Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. T cell responses to the beta cell autoantigen insulin are essential for efficient development of type 1 diabetes (T1D). An important emerging concept related to the essential nature of insulin as an autoantigen is that CD4+ T cell responses develop to unconventional insulin peptides, including hybrid peptides in which amino acids from insulin are linked to those from another protein (or another region of insulin). In the case of beta cell- cytotoxic CD8+ T cells, which are essential for beta cell killing, little is known about their ability to recognize unconventional insulin peptides. By analogy to hybrid peptides recognized by tumor-reactive CD8+ T cells isolated from cancer patients, hybrid insulin peptides presented on class I MHC molecules could be generated by proteasome-catalyzed peptide splicing. We have discovered that an H2-Db-bound hybrid insulin peptide is a ligand for beta cell-cytotoxic T cells in NOD mice, creating the first link between class I MHC-bound hybrid insulin peptides and T1D. Our project will test the hypothesis that spliced insulin peptides, and those derived from other non-canonical sources, are important targets of beta cell-cytotoxic CD8+ T cells in T1D. In Aim 1, we will use mass spectrometry to identify the immunopeptidome of human pancreatic islets expressing HLA-B*39:06 or A*24:02, both of which are predisposing for T1D, or A*02:01, commonly expressed by T1D patients. Limited information is available regarding the peptides presented by class I MHC molecules on primary human pancreatic islet cells. We are uniquely positioned to address this based on our robust protocols for the characterization of MHC-bound peptides, including from small samples, coupled with workflows that allow identification of spliced peptides and those derived from other non-canonical sources. Although our project will be focusing on such unconventional insulin peptides, all class I MHC-bound peptides will be identified. In Aim 2, we will investigate unconventional insulin peptides as CD8+ T cell epitopes in T1D and explore their importance in disease pathogenesis. Our recent development of NOD mouse models transgenically expressing human insulin along with HLA-B*39:06, A*24:02, or A*02:01 will allow us to examine the unconventional human insulin peptides identified in Aim 1 for recognition by islet-infiltrating CD8+ T cells. We will use these models and several complementary approaches to further assess the pathogenicity of the T cell specificities we identify. Islet- infiltrating CD8+ T cells from T1D patients will also be examined.

PROJECT NARRATIVE We will investigate new classes of molecules as targets of the immune response in type 1 diabetes, which is a growing health problem that is associated with significant morbidity and mortality. Our project will identify therapeutic and immune monitoring targets and lay a foundation for the development of an antigen- specific therapy for type 1 diabetes.