Major histocompatibility complex-controlled, antigen-presenting cell-expressed specificity of T cell antigen recognition. Identification of a site of interaction and its relationship to Ir genes.

In previous work (5,6), we have reported studies on a T lymphocyte hybridoma clone and the peritoneal exudate T cells (PETLES) from B10.A(5R) mice primed with the cytochrome c carboxyl terminal peptide (residues 81-103) of the tobacco horn worm moth (Manducca sextus). As expected, since B10.A(5R) is a low responder to pigeon fragment 81-104, it was found that the B10.A(5R) lymphocytes were unable to respond to the pigeon cytochrome c 81-104 fragment presented on syngeneic B10.A(5R) antigen-presenting cells (APC). However, these same T lymphocytes did respond to the pigeon fragment when presented on B10.A APC. Thus, some structural difference between the pigeon and moth peptides had prevented B10.A(5R) APC from effectively presenting the pigeon fragment to moth-primed B10.A(5R) lymphocytes. This structural difference was found to be the deletion of an alanine at position -103 (Ala103) from the pigeon sequence in the moth peptide. Two additional T cell specificities were created by changing residue-99. These T cell populations from the B10.A(5R) showed an identical dependence on the Ala103 deletion when B10.A and B10.A(5R) APC were compared. The relationship of APC-expressed antigen specificity and MHC-linked immune responsiveness differences was also examined. The B10.A(5R) was found to be a high responder to each of three peptides that lack Ala103 but not to the Ala103-containing analogues. B10.A mice, in contrast, respond to both types of peptides. Utilizing allogeneic antigen-presentation to B10.A PETLES by pulsed APC, it was shown that the poor response of the B10.A(5R) to the Ala103-containing peptides was, in two of three cases, not associated with any differences in T cell repertoires but due to two different APC capabilities of B10.A and B10.A(5R). The exception apparently represents a case of T cell repertoire polymorphism between B10.A and B10.A(5R) that can also affect immune responsiveness.

Contribution of antigen-presenting cell major histocompatibility complex gene products to the specificity of antigen-induced T cell activation.

Previous studies from our laboratory showed that B 10.A mice are high responders to pigeon cytochrome c fragment 81-104, whereas'B 10.A(5R) mice are low responders. In the present studies, the C-terminal cyanogen bromide cleavage fragment and homologous synthetic peptides of tobacco horn worm moth cytochrome c were shown to be immunogenic in both B10.A and B10.A(5R) mice. These strains, however, showed different patterns of cross-reactivity when immune lymph node T cells were stimulated with cytochrome c fragments from other species. To examine the two patterns of responsiveness at a clonal level, cytochrome c fragment-specific T cell hybridomas were made and found to secrete interleukin 2 in response to antigen. The patterns of cross- reactivity of these B 10.A and B 10.A(5R) clones were similar to that seen in the whole lymph node population. Surprisingly, when these clones were tested for major histocompatibility complex (MHC)-restricted antigen recognition, they were all found to respond to antigen with both B10.A and B10.A(5R) antigen-presenting cells (APC). Furthermore, the cross-reactivity pattern appeared to be largely determined by the genotype of the APC, not the genotype of the T cell clone. That is, a given T cell clone displayed a different fine specificity when assayed with B10.A or B10.A(5R) APC. This observation indicates that the APC MHC gene product and antigen interact during the stimulation of the T cell response and that as a consequence the specificity of antigen-induced T cell activation is influenced by these MHC gene products. (During the preparation of this manuscript it has come to our attention that results similar to our own, concerning the fine specificity of cytotoxic T cell clones, have been obtained by Dr. T. R. Hunig and Dr. M. J. Bevan, Massachusetts Institute of Technology, Boston, MA. T. R. Hunig and M. J. Bevan. 1981. Specificity of T-cell clones illustrates altered self hypothesis. Nature. 294:460.)

Selection of amino acid sequences in the beta chain of the T cell antigen receptor.

