TGF-β converts naive CD4⁺CD25⁻ T cells to CD4⁺ CD25⁺ anergic/suppressor T cells. (A) Schematic for the experiment. Freshly isolated B6 CD4⁺CD25⁻ T cells were stimulated with anti-CD3 and APCs in the absence and presence of TGF-β for 1 wk. Viable CD4⁺ T cells were stained with FITC–anti-CD25 mAb, and four populations of cells (control 25⁻, control 25⁺, TGF-β 25⁻, and TGF-β 25⁺) were sorted by FACStar^Plus™ Cell Sorter. (B) The individual populations (5 × 10⁴) were restimulated with anti-CD3 and APCs (2 × 10⁵) to monitor their proliferative response. The data are shown as mean of duplicate wells of ³H incorporation (CPM). (C) 1.5 × 10⁴ freshly isolated CD4⁺CD25⁻ responder T cells were stimulated with anti-CD3 and APCs in the absence (naive CD25⁻ alone) or presence of the four individual populations of cells (5 × 10⁴) to examine their suppressive ability for naive responder T cell activation. The data are representative of three experiments.