ATRX loss in IDH1 mutant glioma cells suppresses T cells and induces immune-suppressive M2 macrophages. A. IDH1^R132H/p53_mut+/-ATRX_loss U373 cells were co-cultured with activated CD8⁺ T cells (10:1) and CD8 T-cell apoptosis measured by caspase 3/7 activation. B. IDH1^R132H/p53_mut+/−ATRX_loss U373 cells were co-cultured with activated CD8⁺ T cells (1:5) for 24 h, remaining viable adherent cells shown in phase-contrast photomicrographs (left panel) and quantified by CCK-8 assay (right panel). C. IDH1^R132H/p53_mut+/−ATRX_loss U373 cells were treated +/−TMZ for 48 h prior to culture with activated Jurkat cells +/−anti-PD-L1 antibody and Jurkat cell apoptosis measured by caspase 3/7 activation (left panel). IL2 production as a marker of Jurkat cell activation was measured by ELISA (right panel). D. Activated, CFSE-labeled PBMC-derived CD8⁺ T cells were co-cultured with macrophages pretreated with conditioned medium from ATRX_wt or ATRX_loss cells and T-cell proliferation quantified by flow cytometry (left panel). Histogram shows CD8⁺ T-cell numbers (right panel). E. Expression levels of CD8A and CD68 in clinical ATRX_mut vs. ATRX_wt TCGA-IDHmut-non-1p19q co-deletion astrocytoma datasets (http://gliovis.bioinfo.cnio.es). F. TIMER datasets showing infiltration of CD8⁺ T cells and macrophages in ATRX_mut vs. ATRX_wt glioma samples (http://cistrome.org/TIMER). *P < .05, **P < .01, and ***P < .001.