Kasper M&#248;lgaard&#160;and Anne Rahbech received grants from T&#248;mmermester J&#248;rgen Holm og Hustru Elisa f. Hansens Mindelegat. Kasper M&#248;lgaardalso received a grant from B&#248;rnecancerfonden.

Experiments were carried out under the guidelines from Herlev Hospital and the Capital Region of Denmark.
NOTE: This protocol contains instructions for both an Optimization run and an Assays run. It is clearly written in the text when instructions are for an Optimization run or an Assay run. Run an Optimization run before continuing with the Assay runs
1. Human peripheral blood mononuclear (PBMC) isolation from buffy coats
<ol>
	<li>PBMC isolation
	<ol>
		<li>Collect buffy coats from the appropriate institution (collected in blood collection bags). Buffy coats originate from healthy donors. Exclude donors that have recently used painkillers.</li>
		<li>Spray the blood collection bag containing the buffy coat with 70% ethanol before transferring to a laminar flow cabinet. Always use sterile techniques and sterile hardware for all steps 
		NOTE: Ensure that the correct authorizations for the handling of human blood samples are obtained.</li>
		<li>Transfer the blood to a sterile 50 mL centrifuge tube.</li>
		<li>Dilute the blood at least 10% with non-supplemented RPMI 1640.</li>
		<li>Pour 20 mL of the preferred density gradient medium in a 50 mL centrifuge tube.</li>
		<li>Aspirate 25 mL of the diluted blood into a serological pipette. Set the electric pipette controller to the lowest speed.</li>
		<li>Hold the tube with density gradient medium at a 45&#176; angle and rest the serological pipette tip on the inner side of the 50 mL centrifuge tube. Slowly release 25 mL of the diluted blood from the serological pipette.</li>
		<li>Make sure the diluted blood and the density gradient medium do not mix. Ensure that the diluted blood rests on top of the density gradient medium.</li>
		<li>Repeat this with subsequent density gradient medium tubes until all diluted blood is processed.</li>
		<li>Carefully move the tubes to a centrifuge and centrifuge at 1000 x g for 30 min in a swing-out rotor at room temperature (RT). Ensure that the acceleration and break are at a minimum. 
		NOTE: After centrifugation, various layers should be visible. The top, clear pink-to-orange layer consists of the blood plasma and platelets. The middle white layer consists of the PBMCs followed by a clear layer that consists of the density gradient medium, and finally a dark red layer at the bottom with red blood cells.</li>
		<li>Using a sterile Pasteur pipette, carefully aspirate the PBMC-containing white layer to a 50 mL centrifuge tube. 
		NOTE: Make sure not to transfer the density gradient medium as this will affect downstream purification. Excess plasma will not affect the results.</li>
		<li>Pool all collected PBMCs in the same 50 mL centrifuge tube and top up to 50 mL with RPMI 1640.</li>
		<li>Centrifuge the cells at 500 x g for 5 min at RT (perform the remaining centrifugation steps at this setting unless specified otherwise).</li>
		<li>Aspirate the supernatant and resuspend the cells in 30 mL of RPMI 1640. Count the cells using a hemocytometer or automated cell counter.</li>
		<li>For cryopreservation, resuspend a maximum of 30 million cells per 1 mL of freezing medium.</li>
		<li>Transfer the cells to cryotubes and freeze until -80 &#176;C using a rate-controlled freezing container (-1 &#176;C per min). For long-term storage, move the PBMCs to -140 &#176;C. 
		&#8203;NOTE: Limit the time cells are in the freezing medium at RT. At RT, DMSO is highly toxic to cells.</li>
	</ol>
	</li>
</ol>
2. Culturing of activated human primary T lymphocytes
<ol>
