Natural Killer Cell Killing
Natural Killer (NK) cells are an essential part of the innate immune system and play a crucial role in immune surveillance and anti-tumor responses. Currently, several types of NK cell-related immunotherapeutics are being developed for the treatment of cancers. Tumor-specific antibodies, including mAbs, BiKES, and TRiKES, that are able to induce NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) have proven to be successful against several types of cancer, while advances in the production of CAR-NK cells could allow for even more specific, enhanced killing of tumor cells. A critical process in evaluating new NK cell-mediated therapeutics is the ability to quantify NK cell activation and tumor killing in order to provide insight into potential treatment efficacy. Traditional techniques for measuring NK-mediated killing and activation often:
The iQue® Natural Killer Cell Killing application utilizes the iQue® Human NK Cell Killing Kit for simultaneous, high throughput analysis of target cell killing, NK cell phenotypic and activation marker expression, and quantification of secreted effector proteins and cytokines. Combining the power of iQue® High-Throughput Screening (HTS) by Cytometry, iQue Forecyt® integrated software analysis and phenotypic and functional readout into a single assay provides a simplified solution for the development of Immuno-oncology and NK cell-mediated cellular therapies.
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Figure 1. Illustration of iQue® Human NK Cell Killing Kit assay principles. Target cells are distinguished from effector cells by staining with a fluorescent encoding dye, and tumor cell killing is then determined by staining with a fluorescent cell membrane integrity dye. NK cells are identified using CD3, CD56, and CD16. Their activation state is assessed using CD69 and CD25. Production of the pro-inflammatory cytokine, Interferon gamma (IFNg), and the pro-apoptotic serine protease, Granzyme B, are quantified using 2-plex iQue Qbeads® in a sandwich immunoassay format in the same well. Analysis of additional cytokines are available with Human NK cell companion kits.
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View iQue® Natural Killer Cell Killing Publications
Figure 2. Quantify natural killer cell mediated-antibody-dependent cellular cytotoxicity, cytokine/protein release and marker expression from a co-culture assay. Encoded Raji tumor cells (20K/well) were co-cultured with PBMCs (200K/well) from two separate donors. PBMCs were incubated with one of three anti-CD20 antibodies: Ab-1 (IgG1), Ab-2 (IgG1) or a negative control Ab-3 (IgA2). Concentration range was between 10ug/mL – 0.128 ng/mL.
At 4h, 10 µL samples were analyzed to assess tumor cell killing using the Human NK Cell Killing Kit and the iQue® 3, Granzyme A was also measured using an iQue® Human NK Cell Companion Kit.
(A,B) Target cell killing by two donors show differential response to the antibodies. (C,D) Granzyme A production was both concentration and donor dependent. (E,F) CD16+ expression of natural killer cells decreases with increasing stimulation.
Figure 3. Direct NK-mediated killing of tumor cells using cytokine stimulation can also be quantified. Encoded K562 tumor cells (20K/well) were co-cultured with enriched (negatively selected) human NK cells that had been incubated for 16-18 h in either media alone (Non-activated NK cells) or media containing 200 U/mL IL-2 & 100 ng/mL IL-15 (Activated NK cells) at an Effector:Target ratio of 1:1 or 5:1. At 4 h and 24 h, 10 µL samples were analyzed to assess tumor cell killing using the iQue® Human NK Cell Killing Kit and the iQue® HTS Platform .
(A) Histogram depicting target cell viability following co-culture with non-activated or cytokine activated NK cells for 4 h. (B,C) Summary of percent target cell death following co-culture of tumor cells with non-activated or cytokine activated NK cells for (B) 4 h or (C) 24 h.
Figure 4. Detect different states of NK activation using phenotyping and analysis of cytokine secretion. Encoded K562 tumor cells (20K/well) were co-cultured with enriched (negatively selected) human NK cells that had been incubated for 16-18 h in either media alone (Non-activated NK cells) or media containing 200 U/mL IL-2 & 100 ng/mL IL-15 (Activated NK cells) at an Effector:Target ratio of 5:1. Following 24 h of co-culture, 10 µL samples were removed and analyzed using the iQue® Human NK Cell Killing Kit and the iQue® HTS Platform.
(A) Expression of activation markers, CD69 and CD25. (B) Production of IFNg and Granzyme B secretion. (C) Additional cytokines were assessed in parallel by combining iQue® Human NK Cell Companion Kits with the iQue® Human NK Cell Killing Kit. NK = Natural Killer cells. T = Target K562 tumor cells.
Activation with IL-2 and IL-15 caused increased activation marker expression and secretion of multiple effector proteins, such as Granzyme B and CCL5 (RANTES). These effects were further enhanced when NK cells were cultured with target cells.
Figure 5. Pre-defined gates and multiple analysis features on iQue Forecyt® enable automatic phenotyping of human NK cells.
(A) Example of using positive and negative wells to create an overlay plot of CD69 expression on non-activated NK cells (negative; red) vs. cytokine activated NK cells (positive; green). (B) Example of plate heat map showing the percent CD69+ cells per well. (C) Example of overlay histogram depicting CD69 expression on NK cells. (Non-act. NK = non-activated NK cells, Act. NK = cytokine activated NK cells, T = tumor target cells).
Figure 6. Easy to follow protocol for the analysis of NK cell mediated killing of tumor cells, along with assessment of the NK cell activation state and cytokine release using the iQue® Human NK Cell Killing Kit.
Platform
Compatible with iQue® 3/iQue® Screener Plus VBR configuration.
Available Sizes
Catalog Numbers
1 x 96 well
97082
5 x 96 wells
97083
1 x 384 wells
97084
5 x 384 wells
97085
Catalog No.
Description
97089
97090
97091
97092
97093
97094
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Perform comprehensive, multiplexed analysis of cells, beads and secreted proteins all from the same cells at the same timepoint.
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