Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling and proteomic profiling of human B-LCL cell lines (TAP1/2 +/+ and TAP1/2 -/-) to analyse HLA peptide repertoire

ABSTRACT: The TAP transporter is responsible for transferring cytosolic peptides into the ER where they can be loaded onto MHC molecules. Deletion of TAP results in a drastic reduction of MHC surface expression and alters the presented peptide pattern. Using the TAP deficient cell line LCL721.174 and its TAP expressing progenitor cell line LCL721.45, we have identified and quantified more than 160 HLA ligands, 50 out of which were presented TAP independently. Peptides which were predominantly presented on the TAP deficient LCL721.174 cell line had a decreased MHC binding affinity according to their SYFPEITHI and BIMAS score. About half of the identified TAP independently presented peptides were not derived from signal sequences and may partly be generated by the proteasome. Furthermore, we have excluded that different HLA presentation ratios were due to varying expression of the respective protein or due to changes in the antigen loading complex. Features of TAP-independently presented peptides as well as proteasomal contribution to their generation provides an insight into basic immunological mechanisms. Experiment Overall Design: Two B-LCL cell lines (TAP1/2 +/+ and TAP1/2 -/-) were compared in their gene and protein expression as well as in their HLA peptide repertoire

ORGANISM(S): Homo sapiens

SUBMITTER: Andreas Oliver Weinzierl

PROVIDER: E-GEOD-9437 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The majority of peptides presented by MHC class I results from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides for MHC class I loading non-canonically. We used an im-munopeptidomics workflow combined with a prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect proteasomal processing of immune epitopes presented by A549 cells. Treatment of A549 cells with IFNγ enhanced proteasomal cleavage probability of MHC class I ligands for both, the constitutive proteasome and the im-munoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated HLA allotype-specific proteasomal cleavage probabili-ties for MHC class I ligands, peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. Peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases. The mass spectrometry raw files used in this study were originally produced for a previous publication, where they were utilized to address the influence of cigarette smoke on the antiviral T-cell immune response. Here, we have reanalyzed the data to investigate processing of MHC class I ligands by c20S or i20S, respectively. Original publication: Chen, J.; Wang, X.; Schmalen, A.; Haines, S.; Wolff, M.; Ma, H.; Zhang, H.; Stoleriu, M.G.; Nowak, J.; Nakayama, M.; et al. Antiviral CD8(+) T cell immune responses are impaired by cigarette smoke and in COPD. Eur Respir J 2023, doi:10.1183/13993003.01374-2022.

Dataset Information

Transcription profiling and proteomic profiling of human B-LCL cell lines (TAP1/2 +/+ and TAP1/2 -/-) to analyse HLA peptide repertoire

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