DC-induced Foxp3⁺ T cells are suppressive in vitro. (A) Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured for 7 days with CD11c⁺ spleen DCs (2 × 10⁴), peptide (0.03 μg/mL), and TGF-β (2 ng/mL). Cells were stained with anti-CD4, CD25, and Foxp3 Abs. (B) As in panel A, but live cells were stained with anti-CD4 and CD25 Abs for sorting. The square indicates the gate for sorting. (C) Sorted cells from (B) were fixed and further stained with anti-Foxp3 Ab. The purity of sorted natural CD25⁺CD4⁺ T regs was also shown. (D) CD25⁻CD4⁺ responder T cells (2 × 10⁴) from DO11.10 mice were CFSE-labeled and stimulated with spleen APCs (10⁵) and anti-CD3 mAb. To these, the induced Foxp3⁺ T regs purified as in panel C were added in graded numbers. After 3 days, CFSE dilution was analyzed with flow cytometry. Dead cells were gated out by TOPRO-3 iodide. Data are representative of 2 independent experiments.