H3K27M diffuse midline gliomas (DMGs) are aggressive paediatric brain tumours with a dismal prognosis, for which radiotherapy remains the cornerstone treatment. Despite transient responses, re-irradiation can offer modest benefit with minimal added toxicity, underscoring the continued reliance on radiation in this setting. Beyond its cytotoxic effects, emerging evidence shows that radiation can elicit immunostimulatory responses, including the upregulation of MHC class I molecules and enhancement of antigen presentation.
This study aims to profile radiation-induced changes at the proteomic and immunopeptidomic levels in H3K27M DMGs, and identify radiation-stable HLA-I peptides as potential targets for immune-based therapies. Three H3K27M paediatric patient-derived cell lines (a radiation-naïve biopsy line and two autopsy-derived lines) were irradiated at two doses: 5Gy and 10Gy. Control and irradiated cells were harvested 72 hours post-treatment to assess HLA-I surface expression via flow cytometry. Cell pellets in triplicate (3e^7 and 2e^6) were snap-frozen for HLA-I immunopeptidomics and proteomic analysis. Using our lab’s SAPrIm protocol, HLA class I molecules were isolated. Both analyses were performed on an Orbitrap Astral mass spectrometer (Thermo Fisher) via data-independent acquisition. IFN-γ–treated cells served as internal control.
We confirmed the induction of HLA class I expression in all lines following radiation treatment, with a significant 1.5- to 1.8-fold increase in HLA-I surface expression. Immunopeptidomic analysis revealed that two of the three DMG cell lines exhibited an increase in HLA class I peptide presentation following irradiation indicating an increase in antigen presentation post-irradiation. Across all lines, 10,000 to 35,000 HLA-I peptides were detected with more than 45% of peptides shared between all conditions, indicating a substantial core immunopeptidome despite treatment-specific variations. Irradiation-exclusive peptides were predominantly derived from source proteins involved in DNA repair, cell cycle regulation, and oxidative stress response, suggesting that radiation exposure selectively alters antigen presentation from key stress-related pathways. Notably, some peptides detected in control & IFN-γ–treated conditions were absent post-irradiation, highlighting the need to identify potential immunogenic peptides discovered that can be affected by radiation treatment. In-depth proteomic analysis of more than 10,000 proteins identified revealed that irradiated cells formed distinct clusters from both control and IFN-γ-treated groups within each cell line reflecting underlying reflecting underlying cell line-specific characteristics, such as TP53 mutation status and prior radiation exposure history. Our study demonstrates that radiation enhances HLA-I expression and antigen presentation in paediatric H3K2M DMGs, uncovering both stable and stress-responsive peptides with potential for therapeutic targeting.