In recent years, adoptive transfer of Treg cells has gained major attention as an alternative or complementary therapy to conventional immunosuppressive treatments with the ultimate
aim of reducing the side effects of conventional drugs [12, 13]. Since only 5–10% of the circulating CD4+ cells in an organism are Foxp3+ Treg cells, their potential use for cell therapy seems to be limited and the peripheral population would require expansion . Isolated CD4+CD25+ cells frequently undergo expansion in the presence of aCD3/ aCD28 Ab and IL-2. Allo-specific expanded Treg cells seem to be more potent in suppressing chronic rejection, graft versus host disease (GvHD) and autoimmune diseases than polyclonal Treg cells. EGFR activation For example it was shown that antigen-specific expanded Treg
(alloreactive Treg (aTreg)) cells could suppress experimental autoimmune diabetes more effectively than polyclonally X-396 solubility dmso expanded Treg cells . We have shown previously that in vitro culture of total murine CD4+ or CD25−CD4+ cells in the presence of alloantigen and a nondepleting anti-CD4 antibody results in the enrichment of CD25+CD62L+Foxp3+ T cells effective in controlling graft survival in vivo in an alloantigen-specific manner . Although the in vitro enriched aTreg cells were effective in vivo, the protocol still has some limitations. To obtain almost pure Treg-cell populations, high anti-CD4 antibody concentrations had to be used, which led to a dramatic reduction in absolute cell numbers. Here, we have investigated whether we can reduce the anti-CD4 antibody concentration needed to enrich aTreg cells by adding Treg-favouring agents such as TGF-β  and 6-phosphogluconolactonase retinoic acid (RA)  or rapamycin (Rapa)  and thereby achieve higher numbers of stable and efficient aTreg cells. The addition of both TGF-β and RA or Rapa to suboptimal anti-CD4 antibody concentrations resulted in increased purity and absolute
numbers of Foxp3+ Treg cells. Importantly, aTreg cells generated by the addition of TGF-β+RA displayed the lowest production of inflammatory cytokines and expression of CD40L, but the highest stability and regulatory potential in vitro and in vivo. Interestingly, nearly all of the aTreg cells obtained under these conditions co-expressed Helios and Neuropilin-1. Indeed, aCD4+TGF-β+RA aTreg cells could ameliorate GvHD and delay rejection of skin transplants in very stringent in vivo models. Addition of TGF-β+RA or Rapa to the nondepleting anti-CD4 antibody enhanced aTreg-cell induction in vitro (Fig. 1). The treatment with TGF-β+RA or Rapa increased the frequency of CD4+CD25+Foxp3+ Treg cells compared with that of untreated cultures or cultures only treated with the aCD4. We could detect an average percentage of over 60% of aTreg cells in cultures treated with aCD4+TGF-β+RA or aCD4+Rapa (Fig. 1A) within the CD25+ population.