Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Summary

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Abstract

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

Introduction

As part of the innate immune system natural killer (NK) cells contribute to the first line of defense against virus-infected or malignantly transformed cells. A system of inhibitory and activating receptors enables them to distinguish between healthy and transformed cells without prior antigen priming in contrast to T cells. Upon target cell encounter NK cells release the content of their cytotoxic granules (e.g., perforin, granzymes) into the immune synapse to kill their target. Moreover, NK cells produce and secrete different kinds of cytokines (e.g., interferon- γ: IFN-γ; tumor necrosis factor-α: TNF-α) and chemokines ....

Protocol

This study was carried out in accordance with the recommendations of the local ethics committee of the University of Frankfurt.

1. Culturing of K562 Cells

1. Culture K562 cells in R10 media (RPMI1640 with glutamine medium, 1% penicillin/streptomycin, 10% fetal calf serum) at a density of 0.5-1 x 10⁶ cells per ml in a cell culture flask at 37 °C and 5% CO₂.
2. Harvest K562 Cells 24 hr Before the Start of a New Experiment.
   1. Remove the cell culture flask containing the K562 cells from the incubator. Re-suspend the K562 cells within the culture media by gently pipetting up and down.
   2. Transfer the cultu....

Results

The gating strategy for analyzing the degranulation, cytokine and chemokine production of the whole NK cell population and three different NK cell subsets are illustrated in Figure 1.

Representative results of one healthy donor are illustrated in Figure 2. NK cells without any stimulus produced neither IFN-γ nor MIP-1β and did not express CD107a on their surface (Figure 2A). In .......

Discussion

The described method is an easy, fast and reliable approach to study NK cell functions from whole blood samples of healthy donors or patients. This method offers the great advantage to directly purify NK cells from whole blood, avoiding the time-consuming density gradient centrifugation, which is mandatory for many other purification methods¹⁵. Moreover, it requires a smaller sample size compared to "classical" NK cell isolation/enrichment methods, which makes it a suitable alternative for samples of p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640 + glutamine	Invitrogen	6187-044
penicilin/streptomycin	Invitrogen	15140-122
BD Falcon Round Bottom Tube	BD	352008
fetal calf serum	Invitrogen	10270-106	heat inactivated before use
T-flask	Greiner Bio-One	690195
K562 tumor cell line	DSMZ GmbH	ACC 10
ammonium-chloride-potassium (ACK) lysis buffer	made in house	/	components are listed in the text
Distilled water: Ampuwa Spüllösung 1,000 ml Plastipur	Fresenius Kabi	1088813
Megafuge 40R Centrifuge	Heraeus	/
EDTA blood collector tubes	Sarstedt	386453	S-Monovette 7.5 ml, K3EDTA
UltraPure 0.5 M EDTA, pH 8.0	Life Technologies	15575-020
Hematopoietic media (XVIVO)	Lonza Group Ltd	BE04-743Q
human serum	DRK Blutspendedienst, Frankfurt/M	/	healthy donors with blood type AB; heat inactivated before use
Neubauer-improved counting chamber, bright line	Marienfeld superior/ LO-Laboroptik Ltd.	0640030/ 1110000
trypan blue solution (0.4%)	Invitrogen	15250-061
3% acetic acid with methylene blue	Stemcell Technologies	07060
Corning 96 Well Clear V-Bottom TC-treated Microplates	Corning	3894
Falcon 96 Well Round Bottom Not Treated Microplates	Corning	351177
DPBS (Ca2+- and Mg2+-free)	Gibco Invitrogen	14190-169
BSA	Sigma Aldrich	A2153-100G
NaN3	Sigma Aldrich	08591-1ML-F
phorbol 12-myristate 13-acetate (PMA)	Merck	524400-1MG
ionomycin	PromoKine	PK-CA577-1566-5
interleukin 15 (IL-15)	PeproTech	200-15
Proleukin S (IL-2)	Novartis Pharma	730523
Golgi Stop, Protein Transport Inhibitor (containing Monensin)	BD Biosciences	554724	This product can be used in combination or instead of Golgi Plug. The best combination for the wished experimental setting has to be tested.
Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A)	BD Biosciences	555029
paraformaldehyde	AppliChem	UN2209
saponin	Sigma Aldrich	47036
flow cytometer: Canto10C	BD Biosciences	/
FlowJo	TreeStar Inc.	/
Graph Pad	Graph Pad Inc.	/
MACSxpress Separator	Miltenyi Biotec	130-098-308
MACSxpress NK isolation kit	Miltenyi Biotec	130-098-185
MACSxpress Erythrocyte Depletion Kit, human	Miltenyi Biotec	130-098-196
MACSmix Tube Rotator	Miltenyi Biotec	130-090-753
anti-human CD3 APC	Biolegend	300412
anti-human CD3 V450	BD Biosciences	560366
anti-human CD14 PerCP	Miltenyi Biotec	130-094-969
anti-human CD14 V450	BD Biosciences	560349
anti-human CD16 PE	Biolegend	302008
anti-human CD16 PerCP	Biolegend	302029
anti-human CD19 PE-Cy7	Biolegend	302216
anti-human CD19 V450	BD Biosciences	560353
anti-human CD45 BV510	BD Biosciences	563204
anti-human CD56 FITC	Biolegend	345811
anti-human CD107a PE	Biolegend	328608
anti-human IFN-γ AF-647	BD Biosciences	557729
anti-human MIP-1β APC-H7	BD Biosciences	561280
DAPI	Biolegend	422801
Zombie Violet Fixable Viability Kit	Biolegend	423113	fixable dead cell marker