Analysis of Natural Killer Cell Activity by Flow Cytometry
The
Tamas Racz, MD; Peter Sacks, PhD; Nguyan T. Van, PhD; Dorothy L. Taylor, MS; Greg Young; Samuel Bugis, MD; Howard E. Savage, PhD; Stimson P. Schantz, MD
\s=b\ A flow cytometric assay was used to detect the lytic and binding capacities of both fresh peripheral blood lymphocytes and purified Leu-19+ natural killer cells against head and neck cancer cell lines. Results demonstrated that natural killer cell-mediated cytotoxicity and effector\x=req-\ target conjugate formation evaluated by flow cytometry was significantly correlated with the standard chromium 51 release assay and the single-cell microscopic assay, respectively. The sorted Leu-19 natural killer cells demonstrated higher lytic capacity with a corresponding higher binding rate compared with the unsorted peripheral blood lymphocytes and sorted Leu-19\m=-\ cells. Flow cytometric analysis of natural killer cell activity (a rapid, simple, and quantifiable procedure) is an alternative to the standard chromium 51 release assay. (Arch Otolaryngol Head Neck Surg.
1990;116:440-446)
Accepted for publication December 1,1989. From the Department of Head and Neck Surgery, National Institute of Oncology, Budapest, Hungary (Dr Racz); the Departments of Head and Neck Surgery (Drs Sacks, Savage, and Schantz, Ms Taylor, and Mr Young) and Hematology (Dr Van), The University of Texas MD Anderson Cancer Center, Houston; and the Department of Surgery, Queen's University, Hotel Dieu Hospital, Kingston, Canada (Dr Bugis). Reprint requests to the Department of Head and Neck Surgery, Box 69, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 (Dr Schantz).
basic immunologie defense mechanisms may prevent tumor growth and progression: natural killer (NK) cells, which recognize primitive or nonself cells, and cells, which rec¬ ognize self in an altered state.1"3 The two mechanisms serve the host in rec¬ ognizing and destroying the heteroge¬ neous population of tumor cells that express various degrees of cellular differentiation.45 There are three common and funda¬ mental steps during cell-mediated
Two
cytotoxicity: (1) target-effector conju¬ gate formation, (2) delivery of the lethal hit, and (3) recycling and repeat¬ ing the actions (under appropriate circumstances).6
The most commonly used assays, ie, the chromium 51 (51Cr) release and microscopic single-cell assay, which evaluate these cytotoxic phenomena, have several disadvantages in their use in clinical medicine. They are la¬ bor-intensive, are subject to laborato¬ ry-induced error, and require use of radioisotopes. To overcome these dis¬ advantages, we report here the use of flow cytometry to estimate NK binding and cytotoxicity. The specific goals of this study were to determine the value of flow cytometry in identifying differ¬ ent stages of NK cell-tumor interac¬ tion and to compare these results with standard 51Cr release and single-cell assays. We also investigated whether
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the number of NK cells and their per¬ cent of binding rate can predict their
lytic potential.
PATIENTS AND METHODS Patients
Eighteen patients were assessed for NK cell activity. Sixteen of them had not been treated previously. One patient had a re¬ current tumor previously treated with ra¬ diation and one patient who had no evidence of disease had previously been treated with surgery and radiation. Preparation of Cell Lines (SCC) cell lines, previously established by expiant outgrowth techniques,7 were used. Line Two squamous cell carcinoma
MDA183 was derived from a well-differen¬ tiated (WD) SCC of the tonsil, and line MDA1386 was derived from a lymph node of a patient with a poorly differentiated (PD) SCC of the hypopharynx. The tumors were classified according to their degree of dif¬ ferentiation as determined by the percent¬ age of keratinized cells: WD indicates 75% or more, and PD indicates 25% or less. K562, an erythroleukemia cell line, was maintained in culture in RPMI supple¬ mented with 10% fetal calf serum.
Preparation of Effector Cells
Lymphocyte-rich mononuclear cells were isolated by centrifugation at 500g for 50 minutes on a Ficoll-diatrizoate sodium den¬ sity gradient (specific gravity, 1.1077; Bionetics, Charleston, SC). Monocytes were re¬ moved by their characteristic adherence to plastic.
51Cr Release Assay
90% of Leu-19+ cells
The assay has been described in detail previously.8 Effector cells were mixed with 10000 51Cr-labeled target cells, either the K562 or the squamous cell cancer cell lines, in multiple ratios from 50:1 to 6:1 and cen¬ trifugea for 1 minute at 200gr in flat-bot¬ tomed microtiter plates in triplicates in a total volume of 0.2 mL. Cells were subse¬ quently incubated at 37°C in 5% carbon di¬ oxide, for 6 hours prior to testing. One hun¬ dred microliters was removed from each well, and the radioactivity was counted for 1 minute. Percent of cytotoxicity was computed ac¬ cording to the following formula:
[(Experimental
Mean cpm Spontaneous Mean cpm)/(Maximum Mean cpm Spontaneous Mean cpm)] X 100 -
—
Maximum release was determined by in¬ cubating target cells in 10% Triton-X 100 for 6 hours at room temperature.
