Fig 2: 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.

Fig 3: 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 4: HIF1α is critical for IL-9 induction in Th cells.a, b Heat-map for RNA-Seq analysis of significantly differentially expressed genes in Th9 cells as compared to Th0 cells. c Naïve CD4+ T cells from WT and Egfrflox/floxXCd4-cre mice were differentiated under Th0 and Th9 polarizing conditions for 3 days followed by qPCR analysis of Hif1α expression. Data are representative of mean ± SEM from three independent experiments. d Naïve CD4+ T cells from WT mice were differentiated under Th9 polarizing conditions with or without 1.0 μM gefitinib for 3 days followed by qPCR analysis of Hif1α expression. Data are representative of mean ± SEM from three independent experiments. e Bioinformatics analysis of HIF1α binding motif in IL-9 promoter. ChIP analysis of HIF1α binding to IL-9 promoter in Th9 cells represented as enrichment of HIF1α on IL-9 promoter relative to input. Data are representative of mean ± SEM from three independent experiments. f Luciferase reporter assay for IL-9 promoter activity in the presence of HIF1α plasmid at 0, 100, and 200 ng concentrations. Data are representative of mean ± SEM from three independent experiments. g, h Naïve CD4+ T cells from WT and Hif1αkd mice were differentiated under Th9, Th2, Th17, iTregs polarizing conditions with daily treatment of 1.0 μg/ml Dox for 3 days followed by qPCR analysis of Il9 expression and ELISA for IL-9 production. Data are representative of mean ± SEM from three independent experiments. i Naïve CD4+ T cells from WT mice were differentiated into Th9 cells with or without 5.0 μM acriflavine (ACF) followed by qPCR analysis of Il9 expression. j Naïve CD4+ T cells from OT-II TCR transgenic mice were differentiated into Th9 cells with or without 5.0 μM acriflavine (ACF). At day 4, cells were adoptively transferred into B16-OVA tumor-bearing WT mice, randomized into three groups. Mean tumor volume was measured over time. Data are representative of mean ± SEM from three independent experiments, (n = 5 mice per group). c ***P = 0.0004, using two-way ANOVA followed by Tukey’s multiple comparison test. d ***P = 0.0006, using two-tailed unpaired Student’s t test. e *P = 0.04, **P = 0.009, ***P = 0.0003, using two-tailed unpaired Student’s t test. f ***P = 0.0009, ****P < 0.0001, using one-way ANOVA followed by Tukey’s multiple comparison test. g, h *P = 0.02, **P = 0.0027, ***P = 0.0002, ****P < 0.0001, P = ns (not significant), using two-tailed unpaired Student’s t test. i **P = 0.01, using two-tailed unpaired Student’s t test. j **P = 0.0014, using two-way ANOVA followed by Tukey’s multiple comparison test.

Fig 5: 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.