Mass Spectrometry of Human Leukocyte Antigen Class I Peptidomes Reveals Strong Effects of Protein Abundance and Turnover on Antigen Presentation

Abstract

HLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins – those with at least fivefold higher density score than expected for their abundance – we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use.

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…By integrating exome sequencing, MHC peptide ligand prediction algorithms and targeted MS, Gubin et al recently identified tumor-specific mutant MHC class I peptides as targets of this form of immunotherapy (88). Proteogenomics approaches (139, 140) using DDA MS were also recently used for the identification of mutant as well as polymorphic MHC class I peptides (5,51,(141)(142)(143)(144). Notably, the detection of tumor-specific mutant MHC peptides has not been achieved yet using DIA MS.…”

“…1, middle panel). In fact, this method can be used for the isolation of both class I and class II MHC peptides from a range of biological sources such as cell lines, primary tissues and plasma (36,(51)(52)(53). Moreover, up to ϳ90% of immunopurified peptides were reported to be specific for the MHC molecules (51,54).…”

“…We also highlight selected biological applications and discuss important current technical limitations that need to be solved to accelerate the development of this field. The immunopeptidome is referred to as the collection of peptides associated with and presented by major histocompatibility complex (MHC) molecules (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). MHC-associated peptides are recognized by T lymphocytes that are in turn activated to eliminate abnormal cells such as pathogen-infected and cancer cells.…”

Minimal Information About an Immuno‐Peptidomics Experiment (MIAIPE)

“…In fact, MS/MS spectrum generation and acquisition is even more important in immunopeptidomics as compared to other sample types due to the very diverse physical chemical properties of these endogenous peptides 25. Thanks to tremendous technological development in the MS/MS field over the last decade, large‐scale MHC‐class I and MHC‐class II immunopeptidome analyses have taken off in recent years,6, 26, 27 thus drastically increasing the availability of immunopeptidome datasets.…”

T cell immunotherapy in hepatitis B Virus related hepatocellular carcinoma

“…Ideally, this can be achieved using TCR-like antibodies specific for every HLA/HBV-epitope complex [43,44] but the diversity of complexes makes this approach unfeasible [45] . Peptide elution and mass spectrometry strategies [46] might seem possible, but such techniques is highly specialized and complex, and at the moment restricted primarily to academic research and not clinical application. As a compromise, the detection of HBV proteins, DNA or mRNA would suffice with the assumption that antigen processing and epitope presentation occurs as expected.…”