Upon stimulation by sensitive target cells, NK cells rapidly release cytotoxic proteins by polarized fusion of secretory lysosomes with the plasma membrane. Secretion of chemokines and cytokines is a slower process, requires transcription and de novo protein synthesis, and follows different vesicular pathways. Thus, the temporal kinetics of responses must be taken into consideration when designing experiments that assess distinct NK cell functional parameters.
As a note of caution, although NK cell degranulation is a prerequisite for NK cell cytotoxicity, assessment of degranulation does not necessarily correlate with target cell lysis. Target cell lysis depends not only on the extent of effector cell degranulation, but also on the content of secretory lysosomes, target cell structures that facilitate adhesion, and polarized secretion of secretory granules, in addition to the target cell’s intrinsic sensitivity to NK cell-mediated death pathways (8 (link)).
Here we provide detailed instructions for a 2-hour assay that quantifies human NK cell degranulation by assessing CD107a surface expression, which is based on the use of peripheral blood mononuclear cells (PBMC) and standard target cell lines. This assay has been used for the differential diagnosis of defects in cellular cytotoxicity (10 (link), 12 (link)). Furthermore, instructions are presented for a comprehensive 6-hour assay in which human NK cell degranulation, as evaluated by CD107a surface expression, is assessed simultaneously with chemokine and cytokine production, detected by intracellular staining of MIP-1β, TNF-α, and IFN-γ. Induction of MIP-1β production is generally robust in NK cells and can be measured as early as 30 min after target cell mixing, whereas TNF-α and IFN-γ are delayed. Depending on the available flow cytometer, additional antibodies can successfully be combined, facilitating increasingly detailed analysis of other functional parameters or responses in specific NK cell subsets. Although PBMCs are used as effector cells in the assays described here, the assays are also applicable to purified NK cells.
As a note of caution, although NK cell degranulation is a prerequisite for NK cell cytotoxicity, assessment of degranulation does not necessarily correlate with target cell lysis. Target cell lysis depends not only on the extent of effector cell degranulation, but also on the content of secretory lysosomes, target cell structures that facilitate adhesion, and polarized secretion of secretory granules, in addition to the target cell’s intrinsic sensitivity to NK cell-mediated death pathways (8 (link)).
Here we provide detailed instructions for a 2-hour assay that quantifies human NK cell degranulation by assessing CD107a surface expression, which is based on the use of peripheral blood mononuclear cells (PBMC) and standard target cell lines. This assay has been used for the differential diagnosis of defects in cellular cytotoxicity (10 (link), 12 (link)). Furthermore, instructions are presented for a comprehensive 6-hour assay in which human NK cell degranulation, as evaluated by CD107a surface expression, is assessed simultaneously with chemokine and cytokine production, detected by intracellular staining of MIP-1β, TNF-α, and IFN-γ. Induction of MIP-1β production is generally robust in NK cells and can be measured as early as 30 min after target cell mixing, whereas TNF-α and IFN-γ are delayed. Depending on the available flow cytometer, additional antibodies can successfully be combined, facilitating increasingly detailed analysis of other functional parameters or responses in specific NK cell subsets. Although PBMCs are used as effector cells in the assays described here, the assays are also applicable to purified NK cells.
