The authors thank Dr. Michael N. Sack (National Heart, Lung, and Blood Institute) for support and discussion. This study was supported by the Intramural Research Programs of the National Institutes of Health, National Cancer Institute and National Heart, Lung, and Blood Institute.&#160;JT is supported by the Ramon y Cajal program (grant RYC2018-026050-I) of MICINN (Spain).

All the experiments were performed in accordance with the Declaration of Helsinki&#8217;s ethical principles of medical research. Peripheral blood samples from donors were obtained from the NIH Department of Transfusion Medicine under the 99-CC-0168 IRB approved protocol, with patient written informed consent.
1. Reagent preparation
<ol>
	<li>Reagents for the isolation of NK cells 
	NOTES: Prepare these reagents in a cell culture hood.
	<ol>
		<li>Prepare NK separation buffer: Supplement PBS (pH 7.4) with 1 mM EDTA and 2% Fetal Calf Serum (FCS) that has previously been heat-inactivated (at 56 &#176;C for 30 min).</li>
		<li>Prepare NK culture medium: Supplement Iscove&#39;s Modified Dulbecco&#39;s Media (IMDM) with 10% Human Serum (HS). Sterile filter and store at 2-8 &#176;C. Warm the medium to 37 &#176;C before adding to the cells.</li>
		<li>Resuspend Human IL-15 in PBS at 200 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Reagents for extracellular flux assay
	<ol>
		<li>Prepare assay media: For mitochondrial stress test medium, add 1 mM sodium pyruvate, 2 mM L-glutamine and 10 mM glucose to the base medium; for glycolysis stress test medium, add 2 mM L-glutamine to the base medium. 
		NOTE: The concentrations of glucose, pyruvate and glutamine are provided as recommended by the extracellular flux analyzer manufacturer. However, media composition can be altered by researchers to perfectly match that of the culture medium, if desired.</li>
		<li>Adjust the pH of both media to 7.4 with 0.1 N NaOH using a benchtop pH meter, sterile filter through a 0.2 &#181;M pore size and store at 2-8 &#176;C. Warm media to 37 &#176;C, check pH and readjust to 7.4 before use if required. 
		NOTE: Only ambient CO2 is contained in the atmosphere of the extracellular flux analyzer, the use of media without bicarbonate is critical.</li>
		<li>Prepare stock solutions for reagents: Oligomycin (ATP synthase inhibitor), 10 mM stock solution in DMSO; 2,4-dinitrophenol (DNP, uncoupler), 1 M stock solution in DMSO; antimycin A (complex III inhibitor), 10 mM stock solution in DMSO; rotenone (complex I inhibitor), 10 mM stock solution in DMSO. Make 30 &#181;L aliquots of all reagents and store at -20 &#176;C. 
		NOTE: These guidelines are intended for reagents purchased individually and prepared by the researcher. If reagents are purchased from the analyzer manufacturer instead, follow their guidelines for reagent preparation.</li>
	</ol>
	</li>
</ol>
2. NK cells isolation from peripheral blood
<ol>
	<li>Peripheral blood mononuclear cells (PBMCs) preparation from human blood 
	NOTE: Perform these steps in a cell culture hood. Decontaminate all residues and material in contact with blood with bleach and discard them into the appropriate container to be incinerated.
	<ol>
		<li>Pipette 20 mL of Lymphocyte Separation Medium (LSM) into a 50 mL conical tube.</li>
		<li>Carefully, while keeping the tube at a 30&#730; angle, pipette 20 mL of blood over the LSM, very gently and touching the wall of the tube. Avoid the mixing of blood with the LSM and create a visible and well-defined interphase between the two fluids. 
		NOTE: Peripheral blood or enriched leukapheresis products can be used.</li>
		<li>Centrifuge the tubes for 25 min, 1000 x g at room temperature. Do not use brake, as it could make both phases in the tube (LSM and blood) mix.</li>
		<li>Carefully take out the tubes from the centrifuge and place them in a rack. Check for the presence of a conspicuous layer of cells (mononuclear cells) that will form at the interphase between LSM (clear) and plasma (yellow), while red blood cells pellet at the bottom of the tube.</li>
		<li>Gently aspirate the mononuclear cell layer with a 10 mL plastic pipette (around 5-8 mL) and place it in a new 50 mL conical tube. The lymphocyte interphase of up to 2 different tubes can be pooled together.</li>
		<li>Wash the mononuclear cells 2x by resuspending in 45 mL PBS and centrifuging at 800 x g for 5 min, at room temperature. 
