Fig. 3: Areg promotes EGFR-mediated Th9 cell differentiation.
From: EGFR-HIF1α signaling positively regulates the differentiation of IL-9 producing T helper cells
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.
