Abstract Diffuse midline glioma (DMG) remains among the most lethal paediatric malignancies, characterised by the H3K27M onco-histone mutation and profound resistance to current therapies. Despite remarkable progress in cancer immunotherapy, the antigenic determinants capable of eliciting effective T-cell responses in DMG remain undefined. Here, we delineate the first comprehensive map of H3K27M-derived and H3K27M-induced antigens, spanning canonical and noncanonical sources, and demonstrate their therapeutic tractability across multiple HLA contexts. Using isogenic H3K27M mutant–wild-type DMG cell line pairs, we demonstrated that the onco-histone fundamentally reshapes the HLA class I ligandome. Through an integrated multi-omics and functional pipeline combining deep immunopeptidomics, predictive modelling, and T-cell functional assays we identified and validated five naturally presented, immunogenic H3.3K27M neoepitopes restricted by three distinct HLA supertypes. These neoantigens were confirmed in patient tumours by PRM-targeted mass spectrometry and elicited potent cytotoxicity when targeted by cloned H3K27M-specific TCRs in co-culture assays. To chart the broader antigenic landscape, we profiled 22 patient-derived DMG lines and 19 primary tumours, integrating fractionated data-dependent and data-independent acquisition immunopeptidomics. By filtering tumour ligandomes against benign brain and reference HLA datasets and using a custom proteogenomic database incorporating Ribo-seq-defined noncanonical ORFs, we uncovered a rich layer of cryptic antigens arising from H3K27M-driven chromatin dysregulation. Remarkably, 18% of these tumour-exclusive ligands were shared across samples, indicating recurrent, non-mutational vulnerabilities exploitable by “off-the-shelf” immunotherapies. Functional testing confirmed that selected dark-proteome-derived peptides robustly activated T cells in an HLA-restricted manner. Together, this work defines the first integrated antigenic atlas of DMG, encompassing both mutation-derived and epigenetically induced antigens. It establishes a direct mechanistic link between H3K27M-mediated chromatin remodelling and tumour-specific antigen presentation providing an actionable foundation for next-generation TCR-T and vaccine therapies in a cancer long deemed immunologically silent.
Shamekhi et al. (2026) studied this question.