Fig 2: 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+IFN?+ and CD8+IFN?+. f TILs were isolated from the tumor followed by FACS analysis of intracellular staining for CD8+IFN?+and CD4+IFN?+ 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 3: Succinate enhances HIF1a-mediated Th9 cell differentiation and anti-tumor immunity.a–e Naïve CD4+ T cells from WT and Hif1akd mice were differentiated under Th9 polarizing conditions. Samples were prepared and subjected to metabolomics. Heat-maps showing a global distribution and quantification of metabolites in the cell extracts; b differentially expressed metabolites of glycolytic pathway; c differentially expressed currency metabolites in cell extract; d footprinting quantification of differentially expressed metabolites in the cell culture supernatants, and e differentially expressed metabolites of TCA cycle in the cell extracts. f Naïve CD4+ T cells from WT mice were differentiated to Th0 and Th9 with or without 1.0 mM aKG, followed by NanoString analysis of mRNA expression in Th9 and Th9 + aKG conditions. Fold change in relative expression relative to control as determined by log2 (Th9 + aKG/Th9 WT). g–i Naïve CD4+ T cells from WT mice were differentiated to Th0 and Th9 with or without 1.0 mM aKG followed by g qPCR analysis of Hif1a and Il9 expression. Data are representative of mean ± SEM from three independent experiments. h FACS analysis of intracellular IL-9 and IL-17 staining, and i mRNA expression of Egln1. Data are representative of mean ± SEM from three independent experiments. j–l Naïve CD4+ T cells from WT mice were differentiated into Th9 cells with or without 5.0 mM succinate followed by j qPCR analysis of Il9, Spi1, Irf4, Irf1, and Gata3 expression. k FACS analysis of intracellular IL-9 and IL-17 production and ELISA for IL-9. l qPCR analysis of Hif1a and Egln1 mRNA expression. Data are representative of mean ± SEM from three independent experiments. m Naïve CD4+ T cells from OT-II TCR transgenic mice were differentiated into Th9 cells with or without 5.0 mM succinate. At day 4, cells were adoptively transferred into B16-OVA tumor-bearing WT mice. Mean tumor volume was measured over time. Data represent one of the three experiments with three independently analyzed mice/group (n = 3 mice per group). g *P = 0.009, using one-way ANOVA followed by Tukey’s multiple comparison test. i **P = 0.004, using two-tailed unpaired Student’s t test. j **P = 0.0027, ***P = 0.0002, ****P < 0.0001, *P = 0.011, using two-tailed unpaired Student’s t test. k. P = ns (not significant), using one-way ANOVA followed by Tukey’s multiple comparison test. l **P = 0.0046, ****P < 0.0001, ***P = 0.0001, using one-way ANOVA followed by Tukey’s multiple comparison test. m ****P < 0.0001, *P = 0.033, using two-way ANOVA followed by Tukey’s multiple comparison test.

Fig 4: NO and HIF1a 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 Hif1a expression. Data are representative of mean ± SEM from three independent experiments. d Bioinformatics analysis of HIF1a binding motif in Nos2 promoter followed by ChIP analysis of HIF1a binding to Nos2 promoter in Th9 cells represented as enrichment of HIF1a 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 HIF1a 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 Hif1akd 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 5: ROR?t phosphorylation regulates colonic inflammation(A) Schematic diagram of Th17 cell-induced colitis model (n = 5 animals per group).(B) Body weight change in percentage.(C) FOB score.(D) Diarrhea score.(E) Spleen and colon length.(F) Colon weight-to-length ratio.(G) Representative colonoscopic images.(H) Colonoscopic score.(I) Representative H&E images.(J) Histology score.(K) Real-time PCR analysis of Il17a, Il17f, and Rorc.(L and M) Flow cytometric analysis of IL-17 and IFN-? by adoptively transferred Th17 cells.(N) Lysates from cLPLs were analyzed by immunoblotting with anti-ROR?t and anti-actin antibodies.Data are representative of two independent experiments (n = 5). Dots indicate individual mice. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. N.S., not significant.