Fig 2: Dectin-1-activated DCs enhance Th9 cell differentiation in vitro.Naive CD4+ T cells from spleens of mice (n=3–5) were cocultured with DCs matured with TNF-a/IL-1ß (BMDC), Curlan (CurDC) or Scleroglucan (SclDC) in the presence of anti-CD3 with (Th9) or without (Th0) addition of Th9-polarizing cytokines TGF-ß and IL-4 for 3 days. Culture supernatants and CD4+ T cells separated by the magnetic cell sorting (MACS) were collected for analysis. (a) Cells were stained with anti-CD4 and anti-IL-9 antibodies and subjected to flow cytometry analysis. Numbers in the dot plots represent the percentages of CD4+IL-9+ T cells. Right, summarized results of four independent experiments obtained as at left. (b) ELISA assessed the IL-9 secretion in the cocultures. (c,d) qPCR analysis of Th9-, Th1-, Th2- and Th17-related cytokines (c) and transcription factors (d). Expression was normalized to Gapdh and set at 1 in BMDC-induced Th9 cells. Results shown are the mean±s.d. of 3–5 independent experiments. *P<0.05; **P<0.01 (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+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 4: Areg promotes EGFR-mediated Th9 cell differentiation.a Naïve CD4+ T cells from WT mice were differentiated into Th0 and Th9 followed by qPCR analysis and ELISA for Areg. Data are representative of mean ± SEM from three independent experiments. b–d Naïve CD4+ T cells from WT mice were differentiated into Th0 and Th9 with or without 100 ng/ml Areg followed by b qPCR analysis of Il9 expression. c FACS staining for IL-9 and IL-17 and ELISA for IL-9. d qPCR for Spi1, Irf4, and Egfr expression. Data are representative of mean ± SEM from three independent experiments. e Naïve CD4+ T cells from WT mice were differentiated under Th9 polarizing conditions with or without 10 ng/ml IL-33 or 10 ng/ml TSLP respectively for 3 days followed by qPCR analysis of Areg expression. Data are representative of mean ± SEM from three independent experiments. f Naïve CD4+ T cells from WT mice were differentiated into Th9 in the presence of isotype antibody (Ab) or anti-Areg Ab followed by qPCR analysis of Il9, Egfr expression and ELISA for IL-9. Data are representative of mean ± SEM from three independent experiments. g Sorted naïve human CD4+ T cells were differentiated into Th9 cells with or without 100 ng/ml Areg. qPCR analysis of Il9 and Egfr expression and FACS for IL-9. Data are representative of mean ± SEM from three healthy individuals. h–l Naïve CD4+ T cells from WT and Areg-/- mice were differentiated under Th9 polarizing conditions. h Heat-map of significantly differentially expressed genes in Th9 cells from WT and Areg-/- mice after RNA-Seq analysis. i Heat-map for RNA-Seq analysis of selected significantly differentially expressed genes in Th9 cells from WT and Areg-/- mice. j, k qPCR analysis of Il9, Spi1, Batf, Egfr, and Il33r expression. l ELISA for IL-9 and flow cytometry analysis of intracellular staining for IL-9. Data are representative of mean ± SEM from three independent experiments. m Naïve CD4+ T cells from WT and Egfrflox/floxXCd4-cre mice were differentiated under Th9 polarizing conditions with or without 100 ng/ml Areg for 3 days; qPCR analysis of Il9 expression and ELISA for IL-9. Data are representative of mean ± SEM from three independent experiments. a ****P < 0.0001, *P = 0.04, using two-tailed unpaired Student’s t test. b *P = 0.02, using one-way ANOVA followed by Tukey’s multiple comparison test. c *P = 0.02 using one-way ANOVA followed by Tukey’s multiple comparison test. d ****P < 0.0001, ***P = 0.0003, using two-tailed unpaired Student’s t test. e *P = 0.01, using one-way ANOVA followed by Tukey’s multiple comparison test. f *P = 0.01, P = ns (not significant), **P = 0.005, using two-tailed unpaired Student’s t test. g **P = 0.0044, ***P = 0.0006, using two-tailed unpaired Student’s t test. j, k ****P < 0.0001, *P = 0.02, P = ns (not significant), using two-tailed unpaired Student’s t test. l *P = 0.04, using two-tailed unpaired Student’s t test. m *P = 0.01, **P = 0.001, ***P = 0.0006, using two-way ANOVA followed by Tukey’s multiple comparison test.

Fig 5: Schematic representation of EGFR-HIF1a signaling pathway in Th9 cells.TGF-ß1 and IL-4 initiates the “differentiation” of naïve CD4+ T cells into Th9 cells, which expresses EGFR and produces EGFR ligand, Areg. Areg “amplifies” Th9 cell differentiation via activating EGFR in a feed-forward loop. Upon activation, EGFR triggers downstream “signaling” through HIF1a which transactivates Il9 and Nos2 promoters resulting in enhanced IL-9 induction. Succinate, a TCA cycle metabolite, and nitric oxide (NO) further stabilizes HIF1a potentiating Th9 cell differentiation and anti-tumor effector functions.