Radiotherapy shapes memory T cell dynamics and region-specific PD-1 expression in the glioblastoma microenvironment

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults, where radiation therapy (RT) remains standard care. However, the precise immunomodulatory effects of RT-specifically on T cell checkpoint receptor profiles and memory T cells dynamics -within the tumor microenvironment are not fully elucidated. In this translational study, we utilized an orthotopic patient-derived xenograft (PDX) model, CRT361, engineered with a Luc+ reporter. This system was supported by an HLA-matched peripheral blood mononuclear cell (PBMC) humanization platform, providing a clinically relevant context for immune analysis. CRT361 cells were implanted intracranially into the right hemisphere, and tumors progression was monitored via multimodal imaging, bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). Following successful PBMC engraftment and a single-dose focal irradiation (2 Gy), T cells were harvested from both peripheral blood and distinct brain hemispheres for profiling via spectral flow cytometry. Our results demonstrate that focal irradiation increased the frequency of both CD8 + and CD4 + central memory T cells (T CM ) in both blood and brain. Furthermore, irradiation led to elevated programmed cell death protein 1 (PD-1) expression on both CD8 + and CD4 + T CM and naïve T cells in peripheral blood. Crucially, we observed an inverse response in the central nervous system: CD8 + T CM within the brain exhibited lowerPD-1 expression levels exclusively in the irradiated right hemisphere.These novel findings provide critical insight into the capacity of radiotherapy to dynamically influence T cell phenotypes and checkpoint receptor landscapes in a tissue-, region-, and cell type-specific manner within the GBM microenvironment. This study design and results establish a robust foundation for the rationale design and optimization of combinatorial strategies leveraging both radiation and emerging immunotherapies.