid resistance and, 360 LFA CTL and, 136, 139, 150-152, 154 cytotoxicity and, 271, 272 LFA-1, B lymphocyte formation and, 194 Ligand CTL and, 168, 169 carbohydrate moieties, 154 exon shuffling, 139, 142, 147 HLA class I antigens, 149-152 monoclonal antibodies, 158 cytotoxicity and, 307 T cell activation and, 30 cell surface molecules, 2, 7, 8, 10-13 gene regulation, 28, 30 receptor-mediated signal transduction, 20, 22 synergy, 17 T cell subsets and, 40, 50-53 Ligand-receptor interactions, T cell activation and, 1, 26 Lineage fidelity, B lymphocyte formation and, 195 Lipid B lymphocyte formation and, 197, 213 cell-mediated killing and, 290, 291, 294296, 312, 313 CTL and, 169 cytolysis and, 275, 278-280 membrane attack complex of complement and, 299-303, 305, 306 pore formers, 315, 317, 318 Lipopolysaccharide B lymphocyte formation and, 196, 200, 221, 222, 231 pore-forming protein and, 313 Liposomes CTL and, 154 cytotoxicity and cytolytic proteins, 319 membrane attack complex of complement, 300, 302, 305, 309 pore-forming proteins, 312, 313 Liver B lymphocyte formation and, 181, 182, 224, 225
B cell precursors, 191, 193 bone marrow cultures, 216 Ig genes, 204 lymphohemopoietic tissue organization, 187 population dynamics, 207 CTL and, 149 hybrid resistance and, 335, 351, 356, 363, 366, 373, 386 T cell subsets and, 67, 69 Liver cell grafts, hybrid resistance and, 333 Localization, B lymphocyte formation and, 195, 196 Low density lipoprotein B lymphocyte formation and, 212, 213 cytotoxicity and, 295, -307, 309 Lung, hybrid resistance and, 356, 366, 373 Lymph nodes B lymphocyte formation and, 191, 223 hybrid resistance and, 359, 384, 393 bone marrow cells, 337, 338 lymphoid cells, 352, 353, 355, 356 NK cells, 372 T cells, 381 T cell subsets and H-2 alloantigen recognition, 78, 92 H-2 molecules in thymus, 99 H-%restricted antigen recognition, 6771 Lymphoblasts, CTL and, 139 Lymphochoriomeningitis virus, hybrid resistance and, 355, 390 Lymphocytes complement-mediated cytotoxicity and, see Cytoxicity, lymphocyte and complement-mediated hybrid resistance and antigen expression, 400 bone marrow cells, 348, 350 leukemia/lymphoma cells, 361, 364 lymphoid cells, 353, 355-357 marrow, 387, 390 T cells, 380 T cell subsets and, 70, 81, 110 Lymphohematopoietic cells, T cell subsets and, 107-109 Lymphohemopoietic tissue organization, B lymphocyte formation and, 185-188 Lymphoid cells B lymphocyte formation and, 196, 213, 220, 225, 226
463
INDEX
hybrid resistance and, 333 antibodies, 376, 380 antigen expression, 401, 405 hone marrow cells, 335, 338, 339 in uitro assays, 393, 394 leukemia/lymphoma cells, 359, 369 marrow engraftment, 386 NK cells, 374, 375 normal hemopoietic cells, 351-358 T cells, 380, 383 T cell subsets and, 44, 78, 90, 96, 101, 109 Lymphokines B lymphocyte formation and, 199, 222, 231 cytolysis and, 284, 286 human T lymphocyte antigen and, 1, 6, 20 gene regulation, 27, 29 synergy, 15 T11, 9, 10 hybrid resistance and, 407 T cell subsets and H-2 alloantigen recognition, 94 H-2 molecules in thymus, 107 H-2-restricted antigen recognition, 52, 62--66, 76, 77 T cell trigger, 112 Lympholysis, hybrid resistance and, 388 Lymphoma cells B lymphocyte formation and, 202 cell-mediated killing and, 293 hybrid resistance and, 334, 358-369 antigen expression, 401, 403 NK cellk, 373, 374 Ly mphopoiesis B lymphocyte formation and, 213, 229231, 237, 238 hybrid resistance and, 339 Lymphoproliferation, B lymphocyte formation and, 193, 208 Lymphosarcoma cells, hybrid resistance and, 358 Lymphotoxin B lymphocyte formation and, 236 cytotoxicity and, 284-286, 298 hybrid re:jistance and, 400, 407 Lymphotoxin-like molecules, cytolysis and, 284-286 Lysine, cell-mediated killing and, 297
Lysosomes CTL and, 168 cytotoxicity and, 276, 281, 288
