Fig. 3

AKT-Foxo1 axis regulates the induction of IL-9 in Th9 cells. a–d Naive CD4⁺CD62L⁺CD44⁻ T cells were isolated from Wt mice and then treated under Th9 (TGF-β1 + IL-4) or Th0 (without skewing cytokines) conditions with/without LY294002 (5–10 μM) as indicated. a At 72 h, cells were restimulated, then intracellular expression of IL-9 and IFN-γ was determined by flow cytometry. b At 72 h, ELISA and qPCR for IL-9 was performed. c, d qPCR was performed for determining mRNA expression of Foxo1, Irf4, Spi1, Batf, Irf1 and Klf2. Contour plot is representative of two independent experiments. Bar shows mean + s.d. from combined three b–d. **P < 0.01 ***P < 0.001, ****P < 0.0001 (Two-way variance analysis (analysis of variance) and Student’s t-test). e Balb/c mice were immunized with ova and treated with LY294002, 6–8 days later, spleen and LN cells were restimulated by OVA peptide (n = 7), % of IL-9⁺CD4⁺ T cells and IL-9 concentration were determined by flow cytometry and ELISA. Mean + s.e.m. **P < 0.01 (Student’s t-test). f–h, Naive CD4⁺ T cells were differentiated into Th9 cells and then retrovirally transduced with GFP empty virus or AKT-DN-GFP (dominant-negative AKT) or CA-AKT-GFP (constitutive active AKT). After 72 h, cells were restimulated and then intracellular expression of IL-9 and IL-17 was determined by flow cytometry gated on GFP⁺ cells f. mRNA expression of Il9 was quantified by quantitative PCR (qPCR) f. g percentage of GFP⁺IL-9⁺ CD4⁺T cells, data representative of two different experiments. h mRNA expression of Il9, Foxo1, Irf4, Batf, Irf1 mRNA in GFP sorted retrovirally transduced Th9 cells. i Naive CD4⁺T cells were stimulated in Th9 conditions and transfected with Scr siRNA or Foxo1 siRNA with and without LY294002 (5 μM). Il9, Foxo1, Pu.1, Batf, Irf4, Irf1 mRNA was assessed by qPCR. j Naive CD4⁺T cells were differentiated into Th0 and Th9 with and without LY294002 (5.0 μM) and Foxo1 inhibitor (25 nM), Il9 mRNA was quantified by qPCR and IL-9 concentration in culture supernatants was estimated by ELISA. Bars show mean ± s.d. from combined three i, j, two f, h experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test)