A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry

<div><p>Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The ⁵¹Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.</p></div

Percent specific lysis of K562 cell line using protocol optimized for direct image cytometry assay.

<p>(a) Cytotoxicity against K562 was repeated by image cytometry with reduced target cell number (50,000 cells/well), in a final media volume of 100 μl. After the standard 4-hour incubation the cells were directly resuspended in the 100 μl volume and read using image cytometer. (b) The data shows that with reduced target cell density (Low) the cytotoxicity of NK cells against K562 was significantly higher compared to previous assay with higher target cell density (High) of 100,000 cells/well. The statistical analysis was performed using Wilcoxon matched-pairs signed rank, non-parametric, two-tailed t test and <i>p</i> value of <0.05 was considered significant. The data is plotted as median with range.</p

Determination of target cell lysis by image cytometry.

<p>The figure shows images from the image cytometer and analysis of fluorescence data using FCS express, to derive live target cell counts. The number of live target cells in the culture was determined by plotting fluorescence intensity of target cells from each E:T ratio compared to spontaneous control. The live K562 cells are shown in blue circles and the lysed cells and apoptotic bodies are highlighted in red circles in the images. Representative fluorescence histograms used for deriving live target cell counts are shown for each tumor cell lines (K562, 721.221 and Jurkat cells).</p

Assessment of NK cell cytotoxicity by image cytometry.

<p>Fluorescent images (from Image cytometer) of K562; 721.221 and Jurkat cell lines showing progressive loss of fluorescent (live) cells from the culture with increasing E:T ratio. The images from spontaneous serve as no treatment control. Representative images of target cell killing from one NK cell donor is shown for each cell line and E:T ratio.</p

Comparison of percent specific lysis obtained by calcein release assay and image cytometry.

<p>Standard calcein release assay and image cytometry were performed within the same assay to determine NK cell killing of K562, 721.221 and Jurkat. The data is shown for each tumor cell line using NK cells expanded from 5 donors (n = 5). Cytotoxicity assay was performed in triplicate for each NK cell donor. Each of the replicate is presented in the plots. The statistical analysis was performed using Wilcoxon matched-pairs signed rank, non-parametric, two-tailed t test and <i>p</i> value of <0.05 was considered significant. The data is plotted as median with range.</p

Sensitivity of calcein release assay.

<p>(a) Dynamic range for the calcein release assay was determined for neuroblastoma and leukemia tumor targets (K562, SK-N-BE(2), CHP134, CHLA155, IMR32, Jurkat, 721.221 and Nalm 6). The cell lines were stained with Calcein AM, and seeded in 96 well plates to generate maximum release (lysis with 1% TritonX-100) and spontaneous release data. After 4 hours the supernatant fluorescence was measured. The maximum release was normalized to 100% and the spontaneous release is represented as % of maximum. (b) Illustration of calcein release from target cells upon lysis by NK cells following necrosis-like and apoptotic death. (c) Bright-field and fluorescence overlay images of calcein release from CHP 134 cells undergoing necrosis-like death following interaction with NK cells. (d) Bright-field and fluorescent overlay images of calcein release from some K562 cells undergoing apoptotic death following interaction with NK cells. The images were derived from live Nikon Biostation IQ-M videos.</p