Dataset Information

High-throughput MS-based immunopeptidomics

ABSTRACT: Comprehensive knowledge of the HLA class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their extraction for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential extraction of HLA-I and -II peptides from up to 96 samples in a plate format from cell lines or tissues. Our methodology drastically reduces sample-handling and can be completed within six hours. We report identification and quantification of thousands of peptides with excellent reproducibility (Pearson correlations reaching 0.98 and 0.97, class I and II, respectively). Due to improved sensitivity, as many as 1,846 HLA-I and 2,633 HLA-II peptides were accurately identified from only 107 B-cells. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma. This straightforward method is applicable for basic and clinical applications.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, T Cell

DISEASE(S): Meningioma

SUBMITTER: Michal Bassani-Sternberg

LAB HEAD: Michal Bassani-Sternberg

PROVIDER: PXD006939 | Pride | 2017-12-15

REPOSITORIES: Pride

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Publications

High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome.

Chong Chloe C Marino Fabio F Pak HuiSong H Racle Julien J Daniel Roy T RT Müller Markus M Gfeller David D Coukos George G Bassani-Sternberg Michal M

Molecular & cellular proteomics : MCP 20171214 3

Comprehensive knowledge of the human leukocyte antigen (HLA) class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their purification for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides from up to 96 samples in a plate format, suitable for bot ...[more]

PMID: 29242379

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Project description:Background: Patient derived organoids (PDOs) can be established from colorectal cancers as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFN) or MEK-inhibitors. Methods: Four PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analysed by MS. Results: We identified an average of 9,936 unique peptides per PDO which compares favourably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. Treatment of four PDOs with IFN upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFN-inducible genes. HLA class II presented peptides increased on average 16-fold with IFN treatment. MEK-inhibitor treatment showed no consistent effect on class I or II HLA expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. Conclusions: Only 3 out of 612 non-synonymous mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

Dataset Information

High-throughput MS-based immunopeptidomics

Publications

High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome.

Similar Datasets

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