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.
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.
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 (e.g., macrophage inflammatory protein-1β: MIP-1β) upon target cell interaction or cytokine stimulation1.
Sufficient NK cell functions such as cytotoxicity, chemokine and cytokine production have an important impact on the fate of diverse diseases. Leukemia patients show increased relapse rates if they exhibit a defective NK cell profile at diagnosis consisting of reduced IFN-γ production and reduced expression of activating NK cell receptors2. An early recovery of NK cell numbers and function including cytokine production upon target cell interaction is associated with a reduced relapse and improved survival rate in patients receiving allogeneic stem cell transplantation3. Moreover, upon initiation of interferon therapy in hepatitis C virus-infected patients the degranulation capacity of peripheral NK cells is stronger in early responders than in non-responders4. NK cell numbers (>80/µl) on day 15 after autologous stem cell transplantation (autoSCT) in patients suffering from lymphoma or multiple myeloma are predictive for an improved progression free and overall survival5. In melanoma patients the expression of the T-cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3), an immune-regulatory protein on NK cells, correlates with disease stage and prognosis6.
Scientists have monitored NK cell functions throughout the last decades. The initial analysis of NK cell cytotoxicity against tumor cells without prior priming was addressed using a 51Cr-release assay7. More recently, scientists developed a non-radioactive method to evaluate the cytotoxicity of expanded NK cells8. Cytokine and chemokine production has been frequently evaluated using enzyme-linked immunosorbent assay (ELISA) techniques9,10. During the last decades these methods have been complemented by flow cytometry-based assays. The use of protein transport inhibitors (e.g., brefeldin A and monensin) and cell permeabilization methods in combination with conventional surface staining protocols have enabled scientists to study chemokine and cytokine production in different specific lymphocyte subsets (e.g., T, B or NK cells)11. Moreover, different flow cytometry-based assays have been developed to monitor T and NK cell cytotoxicity. In 2004 Alter et al. described the surface expression of the lysosome-associated protein CD107a (Lamp1) on NK cells upon target cell encounter as a marker for the degranulation of cytotoxic granules12. Since a wide range of different fluorochromes and multi-channel flow cytometers are available in our days, it has become possible to simultaneously monitor diverse NK cell functions (cytotoxicity, cytokine and chemokine production) in different NK cell subsets. This becomes especially important in situations where sample size is limited, e.g., in biopsies or blood samples of patients suffering from leukopenia.
To test global NK cell functions, the different flow cytometry-based assays can be efficiently combined. Theorell et al. stimulated NK cells from healthy donors with the tumor cell line K562 and analyzed NK cell degranulation, inside-out signal and chemokine production via flow cytometry13. Recently NK cell subgroups, phenotypes and functions in tumor patients during autoSCT were analyzed using flow cytometry-based assays. It was demonstrated that NK cells were able to degranulate and produce cytokines/chemokines upon tumor cell recognition at very early time points after autoSCT11.
Here a protocol is described to evaluate NK cell functions upon interaction with tumor cells including degranulation capacity, chemokine and cytokine production using a flow cytometry-based assay that makes it possible to monitor NK cell functions in different subsets simultaneously.
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
2. Isolation of NK Cells
3. Harvesting of the K562 Cells for NK Cell Stimulation
4. Stimulation of NK Cells with the Tumor Cell Line K562 and Cytokines
5. Surface and Intracellular Staining
6. Flow Cytometry Compensation and Acquisition
7. Analysis and Statistics
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 contrast, NK cells stimulated with tumor cells and cytokines produced significant amounts of intracellular IFN-γ and MIP-1β. Moreover, over 20% of them degranulated upon tumor cell interaction indicated by expressing CD107a on their surface (Figure 2C). PMA and Ionomycin stimulation were used as a control (Figure 2B).
