Immunology Today November 1983 T-cell activation by antigen

Promising clues to reception genes and molecules fromJay Berzofsky The mechanism of T-lymphocyte recognition of, and activation by, specific antigens, and the molecular structures involved in this process, have been a major concern of immunologists over the past decade. The latest findings presented in Kyoto suggest that the answers to some of these questions may be just around the corner. However, none of them can be viewed yet as completely solved, and controversies still abound. The problem has been attacked at several levels, including characterization of the antigenic sites, or epitopes, on antigens which are involved in activation of T cells, the role of accessory antigenpresenting cells in processing antigen and in the genetic restriction of T-cell recognition of antigen, and the structure of the antigen-specific receptors on T cells and the genes which encode them. The following are highlights of the progress in these areas which emerged at the congress.

Major histocompatibility complex ( M H C ) restriction Over the decade since the discovery of genetic restrictions on the interaction of T cells with antigen on B cells or antigenpresenting cells (macrophages or dendritic cells), 1'2 uncertainty has prevailed about the mechanism by which such restriction is imposed. The basic observation, as currently viewed, is that many types o f T lymphocytes - including those which mediate help for antibody production by B lymphocytes, those which mediate delayed hypersensitivity, and those which proliferate in response to antigen - are not activated by antigen alone, but only by antigen 'presented' on other cells bearing major histocompatibility antigens of the appropriate haplotype. Moreover, the particular antigenic site or epitope which is stimulatory deponds on the M H C antigens of the anti-

gen-presenting cells. 3 Several studies were presented which amplify this observation, using cloned T-cell lines. In the case of the mouse, where there are two genetic subregions encoding class-II (or Ia) M H C antigens involved in antigen presentation, I-A and I-E, some antigens, such as ( T , G ) - A - L and GLPhe, can be seen only in association with Ia antigens encoded in one or the other of these subregions. I. Berkower,J. Berzofsky and coworkers (Bethesda) found that even for myoglobin, which can be presented in association with either I-A or I-E antigens, all T-cell clones specific for one major epitope were restricted to I-A, whereas ~all those specific for a second epitope were restricted to I-E. Analogous results were reported by D. Eckds and coworkers (Washington) for the case of h u m a n T-cell clones specific for different epitopes of influenza hemagglutinin and restricted by different H L A - D region specificities. However, given the limited n u m b e r of class-II M H C antigens, one would expect some redundancies. A. Reske-Kunz and E. Ri~de (Mainz) found that one Ia epitope was involved in the presentation of more than one epitope of insulin to different T-cell clones. A. Rosenthal (Rahway, New Jersey) used the b m l 2 mutant, which carries mutations in some epitopes of the I-A~ chain and not others, to show that different Ia epitopes on the same Ia molecules were preferentially involved in the presentation of different antigens. These studies amplify, at the clonal level, the observation that presentation of antigen on cells bearing different Ia molecules leads to the preferential activation of T cells specific for different epitopes? However, they leave unresolved the controversy over where and how this selective activation occurs. Currently, the controversy has taken the following form: both sides accept the postulate that

each T cell must simultaneously recognize both the exogenous antigen and an Ia antigen, and that each combination of antigen plus Ia on the presenting cells can be viewed as a distinct antigenic entity (as defined by crossreactions among these combinations and between these combinations and alIo-MHC antigens). However, one side would explain the selective recognition of different antigens in association with different Ia molecules by gaps in the T-cell repertoire (possibly due to self-tolerance) in any given strain. The other side explains the observations by a noncovalent interaction of limited specificity between the Ia molecule on the antigen-presenting cell and the exogenous antigen, such that each Ia molecule can present only certain antigens or epitopes to T cells. As a test of the former hypothesis, Ishii, Nag3' and Klein 4 asked whether presenting cells from a strain nonresponsive to a particular antigen could present that antigen to a T cell of a different strain (which might have a different T-cell repertoire). Using a complicated protocol designed to eliminate alloreactivity and allow primary immunization on allogeneic cells, they reported that such presentation did occur. In view of the observations cited above that different epitopes on the same antigen molecule were preferentially stimulatory in the context of different Ia molecules, it was important to know whether presenting cells from the lowresponder strain were presenting the same epitope that was seen on highresponder presenting cells. J. Klein (Tfibingen) reported in Kyoto that the same epitope of beef insulin, the A loop, that is presented by high-responder presenting cells is also presented to highresponder T cells by low-responder presenting cells. (A similar T-cell clone in the guinea pig which saw insulin on a low-responder presenting cell was re-

