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.

Profound -glycan remodelling accompanies MHC-II immunopeptide presentation.

Immunopeptidomics, the study of peptide antigens presented on the cell surface by the major histocompatibility complex (MHC), offers insights into how our immune system recognises self/non-self in health and disease. We recently discovered that hyper-processed (remodelled) -glycans are dominant features decorating viral spike immunopeptides presented via MHC-class II (MHC-II) molecules by dendritic cells pulsed with SARS-CoV-2 spike protein, but it remains unknown if endogenous immunopeptides also undergo -glycan remodelling. Taking a multi-omics approach, we here interrogate published MHC-II immunopeptidomics datasets of cultured monocyte-like (THP-1) and breast cancer-derived (MDA-MB-231) cell lines for overlooked -glycosylated peptide antigens, which we compare to their source proteins in the cellular glycoproteome using proteomics and -glycomics data from matching cell lines.

Immunolyser: A web-based computational pipeline for analysing and mining immunopeptidomic data.

Immunopeptidomics has made tremendous contributions to our understanding of antigen processing and presentation, by identifying and quantifying antigenic peptides presented on the cell surface by Major Histocompatibility Complex (MHC) molecules. Large and complex immunopeptidomics datasets can now be routinely generated using Liquid Chromatography-Mass Spectrometry techniques. The analysis of this data - often consisting of multiple replicates/conditions - rarely follows a standard data processing pipeline, hindering the reproducibility and depth of analysis of immunopeptidomic data.