Small extracellular vesicles derived from PD-L1-modified mesenchymal stem cell promote Tregs differentiation and prolong allograft survival

We aimed to explore whether programmed cell death protein-1 ligand (PD-L1) modification on small extracellular vesicles (sEVs) could promote T regulatory cells (Tregs) differentiation. In this study, it was confirmed that under physiological conditions, PD-L1 expression was minimal in the MSCs and absent in the MSC-sEVs. A vector harboring the PD-L1 gene was constructed and transfected into bone marrow mesenchymal stem cells (BM-MSCs). By extracting the sEVs of these modified BM-MSCs and monitoring the expression of the PD-L1 protein, however, PD-L1 expression was substantially increased in the MSCs and concentrated in the sEVs. Then, the rat naive CD4 + T cells were cocultured with the sEVs derived from the PD-L1-modified MSCs (sEVs(PD-L1)). By flow cytometry, a higher percentage of Tregs and anti-inflammatory downstream cytokines (including IL-2, IFN-gamma, TGF-beta, IL-10) was detected in the sEVs(PD-L1) group than that in the control group treated by either sEVs in wild type, modified by empty vector, or blank control. Suppressive effect on CD4 + T cell proliferation serves as additional evidence to support the immunoregulation capacity of sEVs(PD-L1). The animal model of vascularized composite allograft further confirmed that PD-L1-modified sEVs induce an immune tolerance, by clinically observation, histopathology, T cell fate and cell product. In conclusion, sEVs(PD-L1) efficiently promotes Treg cell differentiation in vitro and in vivo, which suggests their therapeutic potential in the treatment of allograft rejection.