Expression of Lytic Units (LUs) Data were expressed as the number of LUs per 10 million peripheral blood lym¬ phocytes (PBLs). An LU was defined as the number of PBLs needed to effect 25% cyto¬ toxicity of 10 000 target cells. Immunofluorescence
Labeling and
Cell Sorting The PBLs were labeled with monoclonal antibodies (MAbs) according to manufac¬ turer's specifications for flow cytometric analysis. In each instance, 1 million PBLs or tumor cells were suspended in 50 gL of Dulbecco's phosphate-buffered saline plus 0.2% sodium azide before the respective MAb was added. Isotype-matched fluorescein isothiocyanate- and phycoerythrinconj ugated antibodies were used as controls to exclude Fc-related binding; all proce¬ dures were performed at 4°C. Typically, 10000 cells were analyzed for single-color and two-color immunofluorescence. The MAbs used in cell-surface staining of effec¬ tor cells were CD45 and anti-Leu-19 (Beeton Dickinson, Mountain View, Calif). CD45 is an anti-human leukocyte antibody to the
antigen and is expressed on all hu¬ man leukocytes (clone 2D1). Anti-Leu-19 is a human lymphocyte antibody to the Leu-19 antigen expressed on essentially all NK cells (clone MY 31). For separation of Leu19* cells, 20 million PBLs were stained and HLe-1
sorted with a cell sorter (FACStar Plus, Becton Dickinson, Sunnyvale, Calif). Cell viability after sorting by trypan blue ex¬ clusion exceeded 95%. The purity was at least 90%. Less than 3% contamination of Leu-19+ cells existed within the negative population. Morphologically, more than
were
large granular
lymphocytes. Target Binding Assay and Single-Cell Agarose Assay The method of Bonavida et al9 was used with minor modifications. Lymphocytes and targets were first equilibrated sepa¬ rately at 37°C at a concentration of 2 mil¬ lion per milliliter. Using an effector-totarget ratio of 1:1,150 uL of each was added to 12 X 75-mm glass tubes, which were gently centrifuged at 50gr for 2 minutes and then placed in a 37°C waterbath for 20 minutes. The tubes were centrifuged at 50g for 5 minutes at room temperature, drained over gauze, and wiped with a tissue to 0.5 cm above the pellet. A 2% agarose solution was kept molten at 45 °C and mixed with RPMI to a final agarose concentration of 1.33%. To prepare the slides, 50 ph of RPMI fetal calf serum was run down the side of the tube, quickly followed by 50 uh of molten agarose added directly on top, and then mixed four to five times with a micropipettor. The tube was inverted and the mixture tapped onto a precoated glass slide and spread evenly over the entire slide. After air drying for 1 to 2 minutes, the slide was stored in RPMI sup¬ plemented with 10% fetal calf serum until cells were counted. The percent of binding was calculated by dividing the number of bound effectors by the total number of effectors. At least 200 effectors were counted on each slide.
Cytometry Cytotoxicity and Binding Assay The flow cytometry assay was performed parallel with the 51Cr release assay at the same effector-to-target ratios. Effector Flow
cells were mixed with 10000 target cells in 12 X 75-mm plastic tubes, in triplicates, in a total volume of 0.2 mL. Cells were centri¬ fuged for 1 minute at 200s and subsequently incubated at 37°C in 5% carbon dioxide, for 6 hours. Twenty minutes prior to the end of incubation, the cell pellets were resuspended in propidium iodide (20 uL/mh in phosphate-buffered saline) (Sigma Chemi¬ cal Co, St Louis, Mo). After incubation, the cells were kept on ice until analysis. Sam¬ ples were assayed on the cell sorter and the data were analyzed using the FACStar Re¬ search Software Package (Becton Dickin¬ son). A minimum of 10 000 events were col¬ lected on each sample. The analysis was performed by setting the "gate window" on the target cells on the forward scatter vs side scatter dot plot screen. The markers were set to determine the dead cell back¬ ground on the histogram of the target cells incubated alone, at the same concentration and volume as the test population. The flow cytometry binding assay was
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performed after staining the effector cells with CD45. The lymphocyte and target preparations were treated in the same fash¬ ion as described in the single-cell agarose assay. They were equilibrated
separately at
37°C at a concentration of 2 million cells per milliliter. Using a 1:1 effector-to-target ra¬ tio, 150 uL of each was added to 12 X 75-mm tubes, which were centrifuged at 50g for 2 minutes. After a 20-minute waterbath at 37°C, a minimum of 10 000 events were col¬ lected. The percent of binding was calcu¬ lated by dividing the number of bound effectors by the total number of effectors.
RESULTS Correlation Between Flow Cytometry Analysis of Cell-Mediated Cytotoxicity and 51Cr Release Assay
The effector and target cells (includ¬
ing K562 and both SCC cell lines) could be distinguished according to their size (forward scatter) and density (side scatter) characteristics. Figure 1
shows results with K562 as an illus¬ trative example, and demonstrates that the percentage of the gated nonviable and viable target cells can be calculated through their contour graphs or histograms. The viability of target cells incubated alone for 6 hours gives the spontaneous death or back¬ ground values (Fig 1). Figure 2 shows that the mean ± SEM cytotoxic values of the 51Cr re¬ lease and the flow cytometric assays were not significantly different at any ratio against K562 and MDA1386 tar¬ gets. One representative experiment is shown in Fig 3. Results were similar testing the sorted Leu-19+, Leu-19", and unsorted PBLs against three dif¬ ferent targets, ie, K562, MDA1386, and MDA183 (Fig 4). Using unsorted PBLs, as is custom¬ ary for standard cytotoxicity assays, the correlation between the two assays after transformation of data to LU values against K562 (r .90; < .001) and MDA1386 (r .76; < .001) was highly significant (Fig 5). Because of minimal activity against MDA183, the correlation between the two assays with the latter cell line was not tested. =
=
Correlation Between the Rate of
Effector-Target Conjugate Formation by Flow Cytometry and the Single Cell Assay
After staining the effector cells with CD45 and performing the target cell
10 000 -ï
r 1000
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800
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600
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600
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800
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400
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Fluorescence 2 Height
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Fig 1.—Analysis of the natural killer cell-mediated cytotoxicity by flow cytometry. Top left, the gate is set on the K562 target cells on the for¬ ward scatter (FSC) vs side scatter (SSC) dot plot distribution of the mixed lymphocyte/tumor cell populations. Furthermore, the same gate is used during the analysis. Bottom left demonstrates the dead cell background determination (5% of the total population) for control target
cells (ie, target cells incubated alone for 6 hours) by histogram due to their fluorescence intensity by propidium iodide uptake. Using the same marker position for the effector-target mixture, the determination of the viable vs nonviable target cell after 6-hour incubation at an effector:target ratio of 50:1 are represented in histogram (bottom right) and in dot plot distribution (38% cytotoxicity) (top right).
binding, we easily distinguished three populations (effector cells, target cells, and conjugates) according to size and fluorescence intensity (Fig 6). After the sorting window on the conjugate population was set and the population was sorted, a microscopic analysis con¬ firmed a high proportion of conjugate
formation contained within window 1, as we expected (Fig 6). The results of the single-cell and flow cytometric as¬ says for binding capacity were nearly identical (Table 1). Thus, values from the assays can be used interchange¬
ably. Finally,
mean cytotoxicity values at different ratios determined by flow cytometry and 5,Cr re¬ lease assays. Results represent 12 experiments against K562 and 9 experiments against MDA1386 target cells. Lines over bars indicate SEM.
