Project Summary/Abstract In Type 1 Diabetes (T1D) autoreactive CD4 T cells mediate the destruction of insulin producing beta-cells. The reasons for this misguided attack are poorly understood. Post-translational protein modifications could provide plausible explanations for the existence of autoreactive T cells in T1D. Hybrid insulin peptides (HIPs) are a form of post translationally modified antigens that form in beta-cells through the covalent ligation between proinsulin fragments and other beta-cell peptides. The resulting HIPs contain non-genomic amino acid sequences making them plausible targets for pathogenic T cells in T1D. Various HIP-reactive CD4 T cell clones were shown to trigger diabetes in non-obese diabetic (NOD) mice, a major animal model for the study of T1D. In addition, HIP-reactive CD4 T cells were identified in residual pancreatic islets of T1D organ donors. Significantly elevated levels of HIP-reactive T cells were also be detected in the peripheral blood of recent-onset T1D patients, but not in non-diabetic control subjects. Importantly, we applied mass spectrometric analyses on islets and confidently validated the presence of several HIPs that participate in T1D pathogenies. Autophagy is a cellular mechanism that removes unnecessary or dysfunctional components of the cell through lysosomal degradation. Our data indicate that autophagy plays a critical role on HIP-content in islets. Here we propose to modulate autophagy in human islets through various mechanisms, including cytokine treatment, with the objective to increase cellular HIP content. This will facilitate our identification efforts of novel HIP through mass spectrometric analyses on islets. We will also study the role of metabolic stress that may lead to the accumulation of a new sub-group of HIPs in beta-cells that could provide a trigger for T1D. Furthermore, we will study the mechanism of HIP-formation in human islets. Understanding this mechanism may provide us with new therapeutic targets that could be used to block HIP-formation in beta-cells and remove epitopes for disease- driving T cells in T1D. Additionally, we will use ELISPOT analyses to establish the role of identified HIPs as T cell epitopes in T1D pathogenesis. Such HIP-reactive T cells could serve as T1D biomarkers and allow us to improve current disease-prediction models. Lastly, we will advance and benchmark an innovative computer algorithm for the mass spectrometric identification of hybrid and native peptides. This algorithm may allow us to identify other types of hybrid peptides that cannot be discovered through conventional methodologies. In summary, success in this project will deliver new technologies, biomarkers, and therapeutic targets for the study and prevention of T1D. Identification of novel HIPs will also deliver targets for the induction of antigen specific tolerance induction in T1D, which may be required for the reversal of T1D.