BM-DCs plus TGF-β induce differentiation of Foxp3⁺T regs from DO11.10 RAG−/− Foxp3⁻CD25⁺CD4⁺ T cells. (A) Day-6 BM-DCs were stimulated with or without 50 ng/mL LPS for 16 hours. After washing, 6 × 10³ immature (without LPS) or mature DCs (with LPS) were cultured with Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from DO11.10 RAG^−/− mice for 5 days with peptide in the presence or absence of TGF-β (2 ng/mL). Cells were stained with mAbs to CD4, KJ1.26 clonotype, and CD25-Abs. After fixation, cells were stained with anti-Foxp3 mAb. Plots were gated on CD4⁺ KJ1.26⁺ cells. (B) As in panel A, but the indicated numbers of DCs were cultured with Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from DO11 RAG^−/− mice for 5 days with the indicated doses of peptide in the presence or absence of TGF-β (2 ng/mL). The absolute numbers of Foxp3⁺CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days are shown. (C) As in panel A, but absolute numbers of CD4⁺KJ1.26 clonotype⁺ T cells per culture at 5 days are shown. (D) As in panel A, but freshly isolated CD25⁺CD4⁺T cells from DO11.10 RAG^+/+ mice (2 × 10⁴, > 90% Foxp3+) were cultured with immature or mature BM-DCs plus 0.1 μg/mL peptide in the presence or absence of TGF-β (2 ng/mL) or IL-2 (100 U/mL). (E) As in panel D, but freshly isolated CD25⁺CD4⁺T cells from DO11.10 RAG^+/+ mice were cultured with immature or mature BM-DCs (2 × 10⁴). Geometric mean fluorescence intensity (MFI) for Foxp3 within the circle is shown in the bottom of the plots. Data are representative from 2 to 3 independent experiments.