*Purpose: Studies in B6 (H2^b) mice tolerized to CBA (H2^k) have shown that induced allospecific Tregs make 2 very different forms of immunoregulatory cytokines: 1) water-soluble molecules IL10 and active TGFβ1 that bind to their specific receptors on T effector cells causing immediate immunosuppression of response; and 2) exosomes containing inhibitory microRNA, enzymes CD39 & CD73 that metabolize ATP to produce the inhibitor adenosine, and IL35. The subunits of the latter were found to be exosome bound via the tetraspannin CD81: hence an “exokine”. However, the exokine concept has not been verified in species such as humans and monkeys.

*Methods: We examined two forms of allospecific regulation involving IL35: 1) suppression of “classical” alloreactivity in a human liver transplant recipient (AJ) as well as in a monkey bone marrow transplant recipient; and 2) suppression of “non-classical” allo-MHC-dependent autoreactivity in a human lung transplant recipient(JS).

*Results: We previously reported that AJ’s PBL at year 6 post-LivTx showed linked suppression when exposed to donor MM HLA class I (B7), causing his Treg cells to produce IL10. In pt JS we found a response to an allo (DR15)-restricted peptide of collagen type V (p1049) at year 7, but he survived to year 14, at which time he had an equally strong allo(DR15)-restricted anti-p1049 Treg response producing TGFβ. We found that both the IL10 response of pt AJ to donor HLA, and the TGFβ response of pt JS to donor HLA-restricted col V/p1049, were paralleled by an IL35 response to the same alloantigens. Furthermore, when in vitro culture supernatant of pt AJ PBL stimulated with donor antigen was separated by ultracentrifugation, all the IL35 signal was found in the exosome fraction, while all the IL10 was found in the 100,000 x g supe.Since IL35 dependence of suppressed alloreactivity was also found in Rhesus monkeys tolerized by total lymphoid irradiation and bone-marrow transplantation (TLI/BMTx; Burlingham et al, Transplant Direct 2015), we obtained plasma from a tolerized monkey. When exosomes in the plasma were analyzed by TEM and immunogold beads, the IL35 subunit Ebi3 was present.

*Conclusions: Data from allo-tolerant human and monkey transplant recipients support the findings in allo-tolerized mice, that IL35 is associated with extracellular vesicles/exosomes.

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