M Macrophages B lymphocyte formation and, 186, 208, 234-236, 238 B cell precursors, 191, 192, 194, 195 bone marrow cultures, 212, 213, 218 genetically determined defects, 230232 cytotoxicity and, 269, 285, 293 hybrid resistance and, 340, 356, 366 antigen expression, 402, 405 effector mechanisms, 370-372, 374, 387, 392, 393 T cell subsets and cell surface molecules, 46 H-2 alloantigen recognition, 89, 90,94 H-2 molecules in thymus, 103-106, 108-110 H-2-restricted antigen recognition, 51, 69-71, 74, 75, 77 Magnesium, cytotoxicity and, 271, 294 Major basic protein, cell-mediated killing and, 311, 312 Major histocompatibility complex (MHC) B lymphocyte formation and, 194, 197 cytotoxicity and, 270 human T lymchocyte activation and, 2, 3, 5, 14 hybrid resistance and, 348, 357, 361, 369, 378, 403 T cell subsets and, 39, 71, 78, 81, 101, 108 cell surface molecules, 43, 44, 50 class I molecules, cytotoxic T lymphocytes and, see Cytotoxic T lymphocytes Marrow engraftment, hybrid resistance and hemopoietic cells, 386-388 specific unresponsiveness, 384-386 Marrow microenvironment, hybrid resistance and, 390-393 Mast cell tumors, hybrid resistance and, 361 Melittin, pore formation and, 313-316 Membrane attack complex of complement amphiphilic nature of C5b-9, 299-304 analogues, 307-311 subunit composition, 304-307
464
INDEX
Membrane-bound IL-1, T cell subsets and, 90 Membrane damage cytolysis and, 277-281 pore formation and cytolytic proteins, 316-319 small peptides, 313-316 Metaphase, B lymphocyte formation and, 206 Methotrexate serum, hybrid resistance and, 350 Micelle, cytotoxicity and, 303 &-Microglobulin B lymphocyte formation and, 198 CTL and, 135, 138, 165, 170 exon shuffling, 138, 139, 147 HLA class I antigens, 149, 150 T cell recognition, 154-156 T cell subsets and, 44, 75 Microtubule organizing center (MTOC), cytolysis and, 275, 282 Mitochondria, cell-mediated killing and, 288 Mitogen B lymphocyte formation and, 192, 200, 228, 231 cytotoxicity and, 272, 284, 285 human T lymphocyte activation and, 7, 8, 11, 12, 26, 27 hybrid resistance and, 350, 393, 395 T cell subsets and, 60, 63-66, 111 Mitosis, B lymphocyte formation and, 184 Mixed-lymphocyte reaction, T cell subsets and cell surface molecules, 43 H-2 alloantigen recognition, 81, 83-87 APC, 88-92 H-2 molecules in thymus, 107, 108 Moloney leukemia virus, 364, 400 Moloney sarcoma-leukemia virus, 153 Moloney sarcoma virus, 378 Monensin, 281 Monoclonal antibodies B lymphocyte formation and, 181, 182, 235, 238 B cell precursors, 188, 189 bone marrow cultures, 213 cell size changes, 202 functional assays, 200, 201 Ig genes, 204 Ly-5 family of glycoproteins, 189, 192
lymphomohemopoietic tissues, 187 markers, 193, 194 NZB mice, 227 PI-linked lymphocyte antigens, 198 SCID mice, 225 tumor cell lines, 195 CTL and, 169, 170 amino acid, 163 blocking, 156-158 exon shuffling, 140, 141, 145, 148 HLA class I antigens, 150 Pz-Microglobulin, 155, 156 somatic cell class I variants, 165-167 cytotoxicity and, 285, 291, 307, 309 hybrid resistance and, 336, 374, 377, 378, 386 T cell activation and, 30 accessory molecules, 14 cell surface molecules, 1, 2 gene regulation, 28 IL-1 receptor, 14 receptor-mediated signal transduction, 19-22, 24-26 T cell antigen receptor, 2, 4-8 T1, 13 T11, 9, 10 Thy-1, 11 Tp44,ll T cell subsets and H-2 alloantigen recognition, 79, 84, 89, 93 H-2-restricted antigen recognition, 53 T cell receptor, 40, 43 Monocytopenia, hybrid resistance and, 392 Morphology B lymphocyte formation and, 182, 186, 204, 213, 219, 238 cytotoxicity and, 271 cytolysis, 274, 276, 281-283 cytolytic proteins, 317 granule proteins, 287, 291, 294, 295 membrane attack complex of complement, 300, 304, 305, 309, 311 mRNA CTL and, 148 T cell activation and, 28 T cell subsets and cell surface molecules, 41-43, 45, 47, 50 H-2 molecules in thymus, 97, 99
465
INDEX
