Allogeneic Treg-derived artificial vesicles: A promising therapeutic modality for multiple sclerosis.

INTRODUCTION: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS). CD4(+) CD25(+hi) Tregs, which normally suppress immune responses, exhibit impaired function in MS. Treg-derived extracellular vesicles (EVs) carry immunoregulatory proteins and miRNAs that modulate T-cell activity. However, EVs from MS patients show reduced suppressive capacity, suggesting their dysfunction contributes to MS pathogenesis. This highlights EVs' potential role in MS development and therapy. METHODS: Tregs were differentiated from naïve T cells isolated from peripheral blood mononuclear cells (PBMCs) of healthy donor, then transduced with B2M-shRNA lentivirus to generate HLA class I-knockdown Tregs. Extracellular vesicles-including natural vesicles, cytochalasin B-induced artificial vesicles, and ultrasound-induced artificial vesicles-were isolated from Tregs and characterized by scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA), flow cytometry, and Western blot. Their effects on healthy donor and MS patient PBMCs were evaluated via flow cytometry and ELISA (IL-6, IL-10, IFN-γ). RESULTS: Ultrasonication yielded a higher number оf vesicles enriched with key immunosuppressive proteins, including PD-1 and Tim-3, compared to cytochalasin B. Functional assays demonstrated the ability оf ultrasonication-induced AVs to suppress inflammatory markers, such as IFNγ, and modulate the cytokine profile in both healthy and MS-derived PBMCs. CONCLUSION: Developing effective MS therapies remains challenging. While cellular therapies face limitations like Treg dysfunction and CNS delivery issues, allogeneic EVs offer a promising alternative due to their scalability, low immunogenicity, and blood-brain barrier penetration. We developed Treg-derived artificial vesicles (TrAVs) that maintain immunosuppressive properties and modulate PBMC responses, suggesting therapeutic potential for MS. Further research is needed to optimize production and validate efficacy in disease models.