DC-induced Foxp3⁺T regs have a comparable phenotype to natural T regs. (A) As in Figure 2, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured with CD11c⁺ spleen DCs and 0.03 μg/mL peptide in the presence of TGF-β. These cultured cells (top) or freshly isolated spleen cells from DO11.10 RAG^+/+ mice (bottom) were stained with mAbs to CD4, clonotype (KJ1.26), and the indicated Abs or isotype controls. Plots were gated on CD4⁺ KJ1.26⁺ cells for the induced T regs (top) and on CD4⁺ cells for freshly isolated spleen cells (bottom). (B) Freshly isolated spleen cells from DO11.10 RAG^+/+ mice (non–T regs, natural T regs) or the cultured cells as in panel A but with low peptide (0.03 μg/mL) or high peptide (0.3 μg/mL) were stained with CD4, clonotype (KJ1.26), CD62L, CD103, and Foxp3 Abs. Plots for non-T regs were gated on Foxp3⁻CD25⁻CD4⁺ T cells, and plots for natural T regs were gated on Foxp3⁺CD25⁺CD4⁺ T cells. Plots were gated on Foxp3⁺CD4⁺KJ1.26⁺ cells for cultured cells. Data are representative of 2 to 4 independent experiments.