Murine cytomegalovirus, hybrid resistance and, 3.59 Murine hepatitis virus, hybrid resistance and, 375, 376 Murine system, T lymphocyte activation and, 1 cell surface molecules, 10, 11, 13, 14 receptor- mediated signal transduction, 23, 25 Murine T cells, 64,79 Mutation B lymphocyte formation and, 185 B cell precursors, 195, 198 genetically determined defects, 224, 22!6, 228, 231, 232 CTL and, 138, 169, 170 amino acid, 158-163, 165 carbohydrate moieties, 153, 154 exon shuffling, 141, 142, 145, 148 &-Microglobulin, 155 monoclonal antibodies, 156, 157, 165, 166 hybrid resistance and, 343, 368, 374, 397 T cell activation and, 5, 8, 12, 20 T cell subsets and, 42, 46 H-2 alloantigen recognition, 81, 82, 85, 86, 88, 93 Myeloid cells B lymph,ocyte formation and, 237 B cell precursors, 189-191 bone marrow cultures, 209, 210, 212, 2113, 219, 220 cyclic neutropenia, 229, 230 inducible cell line, 220 lymphohemopoietic tissue organization, 87 NZB mice, 229 SCID mice, 225 soluble mediators, 232, 233 W/W anemic mice, 224 cytolysis and, 285 hybrid resistance and, 373, 376 Myeloma cells B lymphocyte formation and, 220 hybrid resistance and, 378 Myelopoiesis, B lymphocyte formation and, 186, 187, 213, 219 Myosin cytolysis and, 275 hybrid resistance and, 378
N Natural cytotoxic cells, hybrid resistance and, 373, 374 Natural killer cells B lymphocyte formation and, 192, 199, 225, 231, 236, 237 cytotoxicity and, 269-273 cytolysis, 275-277, 280-286 granule proteins, 286, 287, 289, 291, 297, 298 membrane attack complex of complement and, 309 hybrid resistance and, 334 antibodies, 376-380 antigen expression, 402-410 bone marrow cells, 336, 338, 340, 351 effector mechanisms, 372-376 leukemia/lymphoma cells, 361, 363-369 lymphoid cells, 354, 357 macrophages, 371, 372 marrow engraftment, 386-388 marrow microenvironment, 390-393 syngeneic stem cell functions, 388-390 T cells, 382 T cell activation and, 9, 10, 24 T cell subsets and, 49 Natural killer cytotoxic factor, 285, 296 Natural resistance, hybrid resistance and, 334 Neoplasia, hybrid resistance and, 387 Neuraminidase hybrid resistance and, 400 T cell subsets and, 64,91 Neutropenia, hybrid resistance and, 376 Neutrophil B lymphocyte formation and, 229, 230 cytotoxicity and, 269 New antigenic determinants, T cell subsets and, 51 Newcastle disease virus, 166, 167, 389 Nucleotide B lymphocyte formation and, 191, 194, 197, 203 CTL and, 159 T cell activation and, 18, 26 T cell subsets and, 42, 43, 46, 47
0 Oligomerization, cytotoxicity and, 306, 315, 317, 318
466
INDEX
Oligosaccharides, CTL and, 135, 152, 153 Osteopetrosis B lymphocyte formation and, 232 hybrid resistance and, 374, 390, 392, 393 Ovalbumin CTL and, 167, 168 T cell subsets and, 55, 70
P Parasites, cytotoxicity and, 269 Peanut agglutinin, T cell subsets and, 97 Peptide B lymphocyte formation and, 215 CTL and, 136, 137, 167-169 amino acid, 162 exon shuffling, 148 HLA antigens, 152 cytotoxicity and, 279, 280, 309, 313 pore formation and, 313-319 T cell subsets and, 41, 47, 110 H-%restricted antigen recognition, 5255, 57, 58, 74, 75, 77 T lymphocyte activation and, 3 Perforin, cytotoxicity and, 280, 281, 291, 305, 307-309 Phagocytosis B lymphocyte formation and, 212, 213 hybrid resistance and, 340, 357, 370 pore-forming proteins and, 313 Phenotype B lymphocyte formation and B cell precursors, 192, 201 Ig genes, 204, 205 lymphohemopoietic tissues, 187 NZB mice, 227, 229 W/W anemic mice, 224 cytotoxicity and, 273, 287 hybrid resistance and, 338, 374, 382, 400, 401 T cell subsets and cell surface molecules, 49, 50 H-2 alloantigen recognition, 84, 87, 88 H-2 molecules in thymus, 96 H-%restricted antigen recognition, 60, 69, 73 T lymphocyte activation and, 1, 7, 28 Phenylalanine, CTL and, 162, 166 Phorbol esters B lymphocyte formation and, 222 cell-mediated killing and, 14-19, 24, 26
