Background

Being a part of innate immune system, natural killer (NK) cells involve in immune surveillance. They distinguish between healthy and abnormal cells (virus-infected or malignant) through a set of germline-encoded inhibitory and activating receptors (Marcus et al., 2014; Tognarelli et al., 2016). Under normal and healthy conditions, NK cells serve a role that is unlike other blood cells perform, until there is an infection. At that time natural killer cells assume their more recognized role in killing of targeted cells. NK cells exhibit direct cytotoxic effect via granzyme/perforin/death receptor pathways against the cells that lost the expression of HLA class I antigen (Geller and Miller, 2011; Paul and Lal, 2017). Further, NK cells are stimulated to produce IFNγ and TNF-α, which exert cytostatic/cytotoxic effects on specific targets. NK cell deficiency seems a part of larger immunological syndrome.

In general, the most common combination of surface markers used to identify the majority of NK cells is the absence of CD3, along with the expression of CD56 (i.e., CD3^-CD56⁺). However, every NK cells of above phenotype may not be functionally active. It has been shown that Perforin and CD107a combination represent superior NK cells function (Rubin et al., 2017).

Several assays have been established for determination of the activity of NK and cytotoxic T cells, when they recognize the target cells. The ⁵¹Chromium release assay (CRA) was described for the first time (Brunner et al., 1968) to assay NK cells activity, which has been considered the ‘gold standard’ for measurement of cytotoxic effect. This assay indirectly determines dead cell population. Moreover, due to risks of handling and disposal of radioactive substance, several alternative methods have been developed time to time. Two fluorochromes based flow-cytometry assay is proposed for direct determination of dead target cells (Radosevic et al., 1990). In this case, fluorescently stained target cells, if lost viability, are counter stained with a DNA intercalating dye. Another fluorescence-based assay was proposed, which depends on the hydrolysis of Calcein AM (Lichtenfels et al., 1994) by intracellular esterases resulting in the formation of Calcein, a hydrophilic strongly fluorescent compound retained in the cytoplasm of the viable cells. In case of damaged ones, it releases in the medium producing an intense green signal measured by fluorimeter. The caveat of this assay is that the release calcein varies in its dynamic range for different tumor targets, and the calcein could retain within the apoptotic bodies and membrane fragments of the lysed cells results in incomplete release leading to underestimation. To overcome these limitations, a novel cytotoxicity assay using an image flow-cytometer was proposed, which is to be claimed as a simple and direct method (Srinivas et al., 2015). Later, two more assays based on LDH release (Konjevic et al., 1997) and bioluminescence (Karimi et al., 2014) have been proposed. While both of these methods provide indirect assessment, the former one is simple whereas the latter method is a complicated one. In this protocol, we have proposed a flow-cytometry based comprehensive method that not only provides direct value of percentage dead target cells but also analyzes sensitive cells in a heterogeneous population of ascitic or blood-derived cells. The earlier published methods were based on tumor cell lines, which is devoid of other cells present in the biological fluid in vivo. The scope of the present protocol is further extended to find out the efficacy of NK cells in cancer patients as compared to healthy persons. It is known that in cancer patients, depending upon the stages of the disease, the potency of NK cell immune surveillance is compromised (Levy et al., 2011). In an earlier paper, we discussed this protocol (Akhter et al., 2018), which clearly indicates that in ovarian cancer a subtype of tumor cells remains unaffected even in the presence of healthy donor NK cells. Thus, this protocol opens up the possibility to decide on a critical issue in NK cell immune therapy, that is how they should be generated for effective therapy.