DC-induced, differentiating, peripheral Foxp3⁺CD25⁺CD4⁺ T cells undergo substantial proliferative activity. (A) As in Figure 2, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice (2 × 10⁴) were CFSE labeled and cultured with CD11c⁺ DCs (2 × 10⁴) from spleen with or without OVA peptide (0.03 μg/mL) in the presence or absence of TGF-β. At day 3 or day 6, cells were stained with anti-CD4 and KJ1.26 clonotype Abs. After fixation, cells were stained with anti-Foxp3 or isotype control mAb. Foxp3 expression and CFSE dilution were shown gated on CFSE⁺CD4⁺KJ1.26 clonotype⁺ T cells. indicates proliferating cells expressed Foxp3. (B) As in panel A, but CFSE-labeled DO11.10 RAG^−/− Foxp3⁻CD25⁻CD4⁺ T cells were cultured with spleen CD11c⁺ DCs (2 × 10⁴) or DC-depleted spleen cells (CD11c⁻ APC, 2 × 10⁵) at the indicated dose of peptide in the presence of TGF-β (2 ng/mL). Foxp3 expression and CFSE dilution were analyzed at day 6. Data are representative of 2 independent experiments.