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300 ported by Shevach and coworkers 5 but in that case the epitope presented was distinct from that presented by the high responder.) Klein concluded that low responsiveness is not due to an inability of low-responder presenting cells to present the epitope, but rather to a gap in the repertoire o f T cells found in that strain. In support of the alternative hypothesis, R. Schwartz (Bethesda) presented evidence for two adjacent sites on a peptide fragment, 81-104, of cytochrome c. One site (residue 99) was the epitope seen by the T cell. 6 The second site (residue 103) determined which Ia molecule (or which presenting cell) could present this epitope. 6 He dubbed this second site the 'agretope' for 'antigen-restriction site' and concluded that it must be the site which is hypothesized to interact with Ia. Moreover, since residues 94-99 are the same in mouse cytochrome as in the immunogenic cytochromes, mice can respond to a self-epitope, on self-Ia, but only when associated with a non-self agretope which allows it to be presented. This is the other side of the coin from the observation of Shevach and coworkers 5 that guinea pig T cells can respond to an epitope of guinea pig insulin, but only when presented on allogeneic presenting cells (i.e., non-self Ia). Both observations lead to the important conclusion that self-tolerance is at least in part genetically restricted. The observation of these two sites, the epitope and the agretope, which seem to independently determine T-cell specificity and genetic restriction, respectively, taken together with earlier evidence that the same T-ceil hybridoma clone sees cytochrome differendy depending on which Ia antigen is used for presentation, 7 strongly supports the second hypothesis that the Ia of the antigen-presenting cell directly interacts, with some degree of specificity, with the antigen and influences T-cell specificity. Further evidence for such an interaction was presented by B. Benacerraf(Boston) who, with K. Rock, found reversible competition at the antigen-presenting ceil level between two closely related antigens, G T and GAT, but not unrelated antigens, for stimulation o f T cell clones. The results demonstrate specificity in the interaction of antigen with some component of thepresenting cell, possibly Ia. Similar findings have been reported by Werdelin. s With evidence for both hypotheses, the controversy cannot yet be resolved, except perhaps by the conclusion that both mechanisms play a role. Antigen does interact in some specific way with Ia, but in addition, certain combinations cannot be seen by T cells of a given strain because of gaps in the T-ceil repertoire.

Indeed, the observation that selftolerance can be genetically restricted is an example of just such a combination of mechanisms at work. Two extreme ends of the spectrum of hypothetical mechanisms of genetic restriction were ruled out. H. Cantor (Boston) reported that helper T-cell clones specific for the hapten azobenzene arsonate (ABA) could be shown to bind radiolabeled ABA-ovalbumin in a specific fashion without an antigen-presenting cell, but that free non-stimulatory analogues of antigen (without M H C ) , which bound to T cells as well as did ABA-ovalbumin, acted as antagonists rather than agonists and inhibited T-ceil proliferation. Similarly, J. Miller (Melbourne) reported that free hapten could inhibit delayed hypersensitivity produced by oxazalone-specific T-cell clones. These studies, in conjunction with the evidence of Rock and Benacerraf ° that T cells which see antigen plus M H C can also see M H C alone, exclude the extreme neoantigenic determinant hypothesis which suggests that what the T ceil sees is a neoantigenic determinant formed by the interaction of antigen and M H C but resembling neither alone. At the other extreme is the strict dual receptor model which supposes that antigen and M H C are bound independently by two separate receptors on the T cell. R. MacDonald(Lausanne) fused artificial liposomes carrying membrane components of one cytotoxic T lymphocyte with a cytotoxic T lymphocyte of different specificity and genetic restriction. He obtained clones which expressed both specificities, but there was no reassortment of one genetic restriction with the other antigen specificity, as would be expected for independent receptors. An earlier study by Kappler et al. lo using fusions of IL-2-producing T hybridomas with T cells expressing different specificity and restriction also did not detect any reassortment of antigen specificity and genetic restriction in clones expressing both parental specificities and restrictions. These studies exclude the extreme case of independently synthesized receptors for antigen and M H C in the absence of any specific interaction between antigen and M H C . We are left with a middle ground in which a single T-cell receptor (perhaps with two subsites) binds an interaction complex of antigen and Ia, both of which retain their own determinants.

