Authors were supported by the German Cancer Aid (Max Eder Nachwuchsgruppe, Deutsche Krebshilfe; EU), the LOEWE Center for Cell and Gene Therapy Frankfurt (EU, ST) funded by the Hessian Ministry of Higher Education, Research and the Arts, Germany (III L 4- 518/17.004) and by the "Alfred- und Angelika Gutermuth-Stiftung", Frankfurt, Germany (EU). BJ was funded by a Mildred Scheel postdoctoral scholarship from the Dr. Mildred Scheel Foundation for Cancer Research. ST was founded by a GO-IN postdoctoral fellowship (PCOFUND-GA-2011-291776). The authors thank Becton Dickinson (BD) for providing the FACSCanto II and Canto10c Flow Cytometry Analyzers used in this study.

This study was carried out in accordance with the recommendations of the local ethics committee of the University of Frankfurt.
1. Culturing of K562 Cells
<ol>
	<li>Culture K562 cells in R10 media (RPMI1640 with glutamine medium, 1% penicillin/streptomycin, 10% fetal calf serum) at a density of 0.5-1 x 106 cells per ml in a cell culture flask at 37 &#176;C and 5% CO2.</li>
	<li>Harvest K562 Cells 24 hr Before the Start of a New Experiment.
	<ol>
		<li>Remove the cell culture flask containing the K562 cells from the incubator. Re-suspend the K562 cells within the culture media by gently pipetting up and down.</li>
		<li>Transfer the culture media containing the K562 cells into a 15 or 50 ml tube and pellet the cells at 400 x g for 8 min. Discard the supernatant and re-suspend the cells in 5 ml R10 media and mix well.</li>
		<li>Transfer 20 &#181;l of the cell solution into a well of a 96-U-well plate. Add 20 &#181;l trypan blue and mix well by pipetting up and down for at least 5 times. Pipette 10 &#181;l of the solution into a cell counting chamber and count the cells.</li>
		<li>Pellet the cells at 400 x g for 8 min. Re-suspend the cells in R10 media and adjust the K562 cell concentration to 0.5-1 x 106 cells per ml.</li>
		<li>Incubate the K562 cells in a cell culture flask at 37 &#176;C and 5% CO2 until use.</li>
	</ol>
	</li>
</ol>
2. Isolation of NK Cells
<ol>
	<li>According to the approval and guidelines of the local ethics committee, obtain written informed consent of the healthy donor or patient before the collection of 6-10 ml of either EDTA or heparinized peripheral blood.</li>
	<li>Store reagents for the NK cell isolation at 4 &#176;C until the start of the experiment and then use them at room temperature (RT) under sterile conditions. Isolate NK cells from peripheral blood using magnetic microbeads and a specific magnet for cell isolation according to the manufacturer&#39;s instructions.
	<ol>
		<li>Reconstitute one vial of the lyophilized NK cell negative isolation antibody cocktail by pipetting 7.5 ml of the provided buffer A (included within the NK cell isolation kit) into the vial. Mix gently by pipetting up and down 3-4 times. 
		NOTE: Make sure, that the suspension is homogenous before each use.</li>
		<li>Prepare the final cocktail solution by mixing appropriate volumes of the reconstituted pellet from step 2.2.1 and buffer B (included within the NK cell isolation kit). To determine these volumes, define the volume of the whole blood sample in ml as volume = 1.0. Use 0.25 volumes of the reconstituted pellet (from step 2.2.1) and 0.25 volumes of buffer B to process 1 volume of whole blood.</li>
		<li>Transfer the mixture into an adequate tube containing the whole blood sample. Do not pipette up and down to avoid the loss of blood within the pipette. Instead, close the tube and move it carefully up and down until the suspension is homogenous. Do not vortex.</li>
		<li>Further homogenize the sample using a tube rotator for 5 min at RT.</li>
		<li>Under sterile conditions, remove the cap and place the tube into the magnetic separator for 15 min as recommended by the manufacturer. Make sure that the tube labels face towards the backside of the magnet to allow undisturbed visibility of the separation line. Do not move the magnet during separation, as this procedure would be disturbed.</li>
		<li>Carefully transfer the supernatant into a new 15 ml tube and fill up with complete media (CM; hematopoietic cell media, 1% penicillin/streptomycin, 5% human sera). Pellet the cells at 400 x g for 8 min.
		<ol>
			<li>Optional: If the pellet is red, re-suspend the cells in 1ml of erythrocyte lysis buffer (e.g., 1 ml of ammonium-chloride-potassium (ACK) lysing buffer: 155 mM NH4Cl; 0.1 mM EDTA; 10 mM KHCO3 in 1 L distilled water (DW)) and incubate for 8 min at RT. Alternatively, use an erythrocyte depletion kit. 
			NOTE: Keep in mind, that the use of ACK buffer might negatively impact NK cell functions14.</li>
			<li>Rapidly dilute the ACK buffer by adding at least 1 ml of CM to stop the lysis and centrifuge at 400 x g for 8 min to pellet the cells.</li>
		</ol>
		</li>
