H3K27M Mutation Modulates the Antigenic Landscape in Diffuse Midline Glioma

Background/Objectives: Diffuse midline gliomas (DMGs) are among the most aggressive paediatric brain tumours, with the pathognomonic H3K27M mutation present in over 80% of cases. This mutation drives epigenetic dysregulation and transcriptional reprogramming, yet its impact on the tumour antigenic landscape remains poorly understood. Given the low mutational burden of DMG, an expanded search beyond neoantigens to include epigenetically dysregulated tumour-associated antigens (TAAs) is critical for advancing antigen-specific immunotherapies. Methods: We performed immunopeptidomic analysis of two patient-derived H3K27M-mutant DMG cell lines (SU-DIPG13 and BT245) and their isogenic counterparts in which only the mutant H3F3A allele was disrupted by CRISPR editing (H3K27M-KO), leaving wild-type histone H3.3 intact. High-resolution mass spectrometry and bioinformatics were used to define H3K27M-associated changes in the immunopeptidome, and functional T cell assays with HLA-matched healthy donor PBMCs assessed peptide immunogenicity. Results: H3K27M modulated the tumour antigenic landscape in a model-specific manner. Loss of the mutant allele increased HLA-I expression in SU-DIPG13 but not BT245, while immunopeptidomic profiling revealed distinct shifts in tumour-associated peptide presentation in both models, indicating that HLA-I abundance alone does not explain these changes. We identified six peptides derived from SLITRK2, PRAME, XKR5 and CBX2 that elicited T-cell responses in vitro; T cells raised against five of these preferentially recognised the parental H3K27M-mutant DMG cells. Several prioritised peptides were detected only in parental H3K27M-mutant cells, supporting mutation-associated, antigen-selective differences in peptide presentation and tumour antigenicity. Conclusions: This study shows that H3K27M modulates the antigenic landscape in DMG in a model-dependent, antigen-selective manner, uncovering immunogenic tumour-associated peptides that could serve as targets for precision immunotherapy and establishing a foundation for T cell-based therapies in this otherwise treatment-refractory disease.