*Purpose: Lymphatic endothelial cells (LEC) highly expres S1PR1 and S1PR2. The ligand S1P is produced by LEC and regulates T cell migration. The mechanisms of how LEC utilize S1P/S1PRs to regulate T cell migration are not understood. We hypothesized that LEC use S1PRs to regulate CD4 T trafficking from tissues through afferent lymphatics into draining lymph nodes (dLN).

*Methods: CD4 T cells were transferred into mice to measure migration into afferent lymphatics and dLN. Specific pharmacologic and genetic S1PR blockade was employed in vitro and in vivo. Mouse primary LEC and LECs line were used to assess migration, chemokine signals, adhesion molecules and S1PR function in vitro.

*Results: S1P selectively promoted various CD4 T cell subsets (naïve, activated, memory, regulatory) to migrate across LEC. Blocking S1PR2, but not S1PR1, specifically inhibited T cell migration toward S1P across the LEC. Similarly, various CD4 T cells also migrated less efficiently into the lymphatic vessels and dLN of S1PR2 antagonist treated or S1PR2 deficient recipients. S1PR2 blockade increased lymphatic endothelial cell layer integrity and decreased permeability in vivo and in vitro. The S1PR2-ERK pathway controlled adherens tight junction molecules expression (VE-cadherin, zonulin, occluding) and TEM migration. S1PR2 antagonists decreased VCAM-1 expression, and reduced T cell contact with LEC surface VCAM-1⁺ domains, which were important for TEM migration. Treatment with neutralizing antibodies against adhesion molecules demonstrated that S1P driven migration was dependent on VLA4­VCAM­1 interactions. S1P-S1PR2 driven migration preferentially utilized a transcellular instead of a paracellular migration pathway, and S1PR2 antagonists and anti-VCAM-1 treatment reduced T cell transcellular migration.

*Conclusions: While both S1PR1 and S1PR2 are expressed by LEC, and only S1PR2 is used to regulate T cell migration across afferent lymphatics into dLN. S1PR2 downstream ERK signaling regulates VCAM­1 expression, which is required for CD4 T cell transendothelial migration. S1PR2 also regulates VE­cadherin expression as well as LEC junctional integrity and permeability. These findings implicate S1PR2 as a novel and specific drug target for regulating the lymphatic migration of CD4 T cells in immunity and tolerance.

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