Induction of antigen-specific regulatory T cells by engineered extracellular vesicles

Extracellular vesicles (EVs) are emerging as versatile nanocarriers for targeted drug delivery and immune modulation. However, strategies that can induce antigen-specific immune tolerance remain limited, highlighting an unmet need for more precise and effective approaches. To address this challenge, we aimed to develop a modular EV-based system capable of inducing antigen-specific regulatory T cells (Tregs). In this study, we developed engineered antigen-presenting EVs (AP-EVs) that co-display peptide-major histocompatibility complex class II complexes (pMHCII), interleukin-2 (IL-2), and transforming growth factor-<i>β</i> (TGF-<i>β</i>) on their surface. These immunomodulatory molecules were anchored to the EV membrane via CD81 or milk fat globule-EGF factor 8 (MFG-E8) scaffolds to ensure stable and multivalent presentation. AP-EVs induced the differentiation of antigen-specific Tregs from naïve CD4⁺ T cells <i>in vitro</i>, and promoted their proliferation and expression of canonical regulatory markers, including CD25, CTLA-4, PD-L1, and LAG-3. <i>In vivo</i>, the combination of AP-EVs and mTOR inhibition with rapamycin significantly enhanced the generation of Foxp3⁺ Tregs in antigen-specific adoptive transfer models. The Tregs induced by AP-EVs <i>in vitro</i> exhibited suppressive function, highlighting the therapeutic potential of this system. Our findings establish a modular, cell-free EV platform for antigen-specific immune tolerance, with potential applications in the treatment of autoimmune and allergic diseases through targeted immune regulation.