Abstract Background Chimeric antigen receptor (CAR)-T cell therapy remains largely ineffective in glioblastoma (GB), where a highly immunosuppressive microenvironment and tumor heterogeneity impair therapeutic durability. Methods Using a human neocortical brain slice model that preserves the complex GB microenvironment, we profiled interactions between natural killer group 2D (NKG2D) CAR-T cells and tumor ecosystems via PIC-seq, spatial transcriptomics, and gene regulatory network reconstruction. Results CAR-T cells initially suppressed tumor growth but rapidly transitioned to a dysfunctional state marked by exhaustion-associated transcriptional programs. This shift was driven by signaling interactions between CAR-T cells and myeloid cells. Tumor-associated macrophages displayed enhanced phagocytic activity and spatially colocalize with mesenchymal-like GB cells within hypoxic regions. Our gene regulatory network analysis identified MAF and BACH2 as key transcriptional regulators, with MAF promoting CAR CD8 exhaustion and BACH2 preserving CD8 T cells effector function. In silico perturbation confirmed the reciprocal effect of MAF and BACH2 on CD8⁺ T cell fate. Conclusions These findings reveal mechanisms of rapid CAR-T cell dysfunction in GB and identify actionable targets for engineering more durable cellular therapies.
Zhang et al. (Fri,) studied this question.