*Purpose: In addition to serving as fluid conduits, lymphatic vessels are critical portals for immune cell migration. Lymphatic endothelial cells (LECs) constitutively express high levels of programmed death-1 (PD-1) ligand (PD-L1), known as an immune checkpoint protein, suggesting they engage and regulate immune cells. We hypothesized that LECs use PD-L1 to regulate PD-1-expressing immune cells during transendothelial migration (TEM).

*Methods: Dermal LECs from wild type (WT) or PD-L1 knockout (KO) mice, or CRISPR-Cas9 PD-L1 KO mouse dermal LECs were used in analyses of PD-L1 signaling and in vitro TEM assays. Purified human and murine naïve, activated, and regulatory CD4 T cells were migrated across human or mouse LECs in vitro. Recombinant PD-1 Fc and PD-L1 Fc were used to induce PD-L1/PD-1 signaling. Treg in vivo migration was assessed by the footpad migration assay and in the mouse melanoma model.

*Results: Activated T cells expressed substantially elevated levels of both PD-1 and PD-L1. For both human and mouse Tregs PD-1 was expressed more than PD-L1, while effector CD4 T cells (Teffs) expressed PD-L1 in excess of PD-1. PD-1^-/- but not PD-L1^-/-Tregs had reduced migration and motility compared to WT. PD-1 crosslinking induced rapid Akt activation suppressed classical NFκB-p65 signaling in Tregs. WT LECs constitutively expressed high levels of PD-L1. PD-L1^-/- LEC or lymphatic vessels (LV) had increased adhesion and junction proteins VCAM-1 and zonulin-1, and had more zipper junctions. PD-1 triggered PD-L1 signaling in LEC, resulting in strong PI3K/Akt and NFκB-p65 responses that regulated endothelial structure and enhanced TEM. Similar changes to LEC junctions were observed when PD-1^high Tregs migrated across LEC layers, resulting in NFκB-p65 translocation into the LEC nucleus. PD-L1 signaling decreased VE-cadherin expression, which was restored by blocking ERK and PI3K/Akt. Importantly, PD-1-blockade of intra-tumoral Tregs reduced tumor egress and the detained Tregs converted to IFN-γ-producing Th1 Tregs.

*Conclusions: Treg PD-1 engages LEC PD-L1 to regulate lymphatic TEM. Treg PD-1 signals through LEC PD-L1 to induce the PI3K/Akt pathway to modulate cell junctions, and through classical NFκB-p65 to regulate VCAM-1 expression. The modulated LEC structures uniquely affect only Treg but not Teff TEM. These data demonstrate novel roles for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and ultimately Treg suppressive function.

« Back to 2022 American Transplant Congress