Figure 4.

TGF-β and TCR costimulation induces Foxp3 expression in CD4⁺CD25⁻ naive responder T cells. (A) B6 spleen cells were sorted into CD4⁺CD25⁻ (CD25⁻) and T_reg (CD25⁺) populations. cDNA from each population was subjected to nonsaturating PCR using Foxp3 or HPRT-specific primers, and data are presented as Foxp3/HPRT ratio. (B) CD25⁻ cells were cultured with medium or 2 ng/ml TGF-β1 (24 h) or stimulated with platebound anti-CD3 and soluble anti-CD28 in the absence or presence of TGF-β1 (72 h) and assessed for the expression of Foxp3 by RT-PCR. (C) CD25⁻ cells were activated with soluble anti-CD3 and APCs with or without TGF-β for 3 d and assessed for Foxp3 expression. (D) Dose dependence of TGF-β and failure of IL-10 on Foxp3 induction in CD25⁻ naive T cells. CD25⁻ cells were cultured as in B in the presence of indicated concentrations of TGF-β or recombinant murine IL-10. (E) Both TGF-β and IL-10 failed to further enhance Foxp3 expression in T_reg. Freshly isolated T_reg were activated with platebound anti-CD3, soluble anti-CD28, and IL-2 (100 U/ml) with or without TGF-β or IL-10, and Foxp3 expression was assessed by RT-PCR. (F and G) Flow cytometry analysis (F) and Foxp3 expression (G) of CD25⁺ or CD25⁻ T cells sorted from TGF-β– and anti-CD3–costimulated naive CD25⁻ T cells (day 7). (H) No Foxp3 expression in both CD25⁺ and CD25⁻ subsets purified from control (anti-CD3 only) stimulated CD25⁻ naive cells (day 7). The data in the figure are representative of at least three experiments.