Despite the clinical success of programmed cell death protein 1 (PD-1) checkpoint blockade in many cancer types, its efficacy in glioblastoma remains notably limited, underscoring the critical need to uncover primary resistance mechanisms and identify synergistic therapeutic targets. To discover genes modulating αPD-1 immunotherapy response, we performed an in vivo CRISPR-Cas9 screen in immunocompetent mice bearing glioblastoma, which prioritized the glutamine (Gln) transporter SLC38A5 as a candidate modulator of αPD-1 immunotherapy efficacy. In human glioblastoma, SLC38A5 was significantly upregulated compared with normal brain tissue. Ablation of SLC38A5 did not impair glioblastoma cell intrinsic growth but profoundly sensitized glioblastoma to αPD-1 therapy in a CD8+ T cell-dependent manner, leading to enhanced anti-tumor immunity and tumor control. Mechanistically, SLC38A5 deficiency created a Gln-enriched tumor microenvironment by impairing Gln uptake of glioblastoma cells. This metabolic rewiring enhanced CD8+ T cell function via SLC1A5-dependent Gln utilization and promoted MHC-I-mediated antigen presentation in glioblastoma cells through a Gln-glutathione (GSH)-reactive oxygen species (ROS) axis. Furthermore, an SLC38A5-targeting nanobody was developed that efficiently accumulated in orthotopic glioblastoma and potentiated αPD-1 therapy to suppress brain tumor growth in vivo. Overall, this study establishes SLC38A5 as a metabolic immune regulator in glioblastoma and presents a promising Nb-based strategy for overcoming αPD-1 immunotherapy resistance.
