Type 1 diabetes (T1D), a prototypic CD4 T cell mediated autoimmune disease, is believed to have a prolonged pre-clinical phase that lasts many years. Therapeutic intervention during that period of the disease has demonstrated effectiveness in a small subset of patients and for a limited time. However, the determination of a precise disease status in those patients is a missing parameter for the therapeutic decision. Indeed, outside of glycemia, and glucose tolerance tests, the only indication of b cell autoimmunity is the measurement of anti-islet autoantibodies (AIAs). However, AIAs have limited predictive power at the individual level and cannot be used to evaluate therapeutic outcomes. Thus, real time biomarkers of islet autoimmunity are an absolute medical need to allow early targeted therapeutic intervention in at-risk populations, evaluate its efficacy, and eventually pave the way to prevent and cure type 1 diabetes. Because T1D has a strong genetic background and that HLA-class II genes dominate this landscape, it is logical to position anti-islet CD4 T cells at the center of the early pathology. In this respect, we hypothesized that if we could measure anti-b cell CD4 T cell immunity, we would have a biomarker for T1D. We validated this concept in human bearing the high-risk HLA-DQ8 allele. Insulin peptides- HLA-DQ8 tetramers were produced and used to isolate antigen specific CD4 T cells from peripheral blood. Four groups of patients were tested, normal blood donors, just diagnosed, established, and at-risk patients. While frequencies of anti-insulin T cells were uninformative, their state of activation was correlated with disease, and could determine whether at-risk patients had active autoimmunity or not, regardless of their AIA status. Similarly, in established T1D patients, autoimmunity was still ongoing. We will expand on these published results in 2 aims. Aim 1 is the core of the proposal and will evaluate in larger cohorts the profile of activation of anti-islet CD4 and CD8 T cells in longitudinal studies to correlate T cell activation and progression. These studies should formally establish or disprove the relapsing-remitting nature of T1D. The same approach has the potential to allow the monitoring of therapeutic interventions. Aim 2 is focused on mechanistic studies and based on the hypothesis that anti-islet T cells must have a unique phenotype of activation and homing. This phenotype will be deconstructed in mice by comparing antigen specific islet-resident T cells and their blood counterparts. After a step of in vivo Crispr/Cas9 validation, the islet specificity of circulating cells harboring the selected phenotype will be translated and tested in humans. These studies will illuminate basic aspects of islet-immune system relationship and could establish new principles of therapeutic intervention. This ambitious proposal is intended to provide the necessary data to deploy in the clinic a set of tools that measure anti-islet i...