Abstract
The lytic capacity of a NK cell is regulated, in part, by the balance in cell surface expression between inhibitory CD94/NKG2A and activating CD94/NKG2C heterodimers. We demonstrate that, in the absence of DAP12, rhesus monkey NKG2A is preferentially expressed at the cell surface with CD94 due to a single amino acid difference in the transmembrane of NKG2A and NKG2C. Furthermore, in the context of an NKG2A transmembrane, the stalk domain of NKG2C was found to enhance heterodimer formation with CD94 compared with the stalk domain of NKG2A. In the presence of DAP12, the ability of NKG2C to compete for cell surface CD94 heterodimerization is enhanced and approaches that of NKG2A. Finally, allelic differences that affect the ability of rhesus NKG2A to reach the cell surface with CD94 could also be mapped to the transmembrane. These differences in the ability of inhibitory and activating NKG2 molecules to reach the cell surface provide a mechanism for the regulation of NK cell activity.
Highlights
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To explore why NKG2A was found at the cell surface as a heterodimer with CD94 at higher levels than NKG2C, we developed a competition assay to evaluate the relative efficiency of individual NKG2 molecules to heterodimerize and be expressed at the cell surface with CD94. 293T cells were transfected with pNKG2A*01-V5, limiting amounts of plasmid encoding CD94 (pCD94) as determined by titration, and untagged pNKG2A*01 or pNKG2C*04 competitor
To determine the amount of DAP12 DNA to use in the planned experiments, we examined the ability of DAP12 to regulate the cell surface expression of NKG2A and NKG2C. 293T cells were cotransfected with plasmids expressing CD94, either NKG2A*01-V5 or NKG2C*04-V5, and increasing amounts of DAP12 DNA
Summary
ITIM, immunoreceptor tyrosine-based inhibitory motif; MFI, mean fluorescence intensity; TM, transmembrane. CD94/NKG2C cross-linking, a signaling cascade is initiated that leads to activation of NK cell-mediated lysis [16, 17] Both the inhibitory and activating CD94/NKG2 receptor complexes interact with the nonclassical MHC class Ib molecule HLA-E on the surface of target cells. Competition for binding of activating and inhibitory NKG2 molecules to CD94 may regulate the balance between receptors expressed on the cell surface. We evaluated the ability of rhesus monkey NKG2A and NKG2C molecules to compete for heterodimerization and cell surface expression with CD94. We evaluated whether NKG2 allelic differences can alter the balance of receptors at the cell surface through differential abilities to compete for CD94 heterodimerization. We mapped the regions of the NKG2 molecules responsible for preferential association with CD94
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