Major Histocompatibility Complex Restricted Cell-Mediated Immunity

M aj or Histocompatibility Complex Restricted Cel I-Mediated Immunity GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST Immunology Bmnch, National Cancer Institute, Bofhesdo, Maryland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Major Histocompatibility Complex Restriction for Distinct T-Lymphocyte Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. T-Cell-Mediated Cytotoxicity B. Delayed Hypersensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Proliferation in Mixed Lymphocyte Reactions . . . . . . . . . . . . 111. Fine Specificity of Cytotoxic Effector Cells .............................. A. Models. ............................ B. H-2-Restricted Specificity . . . . . . . . . . . C. Infecting or Modifying Agent Specificity . . . . . . . . . . . . . IV. Immune Response Genes for H-2-Restricted Cytotoxicity .

55 57 57 63 64 64 65 66 73 78 83 87

I. Introduction

The major histocompatibility complex (MHC) has been known to be involved in some aspects of immune recognition for more than two decades. The MHC was found to code for cell surface antigens involved in allograft rejection (Gorer et al., 1948; S h r e f i r and David, 1975). This genetic region has also been shown to control immune response potential in a number of animal species (Benacerraf and McDevitt, 1972; McDevitt and Bodmer, 1974). In the mouse, the MHC, known as the H-2 complex, has been divided into four major regions: K, I , S, and D (Shreffler and David, 1975). The K and D regions determine the strong serologically detectable transplantation antigens of the mouse, which are important for tissue graft rejection, the generation of anti-H-2 antibodies, and the target cell antigens for thymus-derived (T-) cell-mediated lympholysis (CML) (Gorer et al., 1948; Alter et al., 1973; Abbasi et al., 1973; Schendel et al., 1973; Nabholz et al., 1974; Brondz et al., 1975; Bevan, 1975a). Recent results obtained using mutant strains of mice in which graft-versus-host and CML reactions can be generated between the mutants and the wild types in the absence of detectable serological differences raise the possibility that the targets of CML may b e distinct from the cell surface products detected ssrologically, although both products are coded within the K and D regions of H-2 (Berke and Amos, 1973; 55

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GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

Widmer et al., 1973; Forman and Klein, 1975; Nabholz et al., 1975). Cytotoxic effector cells can be generated without inducing strong proliferation by culturing mixtures of cells differing only at the K or D region or both, whereas cell mixtures differing only at the Z region induce strong proliferative responses (MLR) in the absence or in the presence of weaker cytotoxic responses (Widmer et al., 1973; Plate, 1974; Wagner et al., 1975; Hodes et al., 1975; Klein et al., 1977).The S region, which maps between the I and D regions, determines the level of a serum a-globulin known as Ss as well as the sex-linked allotypic variant designated SZp (Shreffler and Passmore, 1971). Products of this region appear to be functionally relevant in the complement system (Lachman et al., 1975; Hansen et aZ., 1975). The I region has been divided into at least five subregions, based on mapping studies using recombinant mouse strains for immune response (Zr) gene functional studies (Lieberman et al., 1972; Melchers et al., 1973; Lozner et al., 1974; Benacerraf and Katz, 1975) as well as for the detection of l a antigens on lymphocyte subpopulations (David et al., 1973; Hauptfeld et al., 1973; Sachs and Cone, 1973; Sachs et al., 1975), and suppressor cell functions (Murphy et al., 1976; Tada et al., 1976). These I subregions are currently designated Z-A,I - B , Z-J, Z-E,and Z-C (Shreffler

et al., 1977). One of the more recent functions associated with the murine MHC has been the finding that efficient CML reactions can be generated in uiuo and in uitro to virally infected or chemically ,modified syngeneic cells only when the target cells are H-2 matched with the infected or

modified stimulating or responding cells or both (Zinkernagel and Doherty, 1974a; Doherty and Zinkernagel, 1975; Gardner et al., 1975; Koszinowski and Thomssen, 1975; Shearer, 1974; Shearer et al., 1975a; Forman, 1975; Rehn et al., 1976a). Similar requirements for H-2 homology have been demonstrated for sensitization and lysis of cells expressing weak transplantation antigens (Bevan, 1975b; Gordon et al., 1975), and have been suggested for tumor-associated antigens (Germain et al., 1975; Schrader et al., 19i'S). Products of the K and D regions appear to be involved in the recognition for these selfassociated CML reactions, irrespective of which system was used to demonstrate the phenomenon. The recognition of antigenic determinants in association with self MHC products need not be limited to the cytotoxic examples cited above, and may be a more general aspect of T-cell recognition. This could involve products of different MHC regions, depending on the functional class of the T-lymphocyte involved (Rosenthal and Shevach, 1973; Erb and Feldmann, 1975; Miller et al., 1975; Schmitt-Verhulst et al., 1977). In this report the role of the MHC products in the various MHC-restricted systems will be reviewed, and

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the experimental evidence obtained in these models will b e considered as it relates to the nature (Binz and Wigzell, 1975a,b); Eichman and Rajewsky, 1975) and specificity of the T-cell receptor(s) (Janeway et al,, 1976; Rehn et al., 1976b). it. Major Histocompatibility Complex Restriction for Distinct T-Lymphocyte Functions

Within the syngeneic models, MHC restriction has been demonstrated for a number of T-cell functions. These include: Tcell-mediated cytotoxicity (Zinkernagel and Doherty, 1974a; Shearer, 1974; Doherty and Zinkernagel, 1975; Gardner et al., 1975; Koszinowski and Thomssen, 1975; Shearer et al., 1975a; Forman, 1975; Rehn et at., 1976a); memory for such cytotoxic responses (SchmittVerhulst et al., 1977); delayed hypersensitivity (Miller et al., 1975, 1976); mixed lymphocyte reactions (Schmitt-Verhulst et al., 1977; Thomas and Shevach, 1977) as well as cellular interactions involving helper cells (Katz et al., 1973, 1975; Sprent and von Boehmer, 1976); macrophages (Rosenthal and Shevach, 1973; Erb and Feldmann, 1975);and suppressor cells (Rich and Rich, 1976), as well as suppression factors (Rich and Rich, 1975; Tada et al., 1976; Moorehead, 1977). In this section a review will be presented of T-cell-mediated cytotoxic responses, delayed hypersensitivity, and mixed lymphocyte reactions involving syngeneic cells and the evidence supporting H-2 restriction.

A. T-CELL-MEDIATEDCYTOTOXICITY Murine T-cell-mediated CML reactions have been generated in viuo and in uitro against alloantigens, usually those involving differences between responding and stimulating cells at H-2 (Cerottini et al,, 1970; Henney, 1971; Wunderlich and Canty, 1970; Wagner et al., 1972; Cerottini and Brunner, 1974; Bevan, 1975a). However, if CML is relevant for autologous reactions such as autoimmunity and/or possibly for surveillance against autologous neoplastic cells or against infections, it is important to investigate the genetic parameters and to elucidate the immune mechanisms involved in reactivity against selflike antigens. Until recently some investigations have been reported in which lymphoid cells responded in graft-vs-host reactions (Cohen et al., 1971), mixed lymphocyte cultures (von Boehmer and Adams, 1973; Ponzio et al., 1975), or cytotoxic reactions against differentiation antigens (Ponzio et al., 1975) or tumor-associated antigens (Wagner and Rollinghoff, 1973; Lundak and Raidt, 1973), all which involve nonallogeneic responses. In such studies the possibility of MHC restriction was not investigated. As was mentioned in the intro-

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GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

duction, however, a number of more recent examples of CML reactions have been reported in which H - 2 restriction has been tested and observed between stimulating and target cells, effector and target cells, or among responding lymphocytes, stimulating cells and target cells.

1 . Virally Infected The first published example in which H - 2 restriction was fully realized as being important for T-cell-mediated cytotoxic reactions was the demonstration that T-lymphocytes from lymphocytic choriomeningitis (LCM)-infected mice could lyse LCM-infected targets only if they expressed the same H - 2 haplotype as the donor of the effector cells (Zinkernagel and Doherty, 1974a). Since the LCM virus is a noncytopathic budding RNA virus, other CML studies were performed using the nonbudding cytopathic DNA ectromelia virus. Mice immunized with ectromelia virus also generated cytotoxic T-lymphocytes (CTL), which could lyse only ectromelia-infected targets expressing the same H - 2 haplotype as the immunized donors (Gardner et al., 1974a, b, 1975). Similar H - 2 restriction for cytotoxic effectors was demonstrated in vaccinia virus-infected mice (Koszinowski and Thomssen, 1975; Koszinowski and Ertl, 1975), as well as for Sendai virus (Doherty et al., 1976a). In the examples cited above, the CTL were taken from infected mice. A technique has been recently developed in which H - 2 restricted cytotoxic effector cells can be generated in a primary in vitro culture system against LCMinfected peritoneal cells (Dunlop and Blanden, 1977).Cytotoxic effectors have also been detected by culturing lymph node cells from herpes simplex virus-infected mice (Pfizenmaier et al., 1977). These T-cell effectors were both virus specific and H - 2 restricted on a shortterm cytotoxic assay of macrophage targets. In the tumor virus systems, MHC restriction has been demonstrated in the mouse for cytotoxic effectors against the MSV (Plata et al., 1976; Gomard et aE., 1976).Similarly, mice injected with syngeneic Friend virus-infected tumor cells generated cytotoxic effectors which were restricted to lyse Friend-infected, H - 2 matched, cultured target cells (Blank et al., 1976). The CTL generated during the recovery from Friend disease were also found to be preferentially H - 2 restricted in their killing potential (Cheseboro and Wehrly, 1976).An unusual case of H - 2 restriction involving Friend virus has been recently reported (Kumar and Bennett, 1977a, b). In this system Friend virus (FV) activates a population of T-suppressor cells, which, in turn, can suppress mitogen-responsive cells (Kumar and Bennett, 1976; Kumar et al.,

