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Figure 5: DC subsets additively inhibit the generation of Tregs after α-GC stimulation in vitro. (a) Naive CD4+CD62L+ T cells were isolated from B6 WT mice. These cells (1 × 105 cells/well) were co-cultured in Treg-polarizing conditions with DCs (2 × 104 cells/well) from either PBS- or α-GC-injected mice. In the upper panel, the percentage of iTreg cells among the total CD4+ T cells in culture was evaluated by flow cytometry at the indicated time points. In the lower panel, the frequencies of CD25+Foxp3+ iTreg cells on day 5 are shown (Mean values ± SD, , , ). (b) Naive CD4+CD62L+ T cells were cultured for 5 days under Treg-polarizing conditions with DCs from either PBS- or α-GC-injected mice. LPS injection was performed as a positive control. Neutralizing mAbs specific to IFN-γ (5 μg/mL), IL-12 (5 μg/mL), or TNF-α (5 μg/mL) were added during the culture. The percentage of CD25+Foxp3+ iTreg cells was evaluated by flow cytometry. These data are representative of two independent experiments. (c) Naive CD4+CD62L+ T cells were cultured for 5 days in Treg-polarizing conditions in the presence of either NK1.1+ DCs (NKDCs) or NK1.1 conventional DCs (DCs) from either PBS- or α-GC-injected mice. The percentage of CD25+Foxp3+ iTreg cells was evaluated by flow cytometry. The mean values ± SD are shown ( ). (d) CD11c-DTR Tg recipient mice were i.p. injected with DT (120 ng/mouse) to remove the CD11c+ DC population. One day later, 1.5 × 106 DCs (either total DCs or NKDC-depleted DCs) from WT mice were i.v. transferred to DT-treated CD11c-DTR Tg recipient mice. Subsequently, 1 hour later, α-GC was administered i.p. into both groups of mice. Three days later, splenocytes prepared from both groups were stained with mAbs for flow cytometry. The upper panels are dot plots of iTregs, and the middle panels are histograms of GITR expression on the iTreg population. The lower panels show the percentages of CD25+Foxp3+ iTreg population among CD3+CD4+ T cells. The mean values ± SD are shown ( ).