Figure 1: Gating strategy. Subsequent gates are plotted. First, debris are excluded in a FSC-A/SSC-A plot. Then lymphocytes are identified and doublets are excluded using two plots with SSC-A/SSC-H and FSC-A/FSC-H. Thereafter, a gate including all CD45+ cells and excluding all dead, CD3+, CD14+ and CD19+ cells is set by plotting CD45 / Dump channel. Next, NK cells are identified by gating on CD56+ cells. A plot with CD56/CD16 can be used to identify the main NK cell subsets. Finally, within the whole NK cell population, functional markers are identified by plotting CD56 versus CD107a, IFN-γ and MIP-1β, respectively. This can be done as well for all different kinds of NK cell subsets. Please click here to view a larger version of this figure.
Figure 2: Representative results from one healthy NK cell donor. Using the gating strategy described in Figure 1, NK cell functional markers like CD107a (for degranulation), IFN-γ and MIP-1β can be identified. The left column (A) shows the results using NK cells in the absence of a stimulus. The middle column (B) demonstrates the results in the presence of the positive control PMA/ionomycin. The right column (C) presents results for NK cells stimulated with the tumor cell line K562 and in the presence of the cytokines IL- 2 (100 U/ml) and IL-15 (10 ng/ml). Please click here to view a larger version of this figure.
Table 1: Antibodies for purity check.
Table 2: Antibodies for extra- and intracellular staining.
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 pediatric and/or immune-deficient patients. This protocol can be used to obtain basal values of NK cell functions ex-vivo, but it also allows the further NK culture and expansion, being in this way complementary with other methods previously described8. Nevertheless some critical steps have to be taken into account. Since the assay is based on extra- and intracellular antibody staining, it is crucial to determine the optimal working concentration for each antibody first. Especially for the intracellular staining, a careful evaluation of the used antibodies is highly recommended. A good approach is to test a dilution series of the used antibody (e.g., 1:100 to 1:12.5) in a cell suspension stimulated with or without PMA/ionomycin. Subsequently the ratio of positive events between stimulated and un-stimulated samples are calculated and plotted. The antibody dilution with the highest positive fold change ratio (best signal-to-background ratio) should be used for further experiments16. Moreover, the use of controls like isotype- and FMO controls can prove to be fundamental especially for intracellular staining in order to gate correctly on the positive cell populations.
However, there are some important limitations of the described method. CD107a expression is a degranulation marker and therefore only indirectly indicates NK cell cytotoxicity. NK cell cytotoxicity and degranulation capacity can be different. Up-regulation of the autophagy pathway in tumor cells results in the degradation of secreted granzyme b and reduces cell death upon NK cell interaction17. Therefore an additional DCM (e.g., cleaved caspase 3) might be added to the staining panel in order to monitor cell death events within the target cells18. Additionally, NK cell cytotoxicity is influenced by the amount of cytotoxic proteins within their granules (e.g., perforin, granzyme b), which can be quantified by an intracellular staining19,20.
Furthermore, there are different possibilities to modify the protocol. Instead of isolated NK cells, peripheral blood mononuclear cells (PBMCs) can be used. Though one has to be aware that the absolute NK cell number within the PBMC population differs between various donors and even between samples derived from the same donor at different time points. NK cell degranulation is highly dependent on the E:T ratio. In order to compare NK cell functions between different donors or at various time points, the absolute NK cell number within the PBMC population should be used to establish a constant E:T ratio throughout all experiments. If NK cell functions are monitored at different time points within the same donor, PBMCs can be frozen and the analysis can be done at once for all different time points in order to reduce inter-experimental variations11.
Since staining panels with up to 18 colors are possible, analysis of NK cell functions can be extended to a much greater detail. Using additional surface markers including CD57, NKG2A and killer cell immunoglobulin-like receptors (KIRs), further NK cell subsets can be identified. Educated NK cells expressing at least one self-KIR degranulate stronger upon interaction with K562 cells than uneducated NK cells. In contrast, more differentiated CD57+CD56+ NK cells produce less IFN-γ upon IL-12 and IL-15 stimulation compared to less differentiated CD57–CD56+ NK cells21. In addition, other markers of the NK cell function may be addressed. LFA-1 is an important molecule for NK cell adhesion and is expressed in its open conformation upon NK cell activation. This so-called "inside-out signal" is an early NK cell activation marker22.