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1976). By using cell-mixing experiments, it was demonstrated that mitogen-responsive cells and the suppressor cells that regulate them must share D region H-2 haplotype for suppression to be effective. Surprisingly, the requirement for H-2-restriction was overcome if the suppressor cells were irradiated in uitro, or if they were harvested from either cortisone-treated or infant donor mice (Kumar and Bennett, 1977a, b). It has been postulated for this complicated example of H-2 restriction that the suppressor-mitogen-responsivecell H-2 restriction requirements are determined by a third late-maturing, cortisone- and radio-sensitive cell (Kumar and Bennett, 1977a, b). MHC restriction for T-cell reactivity in such viral systems seems not to be a phenomenon limited to the mouse, since similar results have been reported for Rous sarcoma virus in chickens (Wainberg et al., 1974). In some of these viral systems mapping studies have been performed using H-2 recombinant and mutant strains to establish whether the H-2 restriction observed was limited to a particular region of the MHC. The K and D regions appear to be the regions relevant for H - 2 homology in T-cell-mediated cytotoxicity to LCM and vaccinia virusinfected targets (Zinkernagel and Doherty, 1975; Zinkemagel, 1976a). The use of H-2 mutant mice, such as the Hzl (H-2ba)(Bailey et d . , 1971), which are presumed to be point mutations involving a single cistron at H - 2 K (Klein, 1975), further supports the concept that products of genes mapping within the K region are involved in the restriction of the CML reaction. N o cross-reactivity was observed between H-2K" and H-2K"" for the LCM and vaccinia cytotoxic responses (Zinkernagel, 1976a). Some cross-reactivity was detected for LCM and ectromelia between the H - 2 K h hmutant and BlO.A(5R) while the latter were the effectors and the former were the virus-infected targets, but not vice versa (Blanden et al., 1976a; Doherty et al., 1976a). The H - 2 mutant experiments suggest that the determinants responsible for allogeneic graft rejection and the H-2 component recognized in association with the viral modification are coded b y the same genetic unit, which would b e distinct from the antigenic structure serologically recognized as a private specificity. It has also been demonstrated that the generation of these H - 2 restricted cytotoxic effector T-cell reactions can b e paralleled by in uiuo effects. For example, fatal lymphocytic choriomeningitis could h e transferred to host mice with non Ig-bearing lymphocytes only when the cells shared K or D region haplotypes with the host animals (Doherty et al., 1976a).Furthermore, protection against ectromelia infection (Kees and Blanden, 1976) as well as against LCM infection in the spleen (Zinkernagel and Welsh, 1976) was achieved by adoptive cell

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GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

transfer of immune cells when these cells shared K or I) with the recipient mice. 2. Chemically Modijied Concomitant with the work on the H-2 restriction in the LCM system (Zinkernagel and Doherty, 1974a), an in uitro system was developed in which cytotoxic T-lymphocytes were generated against trinitrobenzene sulfonate-(TNP-)modified autologous cells (Shearer, 1974). These effector cells were able to lyse targets modified with the same agent which expressed the same H-2 haplotype as the responding and modified stimulating cells (Shearer, 1974; Shearer et d., 1975a; Forman, 1975). Other chemically modifying agents that covalently bind to cell surfaces have been used to generate effectors in uitro and have also been demonstrated to exhibit H-2 restriction. These include N-(3-nitro-4-hydroxy-5-iodophenylacetyl-~-alanylglycylglycyl-) azide (N-) (Korenet al., 1975; Rehn et al., 1976a),N-(3-nitro4-hydroxy-5-iodophenylacetyl)azide, N-(2,4,6-trinitrophenyl)-P-alanylglycylglycyl azide (Rehn et al., 1976b), fluorescein isothiocyanate (Starzinski-Powitz et al., 1976b), and dinitrobenzene sulfonate (Forman, 1975, A.-M. Gilheany et al., unpublished results). Cytotoxic reactions exhibiting self-restriction have been recently demonstrated using TNP-modified human peripheral blood leukocytes (Shaw, unpublished observations). Mapping studies for H-2 restriction in the TNP-modified CML system indicated that the cytotoxic effectors efficiently lysed only TNPmodified target cells which expressed the same haplotype as the effector and stimulating cell populations at K (or K-plus Z-A) and/or at D (Sheareret al., 1975a; Forman, 1975).In certain mouse strains, particularly those expressing the k haplotype at K through Z-J, lysis was detected on TNP-modified targets expressing the haplotype of the effectors and stimulating cells at the K end, but not on targets expressing effector haplotype at H-2D (Shearer, 1974; Shearer et d.,1975a; Schmitt-Verhulst and Shearer, 1975). The lack of lysis of TNPmodified H-2D-matched targets by effectors from certain mouse strains that are capable of lysing TNP-modified H-2K-matched targets has been attributed to H-2-linked genetic unresponsiveness in the responding cell population (see Section IV) (Schmitt-Verhulst and Shearer, 1975,1976; Schmitt-Verhulst et al., 1976).Restriction of these cytotoxic effector cells to the K and D regions of H-2 has been independently demonstrated by the blocking of lysis by specific antisera directed against H-2K and H-2D products (Schmitt-Verhulst et al., 1976). Blocking withgnti-Z-region sera in the assay had no detectable

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effect on lysis (Schmitt-Verhulst et al., 1976). The use ofTNP-modified BIO.A(4R) targets for lysis by B1O.A or B1O.BR effectors does not distinguish between homology requirements at K or at K plus I-A. The complete inhibition of lysis with BIO.A effectors by A.TL anti A.AL serum (strains that appear to differ only at H-2K) (Schmitt-Verhulst et al., 1976), and the lysis of TNP-modified SJUJ targets by A.TL effectors (which appear to share only H-2K) (Schmitt-Verhulst and Shearer, 1976) indicate that products of the I-A region are not required in the CML specificity at the lytic phase. See Table I for a listing of a number of inbred and congenic resistant mouse strains with their respective H-2 haplotypes. Partial mapping studies have been performed in the N-modified CML system with similar results (Rehn et al., 1976a; Shearer et aZ., 1976).

3. Weak Transplantation Antigen Associated Cultures of lymphocytes differing only at non H-2-linked minor histocompatibility loci do not elicit cytotoxic T-effector cells. However, CML responses against minor H antigens (Bevan, 1975b,c, 1976) and the sex-linked H-Y antigen (Gordon et al., 1975; Gordon and Simpson, 1976) have been generated using cultured lymphocytes from primed TABLE I DESICNA~O OFNH - 2 HAPLOTYPES IN THE INBRED AND CONGENIC RESISTANT MOUSE STRAINS DISCUSSED IN THIS REVIEW H - 2 region" Mouse strain

CBA, B1O.BR A, BIO.A BALBk, B10.D2 C57BW10 BlO.A(4R) BlO.A(2R) B1O.HTT A.TH A.TL A.AL SJUJ BlO.A(3R) B lO.A(5R) "

K

I-A

I-B

I-]

I-E

1C

S

D

k k d b k k

k k d b k k

k k d b b k

k k d b b k

k k d

k d d b

k d d b

k d d b

$

S

S

S

d k

d k s k k

b d d d d

S

S

k

b b k k

b

S

S

S

S

S

k k

k k

k k

k k

k k

b

b

S

S

S

S

S

S

S

S

b

b b

b b

b k

k k

d d

d d

d d

b

From Klein (1975); Shrefiier et al. (1977).

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GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

donor mice. The responding cell donors differed from the priming and challenging immunogenic cells only at the respective minor loci. Surprisingly, lysis was detected only on target cells that expressed not only the same minor antigens but also the same H-2 haplotype as the stimulating cells. Mapping studies for CML against the minor H antigens indicated H-2 restriction for the D region and for one or more regions to the left ofl-C (Bevan, 1975~). In the H-Y system the lysis b y female effectors of male targets in the C57BW10 responder strain was restricted to H-2Db(Simpson and Gordon, 1977).When the responding cell donors were F, hybrids, the H-2 restricted specificities were associated with H-2Db, H-2Kd in (B10 x Balb/c)F1, H-2Dk in (B10 x CBA)F,, and H-2Kkin (BlOxA)F1(Simpson and Gordon, 1977). Part of this H-2 restriction can be accounted for by immune response genes, which will be considered in Section IV. Similar observations have been recently reported involving HLA-associated cytotoxic responses against human H-Y antigens (Goulmy, et al., 1977).A minor histocompatibility antigenic difference, linked to the ninth chromosome in the region which codes for the Thy 1 antigen, has been described in AKWCu and AKWJ mice, which are both H-2k(Zatz, 1977). Similar to the CML reactions observed for other weak histocompatibility antigens the effectors generated against the Thy l-linked antigen(s) were able to lyse only H-2-matched target cell expressing the relevant weak antigen (Zatz, 1977). The above findings contrast with another report (Peck et al., 1977), in which H-2 restricted CML was described in completely syngeneic secondary in nitro cultures. In these experiments T-cell-enriched lymphocytes were cultured in the presence of fetal bovine serum (FBS) for 5 days and the resulting blast cells were recultured with fresh irradiated spleen cells syngeneic to the responding cell population. Such secondary cultures generated cytotoxic activity that could be detected only on H-2-matched lipopolysaccharide-stimulated spleen cells, whether these targets had ,been cultured in the presence or in the absence of FBS. The latter experiments rule out the possibility that FBS was responsible for the generation of a new antigen on the cell surface (Forni and Green, 1976). When I-region haplotype was shared between target and effector cells, but not K or D haplotypes, no lysis was detected. Although no obvious new antigen was expressed on both “stimulating” and target cells in this system, B-cell-speci fic surface antigens and virus-associated antigens resulting from the activation of endogeneous viruses during cellular stimulation by FBS are possible candidates for the antigenic determinant recognized by the T-effector cell in association with H-2-coded products. H-2 restricted

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syngeneic cytotoxicity had been previously reported in long-term lytic assays of mouse lymphocytes cultured on syngeneic fibroblasts (Ilfeld et al., 1975; Shustik et al., 1976).

