Lymphocyte egress from peripheral lymphoid organs during recirculation is essential for normal immune functions, but the mechanisms regulating this process are still incompletely understood. From the lymph nodes, naïve lymphocytes egress into the lymph in a process that requires lymphocyte-intrinsic expression of the sphingosine-1-phosphate receptor (S1P1), a G-protein coupled receptor (GPCR). Furthermore, lymphocyte egress is dependent on a radiation resistant source that maintains the level of S1P1's ligand, sphingosine-1-phosphate (S1P), in the lymph. The cellular source of lymph S1P and how S1P1 acts to promote egress are not defined.

We investigated the hypothesis that lymphocyte S1P1 functions to overcome signals that retain cells in lymphoid tissues. We found that CCR7-deficient T cells exited lymph nodes more rapidly while CCR7 overexpressing cells were retained longer, as compared to wild-type cells. Using the immunosuppressive drug FTY720 to down-modulate S1P1 function, we showed that the requirement of lymphocyte S1P1 for egress was partially relieved by CCR7 deficiency, and more fully relieved by pertussis toxin treatment (PTX) that inactivates lymphocyte Gαi. PTX treatment also restored egress competence in S1P1-deficient T cells. Furthermore, we found T cell accumulation in the LYVE-1+ cortical sinuses was promoted by S1P1 and antagonized by CCR7 expression intrinsically within the lymphocytes.

Next, we studied the role of lymphatic endothelial cells in generating lymph S1P. We showed that mice with tissue-specific deletion of Sphk1 by Cre expression from the lymphatic vascular endothelium gene-1 (Lyve-1) and lacking Sphk2 had a loss of S1P in the lymph while maintaining normal plasma S1P level. In lymphatic Sphk-deficient mice, egress from lymph nodes and Peyer's patches was markedly reduced, while nodal LYVE-1+ cortical sinuses lacked lymphocytes and appeared collapsed. Treatment with PTX to inhibit lymphocyte-Gαi restored lymphocyte egress in these mice. Furthermore, we found in the absence of lymphatic Sphks, lymphatic vasculature architecture was altered.

Together our findings support a model whereby lymphatic endothelial cells produce lymph S1P, which acts directly on lymphocyte S1P1 to overcome retention signals to promote lymphocyte egress from lymph nodes and Peyer's patches.