Fig 2.—The
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we compared the rate of cell target binding as determined by flow cytometry, as well as the percent¬ age of Leu-19+ positive PBLs, with the
LU values against both K562 and MDA1386 target cells (Table 2). Should either the percentage of bind¬ ing or the percentage of Leu-19+ di¬ rectly relate to the LU value, then all assessments could potentially be used interchangeably. This would greatly
simplify determination of natural im¬
status. The cumbersome cyto¬ toxicity assays could be eliminated. mune
Results of the comparison showed that only the relationship between percent¬ age binding and LU activity against MDA1386 was significant (r .82; =
< .01). No other significant relation¬ ships were noted. Table 3 reveals fur¬ ther that it is the Leu-19+ population that binds and kills most readily the respective target cell population. No significant lysis was mediated by the Leu-19" population.
COMMENT
These
results
offer
alternative
methods, compared with traditional
10
30
20
40
50
60
Effector-to-Target Ratio Fig 3.—One representative experiment of the comparison between flow cytometry and 5,Cr release assay. Each experiment was performed in triplicate wells. Vertical bars represent SEM.
NK cell functional assays, for both a better understanding of the factors that contribute to NK cell-mediated lysis and an improved means of apply¬ ing this clinically relevant informa¬ tion to day-to-day assessment of pa¬ tients with head and neck cancer. The most commonly used method for detecting only the end results of these processes is the 51Cr release assay. Some of the disadvantages of this as¬ say are the use of radioactive material and the difficulty of reproducing data from laboratory to laboratory. Some target cells are refractory to labeling and others (especially fresh tumor cells) may spontaneously release large
Fig 4.—Cytotoxicity values of the Leu-19+ effector cells, unsorted PBLs, and Leu-19 effector cells against K562, MDA1386, and MDA183 target cells. Means of three distinct experiments. Lines over bars indicate SEM.
Effector-to-Target Ratio
Effector-to-Target Ratio
MDA183
MDA1386
| ^ ¡3
Ü
Leu-19* Flow
Unsorted Flow Leu-19- Flow
Leu-19+ 5,Cr Unsorted 5,Cr
6
3
Effector-to-Target Ratio K3562
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Leu-19" 5,Cr
Flow LUs
Flow LUs
K562
MDA1386
Fig 5.—The correlation between the flow cytometry and 51Cr release lytic unit (LU) values with K562 (left) and MDA1386 (right).
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Forward Scatter Histogram
Fig 6.—Left, Dot plot distribution of MDA1386 target cells (window 3), the peripheral blood lymphocytes stained by CD45 monoclonal antibody (window 2), and the conjugates (window 1) formed between periph¬ eral blood lymphocytes and the target cell. Right, Cells in window 1 were sorted and stained with May-Grünwald;the slide shows the conjugate formation.
amounts of 51Cr.10 Another standard assay used for understanding the pro¬
of binding and recycling of killer cells against cancer targets, namely, the direct microscopic single-cell as¬ say, has been developed. This assay provides information on a single-cell level about effector-target cell conjucess
assay is laborious and difficult to quantitate because fewer cells are counted. Past studies have evaluated flow cy¬ tometry as a method to evaluate cellmediated cytotoxicity by examining the release of various fluorescein dyes from the lysed target.1012 Although
gate formation and lysis. The
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flow cytometric assays in these studies provided cytotoxicity results that were comparable with the 51Cr release as¬ say, there was a time limit because of a relatively rapid leakage of the dye with a concomitant high spontaneous release from the target cell. Thus, the period of incubation of the combined
effector-target populations in order to
Table
generate lysis was limited. In addition,
calculation of the results is compli¬ cated, limiting this method's day-to¬ day practicality. Recently, propidium iodide dye ex¬
was used to determine the number of dead cells.13 Propidium iodide dissolves in an isotonic solution, and its entry into living cells is pre¬ vented by the intact cell membrane. Hence, fluorescence is seen only in the nuclei of dead cells. The technique, as has been demonstrated in our study, makes possible the measurement of both live effector and target cells within the same sample and allows the effector-to-target ratio to be more ac¬ curately estimated. In our assay, the success of the method was based on the observation that the target cells in question were distinguishable from the effector cells based on their volume
1.—Comparison of the Binding Results of the Flow Cytometric Technique vs Microscopic Single-Cell Assay* Effector
Leu-19*
clusion
Targets
Microscope
K562
23
Flow
Microscope
26 ± 6
16 ± 5
Leu-19-
Flow
Flow
Microscope 10
15 Data
are
the
SEMs of three distinct experiments of effector (Leu-19*, unsorted peripheral blood Leu-19) and target (K562, MDA1386, and MD183) conjugates.
means ±
lymphocytes [PBLs]
and
Table 2.—Correlation of the Peripheral Blood Lymphocyte Binding Percent of Leu-19+ Cells Within the Peripheral Blood With Units (LUs) Against K562 and MDA1386
Capacity and
K562
ExperimentNo.
%
Leu-19·*
%
16
Lytic
MDA1386
Bindingt
LUs, No.
10
%
Leu-19'*
%
10 34
34
19
38
26
13
23
56
25
13
24
65
15
19
25
33
Binding!
LUs, No.
19
30
24
35
21
62
95
278
Percentage of Leu-19* cells within peripheral blood lymphocytes. tPercentage of peripheral blood lymphocytes that bind to the respective target as determined by flow cytom¬ etry.
Table 3.—Comparison of the Leu-19+, Unsorted Peripheral Blood Lymphocyte (PBL), and Leu-19" Cell Binding With Lytic Capacity Against K562, MDA1386, and MDA183 Target Cells MDA1386
K562
predicted simply through the quantitation of percentage of PBLs binding a specific target; specifically, in this in¬
LUs, No. Experiment No.
stance, the MDA1386. The establish¬
ment of a reliable prediction model for NK cell function would make the cyto¬ toxicity assay unnecessary and would provide a simple method for determin¬ ing the lytic potential of the effector population on a daily basis. In that re¬ gard, only the measure of PBL binding with MDA1386 significantly correlated
Unsorted PBLs
MDA183
characteristics. However, as perhaps in circumstances of interleukin 2 stim¬ ulation of lymphocytes when all popu¬ lations are identical in size, target cells can be identified by staining the target with a fluorescent dye, CFDA11 or
PKH-I.14 The detection of binding capacity was made possible in our study by la¬ beling the effector cells with fluores¬ cein isothiocyanate-conjugated CD45 surface marker, which is a pan-antileukocyte antibody. In our study, CD45 did not inhibit lysis (data not shown). Other investigators also have not found any effect on NK cell function.15 Because conjugate formation largely depends on centrifugation,16 and the percentage of conjugate increases as high speed increases, we strictly main¬ tained the same conditions used for the microscopic single-cell assay. Interest¬ ingly, results from this initial study showed that NK cell activity could be
Cells, % Binding
LUs, No.