Finally, the stimuli for testing NK cell functions can be modified as well. In our experience freshly isolated NK cells only degranulate poorly upon K562 cell interaction if no cytokines are added. Using freshly isolated PBMCs without further cytokine addition circumvent this issue, because of the influence of bystander cells. Moreover, cytokine production, especially IFN-γ and TNF-α, can be initiated upon IL-12 and IL-18 stimulation23. IFN-γ production within the NK cell subsets may differ depending on the used stimulus (cytokine- versus target-induced stimulation)24. Additionally, instead of using K562 cells as target cells, primary tumor cells derived from tumor patients could be used in order to test NK cell functions against autologous tumor cells before or during immune-modulating therapies (e.g., monoclonal antibody treatment or immuno-modulatory drugs (IMiDs)).
Within the past few years, the field of immunotherapy has been evolving rapidly with the clinical approval of immune checkpoint inhibitors (e.g., ipilumumab, nivolumumab, pembrolizumab)25 and successful trials using genetically modified chimeric antigen receptor (CAR)-expressing T cells as well as CAR-NK cells (reviewed in reference26), for the treatment of hematological tumor patients27. Therefore NK cell-based immunotherapy is getting increasingly more into focus. Anti-CD20 antibodies have been a great success in the treatment of malignant lymphomas within the last two decades28. The vast majority of NK cells express the low affinity Fc receptor CD16 enabling the cells to recognize and kill antibody-labeled target cells (antibody-dependent cellular cytotoxicity — ADCC). The antibody's ability to trigger NK cell functions can be tested in vitro using the described protocol29. Terszowski et al. analyzed NK cells' ADCC against a B lymphoblastoid cell line using different clinically approved anti-CD20 antibodies. While ADCC induced upon rituximab treatment (first clinically approved anti-CD20 antibody30) was negatively influenced by KIR/HLA interaction, this effect was not observed when using obinutuzumab (a novel glycoengineered type II CD20 monoclonal antibody)31,32. Moreover, NK cell functions are influenced by different novel anti-tumor drugs like the IMiD lenalidomide33and different tyrosine-kinase inhibitors (TKI)34. Currently NK cell specific checkpoint inhibitors are tested in clinical trials. Examples are the use of anti-KIR35,36 or anti-NKG2A antibodies (NCT02331875) as well as activating agonists like an anti-CD137 antibody (NCT01775631; NCT02110082).
Importantly, adoptive NK cell transfer has been performed in a variety of different malignant diseases with promising clinical effects37. With the aim to select the best donor, NK cell functions against the patient's tumor might be tested in vitro using blood samples from potential NK cell donors.
In summary, the described protocol is designed to analyze diverse NK cell functions from healthy donors or patients. Those functions can be monitored in diverse NK cell subsets upon various stimuli at selected time points.
The authors have nothing to disclose.
Authors were supported by the German Cancer Aid (Max Eder Nachwuchsgruppe, Deutsche Krebshilfe; EU), the LOEWE Center for Cell and Gene Therapy Frankfurt (EU, ST) funded by the Hessian Ministry of Higher Education, Research and the Arts, Germany (III L 4- 518/17.004) and by the “Alfred- und Angelika Gutermuth-Stiftung”, Frankfurt, Germany (EU). BJ was funded by a Mildred Scheel postdoctoral scholarship from the Dr. Mildred Scheel Foundation for Cancer Research. ST was founded by a GO-IN postdoctoral fellowship (PCOFUND-GA-2011-291776). The authors thank Becton Dickinson (BD) for providing the FACSCanto II and Canto10c Flow Cytometry Analyzers used in this study.
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 1000ml Plastipur | Fresenius Kabi | 1088813 | |
Megafuge 40R Centrifuge | Heraeus | / | |
EDTA blood collector tubes | Sarstedt | 386453 | S-Monovette 7,5 ml, K3 EDTA |
UltraPure 0.5M 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 |