Figure 2

Foxo1 is required for Th9 Cell differentiation. (A,B) Naïve CD4⁺ T cells purified from splenocytes of conditional Foxo1 knockout mice (CD4^CreFoxo1^fl/fl) and littermate controls (Foxo1^fl/fl) were differentiated for 4 days under Th0, Th1, Th9 and Th17 cell conditions followed by intracellular cytokine staining (A, left panel) and Luminex analysis of cytokine secretion (B). Four days following Th9 cell polarization, cells were harvested and stimulated with PMA and ionomycin followed by surface staining for CD4 and intracellular staining for IL-9 (x-axis) and IFNγ (y-axis). Average of three experiments from IL-9- and IFNγ-positive T cells in Foxo1−/− and WT Th9 cells are shown (Fig. 2A, right panel). (B) Luminex bead-based cytokine assay of cell-free supernatant collected from Th9 cells 4 days following differentiation. (C) Decrease IRF4 and PU.1 expression in Foxo1−/− Th9 cells. IRF4 and PU.1 (Spi1) mRNA levels in Foxo1−/− and WT Th9 cells measured by Taqman PCR on day 4 after cell differentiation. (D) Effects of IFNγ neutralization on IL-9 expression in Foxo1−/− and WT Th9 cells. Splenic CD4⁺ T cells were isolated from naïve Foxo1−/− and WT mice and were polarized under Th9 cells in the presence or absence of anti-IFNγ neutralizing antibody. IL-9 and IFNγ expression was measured by Luminex assay. Data are representative of three experiments with similar results. **p < 0.01 by Unpaired Student t test; NS: not significant.