MHC CLASS II MOLECULES AND TYPE I IDDM

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DESCRIPTION (Adapted from Investigator's abstract): The overall objective of this research application is to understand the role of MHC class II genes in susceptibility and resistance to IDD in the NOD mouse. In previous published studies, the investigator has characterized the impact of MHC class II gene polymorphisms on IDD susceptibility in NOD utilizing transgenic mice carrying Ab alleles from IDD resistant strains and mutated forms of Abg7. Subsequent analyses by the PI have found that these studies may have been flawed due to the presence of an unexpected effect of MHC class II transgene expression on the B-cell compartment. In subsequent experiments, the PI has developed an Abd transgenic strain that appears to express these transgenes at normal levels without effecting the B-cell compartment. This transgene results in a dramatic decrease in IDD incidence in NOD, consistent with the early studies and supporting the role of Abg7 in IDD pathogenesis. The current application is focused on assessing the role and molecular mechanism by which Abg7 mediates IDD susceptibility. There are 3 specific aims: 1) To isolate T-cell clones and T-cell hybridomas specific for GAD65 and, if feasible, murine preproinsulin restricted to Ag7 and similar sets of T-cell clones and hybridomas restricted to Ag7.PD and Ad. In preliminary studies, the PI reports the successful production of several GAD reactive CD4+ T-cell clones. These T-cells would be utilized to identify the immunodominant peptide epitopes presented by these MHC class II molecules and to determine whether they present different peptides. 2) To measure the binding of the immunodominant GAD65 peptide epitopes for each allele on paraformaldehyde fixed spleen B cells and monocytes. These studies would utilize the peptides defined in Specific Aim 1 to characterize the stability and affinity of the interactions of these peptides with their respective alleles using the peptide binding procedure originally described by Rothbard et al. 3) To characterize the T-cell proliferative response, and pattern of T-cell cytokine production, elicited by each immunodominant peptide bound to its respective I-A allele. These studies are designed to assess the impact of these various peptide-class II allele complexes on the profile of the T-cell response elicited and to correlate this results with IDD pathogenesis in NOD.