Rat DCs, made immature by adenoviral gene transfer of dnIKK2, generated in MLR a unique population of CD4+ T regulatory cells (dnIKK2-Treg) which potently inhibited Tcell response in vitro without need of cell-to-cell contact, and prolonged kidney allograft survival (Transplantation, 2007). Here we tested the hypothesis that the suppressive capacity of dnIKK2-Treg was mediated by microvesicles. We found that dnIKK2-Treg released a larger quantity of CD3+ and CD63+ exosomes (Treg-exo) than resting or activated T cells (Trest-exo, Tact-exo). Treg-exo released from as few as 2.000 Treg completely suppressed proliferation of T cells (1x10E6) in MLR (1,272±782 cpm in MLR+Treg-exo vs 22,295±3,501 of MLR+Trest-exo, and 24,475±4,083 of MLR+Tact-exo, n=5). Results were confirmed by CFSE staining, showing similar suppression of both CD4+ and CD8+ T cell proliferation (44±13% and 41±5% respectively). Western blot analysis revealed a strong specific signal for iNOS protein in Treg-exo. However, the anti-proliferative effect of Treg-exo was only partially reversed by NOS inhibition (9,464±2,415 cpm, n=3). Anti-IL-10 antibody had no effect. Cell cycle analysis (PI staining) revealed that Tcells in MLR+Treg-exo had impaired capability to exit from G0/G1 toward the S and G2 phases (cells in S and G2 phases: 8±2% and 4±2% respectively vs 19±3% and 14±3% in MLR+Tact-exo). Even, most of them (55±8%) were apoptotic (subG1). TUNEL staining confirmed a higher % of apoptotic Tcells at the end of MLR+Treg-exo (58±6%) as compared to MLR+Tact-exo (38±9%). Tcells pre-exposed to Treg-exo, but not to Trest-exo or Tact-exo, potently suppressed T cell proliferation (p<0.05) indicating that Treg-exo induced T cells to become suppressive. As CD4+CD25+ Treg derived miRNA have been shown to mediate T cell suppression (Immunity, 2014), analysis of miRNA profiling expression in Treg-exo, Trest-exo and Tact-exo is ongoing. In vivo immunoregulatory effect of Treg-exo was evaluated in the fully mismatched BN to LW rat kidney transplant model. Treg-exo given into the spleen of 4-day-CsA treated recipient rats, completely prevented acute rejection and prolonged allograft survival (73±34 days post-tx, n=4, p<0.05 vs 4-day-CsA alone 16±3 days, n=3) with 75% of recipient rats achieving long term allograft survival (>60 days post-tx). These results might open new perspectives for Treg-based strategies to control T cell alloreactivity in kidney transplantation.

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