Fig 2: PHD2 and Hypoxia-mediated HIF1α stabilization and IL-9 induction in Th cells.a, b Naïve CD4+ T cells from WT and Phd2kd mice were differentiated under Th9 polarizing conditions with daily treatment of 1.0 μg/ml Dox for 3 days. a qPCR analysis of Hif1α, Spi1, Irf1, Batf and Il9 expression. Data are representative of mean ± SEM from three independent experiments. b ELISA and FACS for IL-9 production. Data are representative of mean ± SEM from three independent experiments. c Pathway analysis depicting the enrichment of pathways associated with HIF1α in WT Th9 cells. d–f Naïve CD4+ T cells from WT mice were differentiated into Th9 under normoxic (21% oxygen) or hypoxic (1.0% oxygen) conditions for 3 days. d qPCR analysis of Il9, Hif1α, Spi1, Gata3, Irf4, and Irf1 expression. Data are representative of mean ± SEM from three independent experiments. e FACS staining for HIF1α. f IL-9 production by flow cytometry and ELISA. Data are representative of mean ± SEM from three independent experiments. a, b ***P = 0.0004, *P = 0.04, **P = 0.0018, using two-tailed unpaired Student’s t test. d, f *P = 0.02, **P = 0.007, ****P < 0.0001, using two-tailed unpaired Student’s t test.

Fig 3: NO and HIF1α synergistically triggers IL-9 induction in Th9 cells.a–c Naïve CD4+ T cells from WT and Nos2−/− mice were differentiated to Th9 cells followed by a qPCR analysis of Spi1, Irf4, Gata3 and Batf expression. Data are representative of mean ± SEM from three independent experiments. b FACS staining for IL-9 and IL-17 in Th9 cells. c qPCR analysis of Hif1α expression. Data are representative of mean ± SEM from three independent experiments. d Bioinformatics analysis of HIF1α binding motif in Nos2 promoter followed by ChIP analysis of HIF1α binding to Nos2 promoter in Th9 cells represented as enrichment of HIF1α on Nos2 promoter relative to input. Data are representative of mean ± SEM from three independent experiments. e Luciferase assay for Nos2 promoter activity in the presence of HIF1α plasmid at 0, 50, and 100 ng concentrations. Data are representative of mean ± SEM from three independent experiments. f Naïve CD4+ T cells from WT and Hif1αkd mice were differentiated under Th9 polarizing conditions with daily treatment of 1.0 μg/ml Dox for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. g Naïve CD4+ T cells from WT mice were differentiated into Th9 cells under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. h Naïve CD4+ T cells from WT and Phd2kd mice were differentiated under Th9 polarizing conditions with daily treatment of 1.0 μg/ml Dox for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. a, c ****P < 0.00001, *P = 0.01, using two-tailed unpaired Student’s t test. d **P = 0.0017, using two-tailed unpaired Student’s t test. e ****P < 0.00001, using one-way ANOVA followed by Tukey’s multiple comparison test. f *P = 0.01, using two-tailed unpaired Student’s t test. g *P = 0.04, using two-tailed unpaired Student’s t test. h **P = 0.0026, using two-tailed unpaired Student’s t test.

Fig 4: EGFR inhibition abrogates the anti-tumor functions of Th9 cells.a, b Naïve CD4+ T cells from WT mice were in vitro differentiated under Th9 conditions with or without 1.0 μM gefitinib for 3 days followed by a. qPCR analysis of Il9 expression. b ELISA for IL-9 and flow cytometry analysis of intracellular staining for IL-9 and IL-17. Data are representative of mean ± SEM from three independent experiments. c–f Naïve CD4+ T cells from OT-II TCR transgenic mice were in vitro differentiated into Th9 with or without 1.0 μM gefitinib for 3 days. Cells were then adoptively transferred into B16-OVA tumor-bearing WT mice, randomized into three groups (n = 5 mice per group). c Mean tumor volume was measured over time shown as tumor growth curve. d, e Spleen and tumor draining lymph nodes (dLN) were harvested and single cell suspensions were made followed by FACS analysis of intracellular staining for CD4+IFNγ+ and CD8+IFNγ+. f TILs were isolated from the tumor followed by FACS analysis of intracellular staining for CD8+IFNγ+and CD4+IFNγ+ cell populations. Data are representative of mean ± SEM from three independent experiments. g, h Sorted naïve human CD4+ T cells were differentiated into Th9 cells with or without 1.0 μM gefitinib. g mRNA expression of Egfr and Il9 was determined by qPCR. Data are representative of mean ± SEM from three healthy individuals. h Intracellular staining for IL-9. a, b ***P = 0.0003, *P = 0.017, using two-tailed unpaired Student’s t test. c ****P < 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison test. d–f ****P < 0.0001, **P = 0.001, ***P = 0.0001, *P = 0.02, using one-way ANOVA followed by Tukey’s multiple comparison test. g *P = 0.014, using two-tailed unpaired Student’s t test.

Fig 5: EGFR pathway is essential for Th9 cell differentiation.a Naïve CD4+ T cells from WT mice were in vitro differentiated into Th0 and Th9 followed by RNA-Seq and ingenuity pathway analysis. b qPCR analysis of Egfr expression. Data are representative of mean ± SEM from three independent experiments. c, d Naïve CD4+ T cells from WT and Egfrflox/floxXCd4-cre mice were in vitro differentiated under Th0 and Th9 polarizing conditions for 3 days followed by c. NanoString analysis. Fold change in relative expression relative to control was determined by log2 (Th9 Egfrflox/floxXCd4-cre/Th9 WT). d qPCR analysis of Il9 expression and ELISA for IL-9 production. Data are representative of mean ± SEM from three independent experiments. e, f Naïve CD4+ T cells from WT mice were in vitro differentiated under Th9 polarizing conditions with or without 10 ng/ml IL-33 or 10 ng/ml TSLP for 3 days respectively; e qPCR analysis of Il9, Egfr expression and f ELISA for IL-9. Data are representative of mean ± SEM from three independent experiments. g Naïve CD4+ T cells from WT and Nos2−/− mice were differentiated under Th9 polarizing conditions followed by qPCR analysis of Il9 and Egfr expression and ELISA for IL-9. Data are representative of mean ± SEM from three independent experiments. h Naïve CD4+ T cells from WT and Egfrflox/floxXCd4-cre mice were differentiated under Th9 polarizing conditions with or without 10 ng/ml IL-33 for 3 days; qPCR analysis of Il9 expression and ELISA for IL-9. Data are representative of mean ± SEM from three independent experiments. i Naïve CD4+ T cells from WT and Egfrflox/floxXCd4-cre mice were differentiated to Th9 followed by qPCR analysis of Il33r expression. Data are representative of mean ± SEM from three independent experiments. b **P = 0.007, using two-tailed unpaired Student’s t test. d *P = 0.033, ****P < 0.00001, ***P = 0.0007, using two-way ANOVA followed by Tukey’s multiple comparison test. e, f *P = 0.04, **P = 0.002, ****P < 0.0001, using one-way ANOVA followed by Tukey’s multiple comparison test. g **P = 0.003, ***P = 0.0009, *P = 0.01, using two-tailed unpaired Student’s t test. h *P = 0.05, ***P = 0.0009, **P = 0.001, ****P = 0.0001, using two-way ANOVA followed by Tukey’s multiple comparison test. i P = ns (not significant), using two-tailed unpaired Student’s t test.