4. Tumor Immunity Cytotoxic effector cells can be generated against syngeneic tumors. Indirect evidence has been provided suggesting that H-2 antigens are recognized in addition to the tumor-associated antigens (Germain et al., 1975; Schrader et al., 1975; Schrader and Edelman, 1976). The lytic phase of murine effector cells generated against H-2-matched lymphoid tumors could b e blocked with specific anti-H-2 sera directed against antigens controlled by the H-2 complex of normal cells (Germain et al., 1975). Similar observations have been reported by others (Schrader et al., 1975; Schrader and Edelman, 1976). These latter investigators have also demonstrated that patching and capping of H-2 antigens on tumor cells resulted in copatching and cocapping of viral antigens (Schrader et al., 1975). Such findings raise the possibility that there may be recognition of self H-2 components in antitumor Tcell-mediated immunity. It would appear, therefore, that products of the MHC may b e involved in the T-cell-mediated aspects of antitumor immunity in a manner similar to the other reported phenomena involving H-2 restricted CML. B. DELAYEDHYPERSENSITIVITY Delayed hypersensitivity (DH) is a type of immune reaction in which T lymphocytes activate an inflammatory response. Characterization of the cell surface antigens on the lymphocytes responsible for DH indicates that this reaction is mediated by a subpopulation of T lymphocytes distinct from cytotoxic effector and suppressor cells (Vadas et al., 1976). Lymphocytes sensitized against soluble protein antigens transferred DH to naive mice only if the donor and recipient were H-2 matched (Miller et al., 1975). Mapping studies revealed that the H-2 region of homology important for this reaction was I-A when the antigen used for sensitization was fowl y-globulin and K , I , or D when dinitrofluorobenzene (DNFB) was the sensitizer (Miller et al., 1976; Vadas et al., 1977). Tolerance to delayed hypersensitivity against DNFB can be effected by suppressor T cells, which release a soluble suppressor factor (Claman et ul., 1977; Moorhead, 1977). This suppressor factor abrogates the ability of sensitized lymphocytes to transfer contact sensitivity to naive mice. The action of this suppressor factor is both antigen specific and H-2-restricted, since it affects only lymphocytes sensitized against

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GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

the same hapten and H-2-matched with the cells from which the soluble suppressor factor was prepared (Moorhead, 1977).

c. PROLIFERATION IN

MIXED LYMPHOCYTEREACTIONS

In a primary in uitro mixed lymphocyte reaction against TNPmodified syngeneic cells, only marginal cellular proliferation was detected (Shearer et al., 1975b). However, a strong proliferative response can be detected against TNP-modified syngeneic cells by restimulating primary cultures with TNP-modified cells syngeneic with the responding and modified primary stimulating cells (Schmitt-Verhulst et al., 1977). It was possible to investigate the specificity of the secondary MLR against TNP-modified syngeneic and allogeneic cells, since primary allogeneic MLC cannot be generated in such secondary cultures (Schmitt-Verhulst et aZ., 1977).Intra-H-2 mapping for the secondary MLR indicated that the relevant regions of homology were Z, D, and K or I< plus Z-A (Schmitt-Verhulst et aZ., 1977). An alternative approach for investigating the specificity of secondary proliferative responses to TNP-modified syngeneic cells in the absence of a primary allogeneic reaction involved the prior elimination of alloreactive clones with bromodeoxyuridine and light (Thomas and Shevach, 1977). Cultures of guinea pig lymphocytes, made unresponsive to allogeneic macrophages, and subsequently primed with TNP-modified syngeneic macrophages, generated secondary proliferative responses only after restimulation with TNP-modified syngeneic and not modified allogeneic macrophages. If, however, the priming was against TNP-modified allogeneic macrophages, secondary stimulation was obtained only with the modified allogeneic macrophages (Thomas and Shevach, 1977). Since the strains 2 and 13 guinea pigs used in this study are considered to differ only in the Z region (B. D. Schwartz et al., 1976), MHC homology involving an Z region product is required between modified primary and secondary stimulating macrophages for such T-cell recognition. Secondary in uitro proliferative responses have been recently obtained using TNPmodified autologous human peripheral blood leukocytes in which proliferation was optimal when primary and secondary modified stimulating cells were from the same donor (Shaw et al., submitted for publication). 111. Fine Specificity of Cytotoxic Effector Cells

The H-2-restricted cytotoxic response for the virally infected and chemically modified systems provides an opportunity to examine the components of immunological recognition. These include the contri-

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butions made to the overall specificity of the effector cells by the agents themselves and by the H-2-coded cell surface products involved. A. MODELS

Two basic models have been proposed to account for the H-2 homology requirements observed for the virally infected, chemically modified, and weak antigen-H-2-restricted autologous CML (Zinkernagel and Doherty, 197411; Dohertyet al., 1976b). These models essentially differ in that one involves a single receptor recognizing self H - 2 products which are either modified or unmodified, but are in close association with the infecting or modifying agent, whereas the other involves two distinct receptors-one specific for the agent and the second specific for self H-2K or H-2D coded cell-surface products. In the single receptor model, the agent could be modifying cell-surface antigenic structures (altered self). Alternatively, a model involving a single receptor recognizing unaltered-self H-2 products in close association or proximity with the infecting or modifying agent is possible. It has been suggested that H-2 molecules serve as adapters that combine with the infecting or modifying antigenic determinants on the cell surface to form an adaptor-antigen complex which is composed of elements of self H-2 coded products and the antigenic determinant (e.g., viral antigen or TNP) (Schrader et al., 1975).For the two-receptor or dual recognition model, one receptor would be specific for the “hapten” or infecting agent. The second receptor would be a responder or effector cell receptor that would function as a responder-stimulator and/or effector-target interaction structure recognizing syngeneic K or D region products. It has also been suggested that genes mapping within the K and D regions of the H-2 complex control the operation of groups of glycosyltransferases (Blanden et al., 1975). Such enzymes would be responsible for attaching sugar residues to the proper sites on syngeneic polypeptides effecting the formatiori of glycopeptides in which the antigenic complex is determined by both the host and viral genome (Blanden et al., 1975). Thus, MHC genes could dictate glycosylation of viral glycopeptides or virus could modify glycosylation of H-2 antigens. However, it is difficult to account for the H2-restricted CML observed for the chemically modified and weak transplantation antigen systems by a glycosylation mechanism. It would be necessary, therefore, to postulate that the glycosylation mechanism would be responsible for the H-2 restriction observed for the virally infected C M L , but not for the other H-2-restricted examples.

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GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

B. H-2-mSTRICTED SPECIFIClTY In most of the H-2-restricted CML models investigated, experiments have been performed using F1hybrid effector cells and virally infected (Zinkernagel and Doherty, 1974b, 1975; Blank et al., 1976), chemically modified (Shearer et al., 1975a; Forman, 1975; Rehn et al., 1976a), or weak antigen-associated (Bevan, 1975b; Gordon and Simpson, 1976) parental stimulating and target cells as a possible approach for distinguishing between the one- and two-receptor models. Without exception, the F! effectors were observed to lyse the target cells of the parental haplotype used for sensitization, but not the targets from the other parental strain. These observations are compatible with the single-receptor model and inconsistent with the dualreceptor model presented in the simplest form. In the altered-self or single-receptor model, clones of effector cells generated by sensitization of responding lymphocytes against modified parental cells of a given H-2 haplotype would lyse modified K or D region matched targets of the same H-2 haplotype only, since the H-2-coded alteredself structures of stimulating immunogen and target antigen would need to be identical. In contrast, the simplest model of dual recognition would predict that either parental haplotype would serve as target cells if H-2 homology is required only between effector and target cells. The restrictions observed in the Fl hybrid-parent experiments would not necessarily exclude the dual receptor model, however, if the following limitations are imposed: First, it must be assumed that receptors for each parental haplotype are clonally expressed in the F1 hybrid responding lymphocytes and that this expression is subject to allelic exclusion; i.e., a given clone of F1 lymphocytes can interact with only a single parental haplotype, both at the responder-stimulator and at the effector-target cell levels. Second, if the H-2 receptor in the dual recognition is required for cell-cell interaction, then this interaction must occur via complementary, but not between like-like, structures, since a like-like interaction through H-2 antigens would necessitate allelic exclusion ofH-2 cell-surface products on the F1cells. It is known that serologically detectable region H-2 antigens are codominantly expressed on F, cell surfaces (Cullen et al., 1972). Distinct clones of cells have been shown to react against K and D regions of allogeneic cells (Nabholz et al., 1974; Bevan, 1975a) and autologous modified cells (Schmitt-Verhulst, et aZ., 1976). Thus, in order for a two-receptor model to be valid, it is necessary to postulate four distinct " clones, each expressing a receptor for virus, hapten," or weak antigens, plus a complementary receptor for a single parental K - or D-region product to account for full F1cytotoxic potential.

