Fig 2: Fenofibrate regulated the gene expression of Th1, Th17, and Treg cell-related transcription factors and cytokines in LGs. (A) qRT-PCR analysis of T-bet, ROR?t, and Foxp3 mRNA levels. (B) IFN-?, IL-17, and TGF-ß1 mRNA levels. Data are representative of three independent experiments. Data are presented as mean ± SD (n = 3–6, Student's t-test, **P < 0.01, ***P < 0.001).

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: ATF3 regulates IL-6-pSTAT3 signaling in intestinal Th17 cells. Flow cytometry analysis of phospho-STAT3 in (A) IL-6 or IL-22 stimulated freshly isolated ileum crypts or IL-6-stimulated peripheral blood mononuclear cell (PBMC) from wild-type mice, or in (B) IL-6-stimulated PBMC from wild-type and ATF3-deficient mice. (C) Flow cytometry analysis of intracellular IL-17A and IL-22 expression in naïve lamina propria T cells from the indicated mice. Cells were treated with PMA, ionomycin and IL-23 in the presence of BFA for 4 h before analysis and gated on live CD45+EpCAM-Lin-CD3+ population as shown. (D) Quantitative real-time PCR analysis of IL-17A and IL-22 mRNA levels in freshly isolated lamina propria (LPL) cells, mesenteric lymph nodes (mLN), or splenocytes. (E) Model of ATF3-mediated mucosal immunity via cross-regulation between IL-22-pSTAT3 signaling in epithelium (associated with AMP production and epithelial fucosylation) and IL-6-pSTAT3 signaling in Th17 cells (associated with signature IL-17A and IL-22 production). “n” refers to the number of mice analyzed. Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig 5: 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.