Specific Instability of HLA-A*03:01 Expression in HEK-293 Cells

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 <i>HLA-A*02:01</i>, -<i>A*03:01</i>, -<i>B*07:02</i>, and -<i>C*07:02</i>. 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 <i>HLA-A*03:01</i>. Flow cytometry experiments confirmed the absence of <i>HLA-A*03:01</i> expression on the surface of HEK-293F cells. Additionally, we generated a HEK-293 transfectant co-expressing the β5i proteasome subunit and the SARS-CoV-2 Spike protein. This transfectant showed selective loss of <i>HLA-A*03:01</i> expression, suggesting that HEK-293 linages tend to specifically lose this allotype. We propose that HEK-293F cells are unsuitable for the identification of <i>HLA-A*03:01</i> ligands or for stimulating T-cell responses restricted to this allele. Moreover, <i>HLA-A*03:01</i> expression should be regularly monitored in HEK-293-derived cells.