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It should be noted in this context that cross-priming for a secondary response to a minor H antigen has been obtained (Bevan, 1976). Lymphocytes from F, mice immunized in uiuo against cells expressing a weak H antigenic difference from the host (but sharing one H-2 haplotype), challenged in uitro with cells expressing the same minor H antigen and the H-2 haplotype of the other parent-generated effectors that were specific for the in uitro immunization, both with respect to H-2 and the minor antigen (Bevan, 1976). These results d o not prove, but are compatible with, the H-2 restriction being expressed in the lytic event between effector and target cells. Another approach for investigating the relative contribution of self H-2-coded products and the foreign antigenic determinants (e.g., virus, hapten, or weak antigen) in the recognition between effector and target cells is the use of nonradioactive targets for blocking the interaction between the effector and “Cr-labeled target cells (“cold target inhibition”) (Zinkernagel and Doherty, 1975; Shearer et al., 1975a, 1976; Forman, 1975; Bevan, 1975b). In such experiments unlabeled target cells were incubated with the effector cells prior to the addition of S’Cr-labeled target cells to the effectors. The results indicated that blocking cells which were H-2-matched with the effector and “Crlabeled target cells inhibited the cytotoxic assay only if they expressed the same antigenic determinant (e.g., virus, hapten, or weak antigen) as that used in sensitization. Furthermore, blocking cells that were not H-2-matched with the effector and targets, but expressed the same antigenic determinant used in sensitization, were likewise ineffective inhibitors of cytolysis (Zinkernagel and Doherty, 1975; Shearer et al., 1975a, 1976; Forman, 1975; Bevan, 1975b). Such results tend to favor a single-receptor model in which both the H-2-coded products and the antigenic determinant contribute to the specificity of the cytotoxic effector cell. A dual recognition model would not necessarily be exclued, however, if it is assumed that the occupation of only one receptor by the relevant H-2-coded product or b y the antigenic determinant does not compete efficiently with a target expressing both antigenic components. The lytic phase of allogeneic CML has been shown to b e blocked b y antisera directed against the K or D region antigens of the target cells used for sensitization (Nabholz et al., 1974). In certain of the H 2-restricted syngeneic CML examples, the involvement in the lytic interaction between effector and target cells of products closely associated on the cell surface with the private antigenic specificity recognized serologically was suggested b y experiments using alloantisera directed against H-2 subregions. Thus, although uninfected or unmodified H-2-matched cells do not serve as cold target inhibitors of

68

GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

cytolysis in these H-2-restricted systems, anti-H-2 sera directed against the H-2 region products involved in sensitization do inhibit the interaction of effector and target cells. In one syngeneic tumor system, antisera directed against products of the entire H-2 region of the target cell inhibited lysis by effectors of the relevant tumor target cells (Schrader et al., 1975). Experiments using alloantisera of either of the two specificities expressed by F1 effector cells sensitized with semisyngeneic tumor cells suggested that blocking of the lytic phase could be achieved only with anti-H-2 sera directed against the target cells, but not with alloantisera directed against the H-2 specificity of the effector cells (Schrader and Edelman, 1976).A similar conclusion had been obtained for allogeneically stimulated effector cells (Schmitt-Verhulst et al., 1976). In another syngeneic tumor system, partial inhibition of lysis was obtained by blocking with antisera directed against products of the D region (Germain e t al., 1975).Blocking with anti-H-2 sera has also been demonstrated in the H2-restricted CML reactions against the vaccinia (Koszinowskiand Ertl, 1975) and MSV (Gomard et al., 1976) viruses. Further analysis of the blocking ofH-2-restricted CML reactions by anti-H-2 sera has been reported using the TNP-modified antigenic cytotoxic model (Schmitt-Verhulst et al., 1976; Burakoff et d.,1976a). The interaction between effectors sensitized against TNP-modified syngeneic lymphocytes and modified target cells was inhibited only by sera directed against the K region of the target cells, but not b y sera directed against Z or D region products in a mouse strain for which the TNP-syngeneic CML response is associated only with the K region of H-2 (Schmitt-Verhulst et id., 1976) (lines 1 through 3 of Table 11). When the lysis by effectors from a different mouse strain which was capable of being sensitized against TNP-self associated with H-2D products was tested on TNP-modified target cells sharing only the I-C, S, D regions ofH-2 with the stimulating cells, inhibition was observed only in the presence of sera directed against I-C, S, D products, but not in the presence of sera directed against products of the K region, or an Z-A region product (Schmitt-Verhulst et al., 1976; and lines 4 and 5 of Table 11).Further evidence for the specificity involved in the blocking of target cells with antisera in the TNP-modified syngeneic CML reaction was indicated by the absence of blocking of the lytic interaction by a strong alloantiserum directed against non-H-2 alloantigens expressed on the TNP-modified tumor cells used as targets as well as by the lack of inhibition by anti-Ia sera on target cells enriched for Zabearing cells (Burakoff et al., 1976b). Anti-TNP antibodies have been found to block the lysis by effector cells generated either by a TNP-modified aiitologous or by unmodified

69

MAJOR HISTOCOMPATIBILITY COMPLEX

TABLE I1 SELECTIVE INHIBITION BY ANTI-ff-2 SERA AND ANTI-TNP SERUM OF THE LYSIS OF TNP-MODIFIED TARGETCELLS BY EFFECXOR CELLS SENSITIZED WITH TNP-MODIFIEn AUTOI.OGOUS STIMULATINGCELLS Responding cells

BIO.A B1O.A B 10.A B 10.D2 BlO.D2 BlO.D2 BlO.D2 BlO.D2 BlO.D2 B1O.BR B1O.BR B1O.BR

Stimulating cells

Target cells

B1O.A-TNP B1O.A-TNP B 1O.A-TNP BlO.D2-TNP BlO.D2-TNP BlO.D&TNP BlO.D2-TNP BlO.D2-TNP BlO.D2-TNP B1O.BR-TNP C57BL/lO C57BLlO

B10.A-TNP B1O.A-TNP B 1O.A-TNP B1O.A-TNP B1O.A-TNP BlO.D2-TNP BlO.D2-TNP B1O.DS-TNP BlO.D2-TNP B10.BR-TNP C57BL/lO-TNP C57BL/lO

'' + + indicates complete inhibition of lysis; - indicates no inhibition of lysis.

Antiserum directed against Kk

( I-C ,S,D)d

(I-A through S)k ( K through I-E)k ( I-C,S,D)d All of H-2d ( K through I-E)d ( I-C,S,D)d Ia-8

TNP-KLH TNP-KLH TNP-KLH

Inhibition of cytolysis"

++ -

++

++ + + -

++ ++ -

+ indicates partial inhibition of lysis;

allogeneic stimulators when assayed on TNP-modified targets (Schmitt-Verhulst et al., 1976; and lines 11 and 12 of Table 11). These observations indicate that some TNP groups are closely associated with unmodified H-2 specificities, since, as shown in line 12 of Table 11, anti-TNP antibodies could block the lysis of TNP-modified targets by effectors that are specific for unmodified alloantigens. The observation that reactivity against or associated with D-end specificities in the TNP-syngeneic CML is blocked only by antiSI-2D-end reagents, but not by anti-H-2K-end reagents suggests that some TNP groups are more closely associated with K or D region products than the K and D region coded antigens are to each other. The blocking studies with anti-H-2 sera directed against the unmodified H-2K or D region products suggest that the lytic interaction involves recognition of products sterically associated with serologically detected H-2 products. However, the CML target cell antigens could be distinct from serologically detected antigens in both the allogeneic and syngeneic H-%restricted systems as suggested by experiments mentioned earlier using H-2 mutant mouse strains that are serologically indistinguishable from wild type, but can generate CML reactions (see Section I). The data obtained using the H-2Kh mutants H-2 H-2 hd, H-2 for the generation of CML against TNP-modified syngeneic cells showed an extensive cross-reactivity between effectors generated in the wildtype syngeneic system and TNP-modified mutant target cells (Forman

70

GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

and Klein, 1976). These results contrast with the data obtained with effector cells from the same mutants sensitized against virally infected syngeneic cells, which were found not to be cross-reactive with the wild-type virally infected target cells (Blanden et al., 1976a; Zinkernagel, 1976a). Both the cross-reactivity found in the TNP-syngeneic system and the absence of cross-reactivity detected in the virally infected system could be obtained by postulating that a single allele at the H-2K locus may control more than one CML determinant on the same molecule. A mutation in this allele would affect only some of these determinants and leave the others unaltered (Forman and Klein, 1976). This interpretation would also account for the observation that although the mutant and wild-type H-2K differ (they react against one another), they still seem to share some determinants in H-2K that can be detected by the stimulation of a strain of a different H-2K haplotype (Forman and Klein, 1975). The use of cell lines that do not express detectable amounts of H-2 coded cell surface antigens provides another way of investigating the importance of MHC products in H-2-restricted T-cell-mediated lympholysis. A murine teratoma cell line which expresses a T / t locus antigen but does not express detectable amounts of H-2 antigens is not lysed as a TNP-modified target of effector cells generated against TNP-modified syngeneic cells (Forman and Vitetta, 1975).The murine teratoma F9 cell line, which does not express H-2 antigens (Artzt et al., 1975), can be infected with LCM virus resulting in the expression of cell-surface viral antigens (Zinkernagel and Oldstone, 1976). Although LCM-infected F9 cells are susceptible to antiviral antibody and complement, they are not lysed by T-cell-mediated cytotoxic effector cells (Zinkernagel and Oldstone, 1976). These results indicate that H-2-coded cell surface products are important in the recognition by T-effector cells leading to lysis of targets. It is unclear from these experiments whether the requirement for H-2 expression is important for LCM-infected target cells only, or for both infected stimulating and target cells. In order to critically investigate whether the H-2 restriction observed in these syngeneic CML systems is due to H-2 homology (a) between effector and target cells, (b) between stimulator and target cells, or (c) between responder and stimulator as well as between effector and target cells, a series of experiments have been performed in which the potential responding cell pool was made unresponsive. Lymphocytes for this purpose have been prepared by at least three techniques. First, chimeras in which lethally irradiated FI hybrid mice were repopulated by T-cell-depleted parental stem cells resulted in re-