Effectors
Binding*
Leu-19* Unsorted PBLs Leu-19^
38
Unsorted PBLs Leu-19-
51Cr Flow
Binding*
s'Cr Flow
117
36
122
133
22
125
111
35
142
111
22
91
105
35
111
95
23
20
26
60
s1Cr Flow
10
10
Leu-19" of PBLs that bind to
Binding*
133
Unsorted PBLs
Percentage
LUs, No. %
34
*
MDA183
respective target by
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flow cytometry.
18
37
with NK cell function against the same cell line in this preliminary study. No predictive measure was identified by simply quantifying the percentage of Leu-19+ lymphocytes, and neither measure was predictive of NK activity against K562. Given that the quantitation of NK cell function has prognostic implica¬ tion for risk of métastases from PD head and neck cancers, the relation¬ ship of binding to the cell line MDA1386 by PBLs to cytotoxic func¬ tion against that cell line may have clinical relevance.17 Binding as mea¬ sured by flow cytometry to this PD head and neck cancer cell line may thus in itself provide prognostic informa¬ tion. This can be performed on a rou¬ tine basis and should be easy to stan-
dardize between laboratories. The insufficient cytotoxic and bind¬ ing ability of the NK cell against cell lines derived from a WD head and neck cancer vs a cell line derived from a PD head and neck cancer extends our pre¬ vious observation regarding the im¬ portance of the recognition, binding, and lytic capacity of the NK cells against dedifferentiated cancer.1720 Previous studies have shown the prognostic information of NK cell function within patients with head and neck cancer.1720 In regard to the devel¬ opment of regional and distant me¬ tastasis, patients with PD SCCs and low NK function had the most prog¬ nostic information, while no relation¬ ship between NK function and risk of metastatic disease could be identified
in those individuals with WD lesions.17 These in vivo observations were ex¬ tended by the in vitro data, demon¬ strating that PD head and neck cancer target cells are highly sensitive to changes in lytic function expressed by
non-major histocompatibility com¬ plex effector cells.20 Future work based on the present study may provide more
information in the cellular level of this phenomenon. Such information not only will allow us to understand better the biology of head and neck cancer, but also may indicate the appropriate clinical use of biologic response modi¬ fiers. This study was supported by the First Indepen¬ dent Investigator Award (R29 CA 46251-01) to Dr Schantz. The Cancer Information Systems Core Facility was funded under National Cancer Insti¬ tute grant CA16672.
References 1. Savary CA, Lotzova E. Phylogeny and ontogeny of NK cells. In: Lotzova E, Herberman RB, eds. Immunobiology of Natural Killer Cells. Boca Raton, Fla: CRC Press Inc; 1986;1:45-63. 2. Grossman Z, Herberman RB. Natural killer cells and their relationship to T-cells: hypothesis on the role of T-cell receptor gene rearrangement on the course of adaptive differentiation. Cancer Res. 1986;46:2651-2658. 3. Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2 deficient lymphoma variants suggest alternative immune defense strategy. Nature. 1986;319:675-678. 4. Stern P, Gidlund M, Orn A, Wigzell H. Natural killer cells mediate lysis of embryonal carcinoma cells lacking MHC. Nature. 1980;285:341\x=req-\ 342. 5. Gidlund M, Orn A, Pattengale PK, Jansson M, Wigzell H, Nilsson K. Natural killer cells kill tumor cells at a given stage of differentiation. Nature. 1981;292:848-850. 6. Kiyohara T, Lauzon R, Haliotic T, Roder JC. Target cell structures, recognition sites and mechanism of NK cytotoxicity. In: Lotzova E, Herberman RB, eds. Immunobiology of Natural Killer Cells. Boca Raton, Fla: CRC Press Inc; 1986:1:107. 7. Sacks PG, Parnes SM, Gallick GE, et al. Establishment and characterization of two new
squamous cell carcinoma cell lines derived from tumors of the head and neck. Cancer Res.
1988;48:2858-2866. 8. Schantz SP, Romsdahl MM, Babcock GF, Nishioka K, Goepfert H. The effect of surgery on natural killer cell activity in head and neck cancer patients: in vitro reversal of a postoperatively
suppressed immunosurveillance system. Laryn-
goscope.
1985;95:588-594.
9. Bonavida B, Bradley TP, Grimm EA. Frequency determination of killer cells by a singlecell cytotoxic assay. Methods Enzymol. 1983; 93:270-280. 10. Pross H, Callewaert D, Rubin P. Assays for NK cell cytotoxicity: their values and pitfalls. In: Lotzova E, Herberman RB, eds. Immunobiology of Natural Killer Cells. Boca Raton, Fla: CRC Press
Inc; 1986;1:1.
11. McGinnes K, Chapman G, Marks R. A fluNK assay using flow cytometry. J Im-
orescence
munol Methods. 1986;86:7-11. 12. Kolber MA, Quinones RR, Gress RE, Henhart PA. Measurement of cytotoxicity by target cell release and retention of the fluorescent dye
bis-carboxyethyl-carboxyfluorescein
(BCECF).
J Immunol Methods. 1988;108:255-264. 13. Shi TX, Tong MJ, Bohman R. The application of flow cytometry inthe study of natural killer cell cytotoxicity. Clin Immunol Immunopathol.
