Endogenous IL-2 from T cells stimulated by CD80/CD86^+/+ DCs is required for the differentiation of Foxp3⁺CD25⁺CD4⁺ T cells. (A) As in Figure 2, but Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured with spleen CD11c⁺ DCs with peptide plus TGF-β in the presence or absence of blocking anti–IL-2 Ab or control Abs (20 μg/mL). (B) Foxp3⁻CD25⁻CD4⁺ T cells from DO11.10 RAG^−/− mice were cultured with spleen CD11c⁺ DCs (2 × 10⁴) or CD11c⁻ APCs (2 × 10⁵) with indicated dose of peptide in the presence or absence of TGF-β. After day 5, culture supernatants were collected, and the concentration of IL-2 measured by Luminex. (C) CD4⁺ T cells from Foxp3-IRES-RFP knock-in OVA OTII CD4 transgenic mice (FIR-OTII) were stained with anti-CD4 and CD25 Abs, and Foxp3⁻CD25⁻CD4⁺ T cells were purified by flow cytometry (left). The purity of Foxp3⁻CD25⁻CD4⁺ T cells after sorting was higher than 99.5% (right; gated on CD4⁺ T cells. (D) The Foxp3⁻CD25⁻CD4⁺ T cells (2 × 10⁴) from FIR-OTII mice were cultured with spleen CD11c⁺ DCs (2 × 10⁴) for 5 days in the presence of peptide (0.03 μg/mL) with or without TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). Cells were gated on CD4⁺ T cells. The absolute numbers of Foxp3⁺CD4⁺T cells per culture from 4 different experiments with spleen DCs or immature BM-DCs are shown. P value is provided by Student t test. (E) As in panel D, but spleen CD11c⁺ DCs or immature BM-DCs were prepared from CD80/CD86^−/− or wild-type (WT) mice in the presence of peptide with or without TGF-β (2 ng/mL) and/or IL-2 (100 U/mL). Cells were gated on CD4⁺ T cells. The absolute numbers of Foxp3⁺CD4⁺ T cells per culture from 4 different experiments with spleen DCs or immature BM-DCs are shown. P value is provided by Student t test. Data are representative of 2 to 4 independent experiments.