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71

population of the Fr host mice with parental immunocompetent cells that were selectively unresponsive to the other parental alloantigens (von Boehmer and Sprent, 1976). Effector cells from such chimeras infected with LCM (Pfizenmaier et al., 1976; Zinkernagel, 197613) or vaccinia (Zinkernagel, 1976b)virus lysed infected target cells expressing the alloantigens to which the chimera was unresponsive. Similarly, parental lymphocytes that expressed only one H - 2 parental haplotype obtained from F1recipients, and were unresponsive to the H - 2 antigens of the other parent, could be sensitized against TNP-modified cells to which the lymphocytes were tolerant (von Boehmer and Haas, 1976; Pfizenmaier et al., 1976).The observation that parental lymphocytes that differentiated in the Fr host environment are unresponsive to the relevant alloantigens but are responsive to the virally infected or TNP-modified alloantigens could b e interpreted as favoring the altered-self model, since such results would be consistent with the CML being directed against new antigenic determinants created b y interaction of self H - 2 products with either viral antigens or TNP. However, these experiments do not formally exclude dual recognition involving a self-recognition structure, since it is possible that such a self-recognition structure could have developed during the differentiation of the parental cells in the F1 host, which could have been associated with development of the unresponsive state in the chimera (Zinkernagel, 1976b). Another technique that has been recently employed to deplete lymphoid populations of specific alloreactive cells is that of negative selection (Sprent and Wilson, 1977). In this technique, T-cells from strain A mice were injected into irradiated (A x B)FI hybrid recipients, and then recovered 1 day later from the thoracic duct lymph. Such recovered cells were almost entirely of donor origin and were unresponsive to the host alloantigens. Although such lymphocytes were incapable of generating CML against the alloantigens of strain B, they did generate CML responses against TNP-modified cells of strain B (Sprent and Wilson, 1977). These data indicate that the reaction against TNP-modified allogeneic cells does not require the presence of lymphocytes responsive to the same alloantigens. Such results raise the possibility that chemical modification of cell surface proteins can result in the formation of new cell-surface antigens, and that distinct lymphocyte populations exist that can respond to these new antigens. This short-term procedure is considered to involve only cell filtration, not the induction of a tolerant state which avoids the possible complication in interpreting the chimera experiments (discussed above). A third approach for obtaining a population of cells specifically depleted of alloreactive cells consisted in the selective in vitro elimina-

72

GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

tion of clones of cells proliferating in response to allogeneic cells (Schmitt-Verhulst, 1977). It was found that when the clones of B1O.BR cells reacting against B10.D2 alloantigens were eliminated from cultured spleen cells after bromodeoxyuridine (BUdR) incorporation and subsequent exposure to light, such cultures were unable to generate cytotoxic effector cells against TNP-modified B 10.D2 allogeneic cells. The same cultures could, however, be sensitized by TNP-modified cells syngeneic to the responder cells. These results indicate that stimulation with BlO.D2 alloantigens in the primary culture activates clones that recognize TNP-modified as well as unmodified B10.D2 stimulators in the secondary cultures. These results do not support the hypothesis that there are separate clones of lymphocytes that are stimulated by modified alloantigens (B1O.DB-TNP)that would not be activated by the unmodified B 10.D2 alloantigens. Similar results have been obtained independently (C. A. Janeway, personal communication). At least two interpretations are possible from these results. First, the clones of cells recognizing TNP-modified alloantigens are among the cells that are sensitized by (and probably bear a receptor for) the alloantigen (favoring a dual-receptor model). Second, the possibility can b e raised that a particular class of cells is required for the recognition of “altered self” and that such cells are not generated in primary in uitro CML against TNP-modified alloantigens. The discrepancy between these in uitro data and the results obtained from the in uiuo negative selection experiments (Sprent and Wilson, 1977) outlined above could be due to an artifact inherent to the in uitro culture conditions. Alternatively, this discrepancy could result from a more drastic elimination of reactive cells in uitro, which would include clones of cells of low affinity for the alloantigen that would not have been selected against by the in uiuo depletion, and would not be detected as alloreactive cells, but could interact with the alloantigen in association with a new cell-surface antigen [see two-receptor model of Janeway et al. (1976)l. It should be noted that a result different from that obtained for elimination of alloreactive clones for mouse CML reactions using BUdR and light has recently been obtained in the guinea pig for MLR against TNP-modified alloantigens (Thomas and Shevach, 1977). Using two inbred strains of guinea pig that diiler at the I-region (B. D. Schwartzet al., 1976),these investigators found that lymphocytes from which a particular population of alloreactive cells had been eliminated by BUdR and light were still capable of proliferating in response to in uitro priming and boosting by TNP-modified allogeneic macrophages expressing the relevant alloantigens (Thomas and

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73

Shevach, 1977). The latter results involve a strong proliferative population of T lymphocytes distinct from the cytotoxic T-effector cells induced in the previously described CML experiments (SchmittVerhulst, 1977). Furthermore, the MHC restriction in the guinea pig MLR system is associated with la alloantigens, not with the analogs of the mouse K and D serologically detected antigens. Therefore, at present no strict contradiction can be claimed between the two types of results. However, one would like to speculate that if MHC-coded products are involved in the cellular interactions leading to differentiated immunocompetent T cells, a common underlying mechanism might be involved irrespective of the T-cell function considered.

c. INFECTING

OR

MODIFYING AGENT SPECIFICITY

The viruses extensively investigated in the H-2-restricted CML systems have belonged to three general classes, which d o not exhibit immunological cross reactivity. Vaccinia and ectromelia which d o exhibit CML cross reactivity are both pox viruses. The LCM virus, an arenavirus, and Sendai, a parinfluenza virus, have not exhibited any CML cross-reactivity between them nor with the pox viruses (Doherty et al., 1976a). Recent studies in which mice were immunized with type A influenza viruses, which are serologically distinct, have resulted in the generation of cytotoxic effectors cells which, although H-%restricted, exhibit considerable viral cross-reactivity (Doherty el a/., 1977).This discrepancy between cross-reactivity at the T cytotoxic effector cell level and lack of serological cross-reactivity raises the possibility that the effectors and antibodies are specific for different viral antigenic components. Such differences could account for the observations that virus-specific antibodies did not block viral T-cell lysis (Doherty et al., 1976a; Blanden et al., 1976a) whereas anti-TNP antibodies did inhibit cytotoxicity in the TNP-modified CML (Schmitt-Verhulst et al., 1976; Burakoff et al., 1976a). One of the potential advantages of the in uith chemically modified syngeneic CML response is that this system can be utilized to investigate the fine specificity of the “haptenic” moiety. No cross-reactivity was detected between effectors generated against TNP-modified stimulating cells when assayed on .i’Cr-labeled N-modified target cells and vice versa (Rehn et al., 1976a). Furthermore, effectors generated b y sensitization against TNP-modified syngeneic cells did not lyse H-2-matched target cells modified by the TNP moiety separated from the cell surface by a /3-alanylglycylglycyl tripeptide (Rehn et aZ., 1976b). These results not only demonstrate the high degree of fine specificity contributed b y the “haptenic” or modifying agent, but they

74

GENE M. SHEARGR AND ANNE-MARIE SCHMITT-VERHULST

also tend to argue against dual recognition involving recognition of the TNP moiety and self H-2 by two independent receptors on the T-effector cell (Rehn el al., 1976b). A similar degree of specificity has been reported for proliferative responses against cells modified with a series of nitrophenyl compounds (Janeway, 1976). It should also be noted that T-effector cells generated against TNP-modified syngeneic stimulating cells do not lyse H-2-matched DNP-modified target cells and vice versa (Forman, 1976; A,-M. Gilheany, et al., unpublished observations). Furthermore, skin painting with dinitrofluorobenzene sensitizes mice for in viuo delayed skin reactions and for in vitro proliferative responses to the dinitrophenyl (DNP) but not to the TNP group (Claman et al., 1977).Thus, a variety of T-cell subpopulations appear to be very specific with respect to the antigens they recognize. It should be noted, however, that early B-cell precursors of antibody-producing cells are also able to distinguish between DNP and TNP (Metcalf and Klinman, 1976), although antibodies against these two haptens are cross reactive (Eisen et al.,

1970). The fine specificity of the TNP-modified syngeneic C M L system has been further investigated by TNP-modification of cells either with

trinitrobenzene sulfonate (TNBS), which covalently modifies cell surface proteins, or with TNP-stearolyl-dextran (TSD), an amphipathic molecule, which can be inserted into the lipid bilayer of the cell membrane and binds to cells b y noncovalent forces (Henkart et al., 1977). Quantitatively equivalent amounts of TNP were detected on the surface of cells modified by both reagents as measured by fluoresceinated anti-TNP antibody. These cell preparations were compared for their ability to: (a) sensitize syngeneic splenic lymphocytes leading to the generation of cytotoxic effector cells; (b) serve as lysable targets in a 4-hour "'Cr-release assay for effector cells generated in (a); and (c) act as blocking cells in the lysis of TNBS-modified targets lysed b y TNP-self effector cells generated in (a). In none of these three experimental systems did TSD-modified syngeneic spleen or H-&matched tumor cells act either as a sensitizing immunogen or as a target antigen. In contrast, TNBS-modified spleen cells sensitized syngeneic lymphocytes to generate effectors against TNBS-modified syngeneic targets. Furthermore, TNBS-modified, H-2-matched cells served as specific lysable targets and as inhibiting cells for such effectors. These results indicate that the manner in which TNP is associated with the cell surface is important in the immunogenicity and antigenicity of hapten-modified syngeneic stimulating cells in generating H2-associated C M L reactions. These findings raise the possibility that a

