Measurement of NK cells

NK cell activity and the number of NK cells are measured in the peripheral blood in humans and in the spleen in rodents. A short-term (4 h) 51Cr-release assay is used to determine NK cell activity, with a standard number (5 X 103) of cultured K562 cells as targets (T) and peripheral blood mononuclear cells (PBMCs) as effectors (E). The latter are adjusted in number to give at least four different E:T ratios, thus allowing for generation of a lytic curve by plotting the per cent specific lysis measured at each E:T ratio. The results of NK cell assay are usually expressed as lytic units (LU) calculated from the lytic curve and defined as the number of effector cells required to kill 5X10' targets in a batch of tested cells. Computer programs are available to facilitate transformation of the per cent specific lysis into LU.

Using variations of this cytotoxicity assay by the addition of antibody or by changing the target, it is possible to measure ADCC or spontaneous LAK activity, respectively. The measurement of the NK cell number should be performed at the same time as the NK cell assay, because both are necessary for an adequate evaluation of natural immunity or for following its changes during disease or therapy. To determine the number of NK cells, PBMCs are stained with labeled monoclonal antibodies specific for NK cell-associated markers (e.g. CD56 or CD16

in humans; NKR-P1 in rats and NK1.1 in mice) and the percentage of positive cells in a population is determined by flow cytometry. This percentage value is then converted into the absolute number of NK cells, based on a simultaneously obtained differential lymphocyte count. A variety of assays are available for assessment of NK cell functions other than lysis. For example, generation of LAK activity in vitro, proliferation, cytokine production, migration, binding to substrates, ability to induce apoptosis or to transduce signals can be accurately measured provided purified NK cells are available. Negative selection with immunobeads is generally used to obtain highly enriched preparations of NK cells for these assays.

See also: Adhesion molecules; Antibody-dependent cellular cytotoxicity; Cytokines; Cytotoxicity, assays for; Cytotoxicity, mechanisms of; Fc receptors; Immune surveillance; Innate immunity; Large granular lymphocytes; Lymphokine-activated killer (LAK) cells; Perforin; Tumors, immune response to; Viruses, immunity to.

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