*Purpose: Antigen presenting cell (APC) CD80 trans-binds CD28 and CTLA4 on T cells, boosting or inhibiting immunity, respectively. CD80 has another ligand, Programmed Death-1 Ligand (PD-L1). Conflicting reports of CD80-PD-L1 interactions have been reported with bidirectional interaction of CD80-PD-L1 inhibiting T cell activation, while APC CD80 cis-binds and competes with PD-1 to abrogate its inhibitory function. These observations suggest additional mechanisms for immune regulation by CD80-PD-L1. We observed CD80 is highly expressed on activated effector CD4 T cells (Teffs) and tested the hypothesis that Teff CD80 interaction with PD-L1 on LEC regulates Teff lymphatic transendothelial migration (TEM).

*Methods: Human or mouse Teffs were migrated across human or mouse LEC, respectively. PD-1-Fc, CD80-Fc, or CTLA4-Fc were used for binding assays. Antibodies to, or Fc fusion proteins of, CD80 or PD-L1 were used to block or stimulate CD80 or PD-L1 on T cells and LECs. Cell signaling and migration were assessed with the footpad migration assay and the B16F10-melanoma model.

*Results: PD-L1^high LECs have no surface PD-1 or CD80. Blockade of CD80^high Teffs with anti-CD80 mAb (1G10), which interrupts only CD80 binding to PD-L1, inhibited Teff TEM in vitro and in vivo. Similar results were observed for human cells in vitro. Ligation of Teff CD80 with immobilized PD-L1-Fc suppressed constitutive ERK phosphorylation and promoted Teff TEM. Protein binding assay showed CD80-Fc but not CTLA4-Fc specifically bound PD-L1 on LECs. CD80-Fc induced strong ERK signaling and modest classical NFκB activation in LECs. CD80 engagement also enhanced LEC VCAM-1 expression, which was abolished by ERK or NFκB-p65 blockade. In contrast, the cell junction protein VE-cadherin was not affected. In the melanoma model, anti-CD80 treatment caused IFNγ-producing Teffs to accumulate in the tumor, showing that CD80 blockade on Teffs inhibited tumor egress and contributed to melanoma regression.

*Conclusions: CD80-PD-L1 signaling licenses Teff TEM across LEC for mice and humans. Teff CD80 trans-interacts with LEC PD-L1 and signals through ERK and classical NFκB pathways to enhance LEC expression of the VCAM-1 adhesion molecule. These data demonstrate novel roles for Teff CD80 and LEC PD-L1 in regulation of lymphatic migration and provide new pathways for regulating immunity and understanding checkpoint molecules and blockade.

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