Fig. 5: 6-phosphogluconate (6PG) is a functional metabolite, directing M-MDSC suppression by inducing IRS-1^S307 phosphorylation and IRS1-PI3K-AKT signaling.

A MDSCs were generated in vitro using IL-6 (40 ng/mL) and GM-CSF (40 ng/mL) from bone marrow (BM) of Pgd^fl/fl and Pgd^fl/flLysM^Cre mice or wild type (WT) mice with 6AN (5 µM) or vehicle. Sorted MDSCs on day 4 were analyzed for 1318 signaling target proteins by Phospho-Explorer Antibody Array (Full moon BioSystem). B Oxidative PPP checkpoints were blocked by shRNA in WT BM-derived MDSCs. Phospho-IRS-1^S307 levels were examined on sorted M-MDSCs. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. C 6PG (10, 100, 1000 µM) was added to MDSC cell free lysates for 30 min and IRS-1^S307 phosphorylation measured by western blot. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. D, E Immunoprecipitated JNK1 and IRS-1 (substrate) were incubated with increasing 6PG concentrations (10, 100, 1000 µM) in an in vitro kinase assay (D). In the same experiment, JNK1-IRS-1 binding was evaluated by co-immunoprecipitation (Co-IP) using anti-JNK1 (E). Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. F Serine to Alanine (S307A) site-specific mutation in IRS-1 was conducted in M-MDSCs (generated as in (A)). Flowcytometry-sorted M-MDSCs were co-cultured with anti-CD3/anti-CD28 activated T cells (CFSE-labeled) at a 1:8 (M-MDSC: T cell) ratio. T cell proliferation was evaluated at 72 h. n = 3; One-way ANOVA. G, H M-MDSCs were generated as in (A, F). Mitochondrial ROS (mROS) production (G) and Arginase 1 expression (H) was examined on M-MDSC by flow cytometry. n = 3; One-way ANOVA. I Sorted M-MDSCs were prepared as in (A, F). Levels of PI3K (top), AKT (middle) and Drp1^S616 (bottom) phosphorylation were determined by western blot analysis. Blots were processed parallel to samples from the same experiment. Result is representative of two repeats. J–L MDSCs were generated as in (A, F), with N-Acetylcysteine (NAC: 0.5 mM) or vehicle (PBS). mROS production in M-MDSCs (J), suppressive capacity in co-culture with T cells (K) and Arginase expression levels (L) determined. n = 3; One-way ANOVA. All data are shown as mean ± SEM.