This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.
NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user's errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3'-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data's integrity.
Natural killer cells are an essential element of the innate immune system. While they are very regulated, they have the capability to recognize and eliminate abnormal cells through cell-to-cell contact and without prior activation1. As such, they form a quick line of defense against infections. Exercise, especially intense, has been shown to transiently depress the immune system2,3,4,5. NK cells are particularly prone to this effect4,6,7, effectively creating a window of enhanced sensitivity to disease. Hence, the study of interventions before, during or after intense exercise with the goal of reducing its impact on NK cell function is of particular interest for the well-being of athletes post-competitions.
However, the study of such interventions is complicated by numerous factors: 1) NK cells are sparse8, at about 1% of the white blood cell compartment; 2) NK cells are very sensitive to stress and rely on constant exposure to physiological conditions to remain viable and stable during experimentation; and 3) standard assays to determine NK cell cytotoxicity, such as Ficoll gradients9 and release assays10, are unreliable and inconsistent. The inherent variability of human samples only compound these issues. Fresh human samples collected during interventions are fairly regulated and difficult to procure, at least when compared to animal samples or immortalized cell lines. This reduces opportunities to repeat experiments or add participants to the study cohort to reach significant statistical thresholds. Collectively, these issues support the need for a streamlined protocol that allows for both high-throughput and a high-reliability analysis of NK cell lytic activity in human samples.
We established a workflow that shortens the time necessary to identify, isolate and test NK cells from human whole blood while minimizing exposure to extraneous factors. The method optimizes the use of two instruments, a magnetic-based cell sorter and an imaging flow cytometer, and an assay-specific, optimized T:E ratio to allow the detection of decreases or increases of NK cell cytotoxicity.
NOTE: All blood collection procedures were conducted in accordance with the guidelines set forth by the Appalachian State University (ASU) Institutional Review Board (IRB).
1. Whole Blood Collection
2. Preparation of DiO-labeled Target Cells
3. Preparation of Controls
4. NK Cell Automated Separation
5. NK Cell Count Following Cell Separation
6. Cytotoxicity Assay Sample Preparation
7. Preparation of Spontaneous ("S") Sample
8. Data Acquisition with Imaging Flow Cytometer
9. Imaging Flow Cytometer Sample Analysis
Figure 1: Representative histograms,scatter plots and images for cytotoxic activity analysis. (A) focus cell analysis. (B) single cell analysis. (C) target cell staining analysis. All determinations are made using the image attached to each event. This can be accessed in analysis software by simply clicking on the event on the graphs. (D) representative image of a doublet event, showing an apoptotic NK cell and a live K562 target. Ch01, Brightfield. Ch02, DiO. Ch05, PI. Please click here to view a larger version of this figure.
Determination of NK cell count
The effect of heavy running on NK cell count in whole blood was measured, using the exercise protocol described in Figure 2. Blood samples were drawn before exercise, immediately after exercise, 1.5 h after exercise, and finally 24 and 48 h after the initial blood draw. The concentration of NK cells per milliliter of whole blood was measured for each runner (Figure 3A) and on average (Figure 3B) for each time point.
Our results (Figure 3A) show that runners 1, 2, and 4 present a similar pattern with a slight increase of NK cell count after exercise, immediately followed by a sharp decrease, before slowly returning to normal after 24 and 48 h. This trend was particularly visible by plotting NK cell averages for the group of runners, but no significant difference from pre-exercise levels was detected. Runner 3 presented a very high count initially, that sharply decreased after exercise and 1.5 h after exercise, before slowly returning to normal after 24 and 48 h. In average, the NK cell count (Figure 3B) decreased 1.5 h after exercise (albeit non significantly) but was back to near normal levels after 24 h.
Determination of NK cell cytotoxicity
After calculating the NK cell count, NK cells were immediately set up to determine their cytotoxic activity as described in section 6 of the protocol. The results are presented in Figure 4A for each individual runner, with Figure 4B depicting the average NK cytotoxicity. Our results show that average NK cytotoxicity decreased slightly after exercise and 1.5 h after exercise (albeit non-significantly), but significantly increased after 24 h. The NK cytotoxicity remained high compared to the pre-exercise levels, albeit non-significantly (in part due to the small number of participants in this pilot project).
Figure 2: Exercise protocol. Blood draws (red arrows) were performed on n = 4 runners.
Figure 3: NK cells counts pre-workout, post-workout, 1.5 h and 21 h post-workout. (A) NK cell count per milliliter of whole blood for each individual runner. (B) average NK cell count per milliliter of whole blood. N = 4; Scale bars = standard error.