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1987;45:356-365. 14. Slezak SE, Horan PK. Cell-mediated cytotoxicity: a highly sensitive and informative flow cytometric assay. J Immunol Methods. 1989; 117:205-214. 15. Deem RL, Shanahan F, Niederlehner A, Targan SR. Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity. Cell Immunol. 1988;117:105-110. 16. Bonavida B, Bradley TP, Grimm EA. The single cell assay in cell mediated cytotoxicity. Immunol Today. 1983;4:196-210. 17. Schantz SP, Ordonez N. Natural killer cell activity predicts metastases from poorly differentiated head and neck cancer. Proc Am Assoc Cancer Res. 1988;29:374. 18. Racz T, Sacks PG, Taylor DL, Schantz SP. Natural killer cell lysis of head and neck cancer. Arch Otolaryngol Head Neck Surg. 1989;115:1322\x=req-\ 1328. 19. Schantz SP, Goepfert H. Multimodality therapy and distant metastases: the impact of natural killer cell activity. Arch Otolaryngol Head Neck Surg. 1987;113:1207-1213. 20. Schantz SP, Racz T, Ordonez NG. Differential sensitivity of head and neck cancer to nonmajor histocompatibility-restricted killer cell activity. J Surg Res. In press.
The
Tamas Racz, MD; Peter Sacks, PhD; Nguyan T. Van, PhD; Dorothy L. Taylor, MS; Greg Young; Samuel Bugis, MD; Howard E. Savage, PhD; Stimson P. Schantz, MD
\s=b\ A flow cytometric assay was used to detect the lytic and binding capacities of both fresh peripheral blood lymphocytes and purified Leu-19+ natural killer cells against head and neck cancer cell lines. Results demonstrated that natural killer cell-mediated cytotoxicity and effector\x=req-\ target conjugate formation evaluated by flow cytometry was significantly correlated with the standard chromium 51 release assay and the single-cell microscopic assay, respectively. The sorted Leu-19 natural killer cells demonstrated higher lytic capacity with a corresponding higher binding rate compared with the unsorted peripheral blood lymphocytes and sorted Leu-19\m=-\ cells. Flow cytometric analysis of natural killer cell activity (a rapid, simple, and quantifiable procedure) is an alternative to the standard chromium 51 release assay. (Arch Otolaryngol Head Neck Surg.
1990;116:440-446)
Accepted for publication December 1,1989. From the Department of Head and Neck Surgery, National Institute of Oncology, Budapest, Hungary (Dr Racz); the Departments of Head and Neck Surgery (Drs Sacks, Savage, and Schantz, Ms Taylor, and Mr Young) and Hematology (Dr Van), The University of Texas MD Anderson Cancer Center, Houston; and the Department of Surgery, Queen's University, Hotel Dieu Hospital, Kingston, Canada (Dr Bugis). Reprint requests to the Department of Head and Neck Surgery, Box 69, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 (Dr Schantz).
basic immunologie defense mechanisms may prevent tumor growth and progression: natural killer (NK) cells, which recognize primitive or nonself cells, and cells, which rec¬ ognize self in an altered state.1"3 The two mechanisms serve the host in rec¬ ognizing and destroying the heteroge¬ neous population of tumor cells that express various degrees of cellular differentiation.45 There are three common and funda¬ mental steps during cell-mediated
Two
cytotoxicity: (1) target-effector conju¬ gate formation, (2) delivery of the lethal hit, and (3) recycling and repeat¬ ing the actions (under appropriate circumstances).6
The most commonly used assays, ie, the chromium 51 (51Cr) release and microscopic single-cell assay, which evaluate these cytotoxic phenomena, have several disadvantages in their use in clinical medicine. They are la¬ bor-intensive, are subject to laborato¬ ry-induced error, and require use of radioisotopes. To overcome these dis¬ advantages, we report here the use of flow cytometry to estimate NK binding and cytotoxicity. The specific goals of this study were to determine the value of flow cytometry in identifying differ¬ ent stages of NK cell-tumor interac¬ tion and to compare these results with standard 51Cr release and single-cell assays. We also investigated whether
Downloaded From: http://archotol.jamanetwork.com/ by a UQ Library User on 06/21/2015
the number of NK cells and their per¬ cent of binding rate can predict their
lytic potential.
PATIENTS AND METHODS Patients
Eighteen patients were assessed for NK cell activity. Sixteen of them had not been treated previously. One patient had a re¬ current tumor previously treated with ra¬ diation and one patient who had no evidence of disease had previously been treated with surgery and radiation. Preparation of Cell Lines (SCC) cell lines, previously established by expiant outgrowth techniques,7 were used. Line Two squamous cell carcinoma
MDA183 was derived from a well-differen¬ tiated (WD) SCC of the tonsil, and line MDA1386 was derived from a lymph node of a patient with a poorly differentiated (PD) SCC of the hypopharynx. The tumors were classified according to their degree of dif¬ ferentiation as determined by the percent¬ age of keratinized cells: WD indicates 75% or more, and PD indicates 25% or less. K562, an erythroleukemia cell line, was maintained in culture in RPMI supple¬ mented with 10% fetal calf serum.
Preparation of Effector Cells
Lymphocyte-rich mononuclear cells were isolated by centrifugation at 500g for 50 minutes on a Ficoll-diatrizoate sodium den¬ sity gradient (specific gravity, 1.1077; Bionetics, Charleston, SC). Monocytes were re¬ moved by their characteristic adherence to plastic.
51Cr Release Assay
90% of Leu-19+ cells
The assay has been described in detail previously.8 Effector cells were mixed with 10000 51Cr-labeled target cells, either the K562 or the squamous cell cancer cell lines, in multiple ratios from 50:1 to 6:1 and cen¬ trifugea for 1 minute at 200gr in flat-bot¬ tomed microtiter plates in triplicates in a total volume of 0.2 mL. Cells were subse¬ quently incubated at 37°C in 5% carbon di¬ oxide, for 6 hours prior to testing. One hun¬ dred microliters was removed from each well, and the radioactivity was counted for 1 minute. Percent of cytotoxicity was computed ac¬ cording to the following formula:
[(Experimental
Mean cpm Spontaneous Mean cpm)/(Maximum Mean cpm Spontaneous Mean cpm)] X 100 -
—
Maximum release was determined by in¬ cubating target cells in 10% Triton-X 100 for 6 hours at room temperature.
