*Purpose: T cells utilize S1PR1 to traffic from thymus into blood, and lymph nodes (LN) into efferent lymphatics. Whether T cell S1PR1 and S1PR4 regulate their migration from tissues into draining LN (dLN) is unknown. We hypothesized that T cells use different S1PRs to traffic from tissues into draining LN through afferent lymphatics.

*Methods: CD4 T cells were adoptively transferred into mice, and migration to afferent lymphatics and dLN measured. Mouse primary lymphatic endothelial cells (LEC) and LEC lines were used to assess migration, chemokine signals, adhesion molecules and S1PR function in vitro. Specific pharmacologic and genetic S1PR blockade was employed in vitro and in vivo.

*Results: S1PR1 and S1PR4 were expressed by all CD4 T cell subsets. S1P enhanced CD4 T cell trans-lymphatic endothelial cell migration, which was both chemotactic and chemokinetic. Pharmacological or genetic blockade of S1P inhibited T cell migration in vitro and in vivo. S1PR1 and S1PR4 antagonist treatment prevented CD4 T cell (naïve, effector and memory) migration in vitro and in vivo. Consistently, S1PR1­-/-­ or S1PR4­-/-­ CD4 T cells failed to migrate toward S1P but not CCL19 in vitro, and into afferent lymphatics and dLN in vivo. In contrast, overexpression of S1PR1 in S1PR1­Tg or S5A transgenic T cells promoted CD4 T cell migration toward S1P in vitro and enhanced homing into dLN in vivo. CD4 T cells utilized S1PR1 and S1PR4 to cross the endothelial cell layer and distribute into the lumen of afferent lymphatics. S1P drove preferential transcellular rather than paracellular trafficking compared to CCL19. S1PR1 and S1PR4 controlled different aspects of T cell motility (distance vs velocity), and had different roles on VCAM-1 binding and release.

*Conclusions: T cell subsets are dependent on an S1P gradient to promote lymphatic transendothelial migration. T cells require S1PR1 and S1PR4 for afferent lymphatic distribution and homing into dLNs. S1PR1 and S1PR4 have distinct roles in T cell adhesion and motility. These results demonstrate unique roles for S1P and S1PRs in regulating T cell migration from tissues into LN, and that distinct receptors and mechanisms are used compared to thymic or efferent lymphatic migration. These findings suggest new and specific drug targets for regulating lymphatic migration in immunity and tolerance.

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