The induction of an immune response in mammals is initiated by specifically reactive T lymphocytes. The specificity of the reaction is mediated by a complex receptor, part of which is highly variable in sequence and analogous to immunoglobulin heavy- and light-chain variable domains. The functional specificity of the T cell antigen receptor is, however, markedly different from immunoglobulins in that it mediates cell-cell interactions via the simultaneous recognition of foreign antigens and major histocompatibility complex-encoded molecules expressed on the surface of various lymphoid and nonlymphoid cells. The relation between the structure of the receptor and its functional specificity was investigated by analyzing the primary sequences of the receptors expressed by a series of T lymphocyte clones specific for a model antigen, pigeon cytochrome c. Within this set of T lymphocyte clones there was a striking selection for amino acid sequences in the receptor beta-chain in the region analogous to the third complementarity-determining region of immunoglobulins. Thus, despite the functional differences between T cell antigen receptors and immunoglobulin molecules, analogous regions appear to be important in determining ligand specificity.

The sites of antigen-T cell and antigen-MHC interactions overlap.

The immune responses of B10.A and B10.A(3R) strains of mice to a synthetic variant of moth cytochrome c 86-103 were compared, and an immune response difference between the two strains was found. When T cell hybridomas were made from both strains it was found that the responsive T cells showed degenerate MHC restriction. The hybridomas from B10.A(3R) mice consistently showed a specificity pattern, when tested with allogeneic B10.A APC, different from that seen when syngeneic B10.A(3R) APC were used. The difference in the immune responses of the two strains of mice could be attributed to this APC-expressed specificity. The results were analyzed to determine whether the portion of the antigen that effected T cell memory (the epitope) and the portion that effected APC-expressed specificity (the agretope) were independent. It was found that a) these sites overlap and b) the APC-expressed specificity (i.e., the specificity of agretope recognition) was dependent on the T cell clonotype. This implies that the agretope is not an independent parameter in the process of MHC-restricted antigen recognition. Therefore its employment as an explanation for various phenomena will be limited by the likelihood of circularity.

The Ia molecule of the antigen-presenting cell plays a critical role in immune response gene regulation of T cell activation.

It is now clear that MHC-encoded molecules influence the immune response by their effects on T cell specificity. However, the mechanism(s) by which this occurs in an antigen-specific manner is not clear. Two broad categories of models have been proposed. One postulates that Ia molecules influence the T cell repertoire during ontogeny and that the clones of T cells capable of recognizing the antigen in association with the nonresponder Ia molecule are deleted or are never assembled. The other postulates that Ia molecules influence the immune response during T cell activation and that the clones of T cells capable of recognizing the antigen in association with the nonresponder Ia molecule are not stimulated because this Ia molecule does not bind or interact well with the antigen. Recent experiments by several laboratories have identified allogeneic T cell clones and populations capable of responding to an antigen in association with nonresponder Ia molecules. This result suggested that nonresponder Ia molecules are capable of interacting with antigen in a functional manner. Therefore it was inferred that Ir gene defects must lie in the T cell repertoire. However, we have recently discovered several mouse T cell clones whose characteristics suggest that this conclusion might not describe the complete picture. These clones have the unusual property of recognizing moth cytochrome c in association with either B10.A or B10.A(5R) Ia molecules, but they recognize pigeon cytochrome c only in associated with B10.A Ia molecules. Thus, the Ia-molecule-antigen interaction appears to be able to affect the fine specificity of the T cell activation process. Interestingly, the failure to respond to pigeon cytochrome c in association with B10.A(5R) Ia molecules correlates with the Ir gene control of the response to this antigen since the B10.A(5R) strain is a nonresponder to pigeon cytochrome c whereas the B10.A strain is a responder. The question we wished to address in this paper was whether the clones we had identified were representative of the T cell repertoires of both strains. If so, this would allow us to conclude that Ia molecule-antigen interactions during T cell activation could account for Ir gene control in certain situations. To answer the experimental question, we first examined several more T cell clones from [B10.A x B10.A(5R)]F1 and B10.A(5R) mice to solidify our previous findings. Then we turned to populations of T cells to determine if these findings could be generalized.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of the diversity of murine antibodies to dextran B1355: N-terminal amino acid sequences of heavy chains from serum antibody.

