Figure 1. BCL6 Is a Negative Regulator of Th9 Cells.

(A) BCL6 is transiently downregulated in polarized Th9 cells. Immunoblot of BCL6 in Th9 cells polarized for 0–72 hours in the absence of exogenous recombinant IL-2. β-actin was used as internal control for protein loading. (B) BCL6 forced expression reduced IL-9 expression. Naïve CD4⁺ T cells were prepared from the spleens of WT mice and cultures were transduced with BCL6-RV-GFP encoding for BCL6-GFP. Cells were differentiated under Th9 cell conditions for 4 days followed by intracellular cytokine staining and BCL6-GFP-positive cells were gated on the basis of the GFP expression. (C) BCL6 knockdown in Th9 cells up-regulates IL-9 production. Naïve CD4⁺ T cells were transfected with siRNA specific for BCL6 or with scrambled (Scr) siRNA using Amaxa followed by polarization under Th9 cell conditions for 4 days. Cytokine analysis of IL-5, IL-9, IL-17 and IFNγ expression in the supernatants was assessed by Luminex. (D) A representative gene expression of Il9, Bcl6 and Stat5a in Th9 cells that were transfected with siRNA specific for BCL6 is shown. Transfection with scrambled siRNA was used as a control. (E) IL-9 production in Th9 cells. Th9 cells were differentiated for 3 days followed by transfection with siRNA specific for BCL6 or with scrambled siRNA. IL-9 release in the culture supernatants was measured by Luminex. Data are representative of three experiments with similar results. **p < 0.005.