		<li>Re-suspend the cell pellet in 1 ml CM and proceed with the counting. Transfer 20 &#181;l of the NK cell solution onto a 96-U-well plate. Add 20 &#181;l of methylene blue to each well and mix well by pipetting up and down for at least 5 times. Pipette 10 &#181;l of the solution into a cell counting chamber and count cells. Alternatively, use 30 &#181;l of undiluted cell suspension for counting with a commercially available cell counter.</li>
		<li>Adjust the cells to a concentration of at least 2 x 104 NK cells per 100 &#181;l CM.</li>
		<li>Perform a surface antibody staining of the isolated NK cell population to check the purity using a flow cytometer.
		<ol>
			<li>Prepare a master mix solution by mixing the volumes of the indicated antibodies as per Table 1.</li>
			<li>Re-suspend the cells in 88.5 &#181;l washing buffer (WB; 0.5% bovine serum albumin (BSA), 0.1% NaN3 in PBS) and add 11.5 &#181;l of the master mix solution. Incubate the cells for 20 min at 4 &#176;C in the dark. 
			Caution: NaN3 is toxic and mutagen. Handle it accordingly to the corresponding Material Safety Data Sheet (MSDS).</li>
			<li>Add 1 ml of WB and pellet the cells at 400 x g for 4 min.</li>
			<li>Re-suspend the cells in 300 &#181;l staining buffer plus DAPI. Acquire the cells using a flow cytometer. 
			NOTE: Proceed further with the experiment only if the NK cell purity is at least 80% of all alive lymphocytes.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Harvesting of the K562 Cells for NK Cell Stimulation
<ol>
	<li>Remove the cell culture flask containing the K562 cells (from step 1.2) from the incubator. Re-suspend the K562 cells within the culture media by gently pipetting up and down.</li>
	<li>Transfer the whole culture media into a 15 or 50 ml tube and pellet the cells at 400 x g for 8 min. Discard the supernatant and re-suspend the cells in 5 ml phosphate-buffered saline (PBS) and mix well.</li>
	<li>Transfer 20 &#181;l of the cell solution into a well of a 96-U-well plate. Add 20 &#181;l trypan blue and mix well by pipetting up and down for at least 5 times. Pipette 10 &#181;l of the solution into a cell counting chamber and count the cells. Alternatively use 30 &#181;l of undiluted cell suspension to count with a commercially available cell counter.</li>
	<li>Pellet the cells at 400 x g for 8 min. Re-suspend cells in CM at a concentration of at least 2 x 104 K562 cells per 100 &#181;l media. 
	NOTE: Use the same K562 and NK cell concentrations in order to incubate both at an effector:target (E:T) ratio of 1:1 later on.</li>
</ol>
4. Stimulation of NK Cells with the Tumor Cell Line K562 and Cytokines
<ol>
	<li>Gently mix the cells by pipetting them up and down for at least 5 times. Distribute 100 &#181;l/well of the NK cell solution into the required wells of a 96-V-well plate.
	<ol>
		<li>Use at least two wells for each donor, one with and one without a stimulus (e.g., K562 tumor cells and cytokines). Whenever possible, perform the experiment at least in duplicate and add a positive control (e.g., phorbol 12-myristate 13-acetate (PMA) and ionomycin; final concentration: 50 nM PMA, 1 &#181;M ionomycin per well). Prepare updated flow cytometry compensations for the date of the experiment. 
		NOTE: Titrate the antibodies prior use (see discussion).</li>
	</ol>
	</li>
	<li>Switch off the light and add the anti-CD107a antibody (2 &#181;l, final concentration: 1:100) to each well with NK cells on the 96-V-well plate.</li>
	<li>Gently mix the K562 cells by pipetting them up and down for at least 5 times.
	<ol>
		<li>From the wells containing the NK cell/anti-CD107a antibody solution, identify the ones planned for stimulation with K562 cells. Add 100 &#181;l/well of the K562 cell solution into these wells. Mix carefully by pipetting up and down for at least 5 times.</li>
		<li>Add interleukin-2 (IL-2; final concentration 100 U/ml) and interleukin-15 (IL-15; final concentration 10 ng/ml) to the wells containing K562 cells and the NK cell/anti-CD107a antibody solution.
		<ol>
			<li>Optional: Test the impact of either K562 cells or cytokines alone on the distributed NK cells to decipher tumor-induced versus cytokine-induced NK cell functions.</li>
		</ol>
		</li>
		<li>Identify the wells on the 96-V-well plate planned as negative controls without stimulus. Add 100 &#181;l/well CM to these wells containing only the NK cell/anti-CD107a antibody solution. Mix carefully by pipetting up and down for at least 5 times.
		<ol>
			<li>Optional: For a positive control, add 100 &#181;l CM containing PMA and ionomycin (final concentration: 50 nM PMA, 1 &#181;M ionomycin per well) to a well with the NK cell/anti-CD107a antibody solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubate the Cells for 3 hr in the Dark at 37 &#176;C and 5% CO2.</li>
