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* These authors contributed equally
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 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.
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 OxPhos2,3. The changes of OxPhos follow the differentiation of T cells to such a degree that even subse....
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
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.......
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.......
Kasper Mølgaard and Anne Rahbech received grants from Tømmermester Jørgen Holm og Hustru Elisa f. Hansens Mindelegat. Kasper Mølgaardalso received a grant from Børnecancerfonden.
....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 |
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