Phorbol myristic acetate, T cell activation and, 8, 9, 11, 12, 14, 19 gene regulation, 28, 30 receptor-mediated signal transduction, 20, 22, 23, 25 Phosphates CTL and, 153 T cell activation and, 16, 24 Phosphatidylcholine, cytotoxicity and, 275, 276 Phosphatidylinositol, B lymphocyte formation and, 197, 198, 202, 222, 239 bone marrow cultures, 215, 216 Phosphatidylinositol hiphosphate, T cell activation and, 15, 16, 23, 25 Phosphatidylinositol-specific phospholipase, B lymphocyte formation and, 198, 214, 215 Phospholipase B lymphocyte formation and, 198, 216, 239 cytotoxicity and, 300, 314 Phospholipase A, cytolysis and, 275, 276 Phospholipase Az, pore formers and, 314316 Phospholipid cytotoxicity and, 276, 300, 302, 303 pore formation, 314, 315, 318 T cell activation and, 15-19 Phosphorylation CTL and, 135, 148 cytotoxicity and, 273 T lymphocyte activation and, 4, 16, 18, 19, 23, 24 Phytohemagglutinin (PHA) B lymphocyte formation and, 198 CTL and, 50 hybrid resistance and, 359, 361 T cell activation and, 8, 14, 22, 26-28 T cell subsets and, 60, 63 Pinocytosis, B lymphocte formation and, 212, 213 Plaque-forming cells, hybrid resistance and, 352 Plasmacytonias, hybrid resistance and, 367, 368 Polarity cytolytic mechanisms and, 273-275, 282 T cell subsets and, 41 Polyinosinic: polycytidylic acid, hybrid resistance and, 371-372, 388-390
INDEX
Polymerization cell-mediated killing and, 290, 292-295, 298 membrane attack complex of complement and, 304, 305, 309, 311 Polymorphism B lymphocyte formation and, 193, 197, 223, 224, 229 CTL and, 135-137, 169 amino acid, 158-163, 165 exon shuffling, 141, 142 HLA class I antigens, 151 monoclonal antibodies, 158, 165 hybrid resistance and, 367, 400, 401, 405 T cell subsets and cell surface molecules, 41, 44-47 H-2 alloantigen recognition, 182, 183 H-2-restricted antigen recognition, 58, 59, 62 Polypeptides B lymphocyte formation and, 190 cell-mediated killing and, 291, 292, 298, 313 CTLand, 168 cytolysis and, 276, 279, 285 cytotoxicity and, 309, 319 pore formers and, 316, 317 T cell activation and, 10, 17, 23 T cell subsets and, 41, 43, 50, 51, 75 Polyperforin 1, cytotoxicity and, 280, 289 Polyperforin 2, cytotoxicity and, 280 Polyribosomf:s, B lymphocyte formation and, 1828 Poly-2-vinylpyridine-N-oxide (PVNO), hybrid resistance and, 371 Population dynamics, B lymphocyte formation and, 205-208 Pore formation, cytotoxicity and, 313-320 Pore-forming protein (PFP), cytotoxicity and, 269, 270, 319 cell-mediated killing, 291-299 cytolytic, 311-319 membrane attack complex of complement, 299, 305, 307-309, 311 Potassium cytotoxicity and, 294, 318 Priming hybrid resistance and, 370 T cell subsets and, 72, 74, 100 Procarbazine serum, hybrid resistance and, 350
467
Proliferation B lymphocyte formation and, 183, 184, 186, 187 B cell precursors, 192, 200, 202 bone marrow cultures, 210, 212 genetically determined defects, 225, 229, 231, 232, 234 inducible cell line, 220 population dynamics, 205-207 cytotoxicity and, 269 hybrid resistance and, 384, 390, 391, 397 bone marrow cells, 335, 336, 338, 340, 349 lymphoid cells, 352, 353 T cell subsets and H-2 alloantigen recognition, 85, 86, 88, 92 H-2-restricted recognition of antigen, 51, 52, 60, 62-66, 70, 72 T lymphocyte activation and, 1, 6, 7, 9, 10, 12-15, 26 Promonocytes, hybrid resistance and, 371 Promyelocytic leukemia cells, hybrid resistance and, 348 Pros taglandin B lymphocyte formation and, 208, 236 hybrid resistance and, 360, 387 Proteases, cytotoxicity and, 282, 298 Protein B lymphocyte formation and, 205, 216, 239 B cell precursors, 191, 197, 198 inducible cell lines, 221, 222 CTL and, 135, 148, 153, 154, 159, 165, 168 cytotoxicity and, 269, 270, 320 cell-mediated killing, 289-295, 297, 311, 312 cytolysis, 277-281, 316-319 mediation, 270-273 membrane attack of complement and, 299, 301, 303, 307, 309-311 small peptides, 314 human T lymphocyte activation and, 1, 4, 8, 18, 23 gene regulation, 29, 30 IL-I receptor, 13, 14 hybrid resistance and, 393 T cell subsets and, 90,97, 99 cell surface molecules, 45-47, 50
468
INDEX
H-2-restriction antigen recognition, 52, 54, 56-58, 62, 70, 74, 75 T cell receptor, 41-43 Protein kinase C B lymphocyte formation and, 222 cell-mediated killing and, 296, 297 T cell activation and, 8, 31 Ca2+, 15-19 gene regulation, 28-30 receptor-mediated signal transduction, 19, 20, 23, 25, 26 T cell subsets and, 65 Proteoglycans, cell-mediated killing and, 298 Proteoliposomes, cytotoxicity and, 274, 278, 303 Proteolysis cytotoxicity and, 275, 276, 301, 305 T cell activation and, 18 T cell subsets and, 53
