This is highly dose-dependent. At a concentration of 5 μg/mL anti-CD4 mAb IFN-γ production was nearly completely abolished. Our combined treatment of anti-CD4 mAb (1μg/mL)+TGF-β+RA reduced the frequency of IFN-γ-producing cells to the same level as the high anti-CD4 mAb

treatment (Supporting Information Fig. 3). However, as stated earlier, anti-CD4 mAb monotherapy using such a high concentration resulted in a dramatically reduced yield of CD4+CD25+Foxp3+ cells as compared to the combined treatment with a lower anti-CD4 mAb concentration. Thus, the combined treatment was superior, as it not only allows generation of Foxp3+ cells but also inhibits differentiation of IFN-γ-producing

Foxp3– effector T cells. Next, we analysed the cytokine profile of aTreg cells upon restimulation with allogeneic CD19+ B cells. Surprisingly, only aCD4+Rapa GPCR Compound Library chemical structure aTreg cells transiently secreted IFN-γ on day 1 after restimulation (Fig. 2B). We could not detect significant differences in the release of IL-17 between the different aTreg-cell populations. CD25+ T cells from aCD4+TGF-β+RA-treated cultures showed reduced TNF-α secretion compared to aTreg cells from all other cultures. To characterise the function of our generated aTreg cells, an in vitro suppression assay was performed. Purified CD4+CD25+ cells from all cultures were able to Ulixertinib concentration suppress proliferation of co-cultured T effector cells even at low aTreg to T effector cell ratios (Fig. 2C). However, aCD4-mAb+TGF-β+RA aTreg cells showed the highest potential. We also assessed specificity of the suppressive capacity of our generated aTreg cells. Therefore, purified CD4+CD25+ T cells were co-cultured with T effector cells and stimulated with either BALB/c (H-2d, cognate alloantigen) or

cytometric bead array (CBA) (H-2k, third party alloantigen) CD19+ B cells. Similar to the proliferation assay, CD4+CD25+ cells purified from all cultures were able to suppress IFN-γ expression by T effector cells stimulated with BALB/c B cells. Again, aTreg cells from aCD4+TGF-β+RA-treated cultures could do that most efficiently up to very low aTreg to T effector ratios (90% inhibition). Although aTreg cells harvested 2-hydroxyphytanoyl-CoA lyase from aCD4+TGF-β+RA-treated cultures could suppress differentiation of IFN-γ-producing responder cells at an aTreg to T effector cells ratio of 1:2 when stimulated with CBA B cells (90% inhibition), the suppressive capacity was dramatically reduced at a lower aTreg to T effector cell ratio (only 50% inhibition) (Fig. 2D). Thus, aTreg cells generated in aCD4+TGF-β+RA-treated cultures show high suppressive capacity in a predominantly antigen-specific manner. In order to test whether our culture conditions primarily favour the expansion of nTreg cells, we performed the cultures using purified CD4+CD25− cells.