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

Publication date: Nov 24, 2025

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 β5i 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.

Semantics

Original Article