		NOTE: After this step, a pellet of peripheral blood mononuclear cells (PBMCs) is obtained and the researcher can proceed to the NK isolation step.</li>
	</ol>
	</li>
	<li>NK isolation from PBMCs
	<ol>
		<li>Count the cells from 2.1.6 and resuspend them in NK Separation Buffer (1x 108 PBMCs/mL).</li>
		<li>Take 10 mL of the cell resuspension (109 PBMCs ) and place them into a 50 mL tube.</li>
		<li>Add 500 &#181;L (50 &#181;L/mL of buffer) of NK cell isolation antibody mix and 10 &#181;L (1 &#181;L/ mL of buffer) of anti-CD3 positive isolation antibody mix to the PBMCs and incubate at room temperature for 10 min.</li>
		<li>Vortex the magnetic beads and add 1 mL to the mix of PBMCs with antibodies (100 &#181;L beads/ml PBMCs). Incubate for 10 min at room temperature with occasionally stirring.</li>
		<li>Add 35 mL of NK isolation buffer (3.5 ml buffer/ml PBMCs), mix and place on the magnet for 15 min (2.2 x 107 PBMCs/mL). After that time, the beads and cells positively selected (all but NK cells) will have adhered to the walls of the tube.</li>
		<li>Carefully collect the supernatant (containing NK cells) with a 50 mL plastic pipette without touching the sides or the bottom of the tube.</li>
		<li>Count the cells using a cell counter and centrifuge at 800 x g for 5 min.</li>
		<li>Resuspend isolated NK cells at 5 x 106 cells/mL in IMDM containing 10% HS and place them in an incubator at 37 &#730;C with 5% CO2 until the experiment is performed. 
		NOTE: This NK isolation protocol states cell numbers and reagent volumes adapted for the use of a 50 mL tube and a big magnet. This protocol can be scaled up (in case more PBMCs are obtained) by repeating the isolation steps in different tubes or scaled down by reducing the volume in the tube. For final volumes of 14-45 mL a 50 mL polystyrene tube is used, for volumes 4-14, a 15 mL polystyrene tube is used and for volumes 1-4 mL, a 5 mL polystyrene tube is used. The magnet is different and fit the appropriate tube in each case. The amount of antibody mix and magnetics beads can also be scaled according to the number of cells and the final volume.</li>
	</ol>
	</li>
	<li>NK cell population staining for flow cytometry
	<ol>
		<li>Take 0.25 x 106 cells per sample from step 2.2.8, remove the medium by centrifuging at 800 x g for 5 min at room temperature and resuspend the cell pellet in 500 &#181;L of PBS.</li>
		<li>Add viability dye according to the manufacturer&#8217;s recommendation (1 &#181;L of DMSO-reconstituted dye to 1 mL of cell resuspension) and incubate at room temperature for 30 min.</li>
		<li>Wash 2x by resuspending in 5 mL PBS and centrifuging at 800 x g for 5 min, at room temperature.</li>
		<li>Stain with the corresponding anti-human antibodies in 100 &#181;L of IMDM containing 10% HS for 30 min on ice protected from light (see Table 1). All the antibodies can be combined and used in a single staining step.</li>
		<li>Analyze the samples by flow cytometry to evaluate the purity of the NK cell population obtained. Use the cell gating and analysis method that have been previously described18,19.</li>
	</ol>
	</li>
	<li>NK cells stimulation with soluble IL-15
	<ol>
		<li>Resuspend 0.75 x 106 cells from steps 2.2.8 or 2.4.3 in 100 &#181;L of IMDM containing 10% HS in a well of a 96 well-plate (round bottom).</li>
		<li>Dilute human IL-15 to 1 &#181;g/mL in IMDM containing 10% HS. Add 100 &#181;L of the diluted human IL-15 to the cells to reach a final concentration of 0.5 &#181;g/mL. 
		NOTE: 0.5 &#181;g/mL is a saturating concentration of IL-15. Lower concentrations of IL-15 or other cytokines such as IL-2, IL-12 or IL-18 may be tested by researchers if desired.</li>
		<li>Place the cells in the incubator at 37 &#176;C and stimulate for 48 h before performing the extracellular flux assay. Resuspend unstimulated (control) cells at the same concentration and volume in IMDM containing 10% HS without IL-15, and place them in the same incubator for 48 h.</li>
	</ol>
	</li>
</ol>
3. Hydration of sensor cartridge
NOTE: The 96 probe tips of the sensor cartridge contain individual solid-state fluorophores for O2 and H+ that need to be hydrated in order to detect O2 and pH changes.
<ol>
	<li>Turn on the analyzer and let it warm up to 37 &#176;C.</li>
	<li>Open the sensor cartridge package and separate the sensor cartridge from the utility plate. Add 200 &#181;L of the calibrant solution in each well of the utility plate and put back the sensor cartridge onto the plate, validating that the sensors are completely submerged in the solution. For optimum results incubate the cartridge overnight at 37 &#176;C in a CO2-free incubator that is properly humidified to prevent evaporation. Prevent bubble formation under the sensors during hydration. 
