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

Summary

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.

Abstract

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.

Introduction

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. Naïve 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 OxPhos^2,3. 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' 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.

Protocol

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

1. PBMC isolation
   1. 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.
   2. 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.
   3. Transfer the blood to a sterile 50 mL centrifuge tube.
   4. Dilute the blood at least 10% with non-supplemented RPMI 1640.
   5. Pour 20 mL of the preferred density gradient medium in a 50 mL centrifuge tube.
   6. Aspirate 25 mL of the diluted blood into a serological pipette. Set the electric pipette controller to the lowest speed.
   7. Hold the tube with density gradient medium at a 45° 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.
   8. 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.
   9. Repeat this with subsequent density gradient medium tubes until all diluted blood is processed.
   10. 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.
   11. 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.
   12. Pool all collected PBMCs in the same 50 mL centrifuge tube and top up to 50 mL with RPMI 1640.
   13. Centrifuge the cells at 500 x g for 5 min at RT (perform the remaining centrifugation steps at this setting unless specified otherwise).
   14. Aspirate the supernatant and resuspend the cells in 30 mL of RPMI 1640. Count the cells using a hemocytometer or automated cell counter.
   15. For cryopreservation, resuspend a maximum of 30 million cells per 1 mL of freezing medium.
   16. Transfer the cells to cryotubes and freeze until -80 °C using a rate-controlled freezing container (-1 °C per min). For long-term storage, move the PBMCs to -140 °C.
       ​NOTE: Limit the time cells are in the freezing medium at RT. At RT, DMSO is highly toxic to cells.

2. Culturing of activated human primary T lymphocytes

1. Thawing of cells (Day 1)
   1. Pre-warm 10 mL of RPMI 1640 per sample to around 37 °C.
   2. Take the desired number of cell ampules and store them temporarily on dry ice.
   3. Resuspend the frozen cells in 10 mL of pre-warmed RPMI 1640.
   4. Centrifuge the cells for 5 min at 500 x g at RT.
   5. Discard the supernatant and wash the cells again by resuspending in 10 mL of RPMI 1640 and centrifuge as described in 2.1.4.
   6. 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).
   7. Plate 2 mL of the cells per well in a 24-well cell culture plate and incubate overnight at 37 °C and 5% CO₂.
2. Activation of cells (Day 0)
   1. Wash CD3/CD28 beads by transferring 12.5 µL of beads per 1 million cells to a microcentrifuge tube. Add 12.5 µL of PBS per 12.5 µL of beads.
      NOTE: It is important to vortex the vial of beads before use.
   2. Place the microcentrifuge tube on a suitable magnet for 1 min.
   3. Discard the buffer and resuspend the beads in the original volume of T cell medium (12.5 µL of T cell medium per 12.5 µL of the original volume of beads).
   4. Add 12.5 µL of beads per million of cells corresponding to a ratio of 1:2 (beads: cells).
   5. Divide the cells into two conditions with around 5 million cells in each.
   6. Add the correct volume of cytokines to the conditions as mentioned in Table 1.
   7. Incubate the cells for 3 days at 37 °C and 5% CO₂.
3. Culturing of cells (Day 3 and 5)
   1. 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.
   2. Add new cytokines to each condition as mentioned in Table 1.

3. Extracellular flux assay

1. Hydration of the sensor cartridge (Day 0)
   1. Unpack the sensor cartridge and carefully remove the sensor cartridge from the utility plate.
   2. Place the sensor cartridge upside-down, taking care not to touch the sensor probes.
   3. Fill the utility plate with 200 µL of calibrant (see Table of Materials for details) and carefully replace the sensor cartridge back into the utility plate.
   4. 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.
   5. Incubate the sensor cartridge plate at 37 °C in a non-CO₂-regulated heating cabinet overnight.
      ​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 °C.
2. Cell coating and preparation of mitochondrial inhibitors (Day 1)
   1. Prepare a coating solution (see Table of Materials) containing NaHCO₃ (pH 8.3, 0.1 M, 1128 µL), Cell-Tak (1 mg/mL, 48 µL) and NaOH (1.0 M, 24 µL).
      NOTE: Coating solution should always be made and used fresh.
   2. Open a fresh XF cell culture plate and add 12 µL of the freshly prepared coating solution to each well. Ensure even distribution of coating solution in the bottom of all the wells.
   3. Incubate the plate at RT with the lid on for 30 min and discard the remaining liquid solution from all the wells.
   4. Wash the plate with 200 µL of sterile water and discard the liquid.
   5. Wash the plate with 200 µL of cell culture grade sterile PBS and discard the liquid.
   6. Leave the plate at RT and let it dry for at least 30 min.
3. Plate T cells in the XF cell culture plate (Day 1)
   1. 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).
   2. Heat to 37 °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).
   3. 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.
   4. 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.
      ​NOTE: The final volume of each well may vary but must be enough to cover the bottom of the well.
   5. Centrifuge the XF cell culture plate at 1000 x g at RT for 10 min to adhere the T cell to the coated surface.
   6. Wash the cells with 200 µL of assay media, discard the media and add 180 µL 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.
   7. Incubate the XF cell culture plate in the non-CO₂ regulated heating cabinet for 30 min to ensure that the plate's temperature is 37 °C.
4. 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.
   1. 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.
   2. Oligomycin working solutions: prepare 5 µM solution (2 mL of assay media + 10 µL of 1 mM oligomycin stock) and 3 µM solution (8 mL of assay media + 24 µL of 1 mM oligomycin stock).
   3. FCCP working solution: Prepare 2 µM solution (2 mL of assay media + 13.2 µL of 300 µM FCCP stock) and a 1.3 µM solution (8 ml of assay media + 34.6 µL of 300 µM FCCP stock)
   4. 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.
   5. Gently knock the edges of the plate on the table to remove potential bubbles in the injection ports.
5. Loading sensor cartridge with oligomycin, FCCP, and antimycin A for Assay run (Day 1)
   1. 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 µM antimycin A solution.
   2. Load 20 µL of either oligomycin or FCCP into injection port A of the sensor cartridge according to plate layout. Add 22 µL of 20 µM antimycin A to injection port B of all wells. The resulting concentration of antimycin A once injected into the well will be 2 µM.
6. Setting up experimental protocol in Flux analyzer software.
   1. Assign the groups and plate map for each condition per plate layout.
   2. 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.
   3. Save the assay set up, fill out the information required to be included for the assay, and press Start.
   4. 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.

Results

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...

Discussion

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 differentiation^1,5. 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...

Materials

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