	<li>Prepare a protein transport inhibitor solution (e.g., brefeldin A and/or monensin) in CM. After 1 hr of incubation, add the solution to each well of the 96-V-well plate with the light switched off (final concentration: 0.5-1&#181;M brefeldin A and 2-3 &#181;M monensin). Mix carefully by pipetting up and down for at least 5 times. Continue the incubation for the remaining 2 hr.</li>
</ol>
5. Surface and Intracellular Staining
<ol>
	<li>After the 3 hr incubation period, mix the cells within the wells of the 96-V-well plate carefully by pipetting up and down for at least 5 times and transfer them into flow cytometry tubes. Add 1 ml/tube of WB. Pellet cells at 400 x g for 4 min.
	<ol>
		<li>Optional: Before centrifugation rinse the wells with 100 &#181;l/well PBS, in order to optimize the cell recovery, by pipetting up and down for at least 5 times before transferring the cells into the respective FACS tube.</li>
	</ol>
	</li>
	<li>Discard the Supernatant and Continue with the Surface Staining.</li>
	<li>Include an amine-reactive fluorescent dye that is non-permeant to live cells with an emission maximum of 423 nm as a fixable dead cell marker (DCM). Perform the staining before (see below) or in parallel with the antibody surface staining depending on the type of the DCM used.
	<ol>
		<li>Re-suspend cells in 99 &#181;l/tube PBS and add 1 &#181;l/tube of the fixable DCM (final concentration: 1:100). Mix cells well using a vortex and incubate them for 15 min at RT in the dark.</li>
		<li>Prepare a master mix solution by mixing together the &#34;surface&#34; antibodies listed in Table 2 (see staining step column highlighted in blue). Use the indicated volumes/tube and multiply them with the number of flow cytometry tubes to be stained.</li>
		<li>After incubation take the flow cytometry tubes with the cells and add 1 ml/tube of WB. Pellet the cells at 400 x g for 4 min.</li>
		<li>Discard the supernatant, re-suspend the cells in 84 &#181;l/tube of WB and add 16 &#181;l/tube of the master mix solution. Mix cells well using a vortex. Incubate cells for 20 min at 4 &#176;C in the dark.</li>
		<li>After 20 min add 1 ml/tube WB and pellet cells at 400 x g for 4 min.</li>
	</ol>
	</li>
	<li>Discard the supernatant and re-suspend the cells in 100 &#181;l/tube of a cold fixation solution (e.g., 2% (final) paraformaldehyde or formaldehyde). Mix cells well using a vortex. Incubate cells for 10 min at 4 &#176;C in the dark.
	<ol>
		<li>Stain the cells for intracellular cytokines/chemokines after this step or store them overnight at 4 &#176;C in WB. 
		NOTE: After overnight incubation, a lower cell recovery may occur. 
		Caution: Paraformaldehyde and formaldehyde are potential carcinogens. Handle them accordingly to the respective Material Safety Data Sheet (MSDS).</li>
	</ol>
	</li>
	<li>Wash cells in 1 ml/tube WB at 400 x g for 4 min and proceed with the permeabilization step.</li>
	<li>Prepare a permeabilization buffer (PB) (e.g., 0.2% saponin, 1% BSA solution in PBS).</li>
	<li>Wash the cells twice in 1 ml/tube of PB at 400 x g for 4 min. Continue with the intracellular staining. 
	Caution: Saponin is potentially irritating. Handle it accordingly to the corresponding Material Safety Data Sheet (MSDS).</li>
	<li>Prepare a master mix solution using the indicated &#34;intracellular&#34; antibody volumes in Table 2 (highlighted in green). Multiply these volumes with the number of tubes to be stained.
	<ol>
		<li>Re-suspend the cells in 90 &#181;l/tube PB and apply 10 &#181;l/tube of the antibody master mix solution. Mix well using a vortex and incubate the cells for 30 min at 4 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Wash Cells Twice in 1 ml/tube of PB at 400 x g for 4 min.</li>
	<li>Discard the supernatant and re-suspend the cells in 400 &#181;l/tube PB. Mix cells well using a vortex. Place cells on ice in the dark until measurement.</li>
</ol>
6. Flow Cytometry Compensation and Acquisition
<ol>
	<li>Select the channels for the different fluorochromes (see Table 2) within the flow cytometry acquisition software in the parameter setup folder. Additionally, choose the channels for FSC-A and -H as well as for SSC-A and -H.</li>
	<li>Load a compensation matrix created for the chosen parameters into the acquisition file.
	<ol>
		<li>Create a compensation matrix using isolated NK cells from step 2.2.8 by performing a single color stain for each used fluorochrome (see Table 2). Use the surface staining protocol described in steps 5.1-5.4. Include an unstained control (without any antibodies) as well as isotype controls and/or fluorescence minus one controls (FMO). 
		NOTE: As an alternative to cell-based compensation, use IgG-binding beads as compensation controls.</li>
		<li>Place each tube for single stain samples and the sample without antibodies into the sample injection port (SIP). Click on the &#34;record&#34; button in the flow cytometry acquisition software and acquire a cell number of at least 5 x 103 NK cells per sample.</li>