	NOTE: The minimum cartridge hydration time is 4 h at 37 &#176;C in a CO2-free incubator. Alternatively, overnight hydration of the sensor cartridge with 200 &#181;L of sterile water at 37 &#176;C in a CO2-free incubator, followed by an incubation of the sensor cartridge with 200 &#181;L of pre-warmed calibrant solution 45 &#8211; 60 min prior to the start of the run, can be used.</li>
</ol>
4. Extracellular flux assay
<ol>
	<li>Preparation of adhesive-coated plates 
	NOTE: Since the measurement of metabolic parameters takes place in a microchamber formed at the bottom of the 96-well assay plate, suspension cells must first be adhered to the bottom of the well. A cell adhesive extracted from the mussel Mytilus edulis is employed. The manufacturer of the cell adhesive recommends a coating concentration of 1 to 5 &#181;g/cm2. The well of the analyzer cell culture microplate has a surface of approximately 0.110 cm2. Thus, for a 5 &#181;g/cm2 concentration, around 0.55 &#181;g of adhesive are required. As 25 &#181;L of the adhesive solution will be used to coat each well, the optimal adhesive solution concentration for the analyzer cell culture microplates is around 22.4 &#181;g/mL (22.4 &#181;g/mL x 0.025 mL = 0.56 &#181;g).
	<ol>
		<li>Prepare 2.5 mL of cell adhesive solution (22.4 &#181;g/mL) in 0.1 M sodium bicarbonate, pH 8.0. Bicarbonate provides the optimum pH for cell adherent performance which, according to the manufacturer, is between 6.5 and 8.0.</li>
		<li>Pipette 25 &#181;L of the cell adhesive solution to each well of the assay plate and incubate at room temperature for 20 min. After that, remove the solution and wash 2x with 200 &#181;L of sterile water/well. Let the wells dry by keeping the plate open for 15 minutes inside a cell culture hood. 
		NOTE: Coated plates may be stored for up to 1 week at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cell seeding in plates coated with adhesive
	<ol>
		<li>Centrifuge cells from step 2.4.3&#160;at 200 x g for 5 min at room temperature. Remove supernatants and wash cells in warmed mitochondrial stress test medium (if a mitochondrial stress test is being performed) or glycolysis stress test medium (if a glycolysis stress test is being performed). Pellet cells again and resuspend to the preferred cell concentration in the same medium (resuspension volume will depend on the cell concentration chosen; since each well will contain 180 &#181;L of the cell suspension, prepare 0.26 x 106, 0.52 x 106, 1.04 x 106, 2.08 x 106, 4.17 x 106 and 8.33 x 106 cells/mL cell suspensions for 0.047 x 106, 0.094 x 106, 0.187 x 106, 0.375 x 106, 0.75 x 106 and 1. 5 x 106 cells per well respectively).</li>
		<li>Plate 180 &#181;L of cell suspension per well along the side of each well. A multichannel pipette is recommended. Use wells A1, A12, H1, and H12 of the analyzer culture plate as control wells for background correction. Add 180 &#181;L of the assay medium in these wells (no cells). Additional control wells can be used if desired and if there is enough space in the plate. 
		NOTE: The presence of serum may cause poor cell attachment.</li>
		<li>Incubate the plate for 30 min at 37 &#176;C in a CO2-free incubator. Prepare 10x compounds in the meantime (see step 4.3 below).</li>
		<li>Change the centrifuge settings to zero braking. Centrifuge the plate at 200 x g for 5 min. Observe the cells under the microscope to check that they form a monolayer at the bottom of the well. Transfer the plates back to the CO2-free incubator for 25 min. For best results, total time after plating should be no greater than around 1 h.</li>
	</ol>
	</li>
	<li>Preparation of 10x working solutions to load into sensor cartridge 
	NOTE: Each of the 96 probe tips of the sensor cartridge harbors 4 ports (A, B, C and D) that can be used to inject compounds sequentially into individual wells.
	<ol>
		<li>To perform mitochondrial stress test, prepare 2.5 mL each of 10 &#181;M oligomycin, 1 mM DNP, and a mixture of 10 &#181;M rotenone and 10 &#181;M antimycin A, in mitochondrial stress assay medium (use the stock solutions from step 1.2.3). Final concentrations in the well after injection will be 1 &#181;M oligomycin, 0.1 mM DNP and 1 &#181;M antimycin A/rotenone.</li>
		<li>To perform glycolysis stress test, prepare 2.5 mL of a mixture of 10 &#181;M rotenone and 10 &#181;M antimycin A in glycolysis stress assay medium (use the stock solutions from step 1.2.3). Dissolve glucose in glycolysis stress test medium for a 100 mM solution and 2-deoxy-glucose (2-DG) in glycolysis stress test medium for a 500 mM solution. Final concentrations in the well after injection will be 10 mM glucose, 1 &#181;M antimycin A/rotenone and 50 mM 2-DG.</li>
		<li>Warm solutions to 37 &#176;C, check pH and readjust to 7.4 if required. Load compounds prepared in step 4.3.1. (for a mitochondrial stress test) or 4.3.2 (for a glycolysis stress test) into ports A, B and C of the hydrated sensor cartridge (from step 3.2) using a multichannel micropipettor and the port-loading guides provided with the cartridge, as shown in Table 2. 
