*Purpose: CD80 is a costimulatory molecule expressed on antigen presenting cells (APCs) that binds CD28 and CTLA4, stimulating or inhibiting immunity, respectively. CD80 also binds Programmed Death-1 Ligand (PD-L1) to transduce coinhibitory signals for T cell activation. Blockade of PD-L1-CD80 with anti-PD-L1 exacerbates allograft rejection, while anti-CD80 mAb has inhibitory and stimulatory effects, suggesting additional mechanisms for regulation of immunity by this interaction. Notably, CD80 is highly expressed on activated effector CD4 T cells (Teffs), and PD-L1 is highly expressed by lymphatic endothelial cells (LEC). We tested whether Teffs-LEC CD80/PD-L1 signaling regulates Teffs function, including lymphatic transendothelial migration (TEM).

*Methods: Human and mouse activated effector CD4 T cells were migrated across human or mouse LEC. Anti-CD80 and anti-PD-L1 mAbs or recombinant CD80-Fc and PD-L1-Fc were used to block or stimulate CD80 or PD-L1 on T cells and LEC. Their effects were assessed for cell signaling and in vitro and in vivo migration.

*Results: Teffs expressed high levels of CD80. Blockade of CD80 on human or mouse Teffs with anti-CD80 mAb (1G10), which interrupts CD80 binding to PD-L1, inhibited human or mouse Teffs TEM in vitro and in vivo. PD-L1-Fc engagement of Teffs CD80 promoted Teffs TEM. Engagement of CD80 on Teffs with immobilized PD-L1Fc suppressed the constitutive phosphorylation of ERK without a specific effect on NFκB or Akt phosphorylation. Blocking LEC with anti-mouse PD-L1 mAb (10F.2H11) which blocks only the CD80-PD-L1 interaction, inhibited Teffs but not regulatory T cell (Treg) TEM. Crosslinking LEC PD-L1 with CD80-Fc induced strong phosphorylation of ERK, modest classical NFκB activation, but did not signal to Akt. CD80 engagement of LEC PD-L1 also enhanced VCAM-1 expression, which is important for TEM, and this increased expression was inhibited by blocking both classical NFκB and ERK signaling. Notably, LEC VE-cadherin expression, which is also important for TEM, was not affected by PD-L1 ligation by CD80-Fc.

*Conclusions: Teff but not Treg uniquely use the CD80/PD-L1 to regulate TEM. Teff CD80 signals through classical NFκB and ERK pathways to enhance LEC expression of the VCAM-1 adhesion molecule. These data demonstrate a novel role for Teffs 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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