*Purpose: The molecules that regulate Treg migration are incompletely identified. Programmed death-1 (PD-1) and its ligand PD-L1 are immune checkpoint proteins which regulate immune cell responses. However, little is known about their functions in T cell migration. Activated Tregs express high levels of PD-1, and its ligand PD-L1 is highly expressed on lymphatic endothelial cells (LEC). Yet how Treg interact with LEC via PD-L1 has not been previously investigated. We tested the hypothesis that Treg PD-1 signals to LEC PD-L1 to regulate lymphatic transendothelial migration (TEM).

*Methods: Human and murine dermal LECs were used in analyses of PD-L1 signaling. Purified human and murine naïve, activated, and regulatory CD4 T cells were migrated across LEC in vitro and lymphatic vessels in vivo. Recombinant PD-1 fused with IgG1 (PD-1 Fc) were used to induce PD-L1 signaling. Signaling was blocked with specific anti-PD-1 or anti-PD-L1 mAbs.

*Results: Human and mouse Tregs had the highest PD-1 expression among T cell subsets. LEC expressed high levels of PD-L1. PD-1 deficiency or blocking PD-1 on Tregs with mAb inhibited in vitro and in vivo lymphatic migration but not non-Treg effector CD4 T cell migration. Treg migration to CCL19 was enhanced by PD-L1 Fc. Crosslinking LEC PD-L1 with PD-1 Fc induced phosphorylation of classical NFκB-p65, PI3K/Akt threonine 308 and extracellular signal-regulated kinase (ERK). Ligation of LEC PD-L1 with PD-1-Fc augmented VCAM-1 expression, which was inhibited by blocking NFκB-p65 but not ERK or PI3K/Akt signaling. Similar changes to LEC junctions were observed when PD-1-high expressing Treg migrated across LEC layers. Tregs also rapidly induced NFκB p65 translocation into the LEC nucleus. PD-1 Fc ligation of LEC PD-L1 also decreased VE-cadherin expression, which was restored by blocking ERK and PI3K/Akt but not NFκB-p65 signaling. Blockade of LEC PD-L1 with anti-PD-L1 mAb inhibited Treg TEM. Importantly, Treg infiltration into melanoma was reduced by treating mice with anti-PD-1 blocking mAb.

*Conclusions: Treg use PD-1/PD-L1 to regulate lymphatic transmigration. Treg PD-1 signals through ERKs and PI3K/Akt pathways to modulate VE-cadherin, and through classical NFκB p65 to regulate VCAM-1 expression on LEC. The modulated LEC structures uniquely affect only Treg but not non-Treg effector CD4 T cell TEM. These data demonstrate a novel role for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and ultimately Treg suppressive function.

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