Summary Type IA or insulin dependent diabetes (T1D) is caused by autoimmune destruction of insulin producing β cells in the pancreatic islets by T lymphocytes. The detection of IgG autoantibodies to insulin and other β cell autoantigens in pre-T1D suggests that T-B lymphocyte interactions are a critical early event in the loss of immune tolerance that leads to T1D. Research in this laboratory is focused on the function of B lymphocytes that recognize the key β cell autoantigen, insulin. By engineering NOD mice to express an insulin autoantibody as a B cell receptor transgene, we discovered that B lymphocytes make unappreciated contributions to the pathogenesis of T1D as antigen presenting cells. Anti-insulin B lymphocytes were found to process and present pathogenic epitopes from insulin B chain to diabetogenic T cells. In all previous studies insulin epitopes were identified by T cell responses to synthetic peptides and natural insulin epitopes on diabetogenic MHCII (IAg7 in NOD and DQ8 in humans) were not know. By combining the novel resource of our anti-insulin B cells with advanced proteomics, we have overcome the barrier to detection of natural insulin epitopes and have discovered an unexpected quantity and quality of insulin-related peptides eluted from IAg7 on anti-insulin B cells. Features of the insulin “immunopeptidome” differ strikingly from synthetic peptides used to mimic epitopes. Pathogenic B chain motifs are in low abundance relative to other insulin-related epitopes and they reside on larger peptides that may support more complex interactions. Surprisingly, a large majority of insulin- related residues from IAg7 reside in multiple epitope clusters encompassing the c-peptide of proinsulin. These findings suggest: i) natural B chain epitopes may reside on larger polypeptides and drive more diverse responses than detected with synthetic peptides (e.g. B9-23); ii) features of c-peptide epitopes eluted from IAg7 indicate anti-insulin B cells capture proinsulin at the site of attack in the islets; iii) properties of proinsulin c-peptide that favor MHCII loading govern thymic education of anti-insulin T cells. These hypotheses will be tested in three aims of the proposal. First, we will identify naturally processed B chain epitopes from IAg7 on B cells that drive unique autoaggressive T cells in T1D. Second, the role of insulitis in loading proinsulin- peptides onto IAg7 in anti-insulin B cells will be determined, and quantitative proteomics will be developed to test this potential biomarker. Third, the functional significance of proinsulin peptides eluted from IAg7 for generation of beneficial T cells that leave the thymus will be assessed. Each of these aims is positioned for rapid translation into human T1D by providing more effective reagents for detection and regulation of autoaggressive T cells, developing a biomarker of early islet invasion, and identifying T cells better suited for maintaining immune tolerance.