TGF-β–induced Tregs release extracellular vesicles that modulate CD4+ T-cell proliferation and phenotype in a Nrp1-dependent manner

Neuropilin-1 (Nrp1) is one of the receptors for TGF-β and is associated with the suppressive function of T regulatory cells (Tregs). Based on previous findings on natural Tregs, this work investigated the role of Nrp1 on induced Tregs (iTregs) and their small extracellular vesicles (sEVs) by using a conditional knockout for Nrp1 on FoxP3+ cells. sEVs derived from iTregs were obtained by ultracentrifugation, purified using size exclusion columns, and characterized by nanoparticle tracking analysis. sEVs' impact on CD4+ T-cell cultures was evaluated using functional modulation assays, including CD4+ T-cell phenotypic analysis by flow cytometry and ELISA. We observed a decrease in iTreg induction when CD4+ T cells came from the Nrp1 knockout (KO) mice, indicating that Nrp1 is required for de novo FoxP3 induction/expression. Second, CD4+ T cells cultured with sEV-derived from wild-type (WT) iTregs reduced CD4+ T-cell proliferation, decreased the expression of CD25, and induced FoxP3 and CD73. In contrast, sEVs derived from Nrp1KO iTregs were unable to suppress CD4+ T-cell proliferation and to favor CD25 and CD73 expression. With respect to cytokine production, both sEVs derived from WT and Nrp1KO iTregs inhibited IL-10 and IFN-γ secretion, indicating that Nrp1 is not required for the regulation of these cytokines by sEV. Overall, this study provides new information into the role of Nrp1 in immunomodulation, revealing that Nrp1 signaling plays a role in iTreg differentiation by permitting FoxP3 expression and that iTreg-derived sEVs depend on Nrp1 to control CD4+ T-cell proliferation and phenotype.