		NOTE: To ensure proper injection in all wells during the assay, each series of ports that are used (e.g., all ports A) must contain the same injection volume across the entire sensor cartridge. This applies to background correction wells and even to those wells not used in the experiment.</li>
		<li>Incubate the loaded sensor cartridge at 37 &#176;C in a CO2-free incubator while setting up the program.</li>
	</ol>
	</li>
	<li>Setting up extracellular flux assay protocols
	<ol>
		<li>Open the extracellular flux analyzer software, and using the Group Definitions and Plate Map tabs indicate groups of wells that have similar conditions (e.g., wells with the same number of cells, or wells with either resting cells or IL-15-stimulated cells). Also, indicate background correction wells (by default A1, A12, H1, and H12 will be set, but additional wells can be used) and empty wells.</li>
		<li>Set up the program described in Table 3 in the software using the Protocol tab.</li>
		<li>Begin the program using the Run Assay tab. Place the sensor cartridge (hydrated and loaded with 10x compounds) and utility plate onto the tray. After the calibration step (15 &#8211; 20 min), when prompted, replace the calibrant plate for the assay plate (without lid) with attached cells. After this, the run is fully automated (the machine will perform all measurements and injections). 
		NOTE: It is possible to perform a mitochondrial stress test and a glycolytic stress test in the same plate, as long as the specific compounds are loaded into the proper ports (oligomycin, DNP and antimycin/rotenone in ports A, B and C respectively of the wells where a mitochondrial stress test is performed; glucose, antimycin/rotenone and 2-DG in ports A, B and C respectively of the wells where a glycolysis stress test is performed), the same volumes for injections are used in each series of ports and wells for each test are properly identified using the Group Definitions and Plate Map tabs of the software.</li>
		<li>After the completion of the run, retrieve the data and analyze them using the software.</li>
	</ol>
	</li>
</ol>
5. Cell number determination
NOTE: Results can be normalized to account for possible differences in cell number. Two major approaches described below can be used.
<ol>
	<li>DNA content determination
	<ol>
		<li>Remove the remaining assay medium with a pipette from each well. When aspirating medium, take care not to disturb the cells. The plate can be stored at -20 &#176;C until analysis. 
		NOTE: The freezing step is important for the efficient cell lysis and DNA content determination.</li>
		<li>Prepare a 1x solution of cell proliferation assay cell-lysis buffer (20x) in distilled water (200 &#181;L/well).</li>
		<li>Add the cell proliferation assay dye stock solution (400x) into the 1x cell-lysis buffer. For the detection of 50 to 50.000 cells per well, use 1x cell proliferation assay dye. For higher cell numbers, using the cell proliferation assay dye at a final concentration higher than 1x is recommended. In this case, use the cell proliferation assay dye at a 5x final concentration (dilute the cell proliferation assay stock solution 80-fold into 1x cell-lysis buffer).</li>
		<li>Add 200 &#181;L of the cell proliferation assay reagent to each well. Incubate the samples for 2&#8211;5 min at room temperature. Protect from light.</li>
		<li>Measure the fluorescence of the samples at excitation: 480 nm; emission: 520 nm using a fluorescence microplate reader according to manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
	<li>Protein content determination
	<ol>
		<li>Carefully, aspirate completely the assay medium from each well without touching the cells and freeze the cells at -20 &#730;C for at least 1 h. Alternatively, the cells can be kept frozen for longer times (up to 1 week) until the analysis&#160;is performed.</li>
		<li>Add 50 &#181;L of radioimmunoprecipitation assay (RIPA) lysis medium supplemented with 1x protease inhibitors (from a 100x stock solution) to each well. Place the plate on a shaker for 5 min at room temperature, and then incubate the plate on ice for 30 min for a complete cell lysis.</li>
		<li>Centrifuge the plate for 5 min at 200 x g at room temperature. This step will pellet cellular debris to prevent interference with the protein measurement.</li>
		<li>Measure protein concentration by bicinchoninic acid (BCA) assay according to manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Long, E. O., Kim, H. S., Liu, D., Peterson, M. E., Rajagopalan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+natural+killer+cell+responses:+integration+of+signals+for+activation+and+inhibition.">Controlling natural killer cell responses: integration of signals for activation and inhibition.</a> Annual Review of Immunology. 31, 227-258 (2013).</li><li>Caligiuri, M. A. <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.">Human natural killer cells.</a> Blood. 112 (3), 461-469 (2008).</li><li>Anton, O. M., Vielkind, S., Peterson, M. E., Tagaya, Y., 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=NK+Cell+Proliferation+Induced+by+IL-15+Transpresentation+Is+Negatively+Regulated+by+Inhibitory+Receptors.">NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.</a> Journal of Immunology. 195 (10), 4810-4821 (2015).