The general aim of this grant proposal is to understand which amino acid structures of the cytoplasmic (Cy) domains of I-A's varied functional roles in an immune response to antigen (Ag).
This aim will be approached both in vitro and in vivo. The in vitro approach will use site-directed mutagenesis to introduce amino acid mutations into the beta chain's Cy domain. Transfected B lymphoma cells, expressing mutant I-A molecules will be tested for their ability to transmembrane signal, translocation of the enzyme protein kinase c (PKC) to the nucleus. The second aspect of the in vitro studies, will be to measure the changes in the translational diffusion (Dlat) of the mutant I-A molecules, compare to wildtype I-A molecules. Mutant molecules that will be analyzed for changes in Dlat will contain either point mutations in the beta chain or truncations of the alpha chain. The ability to translocate PKC and the Dlat of mutant I-A molecules will be correlated. The final in vitro studies will investigate the role the mutations in the beta and alpha chains have on the ability of I-A molecules to present Ag. T-cell hybrids specific for hen egg lysozyme peptides will be used to probe the ability of transfectants to present peptide Ag. The results of changes, in PKC translocation, Dlat and the ability to present Ag will be correlated. The in vivo aspect of this grant proposal will make use of transgenic mice that express mutant I-A molecules that have two distinct phenotypes, one has wildtype Dlat values and altered PKC translocation kinetics, the other has a Dlat value 3-fold above wildtype and an altered PKC translocation response. Using these mice, the affinity of the T lymphocyte's Ag receptor (TCR) will be probed. The rationale for the in vitro studies of Ag presentation is that mutant I-A molecules are better at presenting Ag to cells expressing high affinity TCR. Using the transgenic mice as an environment to mature T lymphocytes in and then generating T-cell hybrids, will allow an assessment of the TCR affinity that is a reflection of the type of I-A it was selected on. The second way to measure changes in the TCR repertoire is to analyze the Vbeta distribution of thymocytes and mature T lymphocytes that have matured through the transgenic thymi. The final in vivo functional assay to determine the affect of mutant I-A molecules on tolerance, will take advantage of Mls system. Normally, mice expressing I-A on an appropriate haplotype background will delete thymocytes that express Vbeta6 as part of their TCR. In these studies mice expressing either wildtype or mutant I-A (described above) will be analyzed for their ability to delete Vbeta6. These combined studies will provide detailed biochemical and functional evidence to identify the amino acids on the beta and alpha chain of I-A, required for Ia's role in an Ag driven immune response.
