Figure 3.

Butyrate sustains cytokine production by differentiated Th9 cells. Naïve CD4+ T cells (CD62L+CD44low) were sorted from spleens of FOXP3 GFP mice and cultured in 96-well flat bottom plates with plate bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of Th9 (TGF-β 2 ng/ml, IL-4 10 ng/ml, and anti-IFNγ 10 μg/ml) differentiation condition for 4 days. At day 4, the medium was replaced by fresh R-10 and butyrate (250 μM) was added in the culture. At day 8, cells were restimulated with with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (5 μg/ml) for 4 h at 37°C and 5% CO₂, and analyzed by flow cytometry (A). The frequencies of FOXP3+ and/or IL-9+ CD4+ T cells (B), FOXP3+ and/or IL-10+ CD4+ T cells (C) and IL-9+ and/or IL-10+ CD4+ T cells (D) are represented by bar graphs. Data are shown as mean ± S.D. Two-way ANOVA and Sidak's multiple comparison test were used for statistical analysis. NS, not significant.