</li><li>Henney, C. S., Kuribayashi, K., Kern, D. E., Gillis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-2+augments+natural+killer+cell+activity.">Interleukin-2 augments natural killer cell activity.</a> Nature. 291 (5813), 335-338 (1981).</li><li>Anton, O. 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=Trans-endocytosis+of+intact+IL-15Ralpha-IL-15+complex+from+presenting+cells+into+NK+cells+favors+signaling+for+proliferation.">Trans-endocytosis of intact IL-15Ralpha-IL-15 complex from presenting cells into NK cells favors signaling for proliferation.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (1), 522-531 (2020).</li><li>Marcais, 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=The+metabolic+checkpoint+kinase+mTOR+is+essential+for+IL-15+signaling+during+the+development+and+activation+of+NK+cells.">The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.</a> Nature Immunology. 15 (8), 749-757 (2014).</li><li>Mookerjee, S. A., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+Maximum+Glycolytic+Capacity+Using+Extracellular+Flux+Measurements.">Determining Maximum Glycolytic Capacity Using Extracellular Flux Measurements.</a> PloS One. 11 (3), 0152016 (2016).</li><li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>Cheng, 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=Mitochondrial+Proton+Leak+Plays+a+Critical+Role+in+Pathogenesis+of+Cardiovascular+Diseases.">Mitochondrial Proton Leak Plays a Critical Role in Pathogenesis of Cardiovascular Diseases.</a> Advances in Experimental Medicine and Biology. 982, 359-370 (2017).</li><li>Terren, I., Orrantia, A., Vitalle, J., Zenarruzabeitia, O., Borrego, F. <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+Metabolism+and+Tumor+Microenvironment.">NK Cell Metabolism and Tumor Microenvironment.</a> Frontiers in Immunology. 10, 2278 (2019).</li><li>Felices, 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=Continuous+treatment+with+IL-15+exhausts+human+NK+cells+via+a+metabolic+defect.">Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect.</a> Journal of Clinical Investigation Insight. 3 (3), 96219 (2018).</li><li>Miller, J. S., 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+First-in-Human+Phase+I+Study+of+Subcutaneous+Outpatient+Recombinant+Human+IL15+(rhIL15)+in+Adults+with+Advanced+Solid+Tumors.">A First-in-Human Phase I Study of Subcutaneous Outpatient Recombinant Human IL15 (rhIL15) in Adults with Advanced Solid Tumors.</a> Clinical Cancer Research. 24 (7), 1525-1535 (2018).</li><li>Conlon, K. 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=IL15+by+Continuous+Intravenous+Infusion+to+Adult+Patients+with+Solid+Tumors+in+a+Phase+I+Trial+Induced+Dramatic+NK-Cell+Subset+Expansion.">IL15 by Continuous Intravenous Infusion to Adult Patients with Solid Tumors in a Phase I Trial Induced Dramatic NK-Cell Subset Expansion.</a> Clinical Cancer Research. 25 (16), 4945-4954 (2019).</li><li>Dubois, S., 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=IL15+Infusion+of+Cancer+Patients+Expands+the+Subpopulation+of+Cytotoxic+CD56(bright)+NK+Cells+and+Increases+NK-Cell+Cytokine+Release+Capabilities.">IL15 Infusion of Cancer Patients Expands the Subpopulation of Cytotoxic CD56(bright) NK Cells and Increases NK-Cell Cytokine Release Capabilities.</a> Cancer Immunology Research. 5 (10), 929-938 (2017).</li><li>Traba, 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=Fasting+and+refeeding+differentially+regulate+NLRP3+inflammasome+activation+in+human+subjects.">Fasting and refeeding differentially regulate NLRP3 inflammasome activation in human subjects.</a> Journal of Clinical Investigation. 125 (12), 4592-4600 (2015).</li><li>Traba, J., Miozzo, P., Akkaya, B., Pierce, S. K., Akkaya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Optimized+Protocol+to+Analyze+Glycolysis+and+Mitochondrial+Respiration+in+Lymphocytes.">An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes.</a> Journal of Visualized Experiments. (117), e54918 (2016).</li><li>Horan, M. P., Pichaud, N., Ballard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+quantifying+mitochondrial+dysfunction+in+complex+diseases+of+aging.">Review: quantifying mitochondrial dysfunction in complex diseases of aging.</a> Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 67 (10), 1022-1035 (2012).</li><li>Tognarelli, S., Jacobs, B., Staiger, N., Ullrich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry-based+Assay+for+the+Monitoring+of+NK+Cell+Functions.">Flow Cytometry-based Assay for the Monitoring of NK Cell Functions.</a> Journal of Visualized Experiments. (116), e54615 (2016).</li><li>Theorell, J., 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=Analysis+of+Intracellular+Ca(2+)+Mobilization+in+Human+NK+Cell+Subsets+by+Flow+Cytometry.">Analysis of Intracellular Ca(2+) Mobilization in Human NK Cell Subsets by Flow Cytometry.</a> Methods in Molecular Biology. 1441, 117-130 (2016).</li><li>Mookerjee, S. A., Gerencser, A. A., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+intracellular+rates+of+glycolytic+and+oxidative+ATP+production+and+consumption+using+extracellular+flux+measurements.">Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements.</a> Journal of Biological Chemistry. 292 (17), 7189-7207 (2017).</li><li>O'Brien, K. L., Finlay, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunometabolism+and+natural+killer+cell+responses.">Immunometabolism and natural killer cell responses.