Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates

JOURNALOF IMMUNOLOGICAL METHODS ELSEVIER

Journal of ImmunologicalMethods 173 (1994) 119-125

Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates Janita L6vgren

a, K a j B l o m b e r g

h,.

a Department of Biochemistry and Pharmacy, .4bo Akademi University, Turku, Finland, b Wallac Biochemical Laboratory, P.O. Box 10, FIN-20101 Turku, Finland

Received 22