R Radioactivity, hybrid resistance and, 349 Radiautoautography, B lymphocyte formation and, 205 Radioresistance, hybrid resistance and, 365, 376, 380, 383, 390, 393, 409 Radiosensitivity, hybrid resistance and, 350, 368 Reactive oxygen metabolism intermediates, cytolysis and, 286 Receptor-mediated signal transduction, T cell activation and, 19-26 Red blood cells, hybrid resistance and, 337, 342, 349, 351, 352, 359 Replication, B lymphocyte formation and, 183-185, 236 B cell precursors, 200 bone marrow cultures, 210, 213, 216, 217 genetically determined defects, 228, 231 population dynamics, 206, 207 soluble mediators, 232-234 Retrovirus B lymphocyte formation and, 195, 233 hybrid resistance and, 378, 400, 403 RNA B lymphocyte formation and, 191, 198, 22 1 hybrid resistance and, 377, 80 T cell activation and, 27
s Sarcomas, hybrid resistance and, 358, 359, 363 Salt, cytotoxicity and, 290, 301 Saccharomyces cereuisiae, cytolysis and, 319 Second messengers B lymphocyte formation and, 197 T cell antigen receptor and, 2, 26, 27 Serine, CTL and, 135, 148 Serine esterases, cytotoxicity and, 267, 288, 297, 298, 310 Serine proteinases, cytotoxicity and, 277, 297, 310 Serology, CTL and, 139-141 Severe combined immunodeficiency disease (SCID) B lymphocyte formation and, 224-226, 237 hybrid resistance and, 351, 365, 366, 379381, 384 antigen expression, 404, 405, 408, 409 Sheep red blood cells cytotoxicity and, 293 hybrid resistance and, 352 T cell subsets and, 67-71, 77, 90, 92 Silica, hybrid resistance and, 350, 368, 371373, 395 Skin allografts, T cell subsets and, 86, 88, 93 Skin graft B lymphocyte formation and, 225 CTL and, 158 hybrid resistance and, 369, 376, 383 Skin graft rejection CTL and, 159, 165 T cell subsets and, 71, 88, 93, 94 Somatic hypermutation, T cell subsets and, 42, 88, 106 Specific unresponsiveness, hybrid resistance and, 384-386 Spleen B lymphocyte formation and, 181, 182, 184, 238 B cell precursors, 198, 200-202 genetically determined defects, 229 inducible cell line, 223 population dynamics, 207, 208 CTL and, 138 hybrid resistance and, 334 antigen expression, 397, 402, 404, 408
469
INDEX
bone marrow cells, 335, 336, 338-340, 342, 345, 347, 348 effector mechanisms, 370 in vitro assays, 394-396 leukemia/lymphoma cells, 358, 361-368 lymphojid cells, 351-356 marrow, 384-388, 391, 393 NK cells, 372, 373, 375 syngeneic stem cells, 388-390 T cells, 380-384 T cell subsets and H-2 akiantigen recognition, 78, 86, 89, 91, 92 H-2 molecules in thymus, 95, 102 H-2-restricted antigen recognition, 67, 69, 72 Spleen colony formation, hybrid resistance and antigen expression, 402, 404 bone marrow cells, 337, 347 syngeneic stem cells, 388-390 T cells, 362 Staphylococcal LX toxin, cytolysis and, 317 Staphylococcal &toxin, cytolysis and, 315, 316 Stem cells B lymphocyte formation and, 181, 182, 184, .187, 206, 237 B cell precursors, 188, 191, 195, 199, 200 bone marrow cultures, 209, 217, 219 genetically determined defects, 220, 224, 225 soluble mediators, 232-234 hybrid resistance and, 352, 362, 367 antigen expression, 402-407 bone m.arrow cells, 336-340, 344, 346 effector mechanisms, 370-373, 377380, 383, 386, 388-391 Steroids B lymphocyte formation and, 209, 219, 236, 238 hybrid resistance and, 348, 387 Streptolysin 0, 317, 318 Stroma, hybrid resistance and, 336, 404 Stromal cell!;, B lymphocyte formation and, 184, 185, 237, 238 B cell precursors, 198, 200, 203 bone marrow cultures, 209, 210, 212-216, 219, 220
genetically determined defects, 227, 229, 231, 232, inducible cell lines, 223 lymphohemopoietic tissue organization, 186-188 soluble mediators, 234 Subclones B lymphocyte formation and, 210 CTL and, 139 Sucrose, cytotoxicity and, 290, 304 Supergenes, cytotoxicity and, 310 Suppression, hybrid resistance and, 380, 381, 384, 387, 389, 395 Supressor cells, hybrid resistance and, 340, 368, 410 marrow, 385-387, 391, 392 Suppressor T cells, T cell subsets and, 102, 107, 110 Synergy B lymphocyte formation and, 222, 234 cytotoxicity and, 284, 296 hybrid resistance and, 406 T cell activation and, 20, 23, 28, 30 Ca2+ ionophores, 15-19 cell surface molecules, 2, 12, 14 Syngeneic preference, hybrid resistance and, 334, 358 Syngeneic stem cell functions, hybrid resistance and, 388-390