		<li>Use the compensation calculation application of the cytometry acquisition software to create a compensation matrix.</li>
	</ol>
	</li>
	<li>Create Dot Plots for Identifying the NK Cell Population.
	<ol>
		<li>Identify single cells using FSC-A/FSC-H and SSC-A/SSC-H dot plots. Click on the &#34;polygon gate&#34; button. Draw a gate around cells, which have a linear distribution pattern in both dot plots. Double-click on the gates to open a new dot plot.</li>
		<li>Choose a FSC-A/SSC-A dot plot to identify the lymphocyte/NK cell population (see Figure 1). Draw a polygon gate around it and double-click on the gate.</li>
	</ol>
	</li>
	<li>Place each sample tube of the NK cell stimulation assay into the SIP. Click on the &#34;record&#34; button and acquire a cell number of at least 5 x 103 NK cells per sample.</li>
</ol>
7. Analysis and Statistics
<ol>
	<li>Open the flow cytometry analysis software program. Click on the loading sample button to open the unstimulated control sample.</li>
	<li>Create a Gating System for the Different NK Cell Subpopulations (see Figure 1).
	<ol>
		<li>Click on the dot plot button to create a FSC-A/SSC-A plot. Click on the rectangle gate button and draw a rectangle gate over all events with a FSC-A value &#62;5 x 104 to exclude debris. Open the gate by double-clicking on the gate.</li>
		<li>Choose again the FSC-A/SSC-A parameters within the new dot plot and click on the polygon gate button. Draw a polygon gate around the lymphocyte population (see Figure 1). Open the gate.</li>
		<li>Select the SSC-A/SSC-H parameter for the new dot plot and draw a polygon gate around all single cells. Single cells demonstrate a linear distribution across the FSC-A/FSC-H and SSC-A/SSC-H parameters (see Figure 1). Open the gate by double-clicking on it.</li>
		<li>Repeat step 7.2.3 using the FSC-A/FSC-H parameters this time.</li>
		<li>Use the parameter CD45 and Dump channel (CD3; CD14; CD19; DCM) to exclude dead cells. Draw a rectangle gate around all CD45 positive and Dump channel negative cells. Open the gate by double-clicking on the gate.</li>
		<li>Identify the whole NK cell population by using the CD56 and Dump channel parameters. Create a rectangle gate around the CD56 positive and Dump channel negative cells. Open the gate by double-clicking on it.</li>
		<li>Draw a rectangle gate around all the three NK cell subsets within a CD56/CD16 dot plot. Identify the different subsets as CD56++CD16-, CD56++CD16+ and CD56+CD16++ NK cell subsets.</li>
	</ol>
	</li>
	<li>Analyze NK Cell Functions for Each Individual NK Cell Subset.
	<ol>
		<li>Click on one of the three NK cell subset gates to open it. Create three different dot plots for CD56/CD107a, CD56/IFN-&#947; and CD56/MIP-1&#946;.</li>
		<li>Draw a rectangle gate for CD56 positive cells, which are positive as well for CD107a, IFN-&#947; or MIP-1&#946; respectively (see Figure 1).</li>
		<li>Repeat step 7.3.1-7.3.2 for each remaining NK cell subset.</li>
	</ol>
	</li>
	<li>Repeat step 7.2 and 7.3 for all stimulated samples. Save all statistical values of each individual sample by clicking on the statistic-exporting button within the analysis software.</li>
	<li>Open the files containing the statistical values of the individual samples to analyze NK cell functions. Select the values for individual NK cell subsets (e.g., CD56++CD16- NK cells) by copying them into another file.
	<ol>
		<li>Subtract the percentage of CD107a positive NK cells, which have not been treated with a stimulus, from the percentage of CD107a positive NK cells, which have been treated with a stimulus. 
		NOTE: Use the result to demonstrate the degranulation capacity of this particular NK cell subset in response to the stimulus.</li>
		<li>Repeat step 7.5.1 for IFN-&#947; and MIP-1&#223;-positive NK cells to demonstrate the cytokine and chemokine production in response to the stimulus.</li>
		<li>Repeat step 7.5.1-7.5.2 for each remaining NK cell subset to evaluate their degranulation capacity and cytokine/chemokine production upon stimulation.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Watzl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+trigger+a+killer:+modulation+of+natural+killer+cell+reactivity+on+many+levels.">How to trigger a killer: modulation of natural killer cell reactivity on many levels.</a> Adv Immunol. 124, 137-170 (2014).</li><li>Khaznadar, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+NK+Cells+in+Acute+Myeloid+Leukemia+Patients+at+Diagnosis+Are+Associated+with+Blast+Transcriptional+Signatures+of+Immune+Evasion.">Defective NK Cells in Acute Myeloid Leukemia Patients at Diagnosis Are Associated with Blast Transcriptional Signatures of Immune Evasion.</a> J Immunol. 195 (6), 2580-2590 (2015).</li><li>Pical-Izard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+natural+killer+cells+in+HLA-matched+HSCT+after+reduced-intensity+conditioning:+impact+on+clinical+outcome.">Reconstitution of natural killer cells in HLA-matched HSCT after reduced-intensity conditioning: impact on clinical outcome.