Abstract This competing renewal application is to continue our studies on the role of hybrid insulin peptides (HIPs) as neo-epitopes for autoreactive CD4 T cells in autoimmune diabetes. During this very productive period we have established that HIPs formed from insulin C-peptide and other β-cell granule proteins are antigens for large numbers of autoreactive CD4 T cells in the pancreas of NOD mice, can be used in strategies to induce antigen-specific tolerance, and are also present in human islets and PBMC of patients with type 1 diabetes (T1D). Our objective in the next period of the award will be to characterize new insulin (Ins) B- chain HIPs and T cells reactive to these HIPs, in both NOD mice and human subjects. We hypothesize that antigenicity of the much studied insulin B chain peptide, B:9-23, is due to hybrid peptide formation between sequences of B:9-23 and cleavage peptide products of other granule proteins. The rationale for pursuing these studies is based on preliminary data indicating that B-chain HIPs containing B9-23 sequences are strong agonists for insulin-reactive T cell clones and that T cells reactive to these HIPs can be identified in the polyclonal T cell population of NOD mice. Our aims for this next project period are to (1) investigate the role of B-chain HIP-reactive T cells in disease pathogenesis; (2) determine whether tolerance can be induced with insulin B-chain HIPs coupled to biodegradable nanoparticles in NOD models of spontaneous disease and islet transplantation; and (3) investigate the presence of B-chain HIP-reactive CD4 T cells in human T1D and develop new human tetramers containing B-chain HIPs. The B-chain HIPs will provide further tools for understanding the autoimmune response to pancreatic β-cell antigens in general, and specifically the contribution of insulin-reactive T cells to the disease process. We predict that these studies will lead to generation of reagents not only for tracking disease-relevant T cells, but also for developing and expanding approaches to antigen-specific therapy.