The investigator proposes to characterize the properties of T-cell responses to GAD65 in NOD mice. Preliminary studies, have produced an extensive panel of GAD65-specific T-cell clones derived from 4 week old female NOD mice. His initial characterizations of these clones, which were isolated using intact GAD65 as the stimulating antigen, predominantly recognize p216-235 (27/28) with a single clone recognizing p280-299 (1/28). Interestingly, all 27 of the T-cell clones recognizing p216-235 exhibit a Th1-like phenotype (IFN-gamma secretion), while the lone clone recognizing p280-299 has a Th2-like phenotype (IL-4 secretion). In addition, characterizations of the Vbeta usage of these clones demonstrate that the response to p216-235 was oligo- or poly-clonal. These findings support the hypothesis that Th1/Th2 of GAD65-specific T-cells are dictated at least in part by the specific peptide epitope recognized. In addition, preliminary data obtained by adoptive transfer of the lone Th2 clone into 10 day old NOD mice indicates that this clone suppresses the development of insulitis, while transfer of one of the Th1 clones in an identical fashion accelerated insulitis. These studies proposes to continue with the goal of characterizing the epitope specificity and functional properties of T-cells spontaneously responding to GAD65 in NOD females.
Three specific aims : Isolate GAD65-specific Th1 and Th2 clones from unimmunized NOD mice representing different stages of disease development and determine the immunodominant epitopes that are recognized. Preliminary data, discusses the isolation of 80 T-cell clones from 4 week old unimmunized NOD females. Clones will be isolated from 10 and 30 week old female NOD. These clones will be characterized with respect to their Th phenotype by cytokine secretion and their TCR will be analyzed. 2. Determine the pathogenic and regulatory functions in IDDM of GAD65-specific Th1 and Th2 clones, respectively. These experiments will involve transfer of clones into young NOD female mice and assessing the acceleration of disease and/or insulitis. 3. Determine the relative binding affinity of GAD65-specific immunodominant epitopes to I-Ag7 and the effect this has on stability and cell surface I-Ag7 half-life. The binding affinities will be done using a competition assay Preliminary results indicate that p216-235 bound with about 10-fold greater affinity than p280-299 to I-Ag7. The half-life studies will be performed The long-term goal of these studies will be to accurately assess the molecular mechanisms mediating the development of autoimmune responses to GAD65 in NOD and determining the role of these responding T-cells in IDDM pathogenesis.
