*Purpose: CCR7 plays critical roles in T cell migration to CCL21 across lymphatic endothelial cells from tissues to lymph nodes (LN). S1PR1 and S1PR4 are also required by T cells for S1P-driven afferent lymphatic migration. However, the distinct roles of CCR7 and S1PRs on T cell LN homing have not been defined yet. We hypothesized that CCR7 and S1PRs have unique roles on T cell transendothelial migration (TEM).

*Methods: CD4 T cells were adoptively transferred into mice, and migration to afferent lymphatics and draining LN (dLN) measured. Mouse primary lymphatic endothelial cells (LEC) and LEC lines were used to assess migration, chemokine signals, adhesion molecules and S1PR and CCR7 function in vitro. Specific pharmacologic and genetic blockade were employed.

*Results: S1P, but not CCL19, promoted T cell interaction with LEC to enhance TEM. VCAM-1, not ICAM-1, promoted T cell migration toward S1P. An S1PR1 antagonist increased migration through VCAM-1, suggesting S1PR1 regulated binding and/or release of T cells from adhesion. In contrast, an S1PR4 inhibitor blocked migration, so S1PR4 was responsible for migration to VCAM-1. S1PR1 regulated migration distances but not velocities, while S1PR4 regulated both distances and velocities. S1P driven TEM was dependent on both chemotaxis and chemokinesis, and both S1PR1 and S1PR4 were required for chemotaxis and chemokinesis. Both S1PR1 and S1PR4 were required for T cell migration toward S1P or S1P plus CCL19. Kinetic analyses showed that S1PR1 and S1PR4 engaged T cell migration at early time points, while CCR7 was required at later time points. Although both S1P and CCL19 regulated T cell mobility, S1P-driven migration of CD4 T cells preferentially utilized transcellular rather than paracellular trafficking compared to CCL19. S1PR1 and S1PR4 antagonist treatment reduced the percentage of transcellular migration toward S1P.

*Conclusions: S1PRs and CCR7 play unique roles in T cell TEM migration. S1PRs initiate T cell-LEC interactions at early time points, while CCR7 drives T cells migration at these and later time points. S1PRs drive a unique transendothelial path compared to CCR7. These findings resolve uncertainties about why so many chemokines and receptors are required for migration, and point to new therapeutic approaches to regulate migration and immunity.

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