</a> Biol Blood Marrow Transplant. 21 (3), 429-439 (2015).</li><li>Ahlenstiel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+changes+in+natural+killer+cell+function+indicate+virologic+response+to+interferon+therapy+for+hepatitis+C.">Early changes in natural killer cell function indicate virologic response to interferon therapy for hepatitis C.</a> Gastroenterology. 141 (4), 1231-1239 (2011).</li><li>Porrata, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+lymphocyte+recovery+predicts+superior+survival+after+autologous+stem+cell+transplantation+in+non-Hodgkin+lymphoma:+a+prospective+study.">Early lymphocyte recovery predicts superior survival after autologous stem cell transplantation in non-Hodgkin lymphoma: a prospective study.</a> Biol Blood Marrow Transplant. 14 (7), 807-816 (2008).</li><li>Silva, I. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+NK-cell+exhaustion+in+advanced+melanoma+by+Tim-3+blockade.">Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade.</a> Cancer Immunol Res. 2 (5), 410-422 (2014).</li><li>Kiessling, R., Klein, E., Wigzell, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Natural"+killer+cells+in+the+mouse.+I.+Cytotoxic+cells+with+specificity+for+mouse+Moloney+leukemia+cells.+Specificity+and+distribution+according+to+genotype.">"Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype.</a> Eur J Immunol. 5 (2), 112-117 (1975).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> J Vis Exp. (48), (2011).</li><li>Mariani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+production+by+natural+killer+cells+from+nonagenarians.">Chemokine production by natural killer cells from nonagenarians.</a> Eur J Immunol. 32 (6), 1524-1529 (2002).</li><li>Reefman, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+secretion+is+distinct+from+secretion+of+cytotoxic+granules+in+NK+cells.">Cytokine secretion is distinct from secretion of cytotoxic granules in NK cells.</a> J Immunol. 184 (9), 4852-4862 (2010).</li><li>Jacobs, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+Cell+Subgroups,+Phenotype,+and+Functions+After+Autologous+Stem+Cell+Transplantation.">NK Cell Subgroups, Phenotype, and Functions After Autologous Stem Cell Transplantation.</a> Front. Immunol. 6, 583 (2015).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J Immunol Methods. 294 (1-2), 15-22 (2004).</li><li>Theorell, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+viable+quantification+of+inside-out+signals+for+LFA-1+activation+in+human+cytotoxic+lymphocytes+by+flow+cytometry.">Sensitive and viable quantification of inside-out signals for LFA-1 activation in human cytotoxic lymphocytes by flow cytometry.</a> J Immunol Methods. 366 (1-2), 106-118 (2011).</li><li>Brander, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+human+NK+cell-mediated+cytotoxicity+by+exposure+to+ammonium+chloride.">Inhibition of human NK cell-mediated cytotoxicity by exposure to ammonium chloride.</a> J Immunol Methods. 252 (1-2), 1-14 (2001).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+NK+cells+from+human+blood+with+the+RosetteSep+kit+from+StemCell+technologies.">Enrichment of NK cells from human blood with the RosetteSep kit from StemCell technologies.</a> J Vis Exp. (8), e326 (2007).</li><li>Lamoreaux, L., Roederer, M., Koup, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+cytokine+optimization+and+standard+operating+procedure.">Intracellular cytokine optimization and standard operating procedure.</a> Nat. Protoc. 1 (3), 1507-1516 (2006).</li><li>Baginska, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B+degradation+by+autophagy+decreases+tumor+cell+susceptibility+to+natural+killer-mediated+lysis+under+hypoxia.">Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia.</a> Proc Natl Acad Sci U S A. 110 (43), 17450-17455 (2013).</li><li>He, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+flow+cytometry-based+cytotoxic+T-lymphocyte+assay+through+detection+of+cleaved+caspase+3+in+target+cells.">A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells.</a> J Immunol Methods. 304 (1-2), 43-59 (2005).</li><li>Sutton, V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serglycin+determines+secretory+granule+repertoire+and+regulates+NK+cell+and+CTL+cytotoxicity.">Serglycin determines secretory granule repertoire and regulates NK cell and CTL cytotoxicity.</a> FEBS J. 283 (5), 947-961 (2016).</li><li>Sch&#246;nberg, K., Hejazi, M., Uhrberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+clonal+analysis+of+NK+cell+effector+functions+by+multi-parameter+flow+cytometry.">Protocol for the clonal analysis of NK cell effector functions by multi-parameter flow cytometry.</a> Methods Mol Biol. 903, 381-392 (2012).