Figure 4: NK cells cytotoxicity pre-workout, post-workout, 1.5 h and 21 h post-workout. (A) NK cell cytotoxicity (expressed as a percentage of dead target cells) for each individual runner. (B) average NK cell cytotoxicity (expressed as a percentage of dead target cells). N = 4; Scale bars = standard error; *: p <0.05; **: p <0.005.
The method described in this study directly measures the specific activity of an individual's NK cells in response to stimuli (in this particular case, prolonged exercise). Typically, NK cells are isolated from one's blood using density gradients or cell sorting by using a combination of markers. While these methods are widely used, they have many drawbacks: they are time consuming, involve multiple manipulations, and are heavily user dependent. As a result, undue stress is placed on the isolated NK cells, which can result in increased variability from experiment to experiment, or even within the same experiment. In this paper, we described a protocol that utilizes magnetic-based isolation. This allows for quick, and reliable sorting of a high number of NK cells in an individual's whole blood, while minimizing blood or NK cell manipulation.
This method is particularly suited for human studies where samples are sparse, but on the flipside the method becomes fairly difficult when too much samples need to be processed together. Indeed, the window of acquisition for cytotoxic assay must be typically within 30 min after the end of the incubation, which in our experience translate into 5 samples at a time at the most. If many samples are to be processed, it is critical to establish a staggering schedule to allow sufficient time for flow cytometry acquisition. Another drawback of this method could be the cost of the reagents that are fairly high and can be consumed quickly if more quantity of blood is processed for higher NK yield. However, we believe that these costs are balanced by the significant amount of time saved.
The critical steps in this protocol are the ones that are the most open to modifications. It is critical to maintain the blood at room temperature and to process within an hour of drawing to avoid NK cell stress. After isolation, cell count is extremely critical for result accuracy, so the method of counting should be constant: for example for trypan blue assay make sure that multiple squares are counted on the hemocytometer, and that the user or lab is using the same identification parameters. The protocol presented here uses 1.5 mL of blood and test NK cell cytotoxicity at a T:E ratio of 1:5, however these parameters can be optimized depending of the desired assay and the available amount of blood: if less blood is available then it is possible to use lower ratios such as 1:4 or even 1:3 to still have reasonable acquisition time. In our experience lower ratios might be insufficient to allow an appropriate cytotoxic activity. Also, because of sample variation, it happens that the NK cell count is much lower than expected; in response, it is necessary to lower the amount of target cells in reaction tubes to maintain the same T:E ratio throughout an experiment. During result analysis, the most critical step is to spend as much time as necessary to obtain a good matrix. This can only be obtained if great care is taken to prepare the controls described in Step 3 of the protocol. Once gates have been established as described in step 9, files can be batched together and analyzed in series with very little variation. If in doubt, the user should always refer to the image gallery by clicking on any event plotted on a graph (seen on Figure 1C), in order to check that all appropriate events are included in the proper gate.
The representative results in Figure 2A showed that 3 runners out of 4 presented a similar pattern. The pattern of the 4th runner was probably specific to that individual and does not represent an erroneous measure. The NK cell count for this individual was at the upper end of the accepted range in whole human blood both pre-exercise and 48 hr post-exercise. This demonstrates one of the strengths of the method described in this paper, where NK cells can be accurately obtained. Additionally, the capability to obtain native cells through one single labelling step, allows for minimal stress and modifications to the NK cells. This is particularly critical when NK cell cytotoxicity is being measured under physiological stress conditions.
Following isolation, NK cell cytotoxicity is typically determined by flow cytometry or a radioactive release assay (chromium assay for example). While release assays are considered a gold standard, they rely on the use of radioactive isotopes and related equipment, which are extremely expensive. Additionally, regulations for the use and disposal of radioactive materials add complexity to experiments that require time-efficiency and precision. The use of a flow cytometry approach makes sense but typical precautions must be followed including the laser settings, and fluorochrome compensation and gating. This is especially true when 2 different types of cells are in the sample, as it is the case for cytotoxic assays. However, in our case the use of an imaging flow cytometer allows an image to be captured for each event, and the size ratio parameter allows the separation of doublets from single cells. This is a critical point, since the cytotoxic assay relies on the physical contact between the effector and the target; very often these 2 types of cells will still be together during flow measurement, introducing false double positives into the results. This very point is illustrated in Figure 1D, which shows a doublet formed by a (dying, PI positive) NK cell, and a (live, DiO positive, PI negative) K562 cells. In traditional flow cytometry, this event would be added to the K562 cell death count but is in fact a false positive. Removing data can indeed be problematic, however it is not, if it is done consistently and using very strict parameters from experiment to experiment (in this case, the "aspect ratio" parameter). In return, the data obtained will be much more stable. Additionally, visualizing all events allows for the precise gating (and count) of each event based on color parameters, without relying on rigid gates that are traditionally established using controls and that do not take into account the natural "data drift" that can occur in flow cytometry. The added capability of imaging every single target event during acquisition, allows for an improved gating of single (DiO only, representing the live target cells) and double-stained population (DiO + PI stained, representing the dead target cells).
