Abstract
Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176–284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and–B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules.
Highlights
The classical Human Leukocyte Antigen (HLA) class I molecules: HLA-A,B, and -C bind and present intracellularly produced peptides on the surface of a wide variety of cells
The cell surface expressions of HLA-A and -B on HEK293T cells were measured by quantitative flow cytometry using allele-specific primary monoclonal antibodies against either HLA-A3 or -B7, as the cells are homozygous for both (Fig 1)
We found that HLA-A3 was highly expressed constitutively with a median of 71,204 molecules per cell (Figs 1B and 2A)
Summary
The classical Human Leukocyte Antigen (HLA) class I molecules: HLA-A,-B, and -C bind and present intracellularly produced peptides on the surface of a wide variety of cells. The peptides may originate from the cell’s own proteome or from an intracellular pathogen, e.g. a virus. Once on the cell surface, the HLA-peptide complex is monitored by specific Cluster of Differentiation (CD8)+ cytotoxic T lymphocytes that recognize foreign peptides and kill the infected cells that present them by inducing apoptosis. Cancer cells can be identified and terminated because of the mutated or aberrantly-expressed peptides they may present. HLA class I molecules consist of an extremely polymorphic transmembrane heavy chain forming the peptide-binding groove and a non-covalently associated beta-2-microglobulin (B2M). Different alleles bind different sets of peptides and certain alleles may influence the course of specific infections. HLA-BÃ57:01 and HLA-BÃ27:05 are associated with slow progression of HIV infection while HLA-BÃ35:03 is associated with rapid progression [1,2,3,4,5]
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