0b0dd019fac77d768c062dad0dfb9566 ijms-26-11357-v2.pdf 30b58af82f1d137d2c2cedc5b8a31e0ba618f1d6 ijms-26-11357-v2.pdf 8d15e53f4328bf31a1ed855efda1ce3ce61c2ef600003dbd4b66d80ef32e8ed5 ijms-26-11357-v2.pdf Title: Specific Instability of HLA-A*03:01 Expression in HEK-293 Cells Subject: HEK-293 is a highly transfectable human cell line widely used as a model for protein expression. Since large amounts of cells are often required for the purification of HLA immunopeptidomes, suspension-growing variants, such as HEK-293F, facilitate the generation of sufficient cell quantities. The HLA class I-typing of these cells is HLA-A*02:01, -A*03:01, -B*07:02, and -C*07:02. HEK-293T cells have been previously used as a source of HLA peptide ligands derived from SARS-CoV-2 proteins. In this study, we purified and analyzed the HLA-I immunopeptidome of HEK-293 and HEK-293F cells using mass spectrometry. Cell surface expression of specific HLA-I allotypes was determined using flow cytometry with allele-specific antibodies. The HLA-I immunopeptidome of HEK-293 cells contained ligands from all three HLA-I allotypes, whereas that of HEK-293F cells lacked peptides derived from HLA-A*03:01. Flow cytometry experiments confirmed the absence of HLA-A*03:01 expression on the surface of HEK-293F cells. Additionally, we generated a HEK-293 transfectant co-expressing the 5i proteasome subunit and the SARS-CoV-2 Spike protein. This transfectant showed selective loss of HLA-A*03:01 expression, suggesting that HEK-293 linages tend to specifically lose this allotype. We propose that HEK-293F cells are unsuitable for the identification of HLA-A*03:01 ligands or for stimulating T-cell responses restricted to this allele. Moreover, HLA-A*03:01 expression should be regularly monitored in HEK-293-derived cells. Keywords: HLA; antigen presentation; immunopeptidome; HEK-293; peptides Author: María Area-Navarro, Alba Pastor-Moreno, Erika Scholz, Américo Cerqueira, Adrián Tirado-Herranz, Miguel Marcilla, Francesc Canals, Manel Juan, José Ramón Palacio and Iñaki Alvarez Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Nov 25 08:59:16 2025 CET ModDate: Tue Nov 25 09:06:59 2025 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1885185 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- FBCVBN+URWPalladioL-Bold Type 1 Custom yes yes yes 140 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 141 0 MHVONT+URWPalladioL-Roma Type 1 Custom yes yes yes 142 0 KBCDUZ+URWPalladioL-Ital Type 1 Custom yes yes yes 143 0 KIINKE+PalatinoLinotype TrueType WinAnsi yes yes no 182 0 SPXLNH+PazoMath Type 1 Builtin yes yes yes 213 0 TSMJFB+CMSY10 Type 1 Builtin yes yes yes 214 0 KIINKE+PalatinoLinotype TrueType WinAnsi yes yes no 200 0 KIINKE+PalatinoLinotype TrueType WinAnsi yes yes no 248 0 KIINKE+PalatinoLinotype TrueType WinAnsi yes yes no 265 0 KIINKE+PalatinoLinotype TrueType WinAnsi yes yes no 297 0 KIINKE+PalatinoLinotype TrueType WinAnsi yes yes no 342 0 KIINKE+PalatinoLinotype TrueType WinAnsi yes yes no 348 0 QKZKVH+VnURWPalladioL Type 1 Custom yes yes yes 408 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-12-19 03:09:46 CET RepresentationInformation: ijms-26-11357-v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-12-18 06:07:11 CET Size: 1885185 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 490 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: Analysis of HEK-293 and HEK-293F Immunopeptidomes Item: Title: HEK-293F Cells Do Not Express HLA-A*03:01 at the Cell Surface Item: Title: RT-PCR Shows a Reduction in HLA-A*03:01 and B*07:02 mRNA Item: Title: Absence of HLA-A*03:01 Expression in a HEK-293 Transfectant Expressing the SARS-CoV-2 Spike Protein Item: Title: Theoretical Binding Affinity of the Peptides Assigned to Each HLA-I Allotype Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Cell Lines, HLA Typing, and Antibodies Item: Title: Flow Cytometry Item: Title: Isolation of the HLA Class I-Bound Peptide Pool Item: Title: LC-MS/MS Analysis Item: Title: MS/MS Ions Search, Peptide Identification, and Binding Motif Deconvolution Item: Title: Subcellular Location Assignation Item: Title: In Silico Theoretical Binding Allele Assignation Item: Title: qPCR of HLA-I Alleles Item: Title: In Silico Prediction of MHC Binding Affinities Item: Title: Western Blot Item: Title: Statistical Analysis Item: Title: Conclusions Item: Title: References Info: Title: Specific Instability of HLA-A*03:01 Expression in HEK-293 Cells Author: María Area-Navarro, Alba Pastor-Moreno, Erika Scholz, Américo Cerqueira, Adrián Tirado-Herranz, Miguel Marcilla, Francesc Canals, Manel Juan, José Ramón Palacio and Iñaki Alvarez Subject: HEK-293 is a highly transfectable human cell line widely used as a model for protein expression. Since large amounts of cells are often required for the purification of HLA immunopeptidomes, suspension-growing variants, such as HEK-293F, facilitate the generation of sufficient cell quantities. The HLA class I-typing of these cells is HLA-A*02:01, -A*03:01, -B*07:02, and -C*07:02. HEK-293T cells have been previously used as a source of HLA peptide ligands derived from SARS-CoV-2 proteins. In this study, we purified and analyzed the HLA-I immunopeptidome of HEK-293 and HEK-293F cells using mass spectrometry. Cell surface expression of specific HLA-I allotypes was determined using flow cytometry with allele-specific antibodies. The HLA-I immunopeptidome of HEK-293 cells contained ligands from all three HLA-I allotypes, whereas that of HEK-293F cells lacked peptides derived from HLA-A*03:01. Flow cytometry experiments confirmed the absence of HLA-A*03:01 expression on the surface of HEK-293F cells. Additionally, we generated a HEK-293 transfectant co-expressing the 5i proteasome subunit and the SARS-CoV-2 Spike protein. This transfectant showed selective loss of HLA-A*03:01 expression, suggesting that HEK-293 linages tend to specifically lose this allotype. We propose that HEK-293F cells are unsuitable for the identification of HLA-A*03:01 ligands or for stimulating T-cell responses restricted to this allele. Moreover, HLA-A*03:01 expression should be regularly monitored in HEK-293-derived cells. 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