Fig. 5: Enzymatic vulnerability of monocyte-derived macrophages enables R-2-HG-driven kynurenine production.
From: Tryptophan metabolism drives dynamic immunosuppressive myeloid states in IDH-mutant gliomas
a,b, l-Trp import and degradation dynamics in immune cell subsets exposed to R-2-HG. Intracellular LC–MS/MS measurements of l-Trp and l-Kyn in T cells and monocyte-derived macrophages exposed to 20 mM R-2-HG and varying doses of l-Trp. Top, linear regression of dose-dependent metabolite accumulation. Bottom, quantification of intracellular l-Trp and l-Kyn levels and the [l-Kyn][l-Trp]−1 ratio. s.e.m is shown as error bands (top) or bars (bottom). n = 3 independent healthy donors. s.e.m. is projected as error bands. Statistical significance was determined by one-way ANOVA in combination with Tukey’s test. c–e, Cell-free assay of IDO1, IDO2 and TDO2 enzymatic activities with increasing concentrations of synthetic R-2-HG. A nonlinear regression model (log (R-2-HG) versus response) is shown. Regression curves are representative of y = 0 + (0.16 − 0)(1 + 10(log(EC50)−x))−1 (EC50, half-maximum effective concentration). Produced l-Kyn levels were measured by absorbance at 321 nm (A321). Gray boxes indicate the concentration range of R-2-HG detected in patient glioma tissues12. n = 2 independent measurements for IDO1, n = 4 independent measurements for IDO2, n = 4 independent measurements for TDO2. f, Expression of Tdo2 in different murine immune cell subsets. RNA-seq data were derived from ref. 27. Tn, naive T cell; Teff, effector T cell; Tem, effector memory T cell; Tgd, γδ T cell; NK, natural killer cell (NK); Mo, monocyte; MG, microglia cell. g, Monocyte-derived macrophage–T cell 3H-dT proliferation assay. Monocyte-derived macrophages from n = 3 Tdo2+/+ and n = 3 Tdo2−/− mice were pretreated with 10 mM R-2-HG or vehicle; 10 ng ml−1 anti-IL-10 and anti-TGF-β antibodies (αIL-10/TGF-β) or an isotype (iso) control; 10 ng ml−1 anti-PD-L1 antibody or isotype control; or 10 mM l-Kyn or vehicle as indicated for 24 h and co-cultured with stimulated C57BL6/J T cells for 24 h. Statistical significance was determined by one-way ANOVA in combination with Tukey’s test. If not mentioned otherwise, all data are represented as mean ± s.e.m.