The N-terminal amino acid sequences of two gamma and two mu chains from normally induced serum antibodies to dextran in BALB/c mice are presented. These heavy chains are derived from antibodies with three distinguishable idiotypes. These variable region (VH) sequences are all identical as far as they have been analyzed (27 to 53 residues). The light chains from these antibodies are all of the lambda type and are identical by isoelectric focusing analysis. Accordingly, the diversity of dextran antibodies appears to reside primarily in the heavy chains. The implications of these observations for antibody diversity are discussed.

Analysis of the diversity of murine antibodies to dextran B1355. I. Generation of a larger, pauci-clonal response by a bacterial vaccine.

Mice immunized with a combination of dextran B1355 in adjuvant followed by three injections of 2 x 10(9) Escherichia coli B organisms produced an average of 14.5 mg/ml of anti-dextran antibodies. It was demonstrated that the stimulating effect of E. coli B was due to antigenic determinants cross-reactive with B1355 and not solely because of adjuvant properties of the organism. The anti-dextran antibodies were distributed among both 7S and 19S components. Isoelectric focusing of the 7S antibodies showed several spectrotypes of antibody, most of which were shared by the majority of the individual sera. The limited spectrotypic heterogeneity of the 7S antibodies was supported by idiotypic studies. Thus, a heterologous, anti-idiotypic serum, rabbit anti-M104, was prepared which distinguished between two closely related myeloma proteins, M104 and J558,with specificity for alpha-(1 leads to 3) dextran. This antiserum demonstrated that some, but not all, of the 7S and 19S anti-dextran antibodies possessed variable region determinants cross-reactive with M104.

The T lymphocyte response to cytochrome c. IV. Distinguishable sites on a peptide antigen which affect antigenic strength and memory.

The murine T cell proliferative response to the carboxyl terminal cyanogen bromide cleavage fragment 81-104 of pigeon cytochrome c (cyt) has been studied. Two interesting properties of this response have been previously described. First, T cells from B10.A mice primed with pigeon cyt 81-104 show more vigorous proliferation when restimulated with moth cyt 81-103 than when stimulated with pigeon cyt 81-104; that is, the B10.A T cell response to pigeon shows heteroclitic restimulation by moth. Second, T cells primed with the acetimidyl derivative (Am) of pigeon cyt 81-104 did not cross-react with the unmodified cyt fragments, but Am-moth cyt 81-103 still stimulated Am-pigeon cyt 81-104 primed T cells better than the Am-pigeon cyt 81-104 fragment. These results raised the issue of whether the antigenic sites on the fragments responsible for the specificity of T cell priming in vivo differed from the residues that contributed to the heteroclitic response of pigeon (or Am pigeon)-primed T cells to moth cyt c fragments. In this paper, synthetic peptide antigens were tested in order to identify which residues caused the heterocliticity of the moth fragment and which residues were involved in the antigenic differentiation of native and derivatized fragments. The heterocliticity of the T cell response to moth fragment 81-103 was found to be due to the deletion of the penultimate residue (Ala103) from the pigeon fragment. However, the ability to cause heterocliticity was not uniquely a property of this deletion. T cells from animals primed with peptides containing substitutions at positions 100 or 102 were also heteroclitically stimulated by the moth-like antigen. The observation that T cells could not be primed for recognition of the changes in peptide sequence that caused heteroclitic stimulation suggests that T cells do not directly recognize determinants in this region. The antigenically significant site of derivatization for T cell priming was found to be Lys99. Furthermore, substitution of a Gln at position 99 also resulted in elicitation of yet a third set of T cell clones specific for the presence of that residue. That is, the specificity of the primed T cell population was found to be altered by changes at residue-99, but no such alterations in specificity were demonstrable when T cells primed with peptides altered at residue-103, residue-102, or residue-100 were compared. Overall, the results demonstrate that the antigen can be divided into two functionally distinct sites that are in close physical proximity.

