Project Abstract T cell exit from a site of inflammation plays key roles in the immune response. At the effector phase of an immune response, T cell exit has been reported to limit tissue inflammation. By contrast, periodic T cell exit from a site of chronic inflammation might prevent T cell exhaustion and sustain the immune response. As memory develops, T cell exit from a site of inflammation limits the formation of resident memory T cells. Whether the T cells that have exited the tissue become a unique form of circulating memory cells is unknown. Overall, while the function of resident memory T cells remaining at a site of inflammation is increasingly well understood, the function of cells that have left a site of inflammation remains unclear. One cue that regulates T cell exit from a site of inflammation is the signaling lipid sphingosine 1- phosphate (S1P). Generally, S1P levels are high in lymph compared to tissues, and S1P guides immune cells out of tissues into lymphatics. This paradigm has been well-established for T cell exit from lymph nodes into efferent lymphatics, and some studies indicate that it holds for T cell exit from non-lymphoid tissues into lymph. Yet we do not understand how S1P gradients are shaped in non-lymphoid tissues during an immune response, or how S1P might interact with other cues that might guide T cell exit from non-lymphoid tissues. A key barrier to answering these questions is our inability to manipulate gene expression with spatial specificity. Thus, we cannot permanently mark T cells at a site of inflammation and follow them through a memory response. Similarly, we cannot disrupt genes that regulate chemotactic cues only at a site of inflammation, avoiding confounding effects on T cell development and differentiation in the thymus and lymph nodes. Conditional gene targeting allows manipulation of gene expression in a cell-type specific and, in some models, inducible manner. However, all murine targeting approaches are currently limited to altering gene expression in entire lineages, and it is not possible to institute permanent changes in cells within a specific location in the animal. To address this problem, we have generated a mouse expressing a light-inducible Cre recombinase (LI- Cre), which allows us to alter gene expression in cells of interest in a location-specific and time-specific manner using blue light. In this grant, we will take advantage of this novel mouse model to answer key questions about the regulation and function of T cell exit from inflamed skin. We will address how S1P receptor 1 cooperates with other candidate exit receptors in two well-defined models of disease, ask how S1P gradients change in the skin during the course of disease, and track the fate of T cells that have exited the skin in the memory phase and in recall responses.