PROJECT SUMMARY Rheumatoid arthritis (RA) is an inflammatory arthritis of autoimmune origin leading to erosive joint damage. While B-cell involvement in RA is well characterized by antibodies to citrullinated protein antigens (ACPAs), much less is known about T-cell reactivity and functionality in the etiology and pathogenesis of RA. Studies have focused largely on the T-cells of the joint and peripheral blood. However, the joint is reflective of long-standing autoimmunity which introduces a bias in adaptive immune responses due to epitope spreading. ACPAs exist systemically and in mucosal sites preceding the onset of joint inflammation, which suggests the originating site of autoimmunity in RA is extra-articular. The lung is one of these sites considered to be a possible location for initiating RA-related autoimmunity. Notably, sputum ACPAs are detectable in over 33% of those at risk for RA and over 66% of those with RA. Furthermore, lung abnormalities are seen in over 70% of those with RA, and lung disease is a common extra-articular manifestation of RA. The goal of this proposal is to characterize the T- cell repertoire of the lung in RA to better understand disease pathogenesis. This involves investigating both phenotype and functionality of RA-related lung T-cells through single cell RNA-seq as well as the antigenic reactivity of these T-cells through IL-2 bioassays when presented with synovial fluid antigens. Studying the T- cell responses of the lung has been a challenge due to sampling limitations. Induced sputum is an inexpensive, non-invasive sample but it typically contains 1% or fewer lymphocytes. We have overcome the limitations of sputum for studying T-cell responses of the lung by the use of a novel microfluidic technique to recover sputum lymphocytes in a completely touch-free and unbiased manner. This allows us to study the T-cells of the lung in RA and compare them to the RA synovium as we hypothesize there will be shared effector states and antigenic reactivity between lung-derived and synovium-derived CD4+ T-cells in RA. Our overall hypothesis is T-cells derived from the lung have common effector states and antigenic reactivity to the T-cell repertoire of the RA synovium. We will assess sputum lymphocytes in RA for the presence of Vβ17+ peripheral helper T (Tph) and Vβ14+ cytotoxic CD4+ T-cells and compare T-cell receptor motifs and antigenic reactivity of T-cells in RA sputum to RA synovium. From the single-cell RNA-seq data, we will also obtain TCR sequences of the RA sputum CD4+ T-cells. We will use previously published datasets containing TCR α and β chain sequences to determine closely related TCRs and motifs between the RA synovium and lung. The findings from our proposal will allow for an improved understanding of T-cell involvement in RA in both the lung as well as the joint.