Beck, Barbara

Abstract We have isolated and characterized four mutant I-Ak-expressing cell lines derived from the B cell-B lymphoma hybrid antigen-presenting cell line TA3. The mutants were isolated by first selecting against expression of one Ak epitope by treatment with a monoclonal antibody in the presence of complement and then selecting for retention of a second Ak epitope by electronic cell-sorting of cells stained for fluorescence with a second monoclonal antibody. The serologic and functional phenotypes of the mutants were characterized by using panels of I-Ak-specific monoclonal antibodies and I-Ak-restricted T hybridomas. We obtained one Ak alpha mutant (J4) that no longer reacts with any Ak alpha-specific antibody and also is incapable of stimulating any I-Ak-restricted T hybridoma. We obtained three Ak beta mutants (LD3, K5, G1) that express a wide range of serologic and functional phenotypes. Correlation of the serologic and functional phenotypes reveals that the serologic epitope Ia.1 may overlap with a major site of T cell recognition, whereas the Ia.17 serologic epitope appears to be only a minor site for T cell recognition.

Abstract Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides.

Abstract The role of IL 1 in the antigen-specific activation of class II-restricted T lymphocytes was examined by using a model system consisting of cloned WEHI 5 B lymphoma accessory cells and class II-restricted, soluble antigen- or alloantigen-reactive T cell clones. The addition of exogenous recombinant IL 1 to the T cell cultures resulted in a significant enhancement of the antigen-specific T cell proliferation response, but at best, only small increases in IL 2 release. Goat IgG anti-IL 1 antibodies were added to the T cell cultures to assess their effect on T cell activation. The IL 1 enhancement of the T cell proliferation response was inhibited by the anti-IL 1 antibodies in a dose-dependent manner. In contrast, only modest levels (10 to 25%) of proliferation inhibition were observed in T cell cultures containing either WEHI 5 or splenocyte accessory cells but no exogenous IL 1. When the anti-IL 1 antibodies were added to primary mixed lymphocyte cultures stimulated by WEHI 5 cells in the absence of exogenous IL 1, no significant inhibition of proliferation was observed. A small but statistically significant proliferation inhibition was observed when anti-IL 1 antibodies were added to mixed lymphocyte reaction cultures stimulated by splenocytes. Two-color cytofluorometric analysis of the effects of IL 1 on antigen-activated T cell clones demonstrated that under suboptimal stimulation conditions, IL 1 stimulated a small but significant increase in the number of T cells bearing IL 2 receptors. In the presence of optimal numbers of WEHI 5 accessory cells, IL 1 enhanced T cell proliferation in the absence of a detectable increase in the number of T cells bearing IL 2 receptors, the number of IL 2 receptors per T cell, or the levels of IL 2 released. Finally, exogenous IL 1 can be added as late as 18 to 24 hr after culture initiation without significantly reducing its ability to enhance the T cell proliferation response. These data indicate that IL 1 has pleiotropic effects on murine T lymphocytes and can function to enhance T cell activation at multiple points during the activation sequence.

Abstract The activation requirements of alloreactive and antigen reactive murine T cells were examined by stimulating class II restricted T cell clones with monoclonal B lymphoma cells. One B lymphoma cell line (T27A) was found to stimulate IL 2 release from some alloreactive T cell clones without stimulating any significant T cell proliferation response. The same B lymphoma cells are capable of stimulating IL 2 release and proliferative responses from other T cell clones. Evidence is presented suggesting that B lymphoma cell stimulation of these T cell clones is largely IL 1 independent and that at least some T cell clones may require activation signals other than Ia, antigen, and IL 1. The addition of exogenous, purified IL 1 to the T cell activation assays was found to have a wide range of stimulatory effects on the proliferative responses of different T cell clones. The absence of comparable IL 1-induced stimulation of IL 2 secretion suggests that IL 1 primarily enhances antigen specific T cell proliferation through mechanisms other than acting as a co-stimulant for IL 2 release.

Abstract The chain specificities of 18 Ak and 26 Ab-reactive anti-Ia monoclonal antibodies have been determined. L cells were transfected with haplotype-matched (A alpha k:A beta k, A alpha b:A beta b) or haplotype-mismatched (A alpha k:A beta b, A alpha b:A beta k) cDNA pairs, lines expressing high levels of surface A complex were selected, and antibody reactivity with a panel of reagents was assessed by cytofluorimetric analysis. Most of the antibodies recognized a determinant specified by one chain, either alpha or (more commonly) beta. A few examples of more complex determinants were also observed. A knowledge of the chain specificities of anti-Ia monoclonal antibodies should prove useful for a variety of studies aimed at dissecting Ia structure-function relationships.

Abstract A series of seven I-Ab-reactive monoclonal antibodies (mAb) has been derived from a BALB/c anti-C57BL/6 immunization. Analysis of the reactivity patterns of these mAb with spleen cells of mice from the independent haplotypes has revealed three groups of mAb: group I mAb react with all haplotypes except d, group II with all except d and k, and group III with all except d, k, and j. In addition, the group I and group II mAb also react with human class II-expressing cells. We have used these mAb to isolate one mutant I-Ab-expressing cell line and three additional I-Ak mutant cell lines. These antibodies have been used, in conjunction with a large panel of I-A-reactive mAb available from others, to extensively characterize our collection of mutant I-A-expressing cell lines. Analysis of the mutant cell lines has allowed us to assign the reactivity of each mAb to either the A alpha- or the A beta-polypeptide. All seven newly isolated mAb appear to react with determinants on the A alpha-polypeptide. Furthermore, analysis of the panel of A alpha k- and A beta k-mutants has allowed us to discriminate at least five epitopes that are separable by mutation on the A beta k-polypeptide, and two epitopes on the A alpha k-polypeptide.

In an effort to characterize the ligand that is bound by T helper lymphocyte antigen receptors, we have begun to identify class II polymorphic residues that comprise part of the allospecific TCR binding sites. Site-directed mutagenesis was used to construct mutant Ak beta (Ak beta*) genes that encode polypeptides into which single or multiple residues of the Ad beta polypeptide have been substituted in the beta 1 domain. A panel of cloned cell lines expressing the mutant Ak beta* Ak alpha or Ak beta* Ad alpha molecules was analyzed for the ability to stimulate Ak or Ad alloreactive T cell hybridomas. Substitution of d for k residues at specific positions in the beta 1 domain resulted not only in the loss of epitopes recognized by Ak-reactive T cells but, more importantly, in the gain of epitopes recognized by Ad-reactive T cells. Some of the polymorphic residues identified as contributing to the T cell epitopes are the same residues that contribute to the serologically immunodominant epitope. Other T cell epitopes map to positions predicted to be located either in an alpha-helix forming one side, or in a beta-pleated sheet forming the bottom of the putative antigen binding site. Thus, unlike serologic epitopes, TCR epitopes can be determined by A beta polymorphic residues in many different regions of the beta 1 domain and frequently depend upon contributions of A alpha polymorphic residues.