Expression of Lytic Units (LUs) Data were expressed as the number of LUs per 10 million peripheral blood lym¬ phocytes (PBLs). An LU was defined as the number of PBLs needed to effect 25% cyto¬ toxicity of 10 000 target cells. Immunofluorescence
Labeling and
Cell Sorting The PBLs were labeled with monoclonal antibodies (MAbs) according to manufac¬ turer's specifications for flow cytometric analysis. In each instance, 1 million PBLs or tumor cells were suspended in 50 gL of Dulbecco's phosphate-buffered saline plus 0.2% sodium azide before the respective MAb was added. Isotype-matched fluorescein isothiocyanate- and phycoerythrinconj ugated antibodies were used as controls to exclude Fc-related binding; all proce¬ dures were performed at 4°C. Typically, 10000 cells were analyzed for single-color and two-color immunofluorescence. The MAbs used in cell-surface staining of effec¬ tor cells were CD45 and anti-Leu-19 (Beeton Dickinson, Mountain View, Calif). CD45 is an anti-human leukocyte antibody to the
antigen and is expressed on all hu¬ man leukocytes (clone 2D1). Anti-Leu-19 is a human lymphocyte antibody to the Leu-19 antigen expressed on essentially all NK cells (clone MY 31). For separation of Leu19* cells, 20 million PBLs were stained and HLe-1
sorted with a cell sorter (FACStar Plus, Becton Dickinson, Sunnyvale, Calif). Cell viability after sorting by trypan blue ex¬ clusion exceeded 95%. The purity was at least 90%. Less than 3% contamination of Leu-19+ cells existed within the negative population. Morphologically, more than
were
large granular
lymphocytes. Target Binding Assay and Single-Cell Agarose Assay The method of Bonavida et al9 was used with minor modifications. Lymphocytes and targets were first equilibrated sepa¬ rately at 37°C at a concentration of 2 mil¬ lion per milliliter. Using an effector-totarget ratio of 1:1,150 uL of each was added to 12 X 75-mm glass tubes, which were gently centrifuged at 50gr for 2 minutes and then placed in a 37°C waterbath for 20 minutes. The tubes were centrifuged at 50g for 5 minutes at room temperature, drained over gauze, and wiped with a tissue to 0.5 cm above the pellet. A 2% agarose solution was kept molten at 45 °C and mixed with RPMI to a final agarose concentration of 1.33%. To prepare the slides, 50 ph of RPMI fetal calf serum was run down the side of the tube, quickly followed by 50 uh of molten agarose added directly on top, and then mixed four to five times with a micropipettor. The tube was inverted and the mixture tapped onto a precoated glass slide and spread evenly over the entire slide. After air drying for 1 to 2 minutes, the slide was stored in RPMI sup¬ plemented with 10% fetal calf serum until cells were counted. The percent of binding was calculated by dividing the number of bound effectors by the total number of effectors. At least 200 effectors were counted on each slide.
Cytometry Cytotoxicity and Binding Assay The flow cytometry assay was performed parallel with the 51Cr release assay at the same effector-to-target ratios. Effector Flow
cells were mixed with 10000 target cells in 12 X 75-mm plastic tubes, in triplicates, in a total volume of 0.2 mL. Cells were centri¬ fuged for 1 minute at 200s and subsequently incubated at 37°C in 5% carbon dioxide, for 6 hours. Twenty minutes prior to the end of incubation, the cell pellets were resuspended in propidium iodide (20 uL/mh in phosphate-buffered saline) (Sigma Chemi¬ cal Co, St Louis, Mo). After incubation, the cells were kept on ice until analysis. Sam¬ ples were assayed on the cell sorter and the data were analyzed using the FACStar Re¬ search Software Package (Becton Dickin¬ son). A minimum of 10 000 events were col¬ lected on each sample. The analysis was performed by setting the "gate window" on the target cells on the forward scatter vs side scatter dot plot screen. The markers were set to determine the dead cell back¬ ground on the histogram of the target cells incubated alone, at the same concentration and volume as the test population. The flow cytometry binding assay was
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performed after staining the effector cells with CD45. The lymphocyte and target preparations were treated in the same fash¬ ion as described in the single-cell agarose assay. They were equilibrated
separately at
37°C at a concentration of 2 million cells per milliliter. Using a 1:1 effector-to-target ra¬ tio, 150 uL of each was added to 12 X 75-mm tubes, which were centrifuged at 50g for 2 minutes. After a 20-minute waterbath at 37°C, a minimum of 10 000 events were col¬ lected. The percent of binding was calcu¬ lated by dividing the number of bound effectors by the total number of effectors.
RESULTS Correlation Between Flow Cytometry Analysis of Cell-Mediated Cytotoxicity and 51Cr Release Assay
The effector and target cells (includ¬
ing K562 and both SCC cell lines) could be distinguished according to their size (forward scatter) and density (side scatter) characteristics. Figure 1
shows results with K562 as an illus¬ trative example, and demonstrates that the percentage of the gated nonviable and viable target cells can be calculated through their contour graphs or histograms. The viability of target cells incubated alone for 6 hours gives the spontaneous death or back¬ ground values (Fig 1). Figure 2 shows that the mean ± SEM cytotoxic values of the 51Cr re¬ lease and the flow cytometric assays were not significantly different at any ratio against K562 and MDA1386 tar¬ gets. One representative experiment is shown in Fig 3. Results were similar testing the sorted Leu-19+, Leu-19", and unsorted PBLs against three dif¬ ferent targets, ie, K562, MDA1386, and MDA183 (Fig 4). Using unsorted PBLs, as is custom¬ ary for standard cytotoxicity assays, the correlation between the two assays after transformation of data to LU values against K562 (r .90; < .001) and MDA1386 (r .76; < .001) was highly significant (Fig 5). Because of minimal activity against MDA183, the correlation between the two assays with the latter cell line was not tested. =
=
Correlation Between the Rate of
Effector-Target Conjugate Formation by Flow Cytometry and the Single Cell Assay
After staining the effector cells with CD45 and performing the target cell
10 000 -ï
r 1000
1000-1
800
800-
1000-1
I
600
c-600
S
100
»400
ü
S
400-
'tí?*'-1'»
co
co
200
1 —\
0
400
600
200
' *|
r
|
|
200
""
1000
800
'
0
200
400
^^
600
1000
800
FSC Histogram
FSC Histogram
o
O
,ì,f,4^,lii, 0
200
400
600
· " '
800
I
1000
200
400
600
' ' ' '
800
I
1000
Fluorescence 2 Height
Fluorescence 2 Height
I_
_I
Fig 1.—Analysis of the natural killer cell-mediated cytotoxicity by flow cytometry. Top left, the gate is set on the K562 target cells on the for¬ ward scatter (FSC) vs side scatter (SSC) dot plot distribution of the mixed lymphocyte/tumor cell populations. Furthermore, the same gate is used during the analysis. Bottom left demonstrates the dead cell background determination (5% of the total population) for control target
cells (ie, target cells incubated alone for 6 hours) by histogram due to their fluorescence intensity by propidium iodide uptake. Using the same marker position for the effector-target mixture, the determination of the viable vs nonviable target cell after 6-hour incubation at an effector:target ratio of 50:1 are represented in histogram (bottom right) and in dot plot distribution (38% cytotoxicity) (top right).
