α-Mannosylated HLA-II glycopeptide antigens dominate the immunopeptidome of immortalised cells and tumour tissues.

Immunopeptides are cell surface-located protein fragments that aid our immune system to recognise and respond to pathogenic insult and malignant transformation. In this two-part communication, we firstly summarise and reflect on our recent discovery documenting that MHC-II-bound immunopeptides from immortalised cell lines prevalently carry N-glycans that differ from the cellular glycoproteome (Goodson, Front Immunol, 2023). These findings are important as immunopeptide glycosylation remains poorly understood in immunosurveillance. The study also opened up new technical and biological questions that we address in the second part of this communication. Our study highlighted that the performance of the search engines used to detect glycosylated immunopeptides from LC-MS/MS data remains untested and, importantly, that little biochemical in vivo evidence is available to document the nature of glycopeptide antigens in tumour tissues. To this end, we compared the N-glycosylated MHC-II-bound immunopeptides that were reported from tumour tissues of 14 meningioma patients in the MSFragger-HLA-Glyco database (Bedran, Nat Commun, 2023) to those we identified with the commercial Byonic software. Encouragingly, the search engines produced similar outputs supporting that N-glycosylated MHC-II-bound immunopeptides are prevalent in meningioma tumour tissues. Consistent also with in vitro findings, the tissue-derived MHC-II-bound immunopeptides were found to predominantly carry hyper-processed (paucimannosidic- and chitobiose core-type) and hypo-processed (oligomannosidic-type) N-glycans that varied in prevalence and distribution between patients. Taken together, evidence is emerging suggesting that α-mannosidic glycoepitopes abundantly decorate MHC-II-bound immunopeptides presented in both immortalised cells and tumour tissues warranting further research into their functional roles in immunosurveillance.