Human thymic Treg-derived extracellular vesicles display a defined proteomic and miRNA signature with donor-variable suppressive activity

Abstract

Extracellular vesicles (EVs) are key mediators of intercellular communication that transfer functional molecules reflective of their parent cells and are increasingly explored as cell-free therapeutic agents. Immunoregulatory cell types, including regulatory T cells (Tregs), produce EVs with potential anti-inflammatory properties. While thymus-derived Tregs (thyTregs) exhibit high stability and potent suppressive activity, their capacity to generate functional EVs remains unknown. EVs were isolated by size exclusion chromatography or by immunocapture from ex vivo–expanded human thyTregs and characterized by electron microscopy, nanoparticle tracking analysis, and Western blotting. Proteomic and miRNA transcriptomic analyses were performed to define EV cargo. Functional activity was assessed by co-culturing EVs with activated allogeneic peripheral blood lymphocytes (PBLs) and evaluating proliferation, activation markers, and cytokine production. Tetraspanin-positive ThyTreg-derived EVs exhibited typical vesicular morphology, a mean size of ~ 200 nm, and expression of canonical EV markers (CD9, TSG101). Proteomic profiling identified 314 proteins enriched in immune-related pathways, including CD25, CTLA-4, TGF-β1, ICOS, and granzyme B, reflecting the parental Treg phenotype. Transcriptomic analysis revealed a distinct miRNA signature dominated by let-7 family members and miR-148a-3p, with predicted targets involved in TGF-β/BMP and MAPK signaling. Functionally, thyTreg-EVs suppressed CD4⁺ and CD8⁺ T cell proliferation in a dose-dependent but heterogeneous manner and, in some donors, induced responder cell death. No significant changes in activation marker expression or cytokine production were observed. Human thyTregs release EVs with immunosuppressive properties and a defined molecular cargo. These findings identify thyTreg-derived EVs as a novel, cell-free platform with potential for immunomodulatory therapies.