HLA Class II Presentation Is Specifically Altered at Elevated Temperatures in the B-Lymphoblastic Cell Line JY

Demmers, Laura C.; Wu, Wei; Heck, Albert J. R.

doi:10.1016/j.mcpro.2021.100089

Cited by 6 publications

(8 citation statements)

References 72 publications

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“…5 ). Demmers 41 . used DDA on an Orbitrap Fusion Lumos Tribrid with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation, and a 90 min LC gradient.…”

Section: Resultsmentioning

confidence: 99%

Thunder-DDA-PASEF enables high-coverage immunopeptidomics and is boosted by MS2Rescore with MS2PIP timsTOF fragmentation prediction model

Gomez-Zepeda,

Arnold-Schild,

Beyrle

et al. 2024

Nat Commun

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Human leukocyte antigen (HLA) class I peptide ligands (HLAIps) are key targets for developing vaccines and immunotherapies against infectious pathogens or cancer cells. Identifying HLAIps is challenging due to their high diversity, low abundance, and patient individuality. Here, we develop a highly sensitive method for identifying HLAIps using liquid chromatography-ion mobility-tandem mass spectrometry (LC-IMS-MS/MS). In addition, we train a timsTOF-specific peak intensity MS2PIP model for tryptic and non-tryptic peptides and implement it in MS2Rescore (v3) together with the CCS predictor from ionmob. The optimized method, Thunder-DDA-PASEF, semi-selectively fragments singly and multiply charged HLAIps based on their IMS and m/z. Moreover, the method employs the high sensitivity mode and extended IMS resolution with fewer MS/MS frames (300 ms TIMS ramp, 3 MS/MS frames), doubling the coverage of immunopeptidomics analyses, compared to the proteomics-tailored DDA-PASEF (100 ms TIMS ramp, 10 MS/MS frames). Additionally, rescoring boosts the HLAIps identification by 41.7% to 33%, resulting in 5738 HLAIps from as little as one million JY cell equivalents, and 14,516 HLAIps from 20 million. This enables in-depth profiling of HLAIps from diverse human cell lines and human plasma. Finally, profiling JY and Raji cells transfected to express the SARS-CoV-2 spike protein results in 16 spike HLAIps, thirteen of which have been reported to elicit immune responses in human patients.

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“…5 ). Demmers 41 . used DDA on an Orbitrap Fusion Lumos Tribrid with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation, and a 90 min LC gradient.…”

Section: Resultsmentioning

confidence: 99%

Thunder-DDA-PASEF enables high-coverage immunopeptidomics and is boosted by MS2Rescore with MS2PIP timsTOF fragmentation prediction model

Gomez-Zepeda,

Arnold-Schild,

Beyrle

et al. 2024

Nat Commun

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show abstract

“…To expand the results obtained in the current study to other HLA-DR and -DP allotypes as well as the HLA-DQ locus, the analysis of the 5856 parental proteins containing HLA class II ligands previously reported − was carried out. Up to 86% of our parental protein data set was present in the pool of these studies.…”

Section: Resultsmentioning

confidence: 99%

Abundance, Betweenness Centrality, Hydrophobicity, and Isoelectric Points Are Relevant Factors in the Processing of Parental Proteins of the HLA Class II Ligandome

et al. 2021

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Adaptive cellular and humoral immune responses to infectious agents require previous recognition of pathogenic peptides bound to human leukocyte antigen (HLA) class II molecules exposed on the surface of the professional antigen-presenting cells. Knowledge of how these peptide ligands are generated is essential to understand the basis for CD4+ T-cell-mediated immunity and tolerance. In this study, a high-throughput mass spectrometry analysis was used to identify more than 16,000 cell peptides bound to several HLA-DR and -DP class II molecules isolated from large amounts of uninfected and virus-infected human cells (ProteomeXchange accession: PXD028006). The analysis of the 1808 parental proteins containing HLA class II ligands revealed that these cell proteins were more acidic, abundant, and highly connected but less hydrophilic than non-parental proteomes. Therefore, the percentage of acidic residues was increased and hydroxyl and polar residues were decreased in the parental proteins for the HLA class II ligandomes versus the non-parental proteomes. This definition of the properties shared by parental proteins that constitute the source of the HLA class II ligandomes can serve as the basis for the development of bioinformatics tools to predict proteins that are most likely recognized by the immune system through the CD4+ helper T lymphocytes in both autoimmunity and infection.