	<li>Thawing of cells (Day 1)
	<ol>
		<li>Pre-warm 10 mL of RPMI 1640 per sample to around 37 &#176;C.</li>
		<li>Take the desired number of cell ampules and store them temporarily on dry ice.</li>
		<li>Resuspend the frozen cells in 10 mL of pre-warmed RPMI 1640.</li>
		<li>Centrifuge the cells for 5 min at 500 x g at RT.</li>
		<li>Discard the supernatant and wash the cells again by resuspending in 10 mL of RPMI 1640 and centrifuge as described in 2.1.4.</li>
		<li>Discard the supernatant and resuspend the cells at 2 x 106 cells per mL of X-VIVO 15 medium + 5% human serum (hereafter: T cell medium).</li>
		<li>Plate 2 mL of the cells per well in a 24-well cell culture plate and incubate overnight at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Activation of cells (Day 0)
	<ol>
		<li>Wash CD3/CD28 beads by transferring 12.5 &#181;L of beads per 1 million cells to a microcentrifuge tube. Add 12.5 &#181;L of PBS per 12.5 &#181;L of beads. 
		NOTE: It is important to vortex the vial of beads before use.</li>
		<li>Place the microcentrifuge tube on a suitable magnet for 1 min.</li>
		<li>Discard the buffer and resuspend the beads in the original volume of T cell medium (12.5 &#181;L of T cell medium per 12.5 &#181;L of the original volume of beads).</li>
		<li>Add 12.5 &#181;L of beads per million of cells corresponding to a ratio of 1:2 (beads: cells).</li>
		<li>Divide the cells into two conditions with around 5 million cells in each.</li>
		<li>Add the correct volume of cytokines to the conditions as mentioned in Table 1.</li>
		<li>Incubate the cells for 3 days at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Culturing of cells (Day 3 and 5)
	<ol>
		<li>Resuspend the cells and split them by transferring half the volume from each well into a new well. Add the same volume of fresh T cell medium to each well.</li>
		<li>Add new cytokines to each condition as mentioned in Table 1.</li>
	</ol>
	</li>
</ol>
3. Extracellular flux assay
<ol>
	<li>Hydration of the sensor cartridge (Day 0)
	<ol>
		<li>Unpack the sensor cartridge and carefully remove the sensor cartridge from the utility plate.</li>
		<li>Place the sensor cartridge upside-down, taking care not to touch the sensor probes.</li>
		<li>Fill the utility plate with 200 &#181;L of calibrant (see Table of Materials for details) and carefully replace the sensor cartridge back into the utility plate.</li>
		<li>Alternatively, to eliminate any bubble formation, incubate the sensor cartridge in sterile ultrapure water overnight and replace it with a pre-warmed calibrant on the morning of the assay.</li>
		<li>Incubate the sensor cartridge plate at 37 &#176;C in a non-CO2-regulated heating cabinet overnight. 
		&#8203;NOTE: It is very important to use a non-CO2 regulated cabinet since excess CO2 will influence the sensor cartridge. Make sure that the Flux analyzer (see Table of Materials for details) is turned on at least a day before use to allow it to warm up to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Cell coating and preparation of mitochondrial inhibitors (Day 1)
	<ol>
		<li>Prepare a coating solution (see Table of Materials) containing NaHCO3 (pH 8.3, 0.1 M, 1128 &#181;L), Cell-Tak (1 mg/mL, 48 &#181;L) and NaOH (1.0 M, 24 &#181;L). 
		NOTE: Coating solution should always be made and used fresh.</li>
		<li>Open a fresh XF cell culture plate and add 12 &#181;L of the freshly prepared coating solution to each well. Ensure even distribution of coating solution in the bottom of all the wells.</li>
		<li>Incubate the plate at RT with the lid on for 30 min and discard the remaining liquid solution from all the wells.</li>
		<li>Wash the plate with 200 &#181;L of sterile water and discard the liquid.</li>
