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

Area of Science:

• Immunology
• Cell Biology
• Mass Spectrometry

Background:

• HEK-293 cells are a standard human cell line for protein expression.
• Suspension-growing variants like HEK-293F are used for large-scale cell culture, essential for immunopeptidome purification.
• HEK-293 cell lines express Human Leukocyte Antigen (HLA) class I allotypes, including HLA-A*02:01, HLA-A*03:01, HLA-B*07:02, and HLA-C*07:02.

Purpose of the Study:

• To analyze and compare the HLA class I immunopeptidome of HEK-293 and HEK-293F cells.
• To investigate the cell surface expression of HLA class I allotypes in these cell lines.
• To determine the suitability of HEK-293F cells for studies involving HLA-A*03:01.

Main Methods:

• Purification and mass spectrometry analysis of HLA-I immunopeptidomes from HEK-293 and HEK-293F cells.
• Flow cytometry using allele-specific antibodies to quantify cell surface HLA-I allotype expression.
• Generation of a HEK-293 transfectant expressing the β5i proteasome subunit and SARS-CoV-2 Spike protein.

Main Results:

• The HLA-I immunopeptidome of HEK-293 cells contained peptides from all expressed HLA-I allotypes.
• HEK-293F cells showed a lack of peptides derived from HLA-A*03:01 in their immunopeptidome.
• Flow cytometry confirmed the absence of surface HLA-A*03:01 expression on HEK-293F cells.
• A generated transfectant also exhibited selective loss of HLA-A*03:01 expression, indicating a tendency in HEK-293 lineages.

Conclusions:

• HEK-293F cells are not suitable for identifying HLA-A*03:01 ligands or for T-cell response studies restricted to this allele.
• The selective loss of HLA-A*03:01 expression in HEK-293F cells necessitates regular monitoring of this allotype in HEK-293-derived cell lines.
• These findings highlight the importance of verifying HLA allotype expression in cell line models for immunological research.