Analysis of Human Natural Killer Cell Metabolism

Summary

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.

Abstract

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 (IFNγ) 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.

Introduction

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 cell^1,2.

Resting human NK cells isolated from peripheral blood can survive....

Protocol

All the experiments were performed in accordance with the Declaration of Helsinki’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

1. Reagents for the isolation of NK cells
   NOTES: Prepare these reagents in a cell culture hood.
   1. 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 °C for 30 min).

Results

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.......

Discussion

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,.......

Materials

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