binding, we easily distinguished three populations (effector cells, target cells, and conjugates) according to size and fluorescence intensity (Fig 6). After the sorting window on the conjugate population was set and the population was sorted, a microscopic analysis con¬ firmed a high proportion of conjugate
formation contained within window 1, as we expected (Fig 6). The results of the single-cell and flow cytometric as¬ says for binding capacity were nearly identical (Table 1). Thus, values from the assays can be used interchange¬
ably. Finally,
mean cytotoxicity values at different ratios determined by flow cytometry and 5,Cr re¬ lease assays. Results represent 12 experiments against K562 and 9 experiments against MDA1386 target cells. Lines over bars indicate SEM.
Fig 2.—The
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we compared the rate of cell target binding as determined by flow cytometry, as well as the percent¬ age of Leu-19+ positive PBLs, with the
LU values against both K562 and MDA1386 target cells (Table 2). Should either the percentage of bind¬ ing or the percentage of Leu-19+ di¬ rectly relate to the LU value, then all assessments could potentially be used interchangeably. This would greatly
simplify determination of natural im¬
status. The cumbersome cyto¬ toxicity assays could be eliminated. mune
Results of the comparison showed that only the relationship between percent¬ age binding and LU activity against MDA1386 was significant (r .82; =
< .01). No other significant relation¬ ships were noted. Table 3 reveals fur¬ ther that it is the Leu-19+ population that binds and kills most readily the respective target cell population. No significant lysis was mediated by the Leu-19" population.
COMMENT
These
results
offer
alternative
methods, compared with traditional
10
30
20
40
50
60
Effector-to-Target Ratio Fig 3.—One representative experiment of the comparison between flow cytometry and 5,Cr release assay. Each experiment was performed in triplicate wells. Vertical bars represent SEM.
NK cell functional assays, for both a better understanding of the factors that contribute to NK cell-mediated lysis and an improved means of apply¬ ing this clinically relevant informa¬ tion to day-to-day assessment of pa¬ tients with head and neck cancer. The most commonly used method for detecting only the end results of these processes is the 51Cr release assay. Some of the disadvantages of this as¬ say are the use of radioactive material and the difficulty of reproducing data from laboratory to laboratory. Some target cells are refractory to labeling and others (especially fresh tumor cells) may spontaneously release large
Fig 4.—Cytotoxicity values of the Leu-19+ effector cells, unsorted PBLs, and Leu-19 effector cells against K562, MDA1386, and MDA183 target cells. Means of three distinct experiments. Lines over bars indicate SEM.
Effector-to-Target Ratio
Effector-to-Target Ratio
MDA183
MDA1386
| ^ ¡3
Ü
Leu-19* Flow
Unsorted Flow Leu-19- Flow
Leu-19+ 5,Cr Unsorted 5,Cr
6
3
Effector-to-Target Ratio K3562
Downloaded From: http://archotol.jamanetwork.com/ by a UQ Library User on 06/21/2015
Leu-19" 5,Cr
Flow LUs
Flow LUs
K562
MDA1386
Fig 5.—The correlation between the flow cytometry and 51Cr release lytic unit (LU) values with K562 (left) and MDA1386 (right).
10 000
11000
800
1000 I
g
s-600 100-4 »-400 10-; îi200
I
0
200
I
I I
400
I
|
I
600
I
I I
|
800
'm.
1000
Forward Scatter Histogram
Fig 6.—Left, Dot plot distribution of MDA1386 target cells (window 3), the peripheral blood lymphocytes stained by CD45 monoclonal antibody (window 2), and the conjugates (window 1) formed between periph¬ eral blood lymphocytes and the target cell. Right, Cells in window 1 were sorted and stained with May-Grünwald;the slide shows the conjugate formation.
amounts of 51Cr.10 Another standard assay used for understanding the pro¬
of binding and recycling of killer cells against cancer targets, namely, the direct microscopic single-cell as¬ say, has been developed. This assay provides information on a single-cell level about effector-target cell conjucess
assay is laborious and difficult to quantitate because fewer cells are counted. Past studies have evaluated flow cy¬ tometry as a method to evaluate cellmediated cytotoxicity by examining the release of various fluorescein dyes from the lysed target.1012 Although
gate formation and lysis. The
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flow cytometric assays in these studies provided cytotoxicity results that were comparable with the 51Cr release as¬ say, there was a time limit because of a relatively rapid leakage of the dye with a concomitant high spontaneous release from the target cell. Thus, the period of incubation of the combined
effector-target populations in order to
Table
generate lysis was limited. In addition,
calculation of the results is compli¬ cated, limiting this method's day-to¬ day practicality. Recently, propidium iodide dye ex¬
was used to determine the number of dead cells.13 Propidium iodide dissolves in an isotonic solution, and its entry into living cells is pre¬ vented by the intact cell membrane. Hence, fluorescence is seen only in the nuclei of dead cells. The technique, as has been demonstrated in our study, makes possible the measurement of both live effector and target cells within the same sample and allows the effector-to-target ratio to be more ac¬ curately estimated. In our assay, the success of the method was based on the observation that the target cells in question were distinguishable from the effector cells based on their volume
1.—Comparison of the Binding Results of the Flow Cytometric Technique vs Microscopic Single-Cell Assay* Effector
Leu-19*
clusion
Targets
Microscope
K562
23
Flow
Microscope
26 ± 6
16 ± 5
Leu-19-
Flow
Flow
Microscope 10
15 Data
are
the
SEMs of three distinct experiments of effector (Leu-19*, unsorted peripheral blood Leu-19) and target (K562, MDA1386, and MD183) conjugates.
means ±
lymphocytes [PBLs]
and
Table 2.—Correlation of the Peripheral Blood Lymphocyte Binding Percent of Leu-19+ Cells Within the Peripheral Blood With Units (LUs) Against K562 and MDA1386
Capacity and
K562
ExperimentNo.