Analysis of the diversity of murine antibodies to dextran B1355. II. Demonstration of multiple idiotypes with variable expression in several strains.

We have developed radioimmunoassays that detect idiotypic (variable region) differences among the alpha(1 leads to 3) dextran-binding meyloma proteins U102, J558, and M104 as well as an assay that detects variable region determinants common to all three proteins. Using these assays, we have examined 7S and 19S anti-alpha(1 leads to 3) dextran antibodies induced in five murine strains of the a1 IgCH linkage group and the recombinant strain BAB/14. All idiotypes were expressed in both 19S and 7S antibodies from all strains, but with considerable strain-specific variability in penetrance. In two strains, one additional type of antibody, which lacked all four idiotypic determinants, generally constituted the bulk of total anti-alpha(1 leads to 3) dextran antibodies.

Expression of identical V alpha V beta gene pairs by IE-alloreactive and IE-restricted, antigen-specific T cells from MHC disparate mice. Evidence for thymic selection of V(D)J combinations.

Alloreactive T cell hybridomas specific for IEk and/or IEb MHC Ag were obtained from IE-nonexpressor (IE alpha b) mice. The TCR V alpha and V beta gene segments used were identified by Northern blot and RNase protection. A large proportion (24 of 80 hybridomas tested) employed the same V alpha genes (V alpha 11.1 or V alpha 11.2) as are utilized in the IEk and IEb restricted response to the Ag cytochrome c. Of these 24 alloreactive hybridomas, 10 also expressed V beta genes utilized in the IE plus cytochrome c repertoire. Structural similarity between the two related sets of TCR indicates that V alpha segments can play a determining role in MHC specificity. These data also suggest that thymic selection based on TCR reactivity with self-MHC products acts on particular V(D)J combinations rather than on V alpha V beta pairings alone.

Immune response deficiency of BSVS mice. II. Generalized deficiency to thymus-dependent antigens.

BSVS mice gave abnormally low IgG responses to 5 thymus-dependent antigens as well as a weak delayed-type hypersensitivity (DTH) response to sheep red blood cells. In contrast to IgG, the IgM antibody responses of these mice were normal to three T-independent antigens as well as to all five T-dependent antigens. The low immune responsiveness of BSVS mice was also reflected in the low levels of IgG(2)a, IgG(2)b and IgG(3) in their normal serum. The low T-dependent immune responses may result from BSVS mice having been selectively bred for susceptibility to infection with St. Louis encephalitis virus and Salmonella. C57BL/6J mice, which are also highly susceptible to Salmonella, gave low immune responses similar to, but genetically distinct from, those of BSVS mice. The levels of Ig-positive and theta-positive cells were normal in BSVS and C57BL/6J mice.

Subclass restriction of murine anti-carbohydrate antibodies.

Examination of the subclass distribution of murine antibodies directed against groups A and C streptococcal carbohydrate, alpha-(1 leads to 3) dextran and phosphocholine yields the surprising observation that these carbohydrate antigens stimulate IgG responses largely restricted to the rare IgG3 subclass. This subclass restriction is particularly impressive in light of the low circulating levels of IgG3 in nonimmune mouse serum and the failure of a variety of other antigens including proteins and aromatic haptens to stimulate IgG3 antibody production. Attempts to alter the subclass restriction of antibodies with carbohydrate specificity by immunization with carbohydrate-coupled protein have been unsuccessful and indicate that immunoregulation of subclass expression probably occurs at the level of the antibody forming (B) cell. It is therefore conceivable that VH regions of murine immunoglobulins may be restricted to particular IgG subclasses. A similar type of subclass restriction has been reported in human and rat anti-carbohydrate antibodies. This recruitment of a minor immunoglobulin isotype by carbohydrate antigens in several species further supports the concept of immunoregulation at the level of subclass, and suggests that these and other mammals may share a structurally similar isotype with perhaps a common evolutionary origin.

