Project Abstract The goal of this proposal is to define novel functions of STING in autoinflammation, cytokine secretion, and immune dysregulation mediated by macrophages, innate lymphoid cells, and radioresistant cells. This work builds on our prior discoveries using our model of STING-associated autoinflammation. The proposed studies utilize several newly generated, already-validated model systems that will allow us to define cell type-specific functions of STING in autoinflammatory disease pathogenesis. STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI), an autoinflammatory disease characterized by T cell cytopenia, interstitial lung disease, Raynaud’s, skin lesions, vasculopathy, and up-regulation of interferon (IFN)-stimulated genes. We previously generated a mouse model of SAVI and discovered that the disease is mediated by type II IFN (IFN-γ) rather than type I or type III IFN. This discovery was unexpected since type I IFN had been predicted to mediate disease. Additionally, we found that innate lymphoid cell functions are altered in STING gain-of-function mice, and that macrophage activation phenotypes and cytokine secretion are perturbed by constitutive STING signaling. We have previously generated and published our floxed-STOP STING mutant mice, which permit cell type-specific expression of STING. However, we have not yet studied cell type-specific functions of the type II IFN receptor and STING in myeloid cells, innate lymphoid cells, and radioresistant cells in our model of STING-induced autoinflammation. Therefore, we will define functions of the type II IFN receptor (Aim 1) and the effects of cell type-specific expression of STING (Aim 2) in SAVI mice. In unbiased, whole-genome CRISPR screening studies, we were led to the discovery that both WT STING and STING gain-of-function can non-transcriptionally regulate pro-inflammatory cytokine secretion by altering the post-Golgi endosomal pathways, and that this occurs without impacting secretion of other proteins. This function of STING requires the protein ArfGAP2. Additionally, we found that ArfGAP2 deletion has no appreciable effect on Golgi structure or on the transit of proteins from the ER to the Golgi when STING is inactive. However, when STING is activated, cytokine trafficking and secretion is greatly diminished in the absence of ArfGAP2, and this effect is non-transcriptional. We generated Arfgap2f/f mice crossed to transgenic Cre animals, and we found that ArfGAP2 is deleted efficiently without impacting survival of target cells. In Aim 3, we will define the immunological and physiological functions of ArfGAP2 in STING-mediated cytokine secretion and autoinflammation (Aim 3).