Foxp3⁺CD25⁺CD4⁺ T cells are induced from Foxp3⁻CD25⁻CD4⁺ T cells more effectively by spleen CD11c⁺ DCs than CD11c⁻ APCs, and DCs from mesenteric lymph nodes differentiate Foxp3⁺T reg in the absence of exogenous TGF-β. (A) Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from DO11.10 RAG^−/− mice were cultured for 5 days with CD11c⁺ DCs (2 × 10⁴) or CD11c⁻ cells (2 × 10⁵) from spleen and indicated doses of OVA peptide in the presence or absence of TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). Cells were stained with mAbs to CD4, clonotype (KJ1.26), CD25, and Foxp3. Plots were gated on CD4⁺ KJ1.26⁺ cells. (B) As in panel A, but absolute numbers of Foxp3⁺CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days were shown. (C) As in panel A, but the absolute numbers of CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days were shown. (D) As in panel A, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured for 5 days at 0.03 μg/mL peptide with CD11c⁺ DCs (2 × 10⁴) from mesenteric or skin draining lymph nodes in the presence or absence of TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). (E) As in panel D, but anti–TGF-β mAb (10 μg/mL) or isotype control was added into the culture. Data are representative of 2 to 4 independent experiments.