Fig. 7: Succinate enhances HIF1α-mediated Th9 cell differentiation and anti-tumor immunity.

a–e Naïve CD4⁺ T cells from WT and Hif1α^kd 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 αKG, followed by NanoString analysis of mRNA expression in Th9 and Th9 + αKG conditions. Fold change in relative expression relative to control as determined by log₂ (Th9 + αKG/Th9 WT). g–i Naïve CD4⁺ T cells from WT mice were differentiated to Th0 and Th9 with or without 1.0 mM αKG followed by g qPCR analysis of Hif1α 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 Hif1α 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.