PROJECT SUMMARY/ABSTRACT Significant data implicate a role for autoantibodies to citrullinated proteins in RA pathogenesis, and autoantibodies to the citrullinating enzyme peptidylarginine deiminase 4 (PAD4) are also found in a subset of patients with the most severe joint disease. Citrullination is the calcium-dependent conversion of peptidyl- arginine to citrulline by the PAD enzymes, but the mechanisms that initiate immune responses to citrullination- associated autoantigens are poorly understood. While different several mechanisms have been proposed, our published and preliminary data implicate cytotoxic lymphocyte-mediated killing of neutrophils in the generation of citrullinated autoantigens and immunogenic PAD4 in a subset of patients with RA. A pathogenic role for cytotoxic T lymphocytes (CTLs) in this process is strongly supported by our preliminary work on patients with T cell large granular lymphocyte leukemia (T-LGLL), a rare form of leukemia in which 20-30% of patients develop RA (T-LGLL/RA). This disease is characterized by clonal expansion of CD8+ T cells, neutropenia and somatic activating mutations in STAT3. Interestingly, we have found striking autoimmune and genetic similarities between T-LGLL/RA and the anti-PAD4 antibody positive subgroup of RA, suggesting a common pathogenic origin for the development of arthritis in these two diseases. Importantly, these findings make T-LGLL/RA a powerful yet simplified human model driven by pathogenic CTLs, in which to study novel pathogenic mechanisms in RA. In this project, we will use unique cohorts of patients with RA and T-LGLL/RA, as well as innovative and state of the art technologies, to examine the novel hypothesis that killing of neutrophils by pathogenic CTLs is a central mechanism promoting the lack of tolerance to autoantigens in a unique serologically distinct RA subset. To address this hypothesis we will define: 1) common pathogenic mechanisms linked to autoreactive CTL expansion and serological profiles indicative of CTL-driven disease in RA and T-LGLL/RA; 2) how the cytotoxic lymphocyte granule pathway shapes the repertoire of autoantigens presented from dying neutrophils to autoreactive CD4+ T cells by antigen presenting cells; and 3) the clonality, specificity and effector functions of autoreactive CD8+ T cells in RA and T-LGLL/RA. Our long-term goal is to apply this knowledge to define precise mechanism-guided preventive and therapeutic interventions in RA.