Additionally, the workflow presented here was optimized to take into account the scarcity of NK cells in whole blood and the need for accurate results. While traditional NK cell cytotoxicity assays explore the lytic activity at several T:E ratios, it is unpractical for a couple of reasons: 1) the amount of available NK cells from whole blood is limited and extremely variable and 2) the amount of target cells needed for the analysis must be large enough to allow for fairly quick acquisition of the data. Several tests at different ratios (not shown here) showed that a 1:5 T:E ratio, which translate in 4 x 104 T/2 x 105 E gives an optimal balance, and allows for great reproducibility. Based on repeated assays with samples from various individuals (not shown here), this ratio also provides an expected cytotoxic activity of 50-70% in normal samples, which offers a significant over and under window to measure the effect of interventions.
The NK cell count was similar to what was found in other works11,12,13, and in-line with the accepted range of frequency of NK cells on blood14. However, the advantage of this new workflow is demonstrated in Figure 3B, where we detected a significant increase of NK cell cytotoxicity after 24 h compared to pre-workout levels, despite a NK cell count that was in average (albeit non significantly) lower at that same time point. This result differs from other published studies where cytotoxic levels were mostly identical (or even lower) to the pre-workout levels. It suggests that instead of being depressed over a long period of time post-exercise, NK cell cytotoxicity might be able to "rebound" to even higher levels after as little as 21 h post-exercise. This observation using a novel methodology with pure, healthy NK cells, if confirmed in follow-up studies, has the potential to change the current perspective that heavy exertion is immunosuppressive. When NK cells are obtained using Ficoll gradients or flow-cytometry-based sorting, the resulting population is either mixed with other cell types, contain multiple damaged NK cells and/or NK cells with modified response. These consequences are to be expected considering the length, harshness and/or imprecision of the aforementioned methods. Removing these issues, and staying close to physiological conditions at all time, will lead to experimental results more representative of the NK cells in vivo behavior.
To summarize, we described an integrated workflow that allows for a fast, accurate isolation and detection of NK cell cytotoxicity from small human blood samples that avoids some of the limitations associated with other methods. While human blood was the focus of this work, it is noteworthy that this methodology can be applied to animal blood samples. This workflow is extremely reproducible and can detect small variations, which is particularly valuable for exercise studies where participant numbers are limited and serial blood measures are acquired. As underscored in the representative results, this improved method has the potential to challenge and improve current knowledge in exercise immunology, and other fields related to NK immuno-surveillance.
The authors have nothing to disclose.
This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2100-68003-30395 from the USDA National Institute of Food and Agriculture.
K-562 lymphoblasts | ATCC | CCL-243 | |
Iscove's Modified Dulbecco's Media | ATCC | 30-2005 | High glucose, with L-Glutamine, with HEPES, Sterile-filtered |
Alpha Minimum Essential medium | ATCC | CRL-2407 | Without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate |
Trypan Blue Solution 0.4% | Amresco | K940-100ML | Tissue culture grade |
Propridium Iodide Staining Solution | BD Pharmingen | 51-66211E | |
Vybranto DiO cell-labeling solution | Vybranto | V-22886 | |
autoMACS Pro Separator | Miltenyi Biotec | 130-092-545 | |
autoMACS Running Buffer | Miltenyi Biotec | 130-091-221 | |
autoMACS Washing Buffer | Miltenyi Biotec | 130-092-987 | |
autoMACS Columns | Miltenyi Biotec | 130-021-101 | |
Whole Blood CD56 MicroBeads, human | Miltenyi Biotec | 130-090-875 | |
ImageStream X Mark II Imaging Flow Cytometer | EMD Millipore | ||
Speedbeads | Amnis Corporation | 400030 | |
0.4-0.7% Hypochlorite (Sterilizer) | VWR | JT9416-1 | |
Coulter Clenz | Beckman Coulter | 8546929 | |
70% Isopropanol (Debubbler) | EMD Millipore | 1.3704 | |
D-PBS (Sheath fluid) | EMD Millipore | BSS-1006-B (1X) | No calcium or magnesium |
INSPIRE Software | EMD Millipore | Version Mark II, September 2013 | |
Ideas Application Software | EMD Millipore | Version 6.1, July 2014 |