Antigen processing The exact molecular mechanism of T cell activation by antigen awaits biochepaical studies of antigen handling by presenting cells and of the T-cell receptor

for antigen. Progress on both fronts was reported at the Congress. E. U n a n u e (Boston) reported that mild paraformaldehyde fixation of antigen-presenting cells before or within 5 minutes of exposure to antigen prevented presentation, but after 1 hour of exposure to antigen, mild paraformaldehyde fixation no longer inhibited presentation. This result suggested that metabolic steps were required early in the handling of antigen by presenting cells, but once this occurred, the cell could be rendered inert and still present antigen to the T cell. However, small peptide fragments of antigen did not require this inhibitable step. Similar results were obtained by Shimonkevitz eta/. u comparing presentation of native ovalbumin or fragments of ovalbumin to T cell hybridomas, and by our own lab (Streicher, Berkower and Berzofsky) using other inhibitors of lysozomal proteases and transport to inhibit presentation of native myoglobin but not a small fragment bearing the same epitope seen by a T-cell done. These results all imply that an epitope on a small peptide can be presented without at least some of the processing steps required for the same epitope on the native protein, and suggest that one of those steps might be proteolysis.

The T-cell receptor for antigen The biochemistry of the T-cell receptor is being studied intensively by a large n u m b e r of laboratories, primarily by the approach of making monoclonal antibodies against T-ceil clones or cloned T-ceil tumors or hybridomas. Antibodies which either inhibit or induce (in the absence of antigen) antigen-specific T-cell function and which are clonespecific (so-called anti-clonotypic antibodies) are tentatively presumed to be specific for the antigen receptor of that clone. This approach was reported at the meeting by P. Marrack (Denver), J. Allison (Smithville, Texas), E. Reinherz (Boston), E. Mozes (Rehovot), H. Cantor (Boston), J. Miller (Melbourne) and M. Bevan (La Jolla), and has been described by Fathman and coworkers, 1~ Janeway and coworkers,t3 Samelson and Schwartz, 14 and Kunkel and coworkersJ 5 Most of these groups have immunoprecipitated molecules of similar size with these antibodies, giving molecular weights of 80-90 000 unreduced and 40-45 000 reduced. Sometimes after reduction two subunitL of slightly different molecular weight can be resolved and sometimes not, but the consensus is that the target of these anticlonotype antibodies is a heterodimer. In one of the more thoroughly studied systems to date, Marrack reported

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further evidence that the structure bound by the anti-clonotypic monoclonal antibodies is the antigen-specific T-cell receptor. These antibodies will inhibit antigen-specific stimulation of one BALB/c ovalbumin-specific clone and not others, even of fairly similar specificity, and, when attached to 'Sepharose', will mimic antigen and activate the clone directly. They do not bind to a subclone which has lost antigen specificity. Conversely, when used to screen a new group of over 400 BALB/c ovalbumin-specific hybridomas from four fusions, this antibody detected only one clone, which had the identical (and unusual) fine specificity pattern, the same genetic restriction, and the same crossreaction with allo-MHC (H-2 b) as the original clone, but could be distinguished because it was derived from a male mouse whereas the original clone came from a female. The immunoprecipitates also gave identical tryptic peptide maps. These results not only strongly support the interpretation that the clonotypic antibody is specific for the T-cell receptor, but also suggest that a single (two-chain) receptor is responsible for antigen specificity, genetic restriction, and cross-reaction with allo-MHC. Furthermore, by isoelectric point and peptide maps, both subunits showed evidence of constant and variable portions. In addition to very similar observations using anti-clonotype antibodies to a h u m a n cytotoxic T-cell clone (including inhibition by free antibody and activation by sepharose-bound antibody and similar molecular weights and peptide-map evidence of variable and constant regions on both chains), Reinherz described comodulation of this receptor with antibody to T3, a 23 000 mol. wt invariant cell surface protein on all mature h u m a n T cells which seems to complex with the antigen-specific receptor. F. Fitch (Chicago) described a monoclonal antibody to a third cellsurface molecule involved in T-cell activation, L3T4, which is a marker for the helper/inducer subset of T cells. Evidence of markers on T cells genetically linked to the immunoglobulin heavy chain allotype locus was described by F. Owen (Boston), T. Taniguchi (Chiba) and K. Okumura (Tokyo). Although these markers have been found on T-cell factors, their relationship to the receptors detected by anti-clonotypic antibodies remains to be determined. Also, T. Tada (Tokyo) described markers on T cells which appear to map genetically in I-A but to be distinct from conventional class II (Ia) molecules found on B cells and macrophages. He suggested that these may actually be receptors for I-A-encoded class-II molecules. A test of this