		<li>Wash the plate with 200 &#181;L of cell culture grade sterile PBS and discard the liquid.</li>
		<li>Leave the plate at RT and let it dry for at least 30 min.</li>
	</ol>
	</li>
	<li>Plate T cells in the XF cell culture plate (Day 1)
	<ol>
		<li>Prepare 50 mL of assay media by mixing suitable XF RPMI media with glucose, pyruvate, and glutamine according to experimental setup (Recommended levels: 4 mM glucose, 1 mM pyruvate and 3 mM glutamine).</li>
		<li>Heat to 37 &#176;C in a non-CO2 regulated incubator and set pH at 7.4. Ensure that there is enough media for plating cells and preparing oligomycin and FCCP solutions (section 3.4).</li>
		<li>Design a plate layout with an increasing number of cells per well for Optimization run or Assay run. Use four 4 wells, filled with media and injected with media, for background measurements.</li>
		<li>Count T cells prepared in section 2.3 (by the preferred method) and pipette the correct number of cells to each well of the XF cell culture plate coated with the coating solution, according to the plate layout. 
		&#8203;NOTE: The final volume of each well may vary but must be enough to cover the bottom of the well.</li>
		<li>Centrifuge the XF cell culture plate at 1000 x g at RT for 10 min to adhere the T cell to the coated surface.</li>
		<li>Wash the cells with 200 &#181;L of assay media, discard the media and add 180 &#181;L of assay media. 
		NOTE: Visually inspect the wells using an inverted light microscope to ensure that the cells are attached and evenly distributed across the well surface.</li>
		<li>Incubate the XF cell culture plate in the non-CO2 regulated heating cabinet for 30 min to ensure that the plate&#39;s temperature is 37 &#176;C.</li>
	</ol>
	</li>
	<li>Loading sensor cartridge with oligomycin and FCCP for Optimization run (Day 1) 
	NOTE: If oligomycin and FCCP concentrations were already optimized, continue at section 3.5.
	<ol>
		<li>Prepare working solutions of oligomycin and FCCP in assay media (prepared in step 3.3.1) as described in steps 3.4.2 and 3.4.3.</li>
		<li>Oligomycin working solutions: prepare 5 &#181;M solution (2 mL of assay media + 10 &#181;L of 1 mM oligomycin stock) and 3 &#181;M solution (8 mL of assay media + 24 &#181;L of 1 mM oligomycin stock).</li>
		<li>FCCP working solution: Prepare 2 &#181;M solution (2 mL of assay media + 13.2 &#181;L of 300 &#181;M FCCP stock) and a 1.3 &#181;M solution (8 ml of assay media + 34.6 &#181;L of 300 &#181;M FCCP stock)</li>
		<li>Load the working solutions of either oligomycin or FCCP into the injection ports of the sensor cartridge (Table 2). 
		NOTE. It is important that no injection ports contain air only. If, for any reason, not all injection ports are used, empty ports need to be filled with assay media.</li>
		<li>Gently knock the edges of the plate on the table to remove potential bubbles in the injection ports.</li>
	</ol>
	</li>
	<li>Loading sensor cartridge with oligomycin, FCCP, and antimycin A for Assay run (Day 1)
	<ol>
		<li>Prepare solutions of oligomycin, FCCP in assay media (prepared in step 3.3.1) according to the optimal concentrations identified in a previous Optimization run. Also, prepare a 20 &#181;M antimycin A solution.</li>
		<li>Load 20 &#181;L of either oligomycin or FCCP into injection port A of the sensor cartridge according to plate layout. Add 22 &#181;L of 20 &#181;M antimycin A to injection port B of all wells. The resulting concentration of antimycin A once injected into the well will be 2 &#181;M.</li>
	</ol>
	</li>
	<li>Setting up experimental protocol in Flux analyzer software.
	<ol>
		<li>Assign the groups and plate map for each condition per plate layout.</li>
