Activated CD4⁺CD25⁺ Tregs affect inhibition of ex vivo CFU-IL3 activity by BMCs. CD4⁺CD25⁺ T cells (Tregs) were prepared (supplemental Figure 1) from spleen and LNC and activated ex vivo with anti-CD3/CD28 beads and rIL2 for 3 days before coculture with syngeneic TCD-BM. After coculture, cells were harvested and plated for CFU activity with rmIL-3 as described. Data are presented as the mean number of CFU ± SD calculated from triplicate wells. P < .05 was considered statistically significant by analysis of variance (ANOVA). Activated syngeneic CD4⁺CD25⁺ T cells prepared from the spleen and LN of BALB/c (A) and CD8^−/− (B) inhibited CFU-IL3 compared with TCD-BM cocultured alone or supplemented with Ab-coated beads and rmIL2. (C) Freshly isolated CD4⁺CD25⁺ T cells as described in panel A from CD8^−/− spleen cells were cocultured at indicated ratios with 2.5 × 10⁴ syngeneic TCD-BMCs in triplicate microwells in medium supplemented with appropriate growth factors. After 72 hours, cells were collected, washed, and plated for CFU-IL3 as described. No inhibition (P > .05) of CFU-IL3 levels was observed. (D) CD4⁺CD25⁺ T cells from B6-CD8^−/− spleen and LN cells were cocultured with anti-CD3/CD28 beads and rIL-2 or rIL-2 only for 3 days. Cells were washed and cocultured in methylcellulose-based medium with syngeneic TCD-BM. Inhibition was observed only in cultures with stimulating beads plus rIL-2–activated Tregs.