PROJECT SUMMARY: Our lab discovered COPA syndrome, a monogenic autoimmune disorder that involves inflammation of the lungs, joints and kidneys. We established a mouse model of COPA syndrome by generating CopaE241K/+ knock-in mice that spontaneously developed clinical and immunologic features of patients, including interstitial lung disease (ILD) and increased levels of activated, cytokine-secreting T cells. We performed bone marrow chimera and thymic transplant experiments to reveal that expression of mutant COPA in the thymic stroma is sufficient to cause a defect in negative selection of CD4+ T cells. Our work revealed that a key step in the initiation of disease in COPA syndrome is a breakdown in central tolerance. The goal of this grant is to unravel the mechanisms by which mutant COPA causes impaired thymic tolerance. COPA syndrome presents in childhood with inflammatory arthritis of the small and large joints and lung disease that manifests as pulmonary capillaritis or ILD. Patients develop clinical features that are observed in systemic lupus erythematosus (SLE) including high-titer anti-nuclear antibodies, autoantibodies to double stranded DNA and immune complex glomerulonephritis. In addition, peripheral blood mononuclear cells from all COPA syndrome subjects exhibit a markedly elevated type I interferon stimulated gene signature, a hallmark of SLE. We recently demonstrated that activation of type I interferon (IFN) signaling in COPA syndrome is caused by constitutive activation of the innate immune adapter molecule STING, which is missorted at the Golgi by mutant COPA. Mutant COPA-mediated STING activation upregulates IFNs in immune cells of CopaE241K/+ mice, and remarkably, also within the thymic epithelium. Taken together, this suggests STING has a functional role in the thymus with the potential to alter thymocyte selection and/or development. We hypothesize mutant COPA breaks central tolerance by causing activation of STING in thymic epithelial cells (TEC). We will use our mouse model to determine how activated STING in thymic epithelial cells alters T cell maturation and selection. We will define the cellular and molecular mechanisms by which mutant COPA impairs autophagic flux and determine whether proteins missorted by mutant COPA are involved in STING-induced autophagy. Finally, we will test whether small molecule STING agonists and inhibitors can fine-tune STING signaling in the thymus to alter thymocyte development and selection. Taken together, our work provides new insight into how activated STING in the thymus shapes the T cell repertoire to cause autoimmunity and immune dysregulation. These findings have important implications for settings in which systemic inflammation causes activation of STING in the thymic stroma including cancer therapy and infections.