		<li>Design the protocol according to the injection strategy (Table 3 and Figure 1). 
		NOTE: When running Assay runs, injection C and D can be omitted.</li>
		<li>Save the assay set up, fill out the information required to be included for the assay, and press Start.</li>
		<li>The flux analyzer will ask for the sensor cartridge prepared in section 3.5. Remove the lid and insert the sensor cartridge as directed by the analyzer. 
		NOTE: The assay will calibrate and check sensors indicated by checkmarks. After successful calibration, the analyzer will request for the XF cell culture plate prepared in section 3.3. The utility plate is ejected from the analyzer and replaced with the XF cell culture plate to start the assay.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>vander Windt, G. J. 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=Mitochondrial+respiratory+capacity+is+a+critical+regulator+of+CD8++T+cell+memory+development.">Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development.</a> Immunity. 36 (1), 68-78 (2012).</li><li>Krauss, S., Brand, M. D., Buttgereit, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+takes+a+breath--new+quantitative+perspectives+on+bioenergetics+and+signal+transduction.">Signaling takes a breath--new quantitative perspectives on bioenergetics and signal transduction.</a> Immunity. 15 (4), 497-502 (2001).</li><li>vander Windt, G. J. 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=CD8+memory+T+cells+have+a+bioenergetic+advantage+that+underlies+their+rapid+recall+ability.">CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (35), 14336-14341 (2013).</li><li>Chang, C. -. 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=Posttranscriptional+control+of+T+cell+effector+function+by+aerobic+glycolysis.">Posttranscriptional control of T cell effector function by aerobic glycolysis.</a> Cell. 153 (6), 1239-1251 (2013).</li><li>vander Windt, G. J. W., Chang, C. -. H., Pearce, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+bioenergetics+in+T+cells+using+a+Seahorse+extracellular+flux+analyzer.">Measuring bioenergetics in T cells using a Seahorse extracellular flux analyzer.</a> Current Protocols in Immunology. 113, 1-14 (2016).</li><li>Buck, M. D., O'Sullivan, D., Pearce, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+metabolism+drives+immunity.">T cell metabolism drives immunity.</a> Journal of Experimental Medicine. 212 (9), 1345-1360 (2015).</li><li>Rivadeneira, D. B., Delgoffe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+T-cell+reconditioning:+Improving+metabolic+fitness+for+optimal+cancer+immunotherapy.">Antitumor T-cell reconditioning: Improving metabolic fitness for optimal cancer immunotherapy.</a> Clinical Cancer Research. 24 (11), 2473-2481 (2018).</li><li>Cieri, 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=IL-7+and+IL-15+instruct+the+generation+of+human+memory+stem+T+cells+from+naive+precursors.">IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.</a> Blood. 121 (4), 573-584 (2013).</li><li>Kenwood, B. 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=Identification+of+a+novel+mitochondrial+uncoupler+that+does+not+depolarize+the+plasma+membrane.">Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane.</a> Molecular Metabolism. 3 (2), 114-123 (2013).</li><li>Alizadeh, D., 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+enhances+CAR-T+cell+antitumor+activity+by+reducing+mTORC1+activity+and+preserving+their+stem+cell+memory+phenotype.">IL15 enhances CAR-T cell antitumor activity by reducing mTORC1 activity and preserving their stem cell memory phenotype.</a> Cancer Immunology Research. 7 (5), 759-772 (2019).</li></ol>