T T cell B lymphocyte formation and, 236 B cell precursors, 189, 191, 192, 198, 199 genetically determined defects, 226, 227, 230, 231 Ig genes, 204 soluble mediators, 233-235 CTL and, 165 cytotoxicity and, 271-273, 277, 297 hybrid resistance and, 337, 364366, 368 antigen expression, 402-405, 408-410 effector mechanisms, 372-374, 376388, 392, 395 T cell activation, 1, 30, 31 cell surface molecules, 1-15 gene regulation, 26-30 intracellular signals, 26
470
INDEX
receptor-mediated signal transduction, 19-26 synergy, 15-19 T cell antigen receptor, 2-8 T cell receptor B lymphocyte formation and, 194, 199, 225, 226, 237 CTL and, 136, 137, 139, 152, 167, 168, 170 cytotoxicity and, 271, 272, 274 T cell subsets and, 40-43, 46, 111 H-2 alloantigen recognition, 79-81, 88 H-2 molecules in thymus, 97, 99, 106, 107 H-2-restricted antigen recognition, 5154, 56, 60-65 T cell subsets in mouse, 39, 110-13 cell surface molecules, 40-50 H-2 alloantigen recognition, 78 alloreactivity, 78-83 antigen-presenting cells, 88-92 effector phase, 92-95 resting T cells subsets, 83-88 H-2 molecules in thymus, 95, 96 development, 96-99 restricted T cells, 99-107 tolerance induction, 107-110 H-2-restricted antigen recognition, 51, 52 effector phase, 75-77 T accessory molecule function, 59-62 triggering of activated T cells and hybridomas, 52-59 triggering of unprimed and resting T cells, 62-75 T helper cells cytotoxicity and, 274, 297 T cell subsets and, 40 H-2 molecules in thymus, 101-103, 105 H-%restricted antigen recognition, 67, 70-72, 75, 77 T killer cells cytolysis and, 280 hybrid resistance and, 382, 383, 400 T cell subsets and, 40, 54 T lymphocytes B lymphocyte formation and, 205, 212, 225 hybrid resistance and, 353, 364 T cell activation and, 17, 19, 21, 22 T cell subsets and, 44
T suppressor cells, hybrid resistance and, 379, 386, 410 Target cells CTL and, 137, 158, 162, 168 carbohydrate moieties, 153, 154 exon shuffling, 142, 144, 149 HLA class I antigens, 149-152 cytotoxicity and, 269-272, 300, 319, 320 cell-mediated killing, 294, 296-298, 312 cytolysis, 273-284 hybrid resistance and, 363, 369, 372, 373, 383 antibodies, 376-378 antigen expression, 400, 404 in vitro assays, 393-396 marrow, 387, 391, 392 T cell subsets and, 76, 94, 95 T lymphocyte activation and, 1, 14, 30 Thiol-activated lysins, 317, 318 Thoracic duct lymphocytes, hybrid resistance and, 356-358 Thymocytes B lymphocyte formation and, 198, 199 cytolysis and, 276, 279 hybrid resistance and, 337, 376, 409 T cell subsets and, 96-98, 105-108, 112 T lymphocyte activation and, 4, 8, 10, 24, 25 Thymus B lymphocyte formation and, 191, 223, 236-238 bone marrow cultures, 212, 214 genetically determined defects, 225227, 231 population dynamics, 207, 208 hybrid resistance and, 337, 352 effector mechanisms, 372-374, 384, 390-392 T cell subsets and, 43, 44, 112 H-2 molecules, 95--10 H-2-restricted antigen recognition, 51, 59, 69 Tolerance induction hybrid resistance and, 356, 361, 409 T cell subsets and, 107-110 Total lymphoid irradiation, hybrid resistance and, 386, 387 Transcription B lymphocyte formation and, 192, 198, 204, 205, 221, 222, 226
471
INDEX
cytotoxiciiy and, 271, 276, 277, 297 hybrid resistance and, 380, 403 T cell subsets and, 41, 43, 48, 97 T lympho'cyte activation and, 3, 27-31 Transfection B lymphocyte formation and, 198, 205 CTL and, 138, 139, 144, 149-152, 163 cytotoxicity and, 272 T cell subsets and, 58, 59, 62, 80, 83 T lymphocyte activation and, 3, 5, 8, 11, 25 Transferrin, B lymphocyte formation and, 193, 212 Translocation, T cell activation and, 17-19, 22, 23 Transmembrane channel, cell-mediated kill ing and, 298 Transmembrane domain, CTL and, 135, 146-148 Trypsin, 2101, 227, 297, 298 Tubulin, 275, 378 Tumor B lymphocyte formation and, 186, 204, 230, 233, 237 B cell precursors, 188, 193, 194, 196 bone m.irrow cultures, 214, 217 inducible cell line, 220, 221 lineage fidelity, 195, 196 CTL and, 135, 138, 139, 154 cytotoxicity and, 270, 284-286, 293, 313 hybrid resistance and, 338 antigen expression, 397, 402-404 effector mechanisms, 375, 378, 380, 382, 387, 393, 394, 396 leukemia/lymphoma cells, 358-369 T cell activation and, 6 T cell sub4jets and, 74, 91
