Tuesday September 15, 2020 - 14:30 to 15:15
Treg use the PD-1-PD-L1 axis for lymphatic transendothelial migration
Wenji Piao1, Lushen Li1, Christina Paluskievicz1, Keli Hippen2, Yigang Zhang2, Vikas Saxena1, Marina Willson Shirkey1, Bruce Blazar2, Leonardo Riella3, Jonathan Bromberg1.
1Surgery, University of Maryland School of Medicine, Baltimore, MD, United States; 2Pediatrics, University of Minnesota Cancer Center, Minneapolis, MN, United States; 3Schuster Family Transplantation Research Center, Harvard Medical School, Boston, MA, United States
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.
NIH 1RO1 AI062765 (JSB).