</a> Nature Reviews: Immunology. 19 (5), 282-290 (2019).</li><li>Ahn, B. H., 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+role+for+the+mitochondrial+deacetylase+Sirt3+in+regulating+energy+homeostasis.">A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14447-14452 (2008).</li><li>Stram, A. R., Payne, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+in+mitochondria:+protein+signaling+in+the+powerhouse.">Post-translational modifications in mitochondria: protein signaling in the powerhouse.</a> Cellular and Molecular Life Sciences. 73 (21), 4063-4073 (2016).</li><li>Armstrong, J. 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=Oxidative+stress+alters+mitochondrial+bioenergetics+and+modifies+pancreatic+cell+death+independently+of+cyclophilin+D,+resulting+in+an+apoptosis-to-necrosis+shift.">Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift.</a> Journal of Biological Chemistry. 293 (21), 8032-8047 (2018).</li><li>Mookerjee, S. A., Goncalves, R. L. S., Gerencser, A. A., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contributions+of+respiration+and+glycolysis+to+extracellular+acid+production.">The contributions of respiration and glycolysis to extracellular acid production.</a> Biochimica et Biophysica Acta. 1847 (2), 171-181 (2015).</li><li>Mookerjee, S. A., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+and+Analysis+of+Extracellular+Acid+Production+to+Determine+Glycolytic+Rate.">Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate.</a> Journal of Visualized Experiments. (106), e53464 (2015).</li><li>Lichtshtein, D., Kaback, H. R., Blume, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+lipophilic+cation+for+determination+of+membrane+potential+in+neuroblastoma-glioma+hybrid+cell+suspensions.">Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (2), 650-654 (1979).</li><li>Michelet, X., 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=Metabolic+reprogramming+of+natural+killer+cells+in+obesity+limits+antitumor+responses.">Metabolic reprogramming of natural killer cells in obesity limits antitumor responses.</a> Nature Immunology. 19 (12), 1330-1340 (2018).</li></ol>

The authors declare that they have no competing financial interests.

In this paper, we have established a protocol for efficiently isolating and culturing pure and viable primary human NK cells from peripheral blood. We have also optimized the conditions for the measurement of the metabolic activity of these NK cells assessed by oxygen consumption rate and extracellular acidification rate by using an extracellular flux analyzer. Compared to other respirometric methods, the extracellular flux analyzer is fast, requires small numbers of cells, and allows high throughput screenings. However,...

Natural Killer (NK) cells are innate lymphocytes that mediate anti-tumor and anti-viral responses. NK cells comprise 5-15% of all lymphocytes in human peripheral blood, and can be also found in spleen, liver, bone marrow and lymph nodes. NK cells do not express polymorphic clonotypic receptors, such as T-cell receptors (TCR) or B-cell receptors (BCR). In contrast, the activation of their cytolytic functions is prompted by the engagement of receptors that recognize invariable ligands on the surface of a target cell1,2.
Resting human NK cells isolated from peripheral blood can survive for several days in culture medium supplemented with human serum. Activation of NK cells by cytokines such as IL-15 or IL-2 drives the cells to proliferation and to an increase of their killing ability, amongst other effects3,4,5. Several studies have shown a direct correlation between NK cell activation and changes in their metabolic activity6. These metabolic changes are destined to meet the particular requirements of the cells in terms of energy and biosynthesis.
Aerobic cells and organisms obtain energy through a series of chemical reactions that involve the catabolism and oxidation of carbohydrates, fat and proteins. Through a combination of glycolysis, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, eukaryotic cells meet the majority of their ATP demand and obtain intermediates required as building blocks for macromolecules essential for cell growth and proliferation. The process of glycolysis (Figure 1A) starts with the entry of glucose in the cell. In the cytosol, glucose is phosphorylated and transformed into pyruvate (with a net production of 2 molecules of ATP per glucose molecule), which can be reduced to lactate or transported into the mitochondria to be transformed into Acetyl-CoA and enter the TCA cycle. The TCA cycle continues cycling fueled with more molecules of Acetyl-CoA and produces CO2 (that eventually will diffuse outside the cell and, by reacting with H2O in the medium, will generate carbonic acid that will lead to the acidification of the medium) and NADH, the molecule in charge of donating electrons to the electron transport chain (ETC). The electrons travel through different protein complexes and are finally accepted by oxygen. These complexes (I, III and IV) also pump H+ from the mitochondrial matrix into the intermembrane space. As a consequence of the electrochemical gradient generated, the H+ will enter again to the matrix through the complex V (ATP-synthase), investing the potential energy accumulated into the generation of ATP.
