{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T22:17:52Z","timestamp":1773699472890,"version":"3.50.1"},"reference-count":19,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T00:00:00Z","timestamp":1763942400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003030","name":"Ag\u00e8ncia de Gesti\u00f3 d\u2019Ajuts universitaris i de Recerca","doi-asserted-by":"publisher","award":["2020PANDE00079"],"award-info":[{"award-number":["2020PANDE00079"]}],"id":[{"id":"10.13039\/501100003030","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011104","name":"Universitat Aut\u00f2noma de Barcelona","doi-asserted-by":"publisher","award":["PPC2024-38"],"award-info":[{"award-number":["PPC2024-38"]}],"id":[{"id":"10.13039\/501100011104","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 \u03b25i 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.<\/jats:p>","DOI":"10.3390\/ijms262311357","type":"journal-article","created":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T14:59:04Z","timestamp":1763996344000},"page":"11357","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Specific Instability of HLA-A*03:01 Expression in HEK-293 Cells"],"prefix":"10.3390","volume":"26","author":[{"given":"Mar\u00eda","family":"Area-Navarro","sequence":"first","affiliation":[{"name":"Department of Cell Biology, Physiology and Immunology, Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2878-3177","authenticated-orcid":false,"given":"Alba","family":"Pastor-Moreno","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Physiology and Immunology, Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erika","family":"Scholz","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Physiology and Immunology, Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Am\u00e9rico","family":"Cerqueira","sequence":"additional","affiliation":[{"name":"Proteomics Unit, Spanish National Biotechnology Centre, 28049 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9324-851X","authenticated-orcid":false,"given":"Adri\u00e1n","family":"Tirado-Herranz","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Physiology and Immunology, Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miguel","family":"Marcilla","sequence":"additional","affiliation":[{"name":"Proteomics Unit, Spanish National Biotechnology Centre, 28049 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Francesc","family":"Canals","sequence":"additional","affiliation":[{"name":"Proteomics Laboratory, Vall d\u2019Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3064-1648","authenticated-orcid":false,"given":"Manel","family":"Juan","sequence":"additional","affiliation":[{"name":"Department of Immunology, Hospital Cl\u00ednic de Barcelona, 08036 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos\u00e9 Ram\u00f3n","family":"Palacio","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Physiology and Immunology, Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9484-0868","authenticated-orcid":false,"given":"I\u00f1aki","family":"Alvarez","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Physiology and Immunology, Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, 08193 Bellaterra, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"801","DOI":"10.37349\/etat.2024.00249","article-title":"Immunopeptidomics in the cancer immunotherapy era","volume":"5","author":"Pongcharoen","year":"2024","journal-title":"Explor. 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