hypothesis using chimeras, and studies of the relationship of these molecules to the T-cell receptors for antigen, are pending. It is clear that rapid progress on the biochemistry of the T-cell receptor for antigen should soon distinguish among the competing models for T-cell activation by antigen suggested from functional studies. At the same time, a promising breakthrough was reported by Mark Davis and Stephen Hedrick (Bethesda) on the genes for the T-cell receptor. They prepared eDNA from m R N A of a T-cell hybridoma and selected for molecules which derived from membrane-bound polysomal m R N A but which did not hybridize with B-cell mRNA. O f 11 distinct genes studied, one cross-hybridized with the Thy 1 gene, and the remainder were screened for rearrangements, based on restriction fragment size, in different T-cell tumors and hybridomas. One cDNA probe detected a gene which was systematically rearranged in eight out of eight T-cell hybridomas but not in liver D N A from different strains of mice. Furthermore; a T hybridoma subelone which had lost its reactivity to antigen displayed a different pattern. The eDNA probe was used to screen a library of normal thymocyte DNA, and three clones were obtained, each with variable segments at the 5' end (by restriction mapping) and about 600 base pairs conserved at the 3' end, consistent with variable and constant regions. The largest of these, when sequenced, had an openreading frame for over 270 amino acid residues. A computer comparison of this sequence with the Dayhoff data bank of protein sequences revealed the 21 best matches as immunoglobulins, although the homology was not great enough to expect D N A hybridization. The homology was greatest around four cysteine residues that define immunoglobulin domains. Thus, although the gene did not encode immunoglobulin, it encoded a membrane-bound structure resembling immunoglobulin, with variable and constant regions, rearranged in different T cells but not other tissues, and not expressed in B cells. These are all properties that one would expect of the gene for the T-cell receptor, but definitive proof is still being sought. Thus, it can be hoped that this long-elusive receptor will soon be characterized at both the protein and the D N A level. T h e r e l a t i o n s h i p b e t w e e n T-cell s p e c i f i c i t y a n d function

Finally, several studies showed a specific relationship between the epitope which activates a T cell and its function. U. Krzych, A. Fowler and E. Sercarz (Los Angeles) showed that of the many

epitopes of /3-galactosidase which stimulated T-cell proliferation, only a few induced helper T cells and a few suppressor T cells. Similar results for help vs proliferation of insulin-specific T cells were reported by P. Hochman and B. Huber (Boston). Furthermore, Berzofsky and H. Kawamura (Bethesda) reported that helper T cells specific for one epitope preferentially help B cells specific for a nearby epitope and thus regulate antibody specificity. Results suggesting T-cell regulation of antibody specificity were also reported 'by F. Celada (Genoa), and similar requirements for proximity between epitopes of /3galactosidase seen by suppressor T cells and by the helper T cells they suppress were described by Krzych and coworkers. Thus, even after the structure of the T-cell receptor and the mechanisms of antigen processing, presentation, and genetic restriction are worked out, the epitope specificity of T-cell activation by antigen will continue to be important because of the major role of this specificity in the subsequent interaction of the T cell with other ceils, in the effector function expressed, and (in the case of antibody responses) in the antibodies ultimately produced. j . A. Berzofsky is a Senior Investigator in the Metabolism Branch, National Canter Institute, National Institutes of Health Bethesda, Maryland 20205, USA.

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