Tumor necrosis factor, 235-237, 285, 298, 374, 400, 401 Tumor necrosis serum, 406 Tyrosine, CTL and, 135, 148, 162
V Venous sinuses, B lymphocyte formation and, 187, 214, 236 Vesicular stomatitis virus, 142, 148, 153, 154, 163, 168 Virus B lymphocyte formation and, 209 CTL and, 135, 142, 153, 154, 165 cytotoxicity and, 269, 270 hybrid resistance and, 375, 378, 390, 400, 402, 403 T cell subsets and, 54, 56, 57, 74-76, 101
W White blood cells, hybrid resistance and, 349, 356 Whitlock-Witte cultures, B lymphocyte formation and, 209, 216-220, 231
Y Yeast, cytotoxicity and, 269, 270, 319
Z Zinc, cell-mediated killing and, 293, 294
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CONTENTS OF RECENT VOLUMES
Volume 3’1
Protein A of Staphylococcus aureus and Related immunoglobulin Receptors Produced by Streptococci and Pneumonacocci JOHN J. LANGONE
The Regulatory Role of Macrophoges in Antigenic Stirnulotion Part Two: Symbiotic Relationship between Lymphocytes and Macrophoges E M I L R. U N A N U E
Regulation of Immunity to the Azobenzenearsonote Hapten MARKI. GREENE, MITCHELL J . NELLES, MAN-SUNS Y , A N D ALFRED NISONOFF
T-cell Growth Factor and the Culture of Cloned Functional T Cells KENDALLA. SMITH A N D F R A N C I S w. RUSCETTI
Immunologic Regulation of Lymphoid Tumor Cells: Model Systems for Lymphocyte Function ABUL K. ABBAS
Formation of B Lymphocytes in Fetal and Adult Life P A U L W. KINCADE Structural Aspects and Heterogeneity of ~mmunoglobdinFc Receptors JAY c. UNKELESS, HOWARD FLEIT, A N D IRAS . MELLMAN
INDEX
The Autologous Mixed-Lymphocyte Reaction MARC E. WEKSLER, CIIAHLES E. M o o i w , Jn., A N D ROBERT W. KOZAK
Volume 33 The CBA/N Mouse Strain: An Experimental Model Illustrating the Influence of the X-Chromosome on Immunity IRWINSCHER
INDEX
Volume 32 Polyclonal B.Cell Activators in the Study of the Regulation of immunoglobulin Synthesis in the Human System THOMAS A. WALDMANNA N D SAMUEI. BHODEH
The Biology of Monoclonal Lymphokines Secreted by T Cell Lines and Hybridomas AMNONALTMANA N D DAVIDH . KATZ
Typing for Human Alloantigens with the Prime Lymphocyte Typing Technique N. MORLINC, B. K. JAKOBSEN,P. PLATZ, L,. P. RYDER, A. SVEJGAARD, A N D M . THOMSEN
Autoantibodies to Nuclear Antigens (ANA): Their lmmunobiology and Medicine ENGM. TAN The Biochemistry and Pathophysiology of the Contact System of Plasma CHARLES G . COCHRANE A N D JOHN H. GRIFFIN
473
474
C O N T E N T S OF R E C E N T VOLUMES
Binding of Bacteria to Lymphocyte Subpapulations MARIUSTEODORESCU ANI) EUGENEP. MAYER
INDEX
Immunoglobulin RNA Rearrangements in B Lymphocyte Differentiation JOHN ROGERSA N D RANDOLPH WALL Structure and Function of Fc Receptors for IgE on Lymphocytes, Monocytes, and Macrophages HANSL. SPIEGELBEHC
Volume 34 T Cell Alloantigens Encoded by the IgT-C Region of Chromosome 12 in the Mouse F. L. OWEN Heterogeneity of H-2D Region Assaciated Genes and Gene Products KEIKO OZATO, A N D TED H. HANSEN, DAVIDH. SAC115 Human Ir Genes: Structure and Function THOMAS A. GONWA,B. MATIJA PETEHLIN,A N D J O H N D. STOBO Interferons with Special Emphasis on the Immune System A N D STEFANIE ROBEHTM. FHIEDMAN N. VOCEL Acute Phase Proteins with Special Reference to C-Reactive Protein and Related Proteins (Pentaxins) and Serum Amyloid A Protein M . B. PEWS A N D MARILYN L. BALTZ Lectin Receptors as Lymphocyte Surface Markers NATHAN SHARON
INDEX
Volume 35
The Murine Antitumor Immune Response and Its Therapeutic Manipulation ROBERTJ. NORTH Immunologic Regulation of Fetal-Mater-