Both glycolysis and mitochondrial respiration can be blocked at different points by using inhibitors. The knowledge and usage of these inhibitors was the basis for the development of the extracellular flux assay. By measuring two simple parameters in real time such as pH and oxygen, the extracellular flux analyzer infers the rate of glycolysis and mitochondrial respiration in a 96-well plate. The glycolysis stress test is performed in a basal medium without glucose (Figure 1B)7. The first measurements of the extracellular acidification rate (ECAR) are indicative of glycolysis-independent acidification. It is referred to as non-glycolytic acidification and correlates with CO2 produced by the TCA that, as explained before, combines with H2O in the medium to generate H+ (TCA-linked ECAR). The first injection is glucose to induce glucose utilization and boost glycolysis. The second injection combines both rotenone, a Complex I inhibitor, and antimycin A, a Complex III inhibitor together, to block the ETC. Cells respond to this dramatic decrease in mitochondrial ATP production by activating glycolysis to maintain cellular ATP levels, and this represents the amount of glycolysis that is not used by the cell in the basal state but could be potentially recruited in response to increases in ATP demand (compensatory glycolysis). The third injection is the glucose analog 2-Deoxyglucose (DG), which competes with glucose as a substrate for the enzyme hexokinase. The product of this phosphorylation, 2-deoxyglucose-6-phosphate cannot be transformed into pyruvate, and therefore glycolysis is blocked, which lowers the ECAR to its minimum. The ECAR measured at this point includes other sources of extracellular acidification that are not attributed to glycolysis or respiratory activity as well as any residual glycolysis not fully inhibited by 2-DG (post 2-DG-acidification).
The mitochondrial stress test is performed in a medium with glucose (Figure 1C)8. The first measurements of the oxygen consumption rate (OCR) correspond to the base line of mitochondrial respiration (basal respiration). The first injection is oligomycin, which inhibits the return of protons through the ATP synthase (complex V), blocking ATP synthesis and thus rapidly hyperpolarize the mitochondrial membrane, which prevents further proton pumping through respiratory complexes, and leads to a decrease in OCR. The comparison between the baseline respiration and the value given by addition of oligomycin represents the ATP-linked respiration. The remaining oligomycin-insensitive rate of oxygen consumption is called proton leak, which represents the flow of protons through the lipid bilayer or proteins in the inner mitochondrial membrane such as the adenine nucleotide translocase9. The second injection is the uncoupler 2,4-dinitrophenol (DNP), an ionophore that induces a massive entry of H+ into the mitochondrial matrix, which leads to depolarization of the mitochondrial membrane and disruption of mitochondrial ATP synthesis. Cells respond to the dissipation of the proton-motive force by increasing the rate of electron transport and oxygen consumption to maximum levels in a futile attempt to recover membrane potential (maximal respiratory capacity). The difference between the maximal respiratory capacity and the basal respiration is the spare respiratory capacity of the cell, which represents the amount of respiration that is not used by the cell to generate ATP in the basal state but could be potentially recruited in response to increases in ATP demand or under conditions of stress8. The third injection is a combination of rotenone and antimycin A. This injection completely stops the ETC and OCR decreases to its lowest level, with the remaining oxygen consumption being non-mitochondrial (caused by NADPH-oxidases, etc.).
Changes in metabolic pathways could somehow predict the functioning of NK cells, as it has been suggested that continuous activation of NK cells with cytokines in vitro could lead to NK cell exhaustion by the study of different metabolic pathways10,11. The correlation between NK cell metabolic status and function is very important from the point of view of cancer immunotherapy. In this field, activation of NK cells with infusion of IL-15, alone or in combination with monoclonal therapeutic antibodies have been tested in order to improve tumor cell killing12,13,14. The knowledge of the metabolic status of the NK cells in response to these treatment strategies would provide a valuable predictor of NK cell activation status and killing function.
The study of metabolic pathways in other myeloid and lymphoid cells such as monocytes, T and B cells has been described15 and optimized methods have been published16. In this protocol we provide a method that combines both an NK isolation protocol that yields high numbers of pure and viable NK cells and an optimized protocol to measure metabolic activity using an extracellular flux analyzer. Here we show that this is a valid method for the study of metabolic changes in resting and IL-15 activated human NK cells. For the extracellular flux assay, parameters such as cell number and drug concentrations have been tested and optimized. Compared with other respirometric methods, the extracellular flux analyzer is fully automated and able to test in real time, with very low quantities of cells, up to 92 samples simultaneously, and thus allows high throughput screenings (with multiple samples and replicates) in a relatively quick manner17.
This method can be used by researchers interested in assessing NK cell function by studying NK cell metabolism. It could be applied as well to cells activated by other cytokines, antibodies or soluble stimuli.

