ID #875 Radiation-Induced Remodeling of the HLA-I Immunopeptidome in Paediatric H3K27M Diffuse Midline Glioma

Abstract

H3K27M diffuse midline gliomas (DMGs) are devastating paediatric brain tumours for which radiotherapy (RT) remains the primary treatment modality. Although responses are transient, re-irradiation can provide modest benefit, underscoring the continued clinical reliance on RT. As most recurrent DMGs arise after prior RT, they frequently exhibit acquired radioresistance, raising questions about how radiation-adapted tumour cells modulate immune-relevant pathways. Beyond cytotoxicity, RT can induce immunostimulatory effects, including upregulation of HLA class I (HLA-I) expression and antigen presentation. However, how radiation reshapes the HLA-I immunopeptidome in H3K27M DMGs remains largely unexplored.

In vitro RT responses were assessed in three paediatric H3.3K27M DMG cell lines (SF7761, SU-DIPG19, SU-DIPG27), comprising one radiation-naïve line and two autopsy-derived, previously irradiated.

Cells were irradiated at 5 Gy or 10 Gy and harvested 72 hours post-treatment. HLA-I surface expression was measured by flow cytometry. Triplicate pellets were processed for HLA-I immunopeptidomics and global proteomics. IFN-γ–treated cells served as an internal control.

Radiation induced a 1.5–1.8-fold increase in HLA-I surface expression across all lines. Across conditions, 10,000–35,000 HLA-I peptides were detected, with >45% shared, indicating a substantial core immunopeptidome that persists despite treatment. Both shared peptides with increased intensity post-radiation and radiation-exclusive peptides were enriched for source proteins involved in DNA repair, cell-cycle regulation, and oxidative stress responses, demonstrating that the immunopeptidome of irradiated cells reflects key features of the radiation-induced stress state. For example, in SF7761, radiation-responsive proteins such as TIGAR, TP53I3 and CDKN1A showed concordant increases in both peptide presentation and protein abundance, illustrating that radiation imprints a stress-associated antigenic signature detectable across the proteome and immunopeptidome. Overall, global proteomic profiling (>10,000 proteins) showed that irradiated cells formed distinct clusters separate from untreated and IFN-γ–treated groups, reflecting cell-line–specific features such as TP53 status and prior RT exposure that shape radiation-responsive antigen presentation.