CFU-IL3 suppression by Tregs is contact-dependent and can be blocked with anti–TGF-β mAb. BALB/c Tregs were activated and cocultured with TCD-syngeneic BMCs. In some cultures, Tregs were physically separated from BMCs by use of transwell plates. In contrast to inhibition after direct coculture, suppression was abolished in the transwell cultures. (A) Activated conventional (CD25⁻) CD4 T cells failed to inhibit CFU-IL3 in this assay. (B) Activated Tregs derived from B6-CD8^−/− mice were cocultured with TCD-B6 BMCs in the absence or presence of anti–TGF-β neutralizing antibody. Chicken anti–TGF-β1 mAb at 6-ng/mL (high [H]) and 3-ng/mL (low [L]) concentrations but not chicken IgY isotype control mAb (Iso H) added during the Treg + BMCs coculture period inhibited levels of CFU-IL3. Statistical analyses: BMCs + Treg versus BMCs + Treg + anti-TGF-β (L) = P <.001; BMCs + Treg versus BMCs + Treg + isotype (H) = P > .05. (C) CD4⁺FoxP3⁺ Tregs mediate regulation of CFU-IL3. B6-Foxp3^gfp Tregs were FACS purified to > 99%. Activation of these cells followed by coculture with lineage-depleted (< 0.5% lineage marker–expressing) B6 BMCs resulted in contact-dependent inhibition of CFU-IL3 levels. Data are presented as the average CFU-IL3 number per 5 × 10³ lineage-depleted BMCs either cocultured in contact with Tregs or separated in transwell cultures.