<table>
	<tbody>
		<tr>
			<td>2-Deoxy-D-glucose (2-DG)</td>
			<td>MilliporeSigma</td>
			<td>D8375-5G</td>
			<td>Glycolyisis stress test injector compound</td>
		</tr>
		<tr>
			<td>2,4-Dinotrophenol (2,4-DNP)</td>
			<td>MilliporeSigma</td>
			<td>D198501</td>
			<td>ETC uncoupler / mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>96 Well Cell Culture Plate/ Round bottom with Lid</td>
			<td>Costar</td>
			<td>3799</td>
			<td>NK cell culture</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>MilliporeSigma</td>
			<td>A8674</td>
			<td>Complex III inhibitor / glycolysis and mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>BD FACSDIVA Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow data acquisition</td>
		</tr>
		<tr>
			<td>BD LSR Fortessa</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow data acquisition</td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>Cell adhesive</td>
		</tr>
		<tr>
			<td>CyQUANT cell proliferation assay</td>
			<td>ThermoFisher Scientific</td>
			<td>C7026</td>
			<td>Cell proliferation Assay for DNA quantification. Contains cell-lysis buffer and CyQUANT GR dye</td>
		</tr>
		<tr>
			<td>EasySep Human CD3 Positive Selection Kit II</td>
			<td>Stemcell technologies</td>
			<td>17851</td>
			<td>NK cell isolation from PBMCs</td>
		</tr>
		<tr>
			<td>EasySep Human NK cell Enrichment Kit</td>
			<td>Stemcell technologies</td>
			<td>19055</td>
			<td>NK cell isolation from PBMCs</td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stemcell technologies</td>
			<td>18001</td>
			<td>NK cell isolation from PBMCs</td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8</td>
			<td>Quality Biological</td>
			<td>10128-446</td>
			<td>NK sell separation buffer</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Falcon-Fisher Scientific</td>
			<td>352235</td>
			<td>Flow cytometry experiment</td>
		</tr>
		<tr>
			<td>Falcon 50 ml Conical tubes</td>
			<td>Falcon-Fisher Scientific</td>
			<td>14-432-22</td>
			<td>NK cell separation</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>NK cell separation buffer</td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow data analysis</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>MilliporeSigma</td>
			<td>G8270</td>
			<td>Component of mitochondrial stress test medium. Glycolysis stress test injector compound</td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>78429</td>
			<td>Protease inhibitor 100X. Use in RIPA lysis buffer</td>
		</tr>
		<tr>
			<td>Human IL-15</td>
			<td>Peprotech</td>
			<td>200-15-50ug</td>
			<td>NK cell stimulation</td>
		</tr>
		<tr>
			<td>Human serum (HS)</td>
			<td>Valley Biomedical</td>
			<td>9C0539</td>
			<td>NK cell culture medium supplement</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td>NK cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>25030-081</td>
			<td>Component of stress test media</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>L34965</td>
			<td>Viability dye for flow cytometry staining</td>
		</tr>
		<tr>
			<td>LSM</td>
			<td>mpbio</td>
			<td>50494X</td>
			<td>PBMCs separation from human blood</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD3 BV711</td>
			<td>BD Biosciences</td>
			<td>563725</td>
			<td>T cell flow cytometry staining</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD56 PE</td>
			<td>BD Pharmingen</td>
			<td>555516</td>
			<td>NK flow cytometry staining</td>
		</tr>
		<tr>
			<td>Mouse anti-human NKp46 PE</td>
			<td>BD Pharmingen</td>
			<td>557991</td>
			<td>NK flow cytometry staining</td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>MilliporeSigma</td>
			<td>75351</td>
			<td>Complex V inhibitor / mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>NK cell separation buffer</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td>For determination of protein concentration</td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Boston BioProducts</td>
			<td>BP-115</td>
			<td>Cell lysis</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>MilliporeSigma</td>
			<td>R8875</td>
			<td>Complex II inhibitor / glycolysis and mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>Seahorse Wave Controller Software</td>
			<td>Agilent</td>
			<td></td>
			<td>Controller for the Seahorse XFe96 Analyzer</td>
		</tr>
		<tr>
			<td>Seahorse Wave Desktop Software</td>
			<td>Agilent</td>
			<td></td>
			<td>For data analysis</td>
		</tr>
		<tr>
			<td>Seahorse XF Base Medium</td>
			<td>Agilent</td>
			<td>102353-100</td>
			<td>Extracellular Flux assay base medium</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Extracellular Flux Analyzer</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent</td>
			<td>102416-100</td>
			<td>Includes 20 XF96 cell culture plates, 18 XFe96 sensor cartridges, loading guides for transferring compounds to the assay cartridge, and 1 bottle of calibrant solution (500 ml).</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>MilliporeSigma</td>
			<td>S5761</td>
			<td>To prepare the Cell-Tak solution</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11360-070</td>
			<td>Component of mitochondrial stress test medium</td>
		</tr>
	</tbody>
</table>

analysis of human natural killer cell metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

Isolation of NK cells from peripheral blood provides a pure and viable population
The extracellular flux assay is based on the measurement of H+ and O2 concentration in the well and will not distinguish among different populations of cells or their viability. For this reason, obtaining a highly pure and viable population of the cell of interest was the key step to succeed in these experiments.
The isolation of NK cells from per...

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Analysis of Human Natural Killer Cell Metabolism

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

analysis-of-human-natural-killer-cell-metabolism

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Immunology and Infection

6.7K Views.  National Institutes of Health. In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

Video: Analysis of Human Natural Killer Cell Metabolism

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 ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT 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 ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...