Extracellular vesicles mediate intercellular transfer of PD-L1 and bystander T-cell suppression

Extracellular vesicles (EVs) carrying programmed death-ligand 1(PD-L1) on their membranes regulate immune responses. While membrane PD-L1 can be exchanged between tumor and immune cells through immune synapse, it remains unclear whether PD-L1 exchange occurs in non-immune cells. Here, we investigated the transfer of PD-L1 onto the surface of various recipient cells via EVs. Genetically engineered 293T PD-L1+ cells, when co-cultured with PD-L1neg isogenic or heterologous cells, showed a ~ 6 to 22-fold increase in surface PD-L1 on 100% of PD-L1neg bystander recipient cells. Purified PD-L1+ EVs retained competency to transfer PD-L1 to recipient PD-L1neg cell surfaces (> six fold increase). Importantly, transferred PD-L1 mediates binding to PD-1 and facilitates suppression of proliferation of activated T cells. Metabolically inert PD-L1neg 293T cells also acquired PD-L1 on their surfaces from PD-L1+ cells, or PD-L1+EVs, indicating a passive transfer mechanism independent of active cellular uptake. Overall, these findings reveal a distinct, passive and functional mechanism of PD-L1 transfer via EVs, defining previously underappreciated cell-physiological pathway that enables immune suppression and evasion by PD-L1neg somatic cells.