</li><li>Bj&#246;rkstr&#246;m, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+NKG2A,+KIR,+and+CD57+define+a+process+of+CD56dim+NK-cell+differentiation+uncoupled+from+NK-cell+education.">Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.</a> Blood. 116 (19), 3853-3864 (2010).</li><li>Bryceson, Y. T., Ljunggren, H. G., Long, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+requirement+for+induction+of+natural+cytotoxicity+and+intersection+of+activation+signals+by+inhibitory+receptors.">Minimal requirement for induction of natural cytotoxicity and intersection of activation signals by inhibitory receptors.</a> Blood. 114 (13), 2657-2666 (2009).</li><li>Cooper, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+natural+killer+cells:+a+unique+innate+immunoregulatory+role+for+the+CD56(bright)+subset.">Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset.</a> Blood. 97 (10), 3146-3151 (2001).</li><li>Fauriat, C., Long, E. O., Ljunggren, H. G., Bryceson, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+NK-cell+cytokine+and+chemokine+production+by+target+cell+recognition.">Regulation of human NK-cell cytokine and chemokine production by target cell recognition.</a> Blood. 115 (11), 2167-2176 (2010).</li><li>Postow, M. A., Callahan, M. K., Wolchok, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+Checkpoint+Blockade+in+Cancer+Therapy.">Immune Checkpoint Blockade in Cancer Therapy.</a> J Clin Oncol. 33 (17), 1974-1982 (2015).</li><li>Glienke, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+applications+of+CAR-expressing+natural+killer+cells.">Advantages and applications of CAR-expressing natural killer cells.</a> Front Pharmacol. 6, 21 (2015).</li><li>Curran, K. J., Brentjens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+for+cancer+immunotherapy.">Chimeric antigen receptor T cells for cancer immunotherapy.</a> J Clin Oncol. 33 (15), 1703-1706 (2015).</li><li>Zappasodi, R., de Braud, F., Di Nicola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoma+Immunotherapy:+Current+Status.">Lymphoma Immunotherapy: Current Status.</a> Front. Immunol. 6, 448 (2015).</li><li>Laprevotte, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+IL-8+acts+as+a+CD16+co-activator+for+natural+killer-mediated+anti-CD20+B+cell+depletion+in+chronic+lymphocytic+leukemia.">Endogenous IL-8 acts as a CD16 co-activator for natural killer-mediated anti-CD20 B cell depletion in chronic lymphocytic leukemia.</a> Leuk Res. 37 (4), 440-446 (2013).</li><li>Maloney, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IDEC-C2B8+(Rituximab)+anti-CD20+monoclonal+antibody+therapy+in+patients+with+relapsed+low-grade+non-Hodgkin's+lymphoma.">IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma.</a> Blood. 90 (6), 2188-2195 (1997).</li><li>Salles, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+1+study+results+of+the+type+II+glycoengineered+humanized+anti-CD20+monoclonal+antibody+obinutuzumab+(GA101)+in+B-cell+lymphoma+patients.">Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients.</a> Blood. 119 (22), 5126-5132 (2012).</li><li>Terszowski, G., Klein, C., Stern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIR/HLA+interactions+negatively+affect+rituximab-+but+not+GA101+(obinutuzumab)-induced+antibody-dependent+cellular+cytotoxicity.">KIR/HLA interactions negatively affect rituximab- but not GA101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity.</a> J Immunol. 192 (12), 5618-5624 (2014).</li><li>Hsu, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunostimulatory+effect+of+lenalidomide+on+NK-cell+function+is+profoundly+inhibited+by+concurrent+dexamethasone+therapy.">The immunostimulatory effect of lenalidomide on NK-cell function is profoundly inhibited by concurrent dexamethasone therapy.</a> Blood. 117 (5), 1605-1613 (2011).</li><li>Salih, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BCR/ABL-inhibitors+imatinib,+nilotinib+and+dasatinib+differentially+affect+NK+cell+reactivity.">The BCR/ABL-inhibitors imatinib, nilotinib and dasatinib differentially affect NK cell reactivity.</a> Int J Cancer. 127 (9), 2119-2128 (2010).</li><li>Benson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+1+trial+of+the+anti-KIR+antibody+IPH2101+in+patients+with+relapsed/refractory+multiple+myeloma.">A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed/refractory multiple myeloma.</a> Blood. 120 (22), 4324-4333 (2012).</li><li>Vey, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+1+trial+of+the+anti-inhibitory+KIR+mAb+IPH2101+for+AML+in+complete+remission.">A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission.</a> Blood. 120 (22), 4317-4323 (2012).</li><li>Terme, M., Ullrich, E., Delahaye, N. F., Chaput, N., Zitvogel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cell-directed+therapies:+moving+from+unexpected+results+to+successful+strategies.">Natural killer cell-directed therapies: moving from unexpected results to successful strategies.</a> Nat Immunol. 9 (5), 486-494 (2008).</li></ol>

