Evaluation of NK cell function by flowcytometric measurement and impedance based assay using real-time cell electronic sensing system

Abstract

Although real-time cell electronic sensing (RT-CES) system-based natural killer (NK) cytotoxicity has been introduced, it has not been evaluated using human blood samples. In present study, we measured flowcytometry based assay (FCA) and RT-CES based NK cytotoxicity and analyzed degranulation activity (CD107a) and cytokine production. In 98 healthy individuals, FCA with peripheral blood mononuclear cells (PBMCs) at effector to target (E/T) ratio of 32 revealed 46.5 ± 2.6% cytolysis of K562 cells, and 23.5 ± 1.1% of NK cells showed increased degranulation. In RT-CES system, adherent NIH3T3 target cells were resistant to basal killing by PBMC or NK cells. NK cell activation by adding IL-2 demonstrated real-time dynamic killing activity, and lymphokine-activated PBMC (E/T ratio of 32) from 15 individuals showed 59.1 ± 6.2% cytotoxicity results after 4 hours incubation in RT-CES system. However, there was no significant correlation between FCA and RT-CES cytotoxicity. After K562 target cell stimulation, PBMC produced profound proinflammatory and immunoregulatory cytokines/chemokines including IL-2, IL-8, IL-10, MIP-1 α β , IFN- γ , and TNF- α , and cytokine/chemokine secretion was related to flowcytometry-based NK cytotoxicity. These data suggest that RT-CES and FCA differ in sensitivity, applicability and providing information, and further investigations are necessary in variable clinical conditions.

Figure 1

Flowcytometric NK cytotoxicity and CD107a degranulation assay. (a) CFSE labeled K562 cells and effector cells (media only, PBMC, or isolated NK cells) from healthy donors were cultured, and CFSE+7-AAD double labeled K562 cells were analyzed. (b) CD107a expression on NK cells after K562 stimulation was measured to analyze NK cell degranulation.

Figure 2

NK cytotoxicity at different E/T ratios. (a) Flowcytometry-based cytolysis of K562 target cells by PBMC (n = 98) and isolated NK cells (n = 15) from healthy donors. (b) RT-CES system-based cytolysis of NIH3T3 target cells by PBMC (n = 46) and lymphokine-activated PBMC (LAK-PBMC) (n = 15) from healthy donors.

Figure 3

Real-time profiling of NK cytotoxicity by RT-CES system. (a) The NIH3T3 target cells were resistant to basal PBMC and NK cells. (b) Addition of IL-2 induced complete NIH3T3 cytolysis, and complete cytolysis by PBMC and NK cells required more than 72 hours and 24 hours, respectively. (c) LAK-PBMC rapidly killed target cells in 2 hours.

Figure 4

Relation among two NK cytotoxicity tests and CD107a degranulation on NK cells. (a) Correlation between two NK cytotoxicity tests by PBMC in FCA and LAK-PBMC in RT-CES system at E/T ratio of 32. (b) Flowcytometric NK cytotoxicity by PBMC at E/T ratio of 16 versus CD107a expression on NK cells after K562 stimulation. (c) NK cytotoxicity by LAK-PBMC in RT-CES at E/T ratio of 16 versus CD107a expression on NK cells after K562 stimulation.

Figure 5

Cytokines/chemokines production after 4 hours incubation of PBMC with K562 cells at E/T ratio of 32. Values represent the mean concentrations from 30 healthy donors.

Figure 6

Relation between flowcytometric NK cytotoxicity and cytokine/chemokine secretion after incubation of PBMC with K562 target cells at E/T ratio 32/1.