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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 OxPhos2,3. The changes of OxPhos follow the differentiation of T cells to such a degree that even subse....

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.......

Representative 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 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.......

Acknowledgements

Kasper Mølgaard and Anne Rahbech received grants from Tømmermester Jørgen Holm og Hustru Elisa f. Hansens Mindelegat. Kasper Mølgaardalso received a grant from Børnecancerfonden.

....

Materials

NameCompanyCatalog NumberComments
24-well tissue culture plateNunc142485
Anti-CD3xCD28 beadsGibco11161D
Antimycin AMerckA8674
Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP)Sigma-AldrichC2920
Cell-TakCorning354240For coating
Dimethyl sulfoxide (DMSO)Sigma AldrichD9170
Human SerumSigma AldrichH4522Heat inactivated at 56 °C for 30 min
IL-15Peprotech200-02
IL-2Peprotech200-15
LymphoprepStemcell Technologies07801
OligomycinMerckO4876
PBSThermo Fisher10010023
RPMI 1640Gibco-Thermo Fisher61870036
Seahorse CalibrantAgilent Technologies102416-100
Seahorse XF 1.0 M glucose solutionAgilent Technologies103577-100
Seahorse XF 100 mM pytuvate solutionAgilent Technologies103578-100
Seahorse XF 200 mM glutamine solutionAgilent Technologies103579-100
Seahorse XF RPMI medium, pH7.4Agilent Technologies103576-100XF RPMI media
Seahorse XFe96 AnalyserAgilent TechnologiesFlux analyzer
Seahorse XFe96 cell culture microplatesAgilent Technologies102416-100XF cell culture plate
Seahorse XFe96 sensor cartridgeAgilent Technologies102416-100
Sodium Bicarbonate concentrate 0.1 M (NaHCO3)Sigma Aldrich36486
Sodium Hydroxide solution 1 N (NaOH)Sigma AldrichS2770-100ML
X-VIVO 15LonzaBE02-060F
T cell beads magnet DynaMag-2 MagnetThermo Fisher12321D
Seahorse waveFlux analyzer software

References

  1. vander Windt, G. J. W., et al. Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. Immunity. 36 (1), 68-78 (2012).
  2. Krauss, S., Brand, M. D., Buttgereit, F.

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