EXTH-52. Spatially Resolved Antigenic Landscapes In Low-grade Glioma Reveal Targets For Personalized Immunotherapy

Abstract

Abstract BACKGROUND Low-grade gliomas (LGGs) exhibit low overall mutational burden and an immunosuppressive microenvironment, contributing to immunotherapy resistance. Intratumoral heterogeneity (ITH) further complicates the targeting of tumor-specific antigens (TSAs), yet its influence on the antigenic and immune landscapes in LGG remains understudied, particularly due to reliance on single-biopsy analyses. METHODS We performed exome and transcriptome sequencing on 70 spatially mapped biopsies from seven IDH-mutant Grade II astrocytoma patients. Tumor purity, immune deconvolution, and neoantigen prediction were conducted to assess regional immune variation and antigenic burden. In one patient (P375), neoantigen-specific CD8+ T cells were isolated using barcoded peptide-HLA multimers. Reactive T-cell receptor (TCR) clonotypes were identified via single-cell TCR sequencing and functionally validated in Jurkat76 and CD8+ T cells. RESULTS Spatial profiling of whole tumors revealed most mutations were biopsy-specific, with a steep drop in mutations shared across multiple regions. RNA-seq–based unsupervised clustering with xCell and DESeq2 revealed two distinct immune microenvironments: immune-hot regions with elevated immune infiltration, and immune-cold regions with minimal immune presence. Notably, mutation-derived n-mer peptides from immune-hot regions showed enhanced predicted immunogenicity relative to those from immune-cold areas. In one patient (P375), neoantigen-specific CD8⁺ TCR clonotypes were identified targeting PRMT5 mutations found in immune-hot regions. A mutant PRMT5-specific TCR demonstrated robust, antigen-dependent activation and dose-dependent cytotoxicity in vitro. This TCR showed high specificity toward the mutant peptide with minimal cross-reactivity to the wild-type sequence. Furthermore, engineered CD8⁺ T cells effectively killed glioma cells endogenously expressing the full-length mutant PRMT5, supporting its candidacy as a viable immunotherapeutic target. CONCLUSIONS Our findings reveal significant 3D spatial and immunologic heterogeneity in low-grade gliomas and demonstrate that neoantigens such as mutant PRMT5 can be selectively targeted by highly specific, cytotoxic TCR-engineered CD8⁺ T cells. These results support the potential of personalized TCR-based immunotherapy for low-grade glioma.