CD4+FoxP3+ T regulatory cells subsets release small extracellular vesicles containing cell death-related proteins as potential mechanism of T cell suppression

Introduction: T regulatory cells (Tregs) are pivotal for immune tolerance. Among the suppression mechanisms attributed to Tregs, the release of small extracellular vesicles (sEV) has been proposed with CD73, Nrp1 and the transfer of key miRNAs playing a key role. Although these findings have been described for natural Tregs (nTregs), little is known on Tregs induced in vitro (iTregs). Methods: Here, we characterized sEV production from three types of Tregs: nTregs and in vitro iTregs produced with TGF-β or with TGF-β plus retinoic acid (RA) (RATregs). Results: Characterization of sEV production indicates that all Tregs produce sEV with similar size and presence of Alix and Tsg101, with RATregs showing the highest production of sEV. Regarding sEV function, nTregs, iTregs and RATregs produce sEV that suppress CD4+ T and CD8+ T cell proliferation. Interestingly, in vitro culture of splenocytes with sEV showed induction of cell death/apoptosis, which is enhanced when adding sEV obtained from nTregs and RATregs, and not from iTregs. Proteomic analysis on sEV obtained from Tregs subsets revealed 163 common proteins, of which some share Tregs biology-related functions such as Apolipoprotein AI, Integrin β2, Lactate Dehydrogenase-A, Thrombospondin 1 and Transferrin Receptor. Depending on the combination of Tregs subsets analyzed, 5 major clusters containing cell death-related proteins were identified, including molecules belonging to the Granzyme/Perforin pathway. Discussion: Although this study does not explain the enhanced cell death observed in cultures with sEV produced by nTregs and RATregs, it does demonstrate that Tregs subsets produce sEV with inhibitory function driven -at least- by cell death, providing new insights on the biology of Tregs.