Project Abstract While environmental entities instigate T1D development, their identity is unknown. Genetics greatly increases risk, but the discordance between identical twins proves an equally critical role for exogenous factors. To identify causal entities, sampling must occur before clinical disease develops. Although labor intensive and costly, prospective longitudinal testing is the only way to illuminate the mysterious events that precede disease. Results from our recent collaboration with the TrialNet Pathway to Prevention study point to a role for CMV infection in T1D development. We found a striking expansion of terminally-differentiated short lived effector CD8 T cells (SLEC) in seroconverted (AA+) at-risk subjects. The frequencies of SLEC are highest in seroconverted subjects that progress to disease. This SLEC subset is identical to the well-characterized CD8 response to CMV. Importantly, we found the SLEC expansion is strongly associated with CMV seropositivity. Our findings demonstrate that an expanded, exhausted antiviral response occurs one year before autoimmune T1D develops. We also determined that SLEC levels correlate strongly with IA-2 autoantibodies, demonstrating a connection between CMV and autoimmunity. T1D results from autoreactive CD8 T cell-mediated attack on pancreatic islets. These autoreactive T cells may arise as a byproduct of the anti- viral response. Our overarching hypothesis is that CMV is an important environmental factor accelerating T1D development in children with genetic risk. The objective of this application is to longitudinally probe the preclinical disease process and unravel the mechanisms connecting CMV and T1D. In Aim 1 we propose to derive kinetic measurements through a longitudinal study. We hypothesize that genetically susceptible pre-T1D children show increased levels of exhausted CD8 T cells prior to the development of autoantibodies and/or clinical autoimmunity. In aim 2 we will measure CMV replication and persistence in at risk subjects that develop autoimmunity. We hypothesize that the accumulation of exhausted CD8 T cells causes viral persistence. In aim 3 we will test mechanisms whereby the virus activates islet specific CD8 T cells. We hypothesize that CMV infection induces the expansion of islet antigen-specific CD8 T cells. An established connection between CMV replication and T1D will point to antiviral therapies as efficacious in subjects with high genetic risk. Furthermore, levels of CMV replication and SLEC represent interlocking biomarkers for stratifying subjects for antiviral and/or vaccine trials. Several CMV vaccines are currently being tested in the clinic. Our results will also facilitate the understanding of other CMV-related human diseases.