Abstract Rheumatoid arthritis (RA) is a destructive inflammatory joint disease associated with increased morbidity and mortality. While biologic therapies have improved treatment response, unmet clinical needs remain due to the refractory nature of RA, and recurrent disease flares despite aggressive treatment. In the prior funding periods, we developed a multidisciplinary translational research program that combined longitudinal near infrared (NIR) imaging of indocyanine green (ICG) with targeted therapies to elucidate how TNF, B cells and lymphatics converge to trigger arthritic flare in murine models and RA patients. Specifically, we found that prior to RA signs and symptoms, there is an increase in lymphatic vessel (LV) contraction frequency to enhance the efflux of CD11b+ myeloid cells from the affected joints, which ultimately gets overwhelmed at early onset. RA disease proceeds with expansion of joint draining lymph nodes from an influx of unactivated-polyclonal CD23+/CD21hi/CD1d hi B cells in inflamed nodes (Bin) and lymph, until a sudden loss of LV contractions is observed along the ipsilateral axis, which results in lymph node collapse and Bin clogging of the sinuses. Interestingly, knee synovitis following popliteal lymph node (PLN) collapse in TNF-Tg mice is ameliorated by anti- CD20 B cell depletion therapy (BCDT), which restores passive but not active lymph flow. Most recently, we demonstrated that loss of LV contractions in TNF-Tg mice is secondary to activated macrophage adherence to the lymphatic endothelial cells (LEC), and subsequent LEC and lymphatic muscle cell (LMC) damage from chronic inflammation. Remarkably, this major defect can be corrected with anti- TNF therapy that ameliorates the inflammatory-erosive arthritis, and restores LEC-LMC integrity. To further understand the role of lymphatics in arthritic flare, we propose three Specific Aims. In Aim 1 we will demonstrate perivascular LMC progenitor incorporation into PLVs during growth and flare in WT growing mice (homeostasis), and TNF-Tg mice with Expanding vs. Collapsed PLN treated with anti- TNF or placebo. We will also confirm their LMC progenitor potential in adoptive transfer studies in vitro and in vivo. In Aim 2 we will demonstrate the role of PDGF signaling in LMC during the Expanding and Collapsed phases of arthritic progression via functional genomic studies, and genetic loss of function in the setting of acute collagen antibody-induced arthritis and chronic TNF-induced arthritis in mice. To correlate these animal studies with human disease, in Aim 3 we will complete a clinical pilot of RA patients receiving anti-TNF therapy for hand flare, to formally demonstrate the utility of NIR-ICG imaging as a biomarker of LV recovery, and its correlation with response to therapy. Completion of these Specific Aims will substantiate our paradigm-shifting hypothesis of RA flare, and may provide novel insights into refractory disease that can be diagnosed by assessing effe...