Figure 5

FFO administration promotes the differentiation of CD4⁺CD25⁺Foxp3⁺Treg. (A) Naïve mice were given 100 mg/kg FFO or NFO in their drinking water, daily, for 20 days. The mice were sacrificed on day 32. (B) Splenocytes were isolated from the spleens of each group and 1.0 × 10⁶ cells/mL were permeabilized with Foxp3 fixation/permeabilization buffer and stained with anti-Foxp3-FITC and (C) anti-CD25-PE and anti-Foxp3-FITC. The Foxp3 and CD25⁺Foxp3⁺ Treg population was analyzed by FACS. Data are from five mice per group. (D) The expression of TGF-β and IL-10 mRNA in the spleen was measured by real-time PCR. (E) Splenocytes (1.0 × 10⁶ cells/mL) were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (0.5 μg/mL). After 3 days, the cells were permeabilized with Foxp3 fixation/permeabilization buffer and stained with anti-Foxp3-FITC. (F) The cells from panel (E) were stained with anti-CD4-FITC and anti-CD25-PE. (G) CD4⁺ T cells were isolated from splenocytes using CD4⁺ T cell isolation beads. The CD4⁺ T cells (1.0 × 10⁶ cells/mL) were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (0.5 μg/mL) for 3 days, after which they were permeabilized with Foxp3 fixation/permeabilization buffer and stained with anti-Foxp3-FITC and anti-CD25-PE. All data (panels A-G except for D) are from FACS analysis. Data are representative of five mice per group. In panel (D), the values are mean ± S.D (n = 5 mice per group). *P < 0.05; and **P < 0.005 compared to mice treated with NFO.