Background: Programmed death-1 (PD-1) attenuates immune responses. Ligand-engagement of PD-1 regulates T cell development, proliferation, cytokine production, and apoptosis. Little is known about PD-1 signaling or functions in T cell migration. We observed that PD-1 is expressed at higher levels on regulatory T cells (Tregs) than non-Tregs, and PD-L1 is highly expressed by lymphatic endothelial cells (LECs). The interplay of Treg PD-1 with LECs PD-L1 has not been previously explored. We investigated the hypothesis that Treg use surface PD-1 to engage PD-L1 on LECs to regulate Treg lymphatic transendothelial migration (TEM).
Methods: Human and murine dermal LECs were used in biochemical, phenotypic, and functional analyses of PD-L1 signaling. Human naïve Tregs and effector T cells and murine wild type (WT) or PD-1^-/- Tregs, naïve, and activated CD4 T cells were migrated across LECs in vitro and in vivo. Recombinant PD-1 or PD-L1 extracellular domains fused with IgG1 (PD-1 Ig and PD-L1 Ig) were used to induce PD-L1 and PD-1 signaling, respectively. Anti-PD-1 or PD-L1 mAbs were used to block PD-1 or PD-L1 signaling.
Results: We demonstrated that PD-1-PD-L1 was involved in Treg TEM into lymphatics. Blockade of Tregs with anti-PD-1 mAb, or blockade of LEC with anti-PD-L1 mAb inhibited Tregs, but not non-Tregs, TEM. However, blockade of Treg PD-L1, or LEC PD-1 with mAb had no effect on TEM. TEM of PD-1^-/-Tregs was decreased compared to WT Tregs. Pretreating Tregs with anti-PD-1 mAb or pretreating mice with anti-PD-L1 mAb inhibited Treg migration into local draining lymph nodes. Human and mouse Tregs highly expressed both PD-1 and PD-L1, while human and mouse LECs had surface expression of PD-L1, but not PD-1. Ligation of human and mouse Treg PD-1 by immobilized PD-L1 Ig enhanced TEM. Crosslinking PD-1 on Tregs triggered extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and PI3K serine/threonine protein kinase Akt activation, but inhibited NFκB-p65 signaling. Crosslinking PD-L1 on mouse LECs with PD-1 Ig induced NFκB-p65, ERK, and Akt activation. Ligation of PD-L1 on LECs by PD-1 Ig decreased VE-cadherin, which was recovered by blocking NFκB-NIK, ERK, and PI3K. LEC PD-L1 ligation enhanced VCAM-1 expression, which was inhibited by blocking NFκB-p65. Tregs but not non-Tregs also caused changes similar to PD-L1 Ig by decreasing VE-cadherin and increasing VCAM-1 at LEC cell-cell junctions.
Conclusions: Treg PD-1 engages LEC PD-L1 to modulate lymphatic endothelial structure, which then promotes Treg lymphatic TEM. Treg PD-1 signals through ERK, JNK, and PI3K/Akt pathways to maintain or increase surface PD-1 expression on Tregs. LEC PD-L1 signals through NFκB-NIK, ERKs, and PI3K/Akt (Thr308) to modulate VE-cadherin, and through classical NFκB-p65 to regulate VCAM-1 expression. These data demonstrate an important and novel role for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and hence Treg suppressive function.