Project Summary The signaling lipid sphingosine 1-phosphate (S1P) plays many roles in the immune response. Most notably, S1P regulates lymphocyte exit from lymphoid organs, where they are initially activated, into circulation, where they can travel to the site of infection. The concentration of S1P is higher in lymph than lymph nodes (LN). This gradient guides lymphocytes out of the LN into lymph and ultimately back to blood. Similarly, the high S1P within blood attracts lymphocytes to exit the spleen into circulation. T cells sense the S1P gradient primarily through the G protein-coupled receptor S1P receptor 1 (S1PR1). FTY720, a drug that targets four S1P receptors including S1PR1, became the first FDA-approved oral therapeutic for multiple sclerosis. By blocking lymphocyte exit from lymphoid organs, FTY720 prevents lymphocytes from accessing the central nervous system. Second-generation drugs that target S1PR1 have also shown promise in Phase II trials for psoriasis and colitis. Unfortunately, use of these drugs is limited because they also target S1PR1 in endothelial cells and cardiomyocytes, resulting in serious side effects. We recently found that the major facilitator superfamily transporter SPNS2 supplies S1P into lymph but not blood, making it a promising new drug target that would enable spatially specific modulation of S1P signaling. Blood S1P levels remain at about 80% of baseline in SPNS2-deficient mice, and these mice exhibit normal lung vascular permeability, unlike FTY720-treated mice. By contrast, S1P levels in lymph are dramatically reduced in SPNS2-deficient mice. In SPNS2-deficient mice, T cells exit the spleen into blood normally but cannot exit LN into lymph, leading to a redistribution of T cells from spleen to LN and a loss of circulating cells. We proposed that targeting SPNS2 could trap T cells in LN without substantial cardiovascular side effects. However, these studies were carried out in homeostasis, and the role of SPNS2 during inflammation or disease is poorly understood. Here, we will investigate how SPNS2 affects T cell function during the effector and memory stages of an immune response. First, we will address whether SPNS2 generates the lymph S1P that guides T cell exit from LN during an immune response, or whether in inflammation other transporters are upregulated and can compensate for SPNS2's loss. This is the key question to determine whether SPNS2 would be an effective drug target. Second, we will test the hypothesis that SPNS2 supplies S1P into the LN parenchyma during an inflammatory response, blunting the gradient that guides exit and slowing (but not halting) T cell transit through an inflamed LN. Third, we will determine whether SPNS2 regulates memory T cell differentiation and survival, extending our surprising finding that SPNS2/S1PR1 signaling regulates mitochondrial content and survival in naïve T cells. The proposed aims will test SPNS2's potential as a target for immunosuppressive therapy, and a...