Project Summary The signaling lipid sphingosine 1-phosphate (S1P) plays critical roles in the immune response. S1P is recognized by five G protein-coupled receptors, which regulate trafficking and cytokine responses of myriad cells including lymphocytes, astrocytes and endothelial cells. Most notably, the abundant S1P in lymph guides T cells out of lymph nodes (LN), where they are initially activated, into circulation, where they can travel to the site of inflammation. FTY720, a drug that targets four of five S1P receptors, was the first FDA-approved oral therapeutic for multiple sclerosis, and second-generation drugs inhibiting S1P signaling have shown promise in psoriasis and colitis. These drugs trap T cells within LN, preventing access to sites of inflammation, and they may have additional important anti-inflammatory effects within diseased tissues. Despite S1P's importance and FTY720's efficacy, many questions remain about how S1P signaling regulates immune responses because we cannot map S1P distribution in tissues. While it is well-established that circulatory S1P directs lymphocyte movement between organs, we understand little about the function of S1P within organs. This is a challenging problem because, in general, we lack tools to chart lipid gradients. A series of elegant studies has shed light on the distribution of protein chemokines by knocking fluorescent reporters into the chemokine- coding locus. But lipids are not encoded genetically, and the complex balance of synthetic enzymes, degrading enzymes, and transporters determines the level of signaling-available lipids. Mass spectrometry has been used to quantify bioactive small molecules, but whole tissue measurements can be misleading because many lipids act both extracellularly as ligands for cell-surface receptors and intracellularly as metabolic intermediates. Moreover, even if interstitial fluid could be extracted from a precise location without inducing inflammation, lipids are generally bound by protein carriers, and it remains unclear which carriers present vs. sequester these lipids from their cognate receptors. To overcome these problems, we have generated a mouse expressing a reporter of signaling-available S1P. To our knowledge, this is the first technique to map signaling lipids in situ. Here, we will use this mouse to address two fundamental questions about S1P gradients within tissues and immune function. In our first aim we will focus on a lymphoid organ. We have chosen LN, where T cells are first activated by tissue infection or auto-antigens before exiting in response to circulatory S1P. We will test the hypothesis that S1P gradients sensed by S1P receptor 5 define LN regions enriched with cells poised to produce IFN?. In our second aim, we will turn to a non-lymphoid tissue, where effector T cells arrive from circulation. Because of FTY720's clinical success, we will map S1P in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We will identify factors that regulate pro-inflammatory S1P in the CNS, which may enable development of spatially targeted therapies that avoid some of FTY720's dangerous side effects.