PROJECT SUMMARY/ABSTRACT The goal of Project 1 is to understand in detail the pathological consequences of ENU-induced mutations implicated by Core B and Core C to modify the T1D phenotype of NOD/NckH mice, and to determine the cellular mechanisms by which those mutations impact the development of T1D. As we have done for Dusp10, a spontaneous mutation bred to homozygosity in our lab based on phenotypic selection, we will analyze each selected mutation to determine which salient immunopathological steps—recognized as hallmarks of T1D initiation and/or progression—are affected. To that end, we will use tools such as analysis of pancreatic islet infiltration (i.e. insulitis); measurement of the numbers and the in vivo functional capacity in various lymphoid organs of diabetogenic or regulatory CD4+FoxP3+ T lymphocytes; and analysis of islet antigen presentation using adoptive transfer of diabetogenic cells expressing the transgenic BDC2.5 T cell receptor (specific for an autoantigenic fusion peptide incorporating insulin and chromogranin A sequences) into 4-week-old recipients. We will pay particular attention to mechanisms of insulin resistance, and to the definition of markers of increased β-cell sensitivity witnessed with particular genetic mutations. In doing so it is plausible that novel immunopathological steps will be discovered. Second, we will investigate the contribution of particular tissues or cell types bearing each mutation to the development of T1D using bone marrow transplantation, adoptive transfer in neonates, islet transplantation, tissue-specific conditional knockout mice, and islet organoid cultures in the presence or absence of immune cells. Results will provide important guidance to Project 2, focused on molecular pathogenesis. We will perform cellular mechanistic studies to understand of the effects of mutations in Paqr8 (protective), and Dusp10, Rapgef1, and Xpnpep1 (exacerbating), identified in our initial collaboration. We expect to actively pursue mechanistic studies of four to six proteins at a time, adding one new protein per year by mutual agreement with Project 2. We expect that strong synergy between Projects will accelerate our progress. An important question we will address is the relevance of our findings to human health. To understand how mutations identified in NOD mice relate to human T1D, we will study the effects of selected mutations in NOD mice bearing a T1D-susceptible humanized immune system modified to contain or lack the orthologous mutations in human hematopoietic stem cells and/or human islet cells. We will also identify human T1D patients with mutations in genes orthologous to those we implicated in our screen of NOD mice. Our findings will uncover new therapeutic targets for treatment of T1D and help to improve prediction of T1D onset in humans.