*Purpose: TGFβ has long been known as a key component in tolerance mediated by Treg cells. Here we show that, unlike IL10, but like CD39/CD73, and the immune-suppressive cytokine IL35, TGFβ produced by allo-specific Tregs was associated with small EV in its latent precursor form (TGFβ /LAP). We investigated the role of small extracellular vesicles (sEV), aka exosomes, in the regulation mediated by T- and B-regs after induction of allo-tolerance. We wished to test the hypothesis that LAP-TGFβ1 was tethered to exosomes produced by T- and B-regs, and could be activated, once these sEV were taken up by conventional T cells, suppressing the local immune response.

*Methods: C57BL/6 mice were tolerized by i.p. injection of CBA/J splenocytes combined with anti-CD40L/CD154 antibody treatment on d. 0,2,&4. At d. 35, spleen and LNs were harvested and SC and LNC were cultured with x-irradiated CBA SC overnight. sEV/exosomes were harvested from plasma and from culture supernatants by ultracentrifugation (100,000 xg) and assayed for 1⁰ and 2⁰ immunosuppression of TT-immunized B6 splenocytes using trans-vivo DTH assay, and for acquisition of TGFβ/LAP and TGFβRIII by flow cytometry.

*Results: We found that, after tolerization induced T& B reg cells secreted TGFb/LAP-coated EV, which was mainly associated with CD81⁺ exosomes.Like CD81-bound IL35, EV-TGFβ became active in BOTH 1⁰ and 2⁰ immunosuppression, the latter being the ability to impart suppression capacity to “naïve” lymphocytes. Small EV/exosomes produced by T & Bregs induced by, and tolerant to, MHC and non-MHC/minor Ags mediate tolerance via TGFβ in 2 major ways— a) In 1⁰ suppression, by directly interfering with T effector functions, &b) In 2⁰ suppression, by uptake and passive surface re-expression of EV components by bystander T cells, which then suppress as “surrogates”

*Conclusions: Like other suppressive components of the Treg exosome which are bound in a latent form, TGFβ/LAP requires processing to become the profoundly suppressive agent of allo-tolerance produced by allo-specific regulatory T cells. We are currently analyzing various mechanisms that might account for tethering the secreted TGFβ/LAP to exosomes.

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