%
Leu-19·*
%
16
Lytic
MDA1386
Bindingt
LUs, No.
10
%
Leu-19'*
%
10 34
34
19
38
26
13
23
56
25
13
24
65
15
19
25
33
Binding!
LUs, No.
19
30
24
35
21
62
95
278
Percentage of Leu-19* cells within peripheral blood lymphocytes. tPercentage of peripheral blood lymphocytes that bind to the respective target as determined by flow cytom¬ etry.
Table 3.—Comparison of the Leu-19+, Unsorted Peripheral Blood Lymphocyte (PBL), and Leu-19" Cell Binding With Lytic Capacity Against K562, MDA1386, and MDA183 Target Cells MDA1386
K562
predicted simply through the quantitation of percentage of PBLs binding a specific target; specifically, in this in¬
LUs, No. Experiment No.
stance, the MDA1386. The establish¬
ment of a reliable prediction model for NK cell function would make the cyto¬ toxicity assay unnecessary and would provide a simple method for determin¬ ing the lytic potential of the effector population on a daily basis. In that re¬ gard, only the measure of PBL binding with MDA1386 significantly correlated
Unsorted PBLs
MDA183
characteristics. However, as perhaps in circumstances of interleukin 2 stim¬ ulation of lymphocytes when all popu¬ lations are identical in size, target cells can be identified by staining the target with a fluorescent dye, CFDA11 or
PKH-I.14 The detection of binding capacity was made possible in our study by la¬ beling the effector cells with fluores¬ cein isothiocyanate-conjugated CD45 surface marker, which is a pan-antileukocyte antibody. In our study, CD45 did not inhibit lysis (data not shown). Other investigators also have not found any effect on NK cell function.15 Because conjugate formation largely depends on centrifugation,16 and the percentage of conjugate increases as high speed increases, we strictly main¬ tained the same conditions used for the microscopic single-cell assay. Interest¬ ingly, results from this initial study showed that NK cell activity could be
Cells, % Binding
LUs, No.
Effectors
Binding*
Leu-19* Unsorted PBLs Leu-19^
38
Unsorted PBLs Leu-19-
51Cr Flow
Binding*
s'Cr Flow
117
36
122
133
22
125
111
35
142
111
22
91
105
35
111
95
23
20
26
60
s1Cr Flow
10
10
Leu-19" of PBLs that bind to
Binding*
133
Unsorted PBLs
Percentage
LUs, No. %
34
*
MDA183
respective target by
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flow cytometry.
18
37
with NK cell function against the same cell line in this preliminary study. No predictive measure was identified by simply quantifying the percentage of Leu-19+ lymphocytes, and neither measure was predictive of NK activity against K562. Given that the quantitation of NK cell function has prognostic implica¬ tion for risk of métastases from PD head and neck cancers, the relation¬ ship of binding to the cell line MDA1386 by PBLs to cytotoxic func¬ tion against that cell line may have clinical relevance.17 Binding as mea¬ sured by flow cytometry to this PD head and neck cancer cell line may thus in itself provide prognostic informa¬ tion. This can be performed on a rou¬ tine basis and should be easy to stan-
dardize between laboratories. The insufficient cytotoxic and bind¬ ing ability of the NK cell against cell lines derived from a WD head and neck cancer vs a cell line derived from a PD head and neck cancer extends our pre¬ vious observation regarding the im¬ portance of the recognition, binding, and lytic capacity of the NK cells against dedifferentiated cancer.1720 Previous studies have shown the prognostic information of NK cell function within patients with head and neck cancer.1720 In regard to the devel¬ opment of regional and distant me¬ tastasis, patients with PD SCCs and low NK function had the most prog¬ nostic information, while no relation¬ ship between NK function and risk of metastatic disease could be identified
in those individuals with WD lesions.17 These in vivo observations were ex¬ tended by the in vitro data, demon¬ strating that PD head and neck cancer target cells are highly sensitive to changes in lytic function expressed by
non-major histocompatibility com¬ plex effector cells.20 Future work based on the present study may provide more
information in the cellular level of this phenomenon. Such information not only will allow us to understand better the biology of head and neck cancer, but also may indicate the appropriate clinical use of biologic response modi¬ fiers. This study was supported by the First Indepen¬ dent Investigator Award (R29 CA 46251-01) to Dr Schantz. The Cancer Information Systems Core Facility was funded under National Cancer Insti¬ tute grant CA16672.
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11. McGinnes K, Chapman G, Marks R. A fluNK assay using flow cytometry. J Im-
orescence
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bis-carboxyethyl-carboxyfluorescein
(BCECF).
J Immunol Methods. 1988;108:255-264. 13. Shi TX, Tong MJ, Bohman R. The application of flow cytometry inthe study of natural killer cell cytotoxicity. Clin Immunol Immunopathol.
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1987;45:356-365. 14. Slezak SE, Horan PK. Cell-mediated cytotoxicity: a highly sensitive and informative flow cytometric assay. J Immunol Methods. 1989; 117:205-214. 15. Deem RL, Shanahan F, Niederlehner A, Targan SR. Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity. Cell Immunol. 1988;117:105-110. 16. Bonavida B, Bradley TP, Grimm EA. The single cell assay in cell mediated cytotoxicity. Immunol Today. 1983;4:196-210. 17. Schantz SP, Ordonez N. Natural killer cell activity predicts metastases from poorly differentiated head and neck cancer. Proc Am Assoc Cancer Res. 1988;29:374. 18. Racz T, Sacks PG, Taylor DL, Schantz SP. Natural killer cell lysis of head and neck cancer. Arch Otolaryngol Head Neck Surg. 1989;115:1322\x=req-\ 1328. 19. Schantz SP, Goepfert H. Multimodality therapy and distant metastases: the impact of natural killer cell activity. Arch Otolaryngol Head Neck Surg. 1987;113:1207-1213. 20. Schantz SP, Racz T, Ordonez NG. Differential sensitivity of head and neck cancer to nonmajor histocompatibility-restricted killer cell activity. J Surg Res. In press.
