Generation of human regulatory T cells de novo with suppressive function prevent xenogeneic graft versus host disease.

Treatment with rapamycin (RAPA) favorably affects regulatory T cells (Treg) in vivo, and RAPA induces the de novo expression of FOXP3 in murine alloantigen-specific T cells. Whether RAPA acts independently or with transforming growth factor beta (TGF-β) to produce ex vivo-induced Treg generation is unknown. Naïve CD4(+) T cells isolated from peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 coated beads in the presence of IL-2 for 5 to 7 days. Ten ng/ml of TGF-β (1 to 100 ng/mL RAPA) was added to some of the cultures. The phenotypes were analyzed with flow cytometry. The conditioned cells were cocultured with CFSE-labeled T cells in different ratios for 5 days. CFSE dilution indicating T response cell proliferation was analyzed by flow cytometry. Xenogeneic graft-versus-host disease (x-GVHD) was induced by transplanting human peripheral blood mononuclear cells into RAG2(-/-) γc(-/-) mice exposed to total body irradiation, and various factors in the subjects were subsequently compared. CD4 cells induced by rapamycin and TGF-β (CD4(RAPA/TGF-β)) expressed the natural Treg phenotypes and trafficking receptors, and no significant cytotoxicity was observed. CD4(RAPA/TGF-β) was anergic and demonstrated potent suppressive activity in vitro. Although the transfer of human peripheral blood mononuclear cells into RAG2(-/-) γc(-/-) mice caused x-GVHD, the cotransfer of CD4(RAPA/TGF-β) decreased human cell engraftment and extended survival in mice. RAPA plus TGF-β induces human naïve T cells to become suppressor cells, a novel strategy for treating human autoimmune diseases and preventing allograft rejection.