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“…The B-lymphoblastoid cell line JY, homozygote in class I alleles (HLA-A*02:01, HLA-B*07:02, HLA-C*07:02) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin. HLA class I peptides were retrieved via immunoaffinity purification, as described previously ( 24 , 27 ). Specifically, HLA class I complexes were immunoprecipitated using the pan-HLA class I mouse monoclonal IgG2a antibody W6/32 ( 28 ), and antigen peptides were separated from HLA molecules by elution with 10% (v/v) acetic acid, and filtration over a 10-kDa molecular weight cutoff membrane (Merck Millipore).…”

Section: Methodsmentioning

confidence: 99%

“…Here, we first revisited the ligandome of the JY B-lymphoblastoid cell line, a model cell line used by several groups active in immunopeptidomics including ours (27,(33)(34)(35), as it has relative high expression of HLA complexes, and is homozygote in its HLA class I alleles, harbouring just HLA-A*02:01, HLA-B*07:02, and HLA-C*07:02 alleles. We have previously shown that several thousand of HLA peptides can be identified when analyzing JY cells.…”

Section: Triggered Ms Detection Of O-glcnacylated Hla Peptide Ligandsmentioning

confidence: 99%

The HLA Ligandome Comprises a Limited Repertoire of O-GlcNAcylated Antigens Preferentially Associated With HLA-B*07:02

et al. 2021

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Mass-spectrometry based immunopeptidomics has provided unprecedented insights into antigen presentation, not only charting an enormous ligandome of self-antigens, but also cancer neoantigens and peptide antigens harbouring post-translational modifications. Here we concentrate on the latter, focusing on the small subset of HLA Class I peptides (less than 1%) that has been observed to be post-translationally modified (PTM) by a O-linked N-acetylglucosamine (GlcNAc). Just like neoantigens these modified antigens may have specific immunomodulatory functions. Here we compiled from literature, and a new dataset originating from the JY B cell lymphoblastoid cell line, a concise albeit comprehensive list of O-GlcNAcylated HLA class I peptides. This cumulative list of O-GlcNAcylated HLA peptides were derived from normal and cancerous origin, as well as tissue specimen. Remarkably, the overlap in detected O-GlcNAcylated HLA peptides as well as their source proteins is strikingly high. Most of the O-GlcNAcylated HLA peptides originate from nuclear proteins, notably transcription factors. From this list, we extract that O-GlcNAcylated HLA Class I peptides are preferentially presented by the HLA-B*07:02 allele. This allele loads peptides with a Proline residue anchor at position 2, and features a binding groove that can accommodate well the recently proposed consensus sequence for O-GlcNAcylation, P(V/A/T/S)g(S/T), essentially explaining why HLA-B*07:02 is a favoured binding allele. The observations drawn from the compiled list, may assist in the prediction of novel O-GlcNAcylated HLA antigens, which will be best presented by patients harbouring HLA-B*07:02 or related alleles that use Proline as anchoring residue.

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HLA Class II Presentation Is Specifically Altered at Elevated Temperatures in the B-Lymphoblastic Cell Line JY

Cited by 6 publications

References 72 publications

Thunder-DDA-PASEF enables high-coverage immunopeptidomics and is boosted by MS2Rescore with MS2PIP timsTOF fragmentation prediction model

Thunder-DDA-PASEF enables high-coverage immunopeptidomics and is boosted by MS2Rescore with MS2PIP timsTOF fragmentation prediction model

Abundance, Betweenness Centrality, Hydrophobicity, and Isoelectric Points Are Relevant Factors in the Processing of Parental Proteins of the HLA Class II Ligandome

The HLA Ligandome Comprises a Limited Repertoire of O-GlcNAcylated Antigens Preferentially Associated With HLA-B*07:02

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