Rearranged beta T cell receptor genes in a helper T cell clone specific for lysozyme: no correlation between V beta and MHC restriction.

The helper T cell clone 3H.25 is specific for hen egg white lysozyme and the class II MHC molecule I-Ab. This TH cell has three rearrangements in the beta-chain gene family-a V beta-D beta-J beta 1 and a D beta 2-J beta 2 rearrangement on one homolog and a D beta 1-J beta 2 rearrangement on the other. These observations demonstrate that this functional T lymphocyte expresses only a single V beta gene segment and, accordingly, exhibits allelic exclusion of beta-chain gene expression. The rearranged 3H.25 V beta gene segment is the same as that expressed in a T helper cell specific for cytochrome c and an I-Ek MHC molecule. Thus, there is no simple correlation between the V beta gene segment and antigen specificity or MHC restriction.

Parallel cross-reactivity patterns of 2 sets of antigenically distinct cytochrome c peptides: possible evidence for a presentational model of Ir gene function.

B10.A mice were immunized with either the carboxyl terminal peptide fragment 81-104 of pigeon cytochrome c or its acetimidyl derivative and an immune response was seen with strong preference for the immunogen. Strain distribution studies and blocking with an anti-Ia monoclonal antibody indicated that the same immune response (Ir) gene and restriction element were utilized in both responses. The specificity of the responses were evaluated by restimulating in vitro with a set of cytochrome c fragments from various species. Even though the derivatized and native fragments were poorly cross-reactive, the same phylogenetic pattern was seen when pigeon cytochrome c fragment 81-104 primed cells were tested with the set of underivatized fragments and when acetimidyl pigeon cytochrome c fragment 81-104 primed cells were tested with the same set of derivatized fragments. Primed cells from a 2nd major histocompatibility complex congenic strain of mice, B10.A(5R), displayed equivalent discrimination between derivatized and native forms but showed a markedly different phylogenetic pattern of cross-reactivity. These data indicate that the immune system recognizes 2 sites on the nominal antigen. One site, which accounts for the common hierarchy and is under Ir gene control, contains residues Gln-100, and possibly other carboxyl terminal residues. The 2nd site, which effects the distinction between native and derivatized fragments, contains at least 1 lysine other than at the carboxyl terminal. The implications of these data for theories of T cell recognition and Ir gene function are discussed.

Analysis of the diversity of murine antibodies to dextran B1355. III. Idiotypic and spectrotypic correlations.

Examination of 19 S and 7 S anti-alpha(1 leads to 3) dextran (Dex) antibodies by serological assays and isoelectric focusing (IEF) has revealed substantial variation of idiotypic and spectrotypic expression between individuals of the same genotype. 7 S antibodies appeared to be more heterogeneous than 19 S by both methods. A strong, but complex, association was found between the IEF patterns of 19 S and 7 S anti-Dex antibodies and their expression of the idiotypic determinant(s) common to both the M104 and J558 dextran-binding myeloma proteins. However, no such relationship was found between the IEF pattern and the expression of idiotypic determinant(s) unique to the M104 myeloma protein. Rather, indistinguishable spectrotypes from different individuals have widely differing levels of expression of the determinant. In addition, this determinant(s) may be present on different spectrotypes of the same isotype. Both the M104 and J558-specific idiotypes were found on antibodies of the IgG3 subclass as well as in pools containing predominantly the IgG2a subclass. These data confirm and extend the picture of substantial structural variation among the antibodies comprising a response of restricted heterogeneity.

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c.

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.

The murine T-cell receptor uses a limited repertoire of expressed V beta gene segments.

Only 10 different V beta gene segments were found when the sequences of 15 variable (V beta) genes of the mouse T-cell receptor were examined. From this analysis we calculate that the total number of expressed V beta gene segments may be 21 or fewer, which makes the expressed germline V beta repertoire much smaller than that of immunoglobulin heavy-chain or light-chain genes. We suggest that beta-chain somatic diversification is concentrated at the V beta-D beta-J beta junctions.