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We identified a subset of natural regulatory CD8+ T cells in peripheral blood of healthy volunteers characterized by the low expression of CD45RC surface molecule. These CD8+Tregs can inhibit allogeneic T cell proliferation in vitro through secretion of IFNg, IL-10, TGFbeta and IL-34. To date, there is no clinical study using CD8+Tregs as a cell-based therapy. Considering their potential for cellular therapy, we set up a process to expand these CD8+Tregs in a short term culture.Tregs were expanded for 14 days with anti-CD3/CD28 mAbs or allogeneic APCs in presence of 1000U/ml IL-2, 10ng/ml IL-15, and with or without cyclosporine A (45ng/ml), rapamycin (45ng/ml), methylprednisolone (500pg/ml), tacrolimus (2ng/ml) or mycophenolate mofetil (1μg/ml). Suppressive function of CD8+Tregs was assessed in vitro on syngeneic CD4+CD25-T cells stimulated by alloAPCs, and in vivo into NSG mice infused with PBMCs and grafted or not with allogeneic human skin for allograft survival and xenogeneic GVHD studies.We demonstrated that natural CD8+Tregs can be efficiently expanded until 2000 fold in 14 days, when stimulated with either allogeneic APCs or anti-CD3 and anti-CD28 mAbs. High dose of IL-2 and IL-15 were required to maintain CD8+Tregs survival. After expansion, CD8+Tregs suppressive capacity was preserved and even increased in vitro, in accordance with their higher expression of immunoregulatory cytokines, while they displayed no cytotoxic activity. Furthermore, transfer of ex-vivo expanded CD8+Tregs allowed to delay GVHD development and allogeneic skin graft rejection in humanized mice models. As usually administered to patients after transplantation, we analyzed the impact of culture medium supplementation with immunosuppressive drugs during expansion. Interestingly, addition of rapamycin improved both CD8+Tregs expansion yield and suppressive activity by 16 and 1.6 fold respectively.We demonstrated that natural CD8+Tregs can be greatly expanded while improved in their suppressive function particularly when cultured in presence of rapamycin, proving their relevance for cell therapy.