Extracellular vesicle-associated PD-L1 induces CD4+ T cell dysfunction and contributes to immunosuppression in sepsis

BACKGROUND: The upregulation of PD-1 on T lymphocytes is one of the key mechanisms driving sepsis-induced immunosuppression. Nevertheless although it is known that PD-L1 on antigen-presenting cells acts as a ligand for PD-1 on T lymphocytes, it remains to be determined whether extracellular vesicle-associated PD-L1 also serves as its ligand. METHODS: Plasma extracellular vesicles (EVs) were isolated via ultracentrifugation. We evaluated the relationship of EV-associated PD-L1 level to immune function and prognosis in septic patients. Additionally, we observed the effect of EV-associated PD-L1 on the proliferation and immune function of co-cultured CD4+ T cells in vitro. We further verified the role of EV-associated PD-L1 by knocking down PD-L1 in a mouse model. RESULTS: The peripheral circulating EV levels were significantly increased in septic patients compared with healthy volunteers. Enhanced PD-L1 protein content on these EVs was evident in septic patients, particularly in those exhibiting adverse prognostic features and pronounced immunosuppression. EVs secreted by LPS-stimulated monocytes were enriched with more PD-L1 than monocytes alone. Co-culture of CD4+ T cells with sepsis-derived EVs significantly downregulated the expression of CD69, IFN-γ and cell viability but upregulated the level of IL-4 and TGF-β. This was accompanied by heightened PD-1 expression and an increased Tregs/CD4+ T cell ratio. Conversely, this effect was partially reversed by inhibition of EV production or knockout of PD-L1 in EVs. Compared with LPS-treated EVs, PD-L1 knockout LPS-treated EVs alleviated the immunosuppressive state in CLP mice. CONCLUSION: The elevated level of circulating EV-associated PD-L1 was closely associated with poor prognosis and immunosuppression in septic patients. EV-associated PD-L1 engaged CD4+ T cells to induce CD4+ T cell dysfunction and may be one of the key mechanisms inducing immunosuppression in sepsis.