The authors have nothing to disclose.

The described method is an easy, fast and reliable approach to study NK cell functions from whole blood samples of healthy donors or patients. This method offers the great advantage to directly purify NK cells from whole blood, avoiding the time-consuming density gradient centrifugation, which is mandatory for many other purification methods15. Moreover, it requires a smaller sample size compared to &#34;classical&#34; NK cell isolation/enrichment methods, which makes it a suitable alternative for samples of p...

As part of the innate immune system natural killer (NK) cells contribute to the first line of defense against virus-infected or malignantly transformed cells. A system of inhibitory and activating receptors enables them to distinguish between healthy and transformed cells without prior antigen priming in contrast to T cells. Upon target cell encounter NK cells release the content of their cytotoxic granules (e.g., perforin, granzymes) into the immune synapse to kill their target. Moreover, NK cells produce and secrete different kinds of cytokines (e.g., interferon- &#947;: IFN-&#947;; tumor necrosis factor-&#945;: TNF-&#945;) and chemokines (e.g., macrophage inflammatory protein-1&#946;: MIP-1&#946;) upon target cell interaction or cytokine stimulation1.
Sufficient NK cell functions such as cytotoxicity, chemokine and cytokine production have an important impact on the fate of diverse diseases. Leukemia patients show increased relapse rates if they exhibit a defective NK cell profile at diagnosis consisting of reduced IFN-&#947; production and reduced expression of activating NK cell receptors2. An early recovery of NK cell numbers and function including cytokine production upon target cell interaction is associated with a reduced relapse and improved survival rate in patients receiving allogeneic stem cell transplantation3. Moreover, upon initiation of interferon therapy in hepatitis C virus-infected patients the degranulation capacity of peripheral NK cells is stronger in early responders than in non-responders4. NK cell numbers (&#62;80/&#181;l) on day 15 after autologous stem cell transplantation (autoSCT) in patients suffering from lymphoma or multiple myeloma are predictive for an improved progression free and overall survival5. In melanoma patients the expression of the T-cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3), an immune-regulatory protein on NK cells, correlates with disease stage and prognosis6.
Scientists have monitored NK cell functions throughout the last decades. The initial analysis of NK cell cytotoxicity against tumor cells without prior priming was addressed using a 51Cr-release assay7. More recently, scientists developed a non-radioactive method to evaluate the cytotoxicity of expanded NK cells8. Cytokine and chemokine production has been frequently evaluated using enzyme-linked immunosorbent assay (ELISA) techniques9,10. During the last decades these methods have been complemented by flow cytometry-based assays. The use of protein transport inhibitors (e.g., brefeldin A and monensin) and cell permeabilization methods in combination with conventional surface staining protocols have enabled scientists to study chemokine and cytokine production in different specific lymphocyte subsets (e.g., T, B or NK cells)11. Moreover, different flow cytometry-based assays have been developed to monitor T and NK cell cytotoxicity. In 2004 Alter et al. described the surface expression of the lysosome-associated protein CD107a (Lamp1) on NK cells upon target cell encounter as a marker for the degranulation of cytotoxic granules12. Since a wide range of different fluorochromes and multi-channel flow cytometers are available in our days, it has become possible to simultaneously monitor diverse NK cell functions (cytotoxicity, cytokine and chemokine production) in different NK cell subsets. This becomes especially important in situations where sample size is limited, e.g., in biopsies or blood samples of patients suffering from leukopenia.
To test global NK cell functions, the different flow cytometry-based assays can be efficiently combined. Theorell et al. stimulated NK cells from healthy donors with the tumor cell line K562 and analyzed NK cell degranulation, inside-out signal and chemokine production via flow cytometry13. Recently NK cell subgroups, phenotypes and functions in tumor patients during autoSCT were analyzed using flow cytometry-based assays. It was demonstrated that NK cells were able to degranulate and produce cytokines/chemokines upon tumor cell recognition at very early time points after autoSCT11.
Here a protocol is described to evaluate NK cell functions upon interaction with tumor cells including degranulation capacity, chemokine and cytokine production using a flow cytometry-based assay that makes it possible to monitor NK cell functions in different subsets simultaneously.

