Extended Data Fig. 4: R-2-HG-driven AHR signaling of GAM.
From: Tryptophan metabolism drives dynamic immunosuppressive myeloid states in IDH-mutant gliomas
a, Intracellular measurements of R-2-HG in primary microglia after in vitro incubation with R-2-HG for 24 h. Nonlinear regression is shown. N = 1 healthy donor. b, Intracellular measurements of R-2-HG in human MΦ. Cells were primed for 2 h with 100 μM Candesartan, 100μM N-(p-Amylcinnamoyl)anthranilic acid (NAA) or vehicle only and treated with 20mM R-2-HG for 48 h. N = 4 healthy donors. Data are represented as mean ± SEM. Statistical significance was determined by one-way ANOVA in combination with Tukey’s test. c, DNA-microarray screen of MΦ from n = 8 healthy donors, treated with exogenous R-2-HG in a matched-pair analysis: procedural overview. d, Quantification of AHR signature expression in IDHwt (grey) and IDHmut (blue) LGG (absolute (left) and normalized to CD11b- (ITGAM-) expression (right)). TCGA dataset, N = 286, n = 68 IDHwt LGG, n = 218 IDHmut LGG. e, AHR target gene expression in human monocyte-derived MΦ treated with increasing concentrations of R-2-HG in vitro as determined by RT-PCR. N = 4 healthy donors. Statistical significance was determined by one-way ANOVA in combination with Tukey’s test. f, Expression levels of AHR in transcripts per million (TPM) in human immune cell subsets as analyzed in the Database of Immune Cell Expression (DICE). g, Cytokine ELISA measurements of IL-10 and TGF-β in human monocyte-derived MΦ following incubation with increasing doses of R-2-HG. Statistical significance was determined by two-tailed student’s t test. N = 4 independent healthy donors evaluated over 6 experimental conditions h. Induction of AHR translocation by supernatant of IDH1-R132H-expressing glioma cells (blue). Supernatant of IDH1-wt-overexpressing GL261 glioma cell line as control (grey). N = 3 IDHwt cell lines, n = 3 IDHmut cell lines. Statistical significance was determined by two-tailed student’s t test. i, AHR translocation reporter assay. Titration of R-2-HG and L-Kyn in DRE-GFP-reporter, n = 3 assay runs. Statistical significance was determined by one-way ANOVA in combination with Tukey’s test.