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75

covalent or at least a stable linkage with cell surface proteins (possibly H-2-controlled products) is important for immunological function. Furthermore, these observations d o not favor the dual receptor model for H-2-restricted syngeneic CML if it is assumed in such a model that one receptor is specific for the TNP moiety exclusively, and the second for unmodified-self major histocompatibility products. Although H-2-restricted specificity has been demonstrated in the CML against TNP-modified syngeneic cells, examples have been published in which some degree of non-H-2-restricted lysis was observed (Shearer et al., 1975a; Burakoff et al., 1976b).Cytotoxic effectors generated against TNP-modified syngeneic cells can lyse TNPmodified non-H-2-matched lymphoid tumor target cells and mitogenstimulated blasts although the level of lysis is not as high as with TNP-modified H-2-matched targets (Shearer et al., 1975a; Burakoff et al., 197613). These observations could b e interpreted as evidence for effector specificity directed: (a) exclusively against the hapten (i.e., TNP group) (Dennert and Hatlen, 1975); (b) against TNP-modified non-H-2-coded cell surface components; (c) against cross-reactive determinants resulting from TNP-modification of different K or D region products (Burakoff et d., 1976b); or (d) against TNP-modified K or D region products, although accompanied by a nonspecific lytic interaction between effector and TNP-modified target (Schmitt-Verhulst and Shearer, 1975; Shearer et al., 1976). It is unlikely that the crossreactivity can be accounted for by (a), since TNP-modified chicken erythrocytes did not block the cross reactivity when the effectors were generated by sensitization with TNP-modified syngeneic cells (Burakoff et al., 197613).Possibility (b) is unlikely based on the observation that the cross-reactive lysis can b e blocked by antisera directed against the H-2 haplotype of the target cells (Burakoff et al., 1976b), although it cannot be excluded that H-2 coded products would be involved even in a nonspecific effector-target cell interaction. At this time results are not available to distinguish between possibilities (c) and (d). Other unexpected cytotoxic reactions have been detected in which a variety of effectors have been reported to lyse TNP-modified target cells syngeneic with the effectors (Schmitt-Verhulst and Shearer, 1975; Burakoff et al., 1976b; Starzinski-Powitz et al., 1976a). First, effector cells generated by sensitization against H-2 alloantigens are capable of lysing TNP-modified targets syngeneic with the effectors (SchmittVerhulst and Shearer, 1975; Lemonnier et al., 1977).Cytotoxic effector cells generated in virally infected mice were also found to lyse not only syngeneic target cells infected with the relevant virus but also

76

GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

TNP-modified syngeneic cells (Starzinski-Powitz et al., 1976a). The extent of lysis detected on the TNP-syngeneic cells was proportional to the lytic activity detected on the relevant virally infected targets. Furthermore, H - 2 restriction was observed for the lysis b y effectors from virally infected mice detected on TNP-modified targets (Starzinski-Powitz et uZ., 1976a). The findings that alloreactive cells and cells reacting against virally infected cells are capable of lysing TNP-modified syngeneic cells have been interpreted as indicating that shared CML specificities exist between TNP-modified K and D region products, allogeneic K and D region products, and syngeneic virally modified K and D gene products (Lemonnier e t aZ., 1977; Starzinski-Powitz et al., 1976a). However, it should be noted that effector cells generated by sensitization with TNP-modified syngeneic stimulators have not been found to lyse unmodified allogeneic target cells of any H - 2 haplotype tested (Schmitt-Verhulst and Shearer, 1975; A.-M. Schmitt-Verhulst and G. M. Shearer, unpublished observations). Furthermore, effector cells generated against TNP-modified syngeneic stimulators were not capable of lysing any of the virally infected target cells tested (Starzinski-Powitz et al., 1976a). Were these “nonspecific’’ effects due to cross-reactive determinants resulting from TNP-modification of K andfor D region products, one would have expected that the cross-reactivity would be demonstrable in both directions, i.e., that TNP-modified syngeneic cells would generate effectors capable of lysing unmodified allogeneic and virally infected targets. It could also be argued that if, on the one hand, TNP modification of K and D region products of one haplotype generates antigenic determinants that are cross-reactive with TNP-modified K and D gene products of a different haplotype (Lemonnier et al., 1977), and, on the other hand, virally infected targets present determinants shared with TNP-modified cells of the same H - 2 haplotype, one might expect cross-reactivity to occur between determinants of one haplotype and TNP-modified H - 2 products of another haplotype. This prediction is contradicted by the observation that effectors generated from LCM-infected mice are H - 2 restricted in their ability to lyse TNP-modified target cells. The possibility that clones of effector cells generated by sensitization against TNP-modified syngeneic cells are cross-reactive with the antigens expressed on unmodified allogeneic cells has been tested by experiments in which the TNP-modified selfreactive clones of cytotoxic precursors were eliminated b y treatment with BUdR and light during primary sensitization. A summary of results typical of such experiments is presented in Table 111. Exposure of B1O.BR responding cells to BUdR and light

77

MAJOR HISTOCOMPATIBILITY COMPLEX

TABLE 111 SELECTIVE ELIMINATION OF CLONES OF BlO.BR RESPONDING CELLS TNP-MODIFIED SELF OR ALLOANTIGENS

AGAINST

EITHER

% Specific lysis 2 SE on

tumor target”

Section

A

B

Elimination of clones reactive with

Restimulation with

B1O.BR-TNP (H-2k-TNP) B1O.BR (H-2’) B1O.BR-TNP C57BL110 (H-2b) C57BUlO-TNP B1O.BR-TNP (H-2’-TNP)” B1O.BR (H-2’) B1O.BR-TNP C57BU10 (H-2’) C57BL/lO-TNP

C

B10.D2 (H-2d)

D

B1O.BR (H-2”)

B1O.BR (H-2’) BlO.BR-TNP BlO.D2 (H-2d) B1O.BR (H-2’) BlO.BR-TNP B10.D2 (H-2d)

RDM-4

RDM-4TNP

e14

(H-2’)

(H-2’-TNP)

(H-2”)

10.01 0.1 f 0.8 5.3 f 0.6 9.2 If: 2.2

10.01 5.2 2 0.8 28.8 2 0.5 39.3 2 2.2 10.01 72.1 2 1.4 38.0 5 1.0 42.9 2 0.5

10.01 0.4 ? 0.8

65.3 ? 1.3 65.6 ? 2.1 10.01 5.3 & 0.9 67.9 f 2.3 66.5 ? 2.3

RDM-4

RDM-4TNP

P815

(H-2’)

(H-2’-TNP)

(H-2d)

10.01 -0.9 2 1.2 2.5 f 1.0

10.01 23.8 f 1.4 N T 10.01 27.2 f 2.7 NT

10.01 NT 2.02 1.0 10.01 NT 44.2? 1.3

L0.01

1.1 ? 0.6 6.0 f 0.8 8.3 2 0.9

ro.01

0.8 f 0.6 8.2 ? 0.5

Primary culture which had not been treated with BUdR and light. of specific lysis was obtained by subtracting % lysis by unsensitized cells (less than 10%) from total % lysis; effector to target ratio was 20: 1 in Sections A and B, 40: 1 in Sections C and D. ‘NT. not tested. “

* Percentage

during primary sensitization against B1O.BR-TNP resulted in the failure to generate cytotoxic effectors which lyse RDM-4-TNP targets after restimulation with B1O.BR-TNP (see Section A, Table 111). In contrast, restimulation of the same BUdR and light-treated cultures with C57BW10 (or C57BWlGTNP) allogeneic cells generated a normal CML response against H - 2 b as detected on the EL-4 targets, as well as a normal “cross-reactive” CML detected on RDM-4-TNP targets (compare with data of Section B, Table 111). Conversely, elimi-

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GENE M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST

nation of B1O.BR clones reactive against B10.D2 alloantigens in cultures treated with BUdR and light was achieved, as shown by the lack of restimulation with BlO.D2 without affecting the ability of the same cultures to be stimulated by B1O.BR-TNP as detected on RDM-4-TNP targets (Sections C and D, Table 111). If the reaction of allogeneically sensitized effectors, which is detected on TNP-modified cells syngeneic with these effectors, were due to the stimulation by these alloantigens of clones of cells cross-reacting with the clones of cells generated by TNP-modified syngeneic cells, one would have expected that preelimination of clones reactive against TNP-modified syngeneic cells would have abolished this cross-reactive lysis. This, however, was not observed, and suggests that lysis detected on TNPmodified syngeneic cells by effectors sensitized against alloantigens is effected by clones of cells that are not cross-reactive with those clones stimulated by TNP-modified syngeneic cells. The results summarized in Sections C and D of Table 111 show that elimination of all clones of cells reactive against one set of “cross-reactive” B 10.D2 alloantigens (Schmitt-Verhulst and Shearer, 1975; Lemonnier et al., 1977) does not affect stimulation with TNP-modified syngeneic cells. This also suggests lack of extensive cross-reactivity between clones of cells specific for alloantigens and for TNP-self. A similar argument can be deduced from the specificities required to restimulate effector cell precursors in secondary in vitro CML reactions (Schmitt-Verhulst e t . al., 1977). These results obtained in secondary CML cultures do not support the hypothesis that the lack of absolute specificity in these CML reactions can be accounted for by clones of T-lymphocytes which recognize altered-self antigens and are cross-reactive with certain alloantigens. More extensive research will be required in this area to test the hypothesis that the repertory of T-cell specificities is generated in the thymus during ontogeny (Jerne, 1971) by encountering modified MHC products (Lemonnier et al., 1977). IV. Immune Response Genes for H-2-Restricted Cytotoxicity

It was observed in the initial studies of the CML response against TNP-modified syngeneic cells that effector cells from certain inbred mouse strains lysed TNP-modified targets expressing either the K or D H - 2 haplotype of the responding and modified stimulating cells, whereas other mouse strains generated effectors capable of lysing TNP-modified targets expressing the K haplotype, but not those expressing the D haplotype of the responder and stimulating cells (Shearer, 1974). Mice that exhibited these differences in D-

