Interrogation of Natural Killer Cell Activity Using High Throughput Advanced Flow Cytometry
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:
- Use labor intensive, indirect measurements of cytotoxicity e.g. Chromium release assay
- Do not allow simultaneous quantification of cytotoxicity, activation state and cytokine/protein release
- Need to use multiple workflows that each require different instruments
- Do not incorporate physiologically relevant environmental conditions
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® Advanced High-throughput Flow Cytometry, iQue® Forecyt integrated software analysis and phenotypic and functional readout into a single assay provides a simplified solution for the development of Immun-oncology and NK cell-mediated cellular therapies.
It is now possible to measure phonotype and function in a single well to improve productivity and gain crucial insight into NK Cell Killing.
- Multiplexed approach to measure NK cell activation state and target cell killing in a single well.
Figure 1. Illustration of iQue® Human NK Cell Killing Kit assay principles. Target cells are distinguished from effector cells by staining with a fluorescent encoder 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.
Gain more biological insights
Enable study of NK cell mediated tumor cell killing in physiologically relevant models.
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® 3.
(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.
Unlock your productivity
Simultaneous measurement of effector cell marker expression and secreted effector proteins in a mixed cells and beads assay format.
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® 3 system.
(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.
Streamline data acquisition
Real time data analysis and novel visualization tools enables rapid data acquisition.
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).
Save time and precious sample
Collapse and streamline traditional workflows into a single miniaturized, multiplex assay with minimal sample manipulation.
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.
iQue® Human NK Cell Killing Kit
Compatible with iQue® 3/iQue® Screener Plus VBR configuration.
1 x 96 well
5 x 96 wells
1 x 384 wells
5 x 384 wells
iQue® Human NK Cell Companion Kits
|iQue® Human MIP 1α NK Cell Companion Kit|
|iQue® Human RANTES NK Cell Companion Kit|
|iQue® Human GM-CSF NK Cell Companion Kit|
|iQue® Human CD178/Fas Ligand NK Cell Companion Kit|
|iQue® Human Granzyme A NK Cell Companion Kit|
iQue® Human TNFα NK Cell Companion Kit