The authors declare no conflict of interest.

Detailed and correct quantification of oxidative phosphorylation is an indispensable tool when describing the energy states of T cells. The state of mitochondrial fitness can be directly related to T cell activation potential, survival, and differentiation1,5. With this protocol, it is possible to determine the various properties of oxidative phosphorylation (see Table 4 for a detailed explanation). Precise quantification of these properties of o...

Correct T cell development and function are essential for the ability of the immune system to recognize and respond to antigens. Mitochondrial oxidative phosphorylation (OxPhos) changes according to the state of the T cell. Na&#239;ve T cells predominantly use OxPhos to produce ATP, whereas activated T cells undergo a metabolic transition where glycolysis becomes dominant1. After the effector phase, the small remaining subset of memory T cells reverts to a metabolic state dominated by OxPhos2,3. The changes of OxPhos follow the differentiation of T cells to such a degree that even subsets of T cells can be differentiated by their specific is OxPhos properties1. Conversely, OxPhos is important for T cells&#39; function, and inhibition of OxPhos has been demonstrated to block proliferation and cytokine production of T cells4. Therefore, the ability to quantify the properties of T cell OxPhos in a precise and reproducible manner is a powerful tool for anyone working with T cells.
In this protocol, properties of T cell OxPhos are measured using an extracellular flux analyzer. The core function of this analyzer is to continuously measure the oxygen content of the growth media of the cells to be analyzed. Oxygen removed from the growth media is assumed to be taken up by the cells. By treating the cells with a variety of OxPhos inhibitors or modifiers, a drop in oxygen uptake is associated with the inhibited or modulated function. For example, inhibition of the ATP synthase will lead to a reduced cellular uptake of oxygen that would otherwise be used to produce ATP by oxidative phosphorylation. Other equipment, including the Clark electrode and the Oroboros instrument, offers similar functionality, and each instrument has different advantages and shortcomings. A wide array of cell types can be used for studies in these devices, but one particularly challenging cell type is human primary T lymphocytes5. Due to their small size, poor survival ex vivo, and non-adherent properties, human primary T cells can be challenging to study.
This is a protocol for studying the mitochondrial respiration of human primary T cells by an extracellular analyzer. The protocol is divided into an Optimization run, where optimal concentrations of cell number per well, as well as the optimal concentration of oligomycin and FCCP, are determined. Furthermore, an Assay run, where the optimized conditions are used.
Using blood-derived human PBMCs and ex vivo primary T cell cultures, this protocol demonstrates the importance of optimal inhibitor concentration and the relevance of using separate instead of a sequential injection of mitochondrial inhibitors when working with sensitive cell types. Finally, it is demonstrated that this assay can robustly detect subtle differences in mitochondrial respiration upon polarization with cytokines IL-2 and IL-15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Nunc</td>
			<td>142485</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD3xCD28 beads</td>
			<td>Gibco</td>
			<td>11161D</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Merck</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>For coating</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D9170</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma Aldrich</td>
			<td>H4522</td>
			<td>Heat inactivated at 56 &#176;C for 30 min</td>
		</tr>
		<tr>
			<td>IL-15</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>Peprotech</td>
			<td>200-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>07801</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Merck</td>
			<td>O4876</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco-Thermo Fisher</td>
			<td>61870036</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse Calibrant</td>
			<td>Agilent Technologies</td>
			<td>102416-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 1.0 M glucose solution</td>
			<td>Agilent Technologies</td>
			<td>103577-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 100 mM pytuvate solution</td>
			<td>Agilent Technologies</td>
			<td>103578-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF 200 mM glutamine solution</td>
			<td>Agilent Technologies</td>
			<td>103579-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF RPMI medium, pH7.4</td>
			<td>Agilent Technologies</td>
			<td>103576-100</td>
			<td>XF RPMI media</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyser</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>Flux analyzer</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 cell culture microplates</td>
			<td>Agilent Technologies</td>
			<td>102416-100</td>
			<td>XF cell culture plate</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 sensor cartridge</td>
			<td>Agilent Technologies</td>
			<td>102416-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate concentrate 0.1 M (NaHCO3)</td>
			<td>Sigma Aldrich</td>
			<td>36486</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide solution 1 N (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>S2770-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Lonza</td>
			<td>BE02-060F</td>
			<td></td>
		</tr>
		<tr>
			<td>T cell beads magnet DynaMag-2 Magnet</td>
			<td>Thermo Fisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse wave</td>
			<td></td>
			<td></td>
			<td>Flux analyzer software</td>
		</tr>
	</tbody>
</table>

real-time monitoring of mitochondrial respiration in cytokine-differentiated human primary t cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

A correct determination of OxPhos properties is an indispensable tool when studying T cells. However, if the assay conditions have not been optimized, there is a substantial risk of misleading or erroneous results. In this protocol, there is a strong focus on the optimization of cell number per well and concentrations of oligomycin and FCCP to be used. In the described setup, oligomycin and FCCP are added incrementally to the same well, increasing the concentration of the mitochondrial modulators. The optimal concentrati...

Watch this Scientific Journal Video about Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells at JoVE.com

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

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3.7K Views.  University Hospital Herlev. Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented. 

Video: Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...