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end-restricted CML differed in the H-2 haplotypes in the left part of the H-2 complex (which included K and a portion of the Z region), although they expressed the same haplotype at H-2D. When the lytic potential of effectors generated by BIO.A and B10.D2 responding cells were compared, the B10.D2 effectors were observed to lyse TNPmodified target cells matched at either the K - orD-end with the cells of the sensitizing phase, whereas B1O.A effectors did not lyse TNPmodified D-end matched targets although they did lyse TNP-modified K-end matched targets (Shearer et al., 1975a; Schmitt-Verhulst and Shearer, 1975; Schmitt-Verhulst et al., 1976). These strain-dependent preferential response patterns to TNPmodified syngeneic cells restricted at the Dregion appear to be regulated by H-2-linked immune response (Zr) genes, and the ability to respond seems to be a dominant trait, since (BIO.A x B10.D2)F1mice are high responders to TNP-H-2Dd (Schmitt-Verhulst and Shearer, 1975). That responding cells from the BIO.A strain are defective in their response potential to “NP-H-2Dd has been verified by CML inhibition experiments in whlch region-specific anti-H-2 sera were used to block the interaction between effector cells and TNP-modified syngeneic target cells (Schmitt-Verhulst et al., 1976). The results indicated that BlO.D2 anti-B1O.BR sera (directed against H-2Kk)severely reduced the lysis by BIO.A effectors, whereas B1O.BR anti-BlO.D2 sera (directed against H-2D d, had no effect on the lysis of B1O.A-TNP targets by BIO.A effectors. In contrast, the B1O.BR anti-BlO.D2 sera (directed against H-2Dd)abolished the lysis of B 10.A-TNP targets by B10.D2 effectors (Schmitt-Verhulst et al., 1976). These results indicate that the TNP-self cultures from B10.D2 donors generated effectors specific for H-2Dd-TNP, whereas cultures from BIO.A donors did not generate effector cells specific for H-2Dd-TNP. Mapping studies for the Zr control of effector cells specific for H-2Dd-TNP have been performed using recombinant mice on the C57BL/10 and A strain backgrounds (Schmitt-Verhulst and Shearer, 1976). As summarized in Table IV, a higher level of responsiveness was found when the s haplotype was expressed to the left of the Z-E subregion as compared with the k haplotype. The A.TH and B1O.HTT were the highest CML responders to H-2Dd-TNP, whereas BIO.A and A.AL were the poorest responders to H-2Dd-TNP, although all four of these strains were high CML responders to their respective TNPmodified H-2K restricted specificities. The A.TL strain, which expresses the high responders haplotype at K and the low responder k haplotype throughout the Z region was an intermediate responder to H-2Dd-TNP, but a high responder to its respective H-2Ks-TNP re-

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TABLE IV

I n Vitro CYTOTOXIC RESPONSES TO TNP-MODIFIEDA~TOLOGOUS CELLS IN DIFFERENT INBRED MOUSE STRAINS ON THE C57BL110 AND A GENETIC BACKGROUNDS"

Responding spleen cells

Stimulating spleen cells

H - 2 haplotype at: K A BJ ECS D

TNP-modified target cells"

% Specific lysis 2 SE

H-2n H-2 H-2k H-2d H-2% H-2k H-2d H-2= H-2 H-2d H-2' H-2' H-2d H-2" H-2'

2.2 2 1.1 5.2 k 1.0 30.6 & 1.7 38.0 & 1.4 49.9 2.6 27.3 2 2.1 8.2 1.6 3.1 2 1.9 32.5 2 4.7 19.9 2 1.1 45.6 2 0.9 11.1 2 2.5 33.3 2 1.4 51.8 2 0.6 11.4-C 1.7

BIO.A

B 10.A-TNP

kkkkkddd

B1O.HTT

B1O.HTT-TNp

s s s s k k k d

A.AL

A.AL-TNP

kkkkkkkd

A.TL

A.TL-TNP

s

A.TH

A.TH-TNP

s s s s s s s d

k k k k k k d

'

*

*

Range

of lysis 1-10 2-16 2246 38-53 50-55 2-8 0-6 23-33 20-23 10-46 0-11 33-44 1652 5-11

Effector: target ratio = 40 : 1. H - 2 d , H-2': and H-2' target cells were 48-hour phytohemagglutinin-stimulatedblast spleen cells from B10.D2, SJL/Jor BlO.S, and B1O.BR donors, respectively. "

I'

stricted specificity (Schmitt-Verhulst and Shearer, 1976).These results are compatible with the regulation of CML responsiveness specific for H-2Dd-TNP being under the control of at least two genes, one mapping to the left of the crossover between K and Z-A (in the A.TL strain)-possibly in the K region-and the second mapping inside the Z-A through I-] subregions. The finding that at least one of the genes controlling responsiveness to H-2Dd-TNP maps to the left of I-A is the first published example of an H-2-linked Zr gene which appears outside of a known Zsubregion. This raises the possibilities that: (a) Zr genes might be randomly distributed so that not all of them would map in the Z region; (b) some Zr genes that are associated with the Z-A subregion are situated to the left of the crossover that occurred in the A.TL strain as a result of this particular recombinant event having actually taken place within Z-A ; or (c) that functionally distinct Zr genes map in different regions of the MHC. It is noteworthy that this is the only mapped Zr gene which controls the generation of cytotoxic T-effector cells (Schmitt-Verhulst and Shearer, 1975, 1976; Schmitt-Verhulst et al., 1976). Heretofore, Zr

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genes mapped within the Z region controlled antibody production (Benacerraf and Katz, 1975), possibly at the level of the interaction between T-helper cells and B cells and/or macrophages. Zr control of in vitro secondary T-cell proliferation in response to soluble antigens also maps within the I region (R. S. Schwartz et al., 1976).The presence of products coded for by the Z-A subregion on soluble factors mediating T-B cooperation (Taussig et al., 1975),and their functional involvement in T-cell proliferation in response to soluble antigens as assessed by antiserum blocking (R. S. Schwartz et al., 1976), suggests that products of genes mapping in the Z region are involved in the cellular interactions required to generate those differentiated lymphoid functions. The differentiation from precursor cells into cytotoxic effector cells could involve different types of cell-surface interaction structures, some ofwhich could b e coded for by genes mapping within H-2 in a region distinct from I . It may be significant that this Ir gene for cytotoxic function appears to map within an H-2 region that controls those cell surface products which are associated with the antigens recognized for cytotoxic function. A leukemogenic model involving Friend virus has been described in which both H-2-restricted antigenic structures and H-2-linked Zr genes appear to be involved in the generation of a cytotoxic response (Blank et al., 1976). BALB.B mice immunized with a syngeneic leukemic cell line ( H F U b ) generated cytotoxic effector cells which lysed H F L h targets, but not the HFWk leukemic line, derived from BALB.K mice, which suggested H-2 restriction. In contrast, BALB.K mice immunized with syngeneic leukemic HFWk cells did not generate effectors detectable on either the H-2-matched HFWk or allogeneic HFWb targets (Blank et nl., 1976).However, (BALB.B x BALB.K)FI mice did generate effectors that lysed HFWk targets after immunization with HFWk, indicating the HFL/k cells are immunogenic and that the FI hybrid between the congenic responder and nonresponder is a responder (Blank et al., 1976). Based on the other mapping studies for recovery from Friend disease (Cheseboro et al., 1974), it might be predicted (if the Friend virus H-2-restricted cytotoxic response is important for recovery) that the H-2 specificity relevant in the cytotoxic response will be H-2Dh. Intra-H-2 mapping of the Ir gene(s) controlling response to leukemic HFL/k has not been reported. A recent study of the cytotoxic effector cells generated b y F, hybrids between AKWJ and a number of H-2 congenic strains against an inoculated AKWJ tumor cell line showed that H-2q’k and H-2”’ hybrids failed to respond, whereas H-2’” hybrids did (Meruelo and McDevitt, 1977; Meruelo, 1977). From the differential CML responsiveness ex-

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hibited by (AKR x B 10.A[5R])F1hybrid mice, the locus conferring the ability to generate CML effectors against the AKWJ tumor line could be mapped in either the Z-J or Z-E subregions of the H-2 complex. The finding that H-2 homozygous but not heterozygous animals responded suggested either that responsiveness was determined by a recessively expressed locus, or that a dominantly expressed locus controls suppression of the response (Meruelo and McDevitt, 1977).Another study has been reported in which differential CML responsiveness and tumor susceptibility has been detected against an AKWJ carcinoma cell line in (AKIUJ x C57BL/6)F* and (AKWJ X DBAI2)FI hybrids (Levy et al., 1976). In these experiments, however, a mechanism other than H-2-linked Zr control of response potential appears to be involved (Levy et uZ., 1976). Zn vivo studies indicated H-2-linked strain differences in the ability of female mice to reject skin grafts from syngeneic males (Gasser and Silvers, 1972). The in vitro generation of an H-&restricted CML by effector cells from female mice stimulated by cells from syngeneic males was found to follow the same H-2-linked pattern of responsiveness (Gordon et aZ., 1975). In other words, females of H-2b haplotype were responders regardless of the non-H-2 background, whereas females of the H-2k and H-2d haplotypes were nonresponders (Gordon et al., 1975). The specificity of the CML effectors generated by H-2b females against cells from syngeneic males was found to be associated with the D region of the H-2 complex. It was recently found (Simpson and Gordon, 1977) that the Fl hybrid between two nonresponder strains (H-2k’d)could generate an in vitro CML against cells from male H-2k’kparents. The H-2 restriction of this CML response was found to be associated with either Kkor Ilk.These results could be interpreted b y the complementation between one or more immune-response genes from each nonresponder parental strain, which would confer responsiveness of females to H-Y antigens (Simpson and Gordon, 1977). However, since the specificity of the CML effector cells not only involves the H-Y antigen, but is also restricted by products coded by the K and/or D regions of the H-2 complex, such a phenotypic complementation need not necessarily result from a genotypic complementation. Indeed it is possible that immune response genes contributed by one of the parents (BALB/C,H-Zd)allow recognition of the H-Y antigen in association with products coded by the H-2 locus corresponding to the haplotype of the other parent (CBA, H-2k)despite the fact that these immune-response genes did not allow responsiveness to H-Y association with H-2d.

