DP106 | EXTRACELLULAR VESICLES RELEASED BY REGULATORY T CELLS EXERT POTENT IMMUNOSUPPRESSIVE EFFECTS AND PREVENT GRAFT-VERSUS-HOST DISEASE IN MURINE MODELS

The adoptive transfer of regulatory T cells (Tregs) alongside conventional T cells (Tcons) represents a validated strategy to control graft-versus-host disease (GvHD) in the setting of HLA-haploidentical hematopoietic stem cell transplantation, while preserving antileukemic activity. While Tregs demonstrate robust immunoregulatory efficacy, their clinical translation is constrained by labor-intensive and costly production. Extracellular vesicles (EVs), endogenously secreted by numerous cell types, are gaining attention as efficient acellular alternatives for immune modulation. We present the first isolation and functional characterization of human Treg-derived EVs, with assessment of their immunoregulatory effects in vitro and in vivo. Tregs (CD4+CD25+ 94.5%±6.3; FoxP3+ 63.7%±11.5; CD127+ 20%±3; suppressive activity 60%±7) were activated with T Cell TransAct and expanded in the presence of rapamycin and IL-2. EVs were isolated using a flow cytometry-based sorting protocol (patent EP19164567A) and characterized by NTA, TEM, AFM, and western blotting for EV markers (CD63, CD81, Flotillin-1). Proteomic profiling revealed clusters of proteins involved in vesicle biology and immune regulation. In suppression assays, FACS-purified EVs achieved 97–98% inhibition of autologous T effector cell proliferation at 3–6×10⁶ EVs/1×10⁵ responder cells, demonstrating threefold greater efficacy than ultracentrifuged EVs. The inhibitory capacity was comparable to that of parental Tregs (p<0.05). A GMP-compliant anti-CD3 magnetic bead-based protocol achieved 70% EV recovery. In vivo, NSG mice were sublethally irradiated and infused with 3×10⁶ Tcons and 1×10⁶ Treg-EVs weekly on days +1, +7, +14, and +21. Controls received Tcons alone or in combination with autologous Tregs. Treg-EV-treated mice showed full protection from GvHD, similar to those treated with Tregs, while Tcon-only mice succumbed to GvHD. All products were cryopreserved prior to infusion, confirming stability. These findings demonstrate that EVs derived from expanded human Tregs exhibit robust and reproducible immunosuppressive activity, supporting their potential as novel acellular therapeutics for the prevention of GvHD.