Extended Data Fig. 2: AOA chemically reacted with keto-acids.

(a-b) Targeted metabolomics analysis of WT, AOA (250 µM) treated WT or GOT1^−/− T_H17 cells. (a) Principal component analysis (PCA) plot. (b) Heatmap showing top 30 differentially expressed metabolites. (c-d) WT and GOT1^−/− naive CD4⁺ T cells were differentiated into T_H17 cells. (c) MS measurements of aspartate and glutamate levels. ***P = 0.0004, **P = 0.0012. (d) Pyrosequencing analysis of Foxp3 CNS2 region CpG methylation status. (e) WT naive CD4⁺ T cells were differentiated into T_H17 cells without treatment (NT), or with AOA (250 µM) or AOA together with 2 mM dimethyl-ketoglutarate (DMK). Flow cytometry analysis of IL17A production upon PMA/Ionomycin stimulation and FOXP3 expression were shown. Representative flow plots of IL17A (top) and FOXP3 (bottom) on the left and statistical analysis on the right. ****P < 0.0001, ***P = 0.0003. (f-i) AOA (300 μM) was left alone or co-incubated with (f) pyruvate (Pyr), (g) oxaloacetate (OAA), (h) α-ketobutyrate (αKB) or (i) dimethyl-ketoglutarate (DMK) at indicated concentrations in PBS overnight. MS analysis of AOA and adducts formed between AOA and keto-acids were shown (left). Statistical analysis (right). ****P < 0.0001, ***P = 0.0003 (AOA vs. AOA + 100 µM DMK, AOA group), ns = 0.2885 (AOA vs. AOA + 100 µM DMK, AOA∙DMK group), ***P = 0.0001 (AOA vs. AOA + 200 µM DMK, AOA∙DMK group). (j) RNA sequencing analysis of WT and GOT1^−/− T_H17 cells. Gene set enrichment analysis (GSEA) plots of glycolysis/gluconeogenesis (top), Oxidative Phosphorylation (middle) and fatty acid metabolism (bottom) genes. Normalized enrichment scores in the gene set were shown. Unpaired t test, two-tailed (c), two-way ANOVA with Sidak’s multiple comparisons test (e), one-way ANOVA with Dunnett’s multiple comparisons test (f-i). Mean ± s.e.m. values were shown. Representative data from n = 3 (a-c, e-i) or n = 2 (d) or n = 1 (j) independent experiment(s) with similar results.