Introduction: Sphingosine 1-phosphate (S1P) receptors 1 (S1PR1) and S1PR2 are highly expressed by lymphatic endothelial cells (LEC), while T cells express S1PR1 and S1PR4. S1P is produced by LEC and regulates T cell migration. The mechanisms of how S1P regulate CD4 T cell migration across LEC are not fully understood. We hypothesized that S1PRs are utilized by LEC to regulate CD4 T trafficking from tissues through afferent lymphatics and into draining lymph nodes (LN).

Methods: CD4 T cells were adoptively transferred into mice to measure T cell migration into afferent lymphatics and draining LN (dLN). Specific pharmacologic and genetic S1PR blockade was employed in vitro and in vivo. Mouse and human primary LEC, blood endothelial cell (BEC), and LEC line were used to assess migration, chemokine signals, adhesion molecules and S1PR function in vitro.

Results:S1P selectively promoted human and murine CD4 T cell migration across LEC, but not BEC. Selective inhibition of S1PR1 or S1PR2 indicated that migration was mediated by S1PR2, but not S1PR1, in LEC. S1PR2 antagonists inhibited T cell transendothelial migration in vitro and decreased T cell migration into lymphatic vessels and dLN in vivo. CD4 T cells also migrated less into the lymphatic vessels and dLN of S1PR2^-/- recipients. Treatment with neutralizing antibodies against adhesion molecules demonstrated that S1P driven migration was dependent on VLA4-VCAM-1 interactions. S1PR2 antagonists decreased VCAM-1 expression by LEC, which resulted in decreased interaction of T cells with VCAM-1⁺ domains of the LEC surface membrane. The S1PR2 antagonist inhibited S1P-induced phosphorylation of ERK to control VCAM-1 expression. The S1PR2 antagonist also increased cell-cell junction integrity of LEC by enhancing VE-cadherin expression, thus further impairing T cell movement across endothelial layers.

Conclusions: While both S1PR1 and S1PR2 are expressed by LEC, only S1PR2 is used to regulate T cell migration across afferent lymphatics into draining LN. S1PR2 downstream ERK signaling regulates VCAM-1 expression, which is required for CD4 T cell transendothelial migration. S1PR2 also regulates VE-cadherin expression as well as LEC junctional integrity. These findings implicate S1PR2 as a novel and specific drug target for regulating the lymphatic migration of CD4 T cells in immunity and tolerance.

CITATION INFORMATION: Xiong Y., Piao W., Brinkman C., Li L., Kulinski J., Olivera A., Cartier A., Hla T., Schwab S., Hippen K., Blazar B., Bromberg J. Lymphatic Endothelium Requires S1PR2 for T Cell Transendothelial Migration Am J Transplant. 2017;17 (suppl 3).

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