Fig. 1.

Splenic DCs efficiently induce Foxp3 expression from naïve CD4⁺CD25⁻ T cells. (A) Cell surface expression of CD40, CD86, and MHC class II (I-A^g7) of freshly isolated NOD splenic CD11c⁺ DCs. (B) Foxp3 expression by precultured sorted CD4⁺CD25⁻CD62L⁺ BDC2.5 T cells and induction in T cells after culture with or without 2 ng/ml TGF-β1 on day 6 of culture. Expression of CD62L by precultured CD4⁺CD25⁻ BDC2.5 T cells is also shown. (C) Time-course of induction of CD4⁺CD25⁺Foxp3⁺ BDC2.5 T cells from naïve CD4⁺CD25⁻Foxp3⁻ BDC2.5 T cells in the presence of 2 ng/ml TGF-β1. (Upper) Total number of Foxp3⁺ T cells per well. (Lower) Percentages of Foxp3⁺ T cells determined by intracellular staining on days 2, 3, 4, 5, 6, and 10 of DC-T cultures. The isotype control for day 3 is shown. (D) Quantification of Foxp3 mRNA by real-time RT-PCR. Samples were prepared from enriched CD25⁺ fractions of the resulting T cells from cocultures with or without 2 ng/ml TGF-β1 or freshly isolated CD4⁺CD25⁺ and CD4⁺CD25⁻ BDC2.5 T cells. Values were standardized by 18s RNA and expressed as fold of increase compared with precultured freshly isolated CD4⁺CD25⁻ cells. (E) Dose-response of TGF-β1 determined on day 6 of DC-T cocultures at indicated concentrations of TGF-β1. The isotype control for the 0.01 ng/ml dose is shown. All results are representative of two to four separate experiments.