Both CD4+ and CD8+ Tregs play a critical role in the control of immune responses and immune tolerance; however, our understanding of CD8+ Tregs is limited while they are particularly promising for therapeutic application. We report here existence of highly suppressive human CD8+CD45RClow/− Tregs expressing Foxp3 and producing IFNγ, IL-10, IL-34, and TGFβ to mediate their suppressive activity. We demonstrate that total CD8+CD45RClow/− Tregs can be efficiently expanded in the presence of anti-CD3/28 mAbs, high-dose IL-2 and IL-15 and that such expanded Tregs efficiently delay GVHD and human skin transplantation rejection in immune humanized mice. Robustly expanded CD8+ Tregs displayed a specific gene signature, upregulated cytokines and expansion in the presence of rapamycin greatly improved proliferation and suppression. We show that CD8+CD45RClow/− Tregs are equivalent to canonical CD4+CD25highCD127low/− Tregs for suppression of allogeneic immune responses in vitro. Altogether, our results open new perspectives to tolerogenic strategies in human solid organ transplantation and GVHD.

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Allogeneic bone marrow transplantation (BMT) is a widely spread treatment of many hematological diseases, but its most important side effect is graft-versus-host disease (GVHD). Despite the development of new therapies, acute GVHD (aGVHD) occurs in 30% to 50% of allogeneic BMT and is characterized by the generation of effector T (Teff) cells with production of inflammatory cytokines. We previously demonstrated that a short anti-CD45RC monoclonal antibody (mAb) treatment in a heart allograft rat model transiently decreased CD45RChigh Teff cells and increased regulatory T cell (Treg) number and function allowing long-term donor-specific tolerance. Here, we demonstrated in rat and mouse allogeneic GVHD, as well as in xenogeneic GVHD mediated by human T cells in NSG mice, that both ex vivo depletion of CD45RChigh T cells and in vivo treatment with short-course anti-CD45RC mAbs inhibited aGVHD. In the rat model, we demonstrated that long surviving animals treated with anti-CD45RC mAbs were fully engrafted with donor cells and developed a donor-specific tolerance. Finally, we validated the rejection of a human tumor in NSG mice infused with human cells and treated with anti-CD45RC mAbs. The anti-human CD45RC mAbs showed a favorable safety profile because it did not abolish human memory antiviral immune responses, nor trigger cytokine release in in vitro assays. Altogether, our results show the potential of a prophylactic treatment with anti-human CD45RC mAbs in combination with rapamycin as a new therapy to treat aGVHD without abolishing the antitumor effect.