*Purpose: Programmed death-1 (PD-1) attenuates immune responses. Engagement of PD-1 by its ligands PD-L1 or PD-L2 regulates T cell development, proliferation, cytokine production, and apoptosis. However, little is known about PD-1 signaling or function in Treg. Since we showed that Treg express higher levels of surface PD-1 than naïve CD4 T cells, and since PD-L1 is highly expressed by lymphatic endothelial cells (LEC), we hypothesized that Treg-LEC PD-1/PD-L1 signaling regulates transendothelial migration and Treg function.

*Methods: Human Treg, naïve and activated CD4 T cells, and murine wild type and PD-1^-/- Treg were isolated and migrated across human or mouse LEC in a transwell based in vitro assay. Anti-PD-1 and anti-PD-L1 mAbs and recombinant PD-1-Ig and PD-L1-Ig were used to block or stimulate PD-1 or PD-L1 on T cells and LEC. Their effects were assessed for signaling and migration.

*Results: Treg expressed higher levels of PD-1 than non-Treg CD4 T cells. Treg PD-1 expression was enhanced by migration across LEC. In contrast, PD-1 expression was not enhanced on other CD4 T cells that migrated across LEC or on Treg that migrated only across plastic membranes. Blockade of PD-1 by anti-PD-1 mAb on human or mouse Treg, but not non-Treg, inhibited lymphatic transendothelial migration in vitro and in vivo. Treg also expressed PD-L1, yet blockade of PD-L1 by anti-PD-L1 mAb on human or mouse Treg did not affect Treg migration. However, blocking PD-L1 on human or mouse LEC inhibited Treg but not non-Treg migration in vitro and in vivo. PD-1^-/- Treg also had decreased transendothelial migration in vitro. PD-1^-/- Treg had limited migration to local LNs in vivo compared to wild type. Immobilized PD-L1-Ig activated Treg PD-1 signaling and promoted Treg transendothelial migration. Immunoblotting showed crosslinking PD-1 on Treg triggered extracellular-signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), as well as PI3K serine/threonine protein kinase Akt (Thr308) activation but inhibited classical NFκB (p-65) signaling on Treg.

*Conclusions: Treg PD-1 signals through MAPKs and PI3K/Akt pathways to maintain or increase surface PD-1 expression on Treg. Treg PD-1 interacts with its ligand PD-L1 on LEC to promote Treg transendothelial migration. PD-1 is required for the enhanced Treg migration in vitro and in vivo, demonstrating a unique and novel function for these molecules.

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