PROJECT SUMMARY/ABSTRACT Pulmonary fibrosis is a poorly understood process that is thought to involve a preceding stage of immune infiltration and inflammation. Bleomycin, a DNA damage inducing agent, is often used to experimentally induce pulmonary fibrosis in mice, indicating that immune sensing of nucleic acids may be tied to the etiology of fibrotic lung disease. cGAS-STING is a cytosolic dsDNA sensing pathway, which is tightly regulated to preserve immune homeostasis by discriminating immune response to danger signals like pathogens or genotoxic stress and immune tolerance to inert physiologic signals. This regulation is broken by constitutively active STING mutations, which cause an autoinflammatory syndrome known as STING Associated Vasculopathy with onset in Infancy (SAVI), in which patients develop immune abnormalities. Consistent with the notion that dysregulated nucleic acid sensing promotes pulmonary fibrosis; SAVI patients rapidly succumb to treatment resistant inflammatory lung fibrosis. To address the urgent need for SAVI lung disease therapy, we have developed gene-targeted mice that express the SAVI mutation STINGV154M(VM) and found that these mice recapitulate aspects of human disease including inflammatory lung fibrosis. The utility of studying our SAVI mouse model lies not only in its impact on studying SAVI disease, but broadly for understanding the immune defects that drive pulmonary fibrosis, a topic that has thus far been constrained by the limitations of current fibrotic lung disease models. Our central hypothesis is that the acquisition of SAVI fibrotic lung disease is mediated by autoreactive B cells. The following key findings support this hypothesis: (i) VM mice possess severe immune abnormalities of lymphocytes characterized by lymphopenia with concomitant hyperactivation of remaining lymphocytes, a feature also seen in SAVI patients. (ii) Using genetic ablation of B and T lymphocytes by Rag1 deficiency, we found that lymphocytes are required for lung disease in VM mice; however, when we specifically ablated αβ T cells by TCRβ deficiency in VM mice, we found that these mice persisted in developing fulminant lung disease. (iii) Additionally, our preliminary data indicates that VM B cells accumulate in the lung extravascular space and become activated independent of αβ T cells. In this proposal, Aim 1 will determine the contribution of B cells to VM SAVI fibrotic lung inflammation using mouse genetic models of B cell deficiency and pharmacologic depletion of B cells by targeted antibody treatment. Aim 2 will determine whether VM SAVI lung B cells promote disease through autoreactivity using mouse models that restrain the BCR repertoire to foreign antigens and by characterizing the BCR repertoire and antibody reactivity in VM mice. The approach includes: survival studies, lung histopathologic analysis, pulmonary function testing, flow cytometry, BCR repertoire sequencing, and antibody staining of mouse lung sections. Ou...