<table><tbody><tr><td>RPMI 1640 + glutamine</td><td>Invitrogen</td><td>6187-044</td><td></td></tr><tr><td>penicilin/streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>BD Falcon Round Bottom Tube</td><td>BD</td><td>352008</td><td></td></tr><tr><td>fetal calf serum</td><td>Invitrogen</td><td>10270-106</td><td>heat inactivated before use</td></tr><tr><td>T-flask</td><td>Greiner Bio-One</td><td>690195</td><td></td></tr><tr><td>K562 tumor cell line</td><td>DSMZ GmbH</td><td>ACC 10</td><td></td></tr><tr><td>ammonium-chloride-potassium (ACK) lysis buffer</td><td>made in house</td><td>/</td><td>components are listed in the text</td></tr><tr><td>Distilled water: Ampuwa Sp&amp;uuml;ll&amp;ouml;sung 1,000 ml Plastipur</td><td>Fresenius Kabi</td><td>1088813</td><td></td></tr><tr><td>Megafuge 40R Centrifuge</td><td>Heraeus</td><td>/</td><td></td></tr><tr><td>EDTA blood collector tubes</td><td>Sarstedt</td><td>386453</td><td>S-Monovette 7.5 ml, K&lt;sub&gt;3&lt;/sub&gt;EDTA</td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Hematopoietic media (XVIVO)</td><td>Lonza Group Ltd</td><td>BE04-743Q</td><td></td></tr><tr><td>human serum</td><td>DRK Blutspendedienst, Frankfurt/M&amp;nbsp;</td><td>/</td><td>healthy donors with blood type AB; heat inactivated before use</td></tr><tr><td>Neubauer-improved counting chamber, bright line</td><td>Marienfeld superior/ LO-Laboroptik Ltd.</td><td>0640030/ 1110000</td><td></td></tr><tr><td>trypan blue solution (0.4%)</td><td>Invitrogen</td><td>15250-061</td><td></td></tr><tr><td>3% acetic acid with methylene blue</td><td>Stemcell Technologies&amp;nbsp;</td><td>07060</td><td></td></tr><tr><td>Corning 96 Well Clear V-Bottom TC-treated Microplates</td><td>Corning&amp;nbsp;</td><td>3894</td><td></td></tr><tr><td>Falcon 96 Well Round Bottom Not Treated Microplates</td><td>Corning&amp;nbsp;</td><td>351177</td><td></td></tr><tr><td>DPBS (Ca&lt;sup&gt;2+&lt;/sup&gt;- and Mg&lt;sup&gt;2+&lt;/sup&gt;-free)</td><td>Gibco Invitrogen</td><td>14190-169</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A2153-100G</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>08591-1ML-F</td><td></td></tr><tr><td>phorbol 12-myristate 13-acetate (PMA)&amp;nbsp;</td><td>Merck</td><td>524400-1MG</td><td></td></tr><tr><td>ionomycin&amp;nbsp;</td><td>PromoKine</td><td>PK-CA577-1566-5</td><td></td></tr><tr><td>interleukin 15 (IL-15)</td><td>PeproTech</td><td>200-15</td><td></td></tr><tr><td>Proleukin S (IL-2)&amp;nbsp;</td><td>Novartis Pharma</td><td>730523</td><td></td></tr><tr><td>Golgi Stop, Protein Transport Inhibitor (containing Monensin)</td><td>BD Biosciences</td><td>554724</td><td>This product can be used in combination or instead of Golgi Plug. The best combination for the wished experimental setting has to be tested.</td></tr><tr><td>Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A)</td><td>BD Biosciences</td><td>555029</td><td></td></tr><tr><td>paraformaldehyde</td><td>AppliChem</td><td>UN2209</td><td></td></tr><tr><td>saponin</td><td>Sigma Aldrich</td><td>47036</td><td></td></tr><tr><td>flow cytometer: Canto10C</td><td>BD Biosciences</td><td>/</td><td></td></tr><tr><td>FlowJo</td><td>TreeStar Inc.</td><td>/</td><td></td></tr><tr><td>Graph Pad</td><td>Graph Pad Inc.</td><td>/</td><td></td></tr><tr><td>MACSxpress Separator</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-308</td><td></td></tr><tr><td>MACSxpress NK isolation kit</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-185</td><td></td></tr><tr><td>MACSxpress Erythrocyte Depletion Kit, human</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-196</td><td></td></tr><tr><td>MACSmix Tube Rotator&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-090-753</td><td></td></tr><tr><td>anti-human CD3 APC</td><td>Biolegend</td><td>300412</td><td></td></tr><tr><td>anti-human CD3 V450</td><td>BD Biosciences</td><td>560366</td><td></td></tr><tr><td>anti-human CD14 PerCP</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-094-969</td><td></td></tr><tr><td>anti-human CD14 V450</td><td>BD Biosciences</td><td>560349</td><td></td></tr><tr><td>anti-human CD16 PE</td><td>Biolegend</td><td>302008</td><td></td></tr><tr><td>anti-human CD16 PerCP</td><td>Biolegend</td><td>302029</td><td></td></tr><tr><td>anti-human CD19 PE-Cy7</td><td>Biolegend</td><td>302216</td><td></td></tr><tr><td>anti-human CD19 V450</td><td>BD Biosciences</td><td>560353</td><td></td></tr><tr><td>anti-human CD45 BV510</td><td>BD Biosciences</td><td>563204</td><td></td></tr><tr><td>anti-human CD56 FITC</td><td>Biolegend</td><td>345811</td><td></td></tr><tr><td>anti-human CD107a PE</td><td>Biolegend</td><td>328608</td><td></td></tr><tr><td>anti-human IFN-&amp;gamma; AF-647</td><td>BD Biosciences</td><td>557729</td><td></td></tr><tr><td>anti-human MIP-1&amp;beta; APC-H7</td><td>BD Biosciences</td><td>561280</td><td></td></tr><tr><td>DAPI</td><td>Biolegend</td><td>422801</td><td></td></tr><tr><td>Zombie Violet Fixable Viability Kit</td><td>Biolegend</td><td>423113</td><td>fixable dead cell marker</td></tr></tbody></table>

flow cytometry-based assay for the monitoring of nk cell functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

The gating strategy for analyzing the degranulation, cytokine and chemokine production of the whole NK cell population and three different NK cell subsets are illustrated in Figure 1.
Representative results of one healthy donor are illustrated in Figure 2. NK cells without any stimulus produced neither IFN-&#947; nor MIP-1&#946; and did not express CD107a on their surface (Figure 2A</str...

Watch this Scientific Journal Video about Flow Cytometry-based Assay for the Monitoring of NK Cell Functions at JoVE.com

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

flow-cytometry-based-assay-for-the-monitoring-of-nk-cell-functions

Research

JoVE Journal

Immunology and Infection

20.8K Views.  Johann Wolfgang Goethe-University. A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Video: Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Cancer Research

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

Medicine

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Source: McBride, J. E., et al. <a href="https://app.jove.com/v/54779">Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.</a> J. Vis. Exp. (2017).
This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.