Strain A/J mice made secondary indirect plaque-forming cell (PFC) responses to azobenzenearsonate (ABA) conjugates of giant keyhole limpet hemocyanin (KLH), a thymic-dependent antigen, but not to conjugates of Ficoll, a T-independent antigen. ABA-Ficoll was also unable to elicit a response in animals primed with ABA-KLH, which have an expanded anti-ABA memory cell pool. On the other hand, ABA-Ficoll rendered mice unresponsive to ABA-KLH when administered before priming or boosting with the T-dependent immunogen. Hence, the T-independent antigen was able to tolerize but unable to trigger B-memory cells responsive to the T-dependent antigen. A/J mice immunized with dinitrophenyl conjugates of Ficoll or bovine IgG (BGG) made vigorous IgM and IgG PFC responses. PFC responses to ABA-KLH and 2,4-dinitrophenyl (DNP)-BGG were abrogated by depleting mice of C3 with cobra venom factor, whereas the IgM and IgG PFC responses to DNP-Ficoll were unaffected. B lymphocytes were fractionated on the basis of receptors for C3 and the subpopulations were assayed for in vitro PFC responses to DNP-Ficoll. Very little response was obtained from complement receptor lymphocyte [CRL(+)] B cells, whereas CRL(-) cells were more responsive than unfractionated B cells. Both populations responded to a polyclonal B-cell mitogen (lipopolysaccharide). On the other hand, the in vitro PFC response to a T-dependent antigen (sheep erythrocytes) correlated with the presence of CRL(+) B cells in the cultures. However, a minor component of this response, sensitive to anti-Thy-1 serum, was made by CRL(-) B cells, indicating the existence of subpopulations of T-dependent B cells with different signalling requirements. The results suggest that most B cells responsive to T-dependent antigens possess receptors for C3 and that C3 plays an obligatory role in the response of these cells. A distinct subpopulation of B cells which lack C3 receptors respond to T-independent antigens. The precursors of PFC for the ABA epitope reside largely or exclusively in the CRL(+) compartment in A/J mice, whereas precursors for the DNP determinant are found in both compartments.
Numerous antigens are now known that can induce antibodies bearing similar or identical variable region determinants (idiotypes) in all individuals of one or more strains of inbred mice (1-3). In many cases, the immune response to such antigens is of a highly restricted character, as judged by isoelectricfocusing (IEF) 1 of induced antibodies or other criteria. A curious feature is that the fraction of antibodies bearing a particular cross-reactive idiotype (Id) varies markedly between the known systems. Characteristic values indicating the extent of idiotypic dominance in the various systems can be tabulated (4). For example, ~30-35% of induced antibodies to group A streptococcal carbohydrate in A/J mice bear a common Id, designated A5A (5). In contrast, the response of BALB/c mice to the phosphorylcholine (PC) determinant, presented on T-independent (6) or T-dependent (7) carriers, is almost entirely (>--90%) dominated by antibodies bearing the T15 Id. Many of the factors that determine the extent of dominance of a paricular Id in a given response are obscure, although the clonal heterogeneity of B cells capable of responding to the epitope is likely to be important.The enumeration of characteristic values of clonal dominance implies a certain degree of stability. However, situations are known in which the representation of particular Ids changes considerably during the course of a response or during a multiple immunization regimen. MacDonald and Nisonoff (8) reported that crossreactive idiotypic specificities, present in the sera of individual rabbits early in the course of a hyperimmunization schedule with a p-azobenzoate conjugate, were replaced, at 2-4 mo, by a new set of cross-reactive specificities. A better-defined system, the NP-b Id present on anti-(4-hydroxy-3-nitrophenyl)-acetyl (NP) antibodies of C57BL/6 mice, has been analyzed by M~ikel~i and Karjalainen (9) and Jack et al.
Thymus-derived lymphocytes exert a number of regulatory and effector activities in the immune system. Evidence has accumulated in recent years that these activities are largely executed by distinct subsets of T cells that can be distinguished by particular cell surface antigens (1, 2). This has led to the concept of a complex network of T cell types, each endowed with its own specific function. Rigorous proof of this concept has been difficult to achieve with heterogeneous cell populations. Recently, it has been possible to establish and maintain for prolonged periods in culture antigen-specific T cell lines (3, 4), paving the way to a more definitive approach to this question. Homogeneous populations of antigen-specific T cells should also prove extremely useful for studies of the chemical nature of the antigen in its activating form, as well as the nature of the T cell antigen receptor itself. In addition, comparison of these parameters between different functional subsets of T cells specific for the same antigenic determinant should be possible.We report here the establishment, genetic control, and preliminary characterization of the antigen specificity of normal T cell lines specifically responsive to a structurally defined epitope, L-tyrosine-p-azobenzenearsonate (ABA-Tyr)) It had been established previously (5, 6) that this simple synthetic compound is immunogenic in guinea pigs and mice. Accordingly, A/J mice were immunized with ABA-Tyr, and lymph node cells from the immune animals were used to establish antigen-reactive T cell lines. Strain A/J mice were chosen for this purpose because their anti-ABA antibody response is dominated by a major cross-reactive idiotype (7), providing the possibility of useful markers for the purification and characterization of T cell antigen-specific molecules as well as studies of the regulation of idiotype expression in antibody responses. The functional activity of these ABA-Tyr-specific T cell lines will be the subject of another communication.
Despite recent advances in our understanding of T cell antigen receptor structure, relatively little is known about the role of this receptor in MHC-restricted antigen recognition. To study this problem, we have developed a panel of ABA-Tyr-reactive, I-Ak-restricted T cell clones that differ in their ability to recognize structural analogs of ABA-Tyr. Three fine specificity groups have been defined. In each group, ABA-Tyr elicited the strongest response of any of the antigens tested. Group I clones responded to ABA-conjugated hydroxyphenyl-ethanol (ABA-HPE). Group II clones responded to ABA-conjugated hydroxyphenyl-methanol (ABA-HPM) but not to ABA-HPE, and group III clones responded only to ABA-Tyr. These studies show that differences as small as a single methylene group can dramatically affect fine specificity. Because these clones are all I-Ak-restricted, it was possible to correlate receptor serology with fine specificity. To this end, monoclonal anti-clonotypes were made against clone 16-F2 from group I and used to study the relationship between fine specificity and clonotype expression. A panel of 15 T cell clones studied with four anti-clonotype antibodies showed a strict correlation between clonotype expression and fine specificity. Taken together, these data suggest that the structure recognized by the anti-clonotype antibodies is a determinant of receptor fine specificity.
Class II-restricted murine T cell clones specific for the immunogenic determinant L-tyrosine-p-azobenzenearsonate failed to proliferate to Ag presented by L cell lines transfected with and expressing the appropriate class II genes, but are activated to kill the APC in an Ag-dependent, MHC-restricted manner. Inhibition of APC proliferation was used as an assay to determine the relative contributions of polymorphic sites on the class II alpha- and beta-chains to MHC-restricted activation of I-A beta k-restricted cloned T cells. Transfectants expressing A beta k in conjunction with the alpha chain of k, u, or d were equally effective APCs, whereas transfectants expressing A beta u were completely ineffective, implicating the beta-chain as more critical for the presentation of L-tyrosine-p-azobenzenearsonate. Site-directed mutagenesis of polymorphic positions in the beta chain revealed a remarkable stringency for the k haplotype, in contrast to the relaxed alpha-chain requirement. These results, in conjunction with others, indicate that the relative contribution of polymorphic sites on class II alpha- and beta-chains to T cell Ag recognition can differ markedly, and, furthermore, may vary as a function of the Ag.
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