rial ~~l~~~~ DAVIDR. JACOBY, LARSB. OLDIN(;, MICIIAELB. A. OLDSTONE
AND
The Influence of Histamine on Immune and Inflammatory Responses J. BEER,STEVENM. DENNIS, MATLOFF,A N D Ross E. ROCKLIN
INDEX
Volume
36
Antibodies of Predetermined Specificity in Biology and Medicine RICIIAHIJALANLEHNEH A Molecular Analysis of the Cytolytic Lymphocyte Response STEVENJ. BUHAKOFP, OPHA WEINBEHGEH, ALAN M . KHENSKY,A N D CAHOLS. REISS
The Human Thymic Microenvironment BARTON F. HAYNES The Generation of Diversity in Phosphorylcholine-Binding Antibodies Aging, ldiotype Repertoire Shifts, and ROGER M . PEHLMUTTEH, STEPHENT. Compartmentalization of the Mucosal-AsCHEWS,RICIIARDDOUGLAS, GREG SORENSEN, NELSONJ O H N S O N , NADINE sociated Lymphoid System ANDREW W. WADEA N D MYRONR J. GEARHART, AND NIVERA,PATRICIA LEROYHOOD SZEWCZUK
CONTENTS OF R E C E N T V O L U M E S
A Major Role of the Macrophage in Quantitative Genetic Regulation of Immunoresponsivenessand Antiinfectious Immunity GUIDOBIOZZI,DENISEMOUTON, CLAUDESTIFFEL,A N D YOLANDE BOUTIIILLIEH
INDEX
Volume 37 Structure, Function, and Genetics of Human Class II Molecules ROBERTC. GILESA N D J. DONALD CAPHA
475
Volume 38 The Antigen-Specific, Major Histocompatibility Complex-Restricted Receptor on T Cells PHILIPPAMARRACK A N D JOHN
KAPPLER Immune Response ( I r ) Genes of the Murine Major Histocompatibility Complex RONALDH. SCHWARTZ The Molecular Genetics of Components of Complement A. D. CAMPBELL, M. C. CARROLL, A N D R. R. PORTER Molecular Genetics of Human B Cell Neoplasia CARLOM. CROCEA N D PETERC. NOWELL
The Complexity of Virus-Cell Interactions in Abelson Virus Infection of Lymphoid and Other tiematopoietic Cells CIIEHYL A. WIIITLOCK AND OWEN N . WllTE
Human Lymphocyte Hybridomas and Monoclonal Antibodies DENNISA. CARSONA N D BRUCED
Epstein-Barr Virus Infection and Immunoregulation in Man GIOVANNA TOSATOA N D R. MICHAEL BLAESE
Maternally Transmitted Antigen JOHNR. RODGERS, ROGER SMITH111, MARILYN M. HUSTON,A N D ROBERTR. RICH
The Clossiccil Complement Pathway: ACtivation ond Regulation of the First Complement Component NEIL R. COOPER Membrane Complement Receptors Specific for Bound Fragments of C3 GORDOND. Ross A N D M. EIIWARD MEDOF Murine Models of Systemic Lupus Erythemator,us ARGYHIOS N . THEOFILOPOULOS A N D FRANK .I. DIXON
INDEX
FREIMARK
Phagocytosis of Particulate Activators of the Alternative Complement Pathway: Effects of Fibronectin JOYCEK. CZOP
Volume 39 Immunological Regulation of Hematopoietic/Lymphoid Stem Cell Differentiation by lnterleukin 3 JAMESN. IIILEA N D YACOB WEINSTEIN Antigen Presentation by B Cells and Its Significance in T-B Interactions ROBERT W. C I I E S N U T A N D HOWARD M . GREY
476
C O N T E N T S OF R E C E N T V O L U M E S
Ligand-Receptor Dynamics and Signal Amplification in the Neutrophil LARRY A. SKLAH Arachidonic Acid Metabolism by the 5Lipoxygenase Pathway, and the Effects of Alternative Dietary Fatty Acids TAKH. LEE A N D K. FRANK AUSTEN The Eosinophilic Leukocyte: Structure and Function GEALV J. GLEICll A N C i l E H Y L R. ADOLPIISON ldiotypic Interactions in the Treatment of Human Diseases RAIF S. GEHA Neuroimmunology DONALD G. PAYAN, JOSEPII P. MCGILLIS, A N D EDWAHV J. GOETZL
Index
Volume 40 Regulation of Human B Lymphocyte Activation, Proliferation, and Differentiation DIANEF. JELINEKA N D PETERE.
LIPSKY
Biological Activities Residing in the Fc Region of Immunoglobulin EDWAHDL. MoRAN A N V W I L L I A M 0. WEICLE Immunoglobulin-Specific Suppressor T Cells RICIIAHDG . LYNCII Immunoglobulin A (IgA): Molecular and Cellular Interactions Involved in IgA Biosynthesis and Immune Response JIHI MESTECKY A N D JEHHYR. MCGIIEE The Arrangement of Immunoglobulin and T Cell Receptor Genes in Human Lymphoproliferative Disorders TIIOMAS A. WALVMANN Human Tumor Antigens RALPH A. REISFELD A N D DAVIVA CIIEHESII Human Marrow Transplantation: An Immunological Perspective PAULJ. MARTIN,JOIIN A. HANSEN, RAINEHSTOHB,A N D E. DONNALL TIIOMAS
Index