Project Summary In Type 1 Diabetes (T1D) it is well recognized that autoreactive CD4 and CD8 T cells infiltrate the pancreatic islets leading to the destruction of insulin-producing β-cells. We recently found that CD4 T cell clones isolated from non-obese diabetic (NOD) mice and humans with T1D recognize hybrid insulin peptides (HIPs) that consist of proinsulin peptides fused to other peptides present in β-cell secretory granules. Our data suggest that HIPs play a central role in the pathogenesis of T1D and we hypothesize that a loss of immune tolerance in HIP-reactive T cells is responsible for loss of tolerance to the pancreatic islets as a whole. However, the factors that cause a loss of tolerance in T cells targeting HIPs are unknown. We also predict that HIPs will be optimal for use in antigen-specific tolerance induction strategies compared to conventional β-cell autoantigens. Our aims will be to i) investigate how disease-susceptibility background genes contribute to a loss of tolerance in HIP-reactive CD4 T cells, and ii) determine if induction of tolerance to HIPs can prevent and reverse diabetes in NOD mice. To investigate how susceptibility genes other than MHC loci contribute to a loss of tolerance we will compare HIP-reactive T cell responses in NOD and diabetes-resistant mice that express the NOD MHC class II haplotype H-2g7 but do not carry susceptibility background genes (B6g7 mice). Understanding how HIP- reactive T cells are regulated in non-autoimmune B6g7 mice and how this fails in NOD mice is critical to understanding the pathophysiology of T1D. To determine if HIPs can be effectively used for antigen-specific tolerance induction, we are developing biodegradable nanoparticles loaded with HIPs. We plan to test the ability of HIP-loaded nanoparticles to prevent disease transfer by diabetogenic T cells and reverse spontaneous disease in NOD mice. These studies are likely to have a significant and lasting impact on T1D research and could potentially lead to the development of novel antigen-specific therapeutics.