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The mechanism(s) by which these l r genes influence the generation of cytotoxic effector cells is unknown. Since it appears that interacting cell types are involved in the generation of effectors at least for the autologous TNP-modified CML (Hodes et al., 1975), it is possible that these genes exert their influences on distinct cell populations and/or their interactions. The cellular expression of such genetic parameters could be exerted on: (a) a population of cytotoxic precursor cells; (b) on helper cells or cells that selectively augment an H-2D- or H 2K-restricted response; and/or (c) on suppressor cells that could regulate the level of cytotoxicity generated. V. Conclusions and Speculation

The published examples for which H-2-restricted CML reactions have been repeatedly observed can be classed into three broad categories, i.e., virally infected, chemically modified, and weak transplantation antigen-associated. The immunogenetic similarities exhibited b y these three systems suggest that they may share certain common mechanistic features as well. However, before making such a conclusion it may be worth discussing the different ways in which the respective antigenic determinants (e.g., virus, hapten, or minor antigen) could be presented in association with H-2-coded products on the cell surface. In the H-2-restricted viral systems, it is possible that certain viral antigenic determinants could have a selective physical association with H-2-determined antigens on the cell surface. Compatible with this hypothesis is the observation that the vesticular stomatitis virus exhibited H-2 antigenic activity common with the H-2 haplotype of the cells that were infected (Hecht and Summers, 1972). Furthermore, in murine leukemogenic studies viral antigens and private H-2 serological specificities have been observed to modulate simultaneously, suggesting some type of physical interaction between H 2-coded and viral antigens (Lilly, 1972). It has also been postulated with some supporting evidence that viruses can induce changes in cell surface H-2-coded antigens, which could be accounted for by a mechanism allowing derepression of genes controlling expression of H-2 products corresponding to foreign haplotypes (Garrido et al., 1976). This latter mechanism would appear not to be responsible for the H-2-restricted syngeneic CML considered here, since the H-2 specificities of infected cells appeared to be unchanged and could not account for the specificity observed in these systems (Doherty et al.,

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1976a,b). In this context it should be noted that cytotoxic effector cells against Sendai virus have been generated in vitro from spleen cells of mice immunized against UV-inactivated Sendai (Schrader and Edleman, 1977). These effectors were restricted to lyse H-2-matched tumor cell targets externally coated with inactivated Sendai virus by short-term incubation. The results suggest that viral components can interact with murine cell surface structures in the absence of viral replication and protein synthesis (Schrader and Edelman, 1977). In the chemically modified system, a random modification of the free amino groups of cell surface proteins (Okuyama and Satake, 1960) results in a nonselective “haptenation” of those proteins including the serologically detectable H-2K and H-2D coded antigens (Forman et al., 1977), which represent only a small portion of the cell surface proteins. An altered-self hypothesis where the actual new antigen consists of modified K or D region products is readily conceivable in such a system (Shearer, 1974),but does not account for the preferential H-2 association of the recognition. In contrast, the concept of physical modification ofH-%coded products is less likely for the system involving the H-2-associated recognition of weak transplantation antigens. More probable is the possibility that the minor transplantation antigens which elicit T-lymphocyte responses are in some way closely associated on the cell surface with the relevant major histocompatibility products. In the generation of cytotoxic effector cells, the involvement of H2-coded products could be important at sensitization and/or during the lytic phase of the CML. An absolute requirement for H-2 homology between effector and target cells or between responding and stimulating cells does not appear to be necessary. This has been shown b y the ability of effector cells from a lymphoid pool tolerized to an alloantigen to generate effectors capable of being sensitized against infected or modified cells expressing the H-2 haplotype of the tolerogen (Pfizenmaier et al., 1976; von Boehmer and Haas, 1976; Zinkernagel, 1976b). These results could be interpreted as indicating that different clones of cells recognize these alloantigens and the new antigens resulting from the interaction of virus or TNP with these alloantigens. This suggests that receptors recognizing alloantigens and antigenic entities associated with these alloantigens are distinct. However, these experiments do not necessarily exclude the possibility that the H-2 homology requirement could function through a complementary interaction structure that might be acquired during the tolerization against the alloantigen, by a process similar to the natural acquisition during ontogeny of tolerance to self H-2-coded products.

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Two other experimental approaches involving the selective induction of unresponsiveness have been employed to investigate this problem. The in uiuo results obtained with negative selection of alloreactive clones indicate that sequestration of clones reactive with a given allogeneic haplotype abrogates that allogeneic response without affecting the CML response against that particular TNP-modified alloantigen (Sprent and Wilson, 1977). In contrast, the in uitro elimination from cultured lymphocytes of clones proliferating in response to an alloantigen did not allow sensitization of CML effectors against the same alloantigen modified with TNP (Schmitt-Verhulst, 1977; C. A. Janeway, personal communication). The selective in uiuo sequestration of alloreactive clones in thoracic duct lymph (Sprent and Wilson, 1977) indicates that distinct clones of responding cells recognized alloantigen and TNP-modified alloantigen. However, in order to account for the in uitro elimination of clones reactive against alloantigen and TNP-modified alloantigen (Schmitt-Verhulst, 1977), it might be necessary to postulate affinity diflerences between clones of lymphocytes recognizing alloantigens and modified alloantigens. More explicitly, the in uiuo sequestration of alloreactive cells might filter out only alloreactive lymphocytes of high affinity, leaving behind the lymphocyte clones that can be activated only through the recognition of an additional antigen by a second receptor (dual recognition model). It would have to be presumed that both types of ,clones would be eliminated under the conditions of optimal in uitro allogeneic stimulation. It is worth noting in this context that the generation of an allogeneic and TNP-modified syngeneic CML are dependent upon distinct subpopulation of lymphocytes as determined b y Ly type (Cantor, 1977). This also raises the possibility that the alloreactive cells and cells reacting against modified allogeneic cells would belong to different subpopulations of T-lymphocytes. One advantage that the chemically modified CML model has over the viral and weak transplantation antigen models is the potential for investigating the antigenic contribution made by the modifying agent to the fine specificity of the T-cell recognition. It would be expected from the dual-recognition model (in its simplest form) that some dissociation could be made between the receptors for the TNP “hapten” and those recognizing unaltered self. In the chemically modified CML system experiments have been designed and performed to test the model b y introducing slight changes in the presentation of the “ h a p ten” on the syngeneic cell surface (Rehn et al., 1976b; Henkart et al., 1977). Cells modified by TNP separated from the cell surface b y a tripeptide did not act as targets for effectors generated by sensitization

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against TNP-modified cells (Rehn et al., 1976b). Furthermore, cells modified with TNP-stearoyldextran presenting quantitatively similar amounts of TNP groups on the cell surface as TNBS-modified cells did not serve as targets for effectors generated by sensitization with TNBS-modified syngeneic cells (Henkart et al., 1977). If T-cell recognition occurs via two independent receptors-one recognizing the TNP group and the other recognizing self H-2-coded products, either or both of the targets modified by these slight variations in TNP presentation on the cell surface should have been recognized by effector cells specific for TNP-self. These findings are more compatible with recognition by a single receptor that would recognize either “hapten”-modified H-2 products or “hapten” in close association with unaltered-self H-2 products. These results can be most readily interpreted either b y the altered-self model or b y an “altered” dualrecognition model-since if two receptors d o exist, they appear not to be independent from one another. For the TNP-self system the “altered” dual-recognition model could be valid if it assumed that the receptor for hapten recognizes the haptenic moiety plus a portion of the adjacent amino acids. The chemically modified self CML provides another advantage for investigating the possibility that TNP-modified soluble proteins could b e antigenic when presented in association with self H-2 products. Hypothetically, such a system could operate through the presentation of TNP groups by syngeneic macrophages, if sensitization and lysis were to occur through dual recognition. Presumably, this system would involve the recognition of the TNP groups by the T lymphocytes, and the TNP-bearing macrophages would specifically interact with the relevant T lymphocytes via the self H-2-coded products. Furthermore, the development of the TNP-modified system for proliferative and cytotoxic responses with human lymphocytes could be useful for elucidating the role of MHC restriction and Zr genes in man (Shaw, unpublished observations). Although the in uitro chemically modified syngeneic system offers some advantages for manipulation and dissection of cell-mediated immune processes, TNP is not a pathogenic agent (Doherty, personal communication). In contrast, certain of the viral systems investigated are pathogenic in man and could be more relevant for elucidation of immune processes potentially relevant for d’isease. The final answer to the one-receptor or two-receptor question will probably come from characterization of the T-cell receptor. If it is assumed that all receptors possess idiotypic determinants, recognition should be blocked b y either of the two idiotypes (Binz and Wigzell, 1975a; Janeway et al., 1976; Doherty et al., 1976b).

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The fact that some of the H-2-restricted syngeneic CML models are under the control of H-2-linked Ir genes as well as being restricted with K and D region products, suggests multiple functional roles for H-2-coded gene products in cell-mediated immunity. If the immune phenomena reviewed here are relevant for natural immunity, MHC products may be simultaneously important for recognition by T lymphocytes (under the control of Ir genes) and for the H-2-restricted structures which they recognize (the K , I , or D region products associated with the infectious agent). ACKNOWLEDGMENTS We are grateful to Dr. William D. Terry, who has supported our research efforts. We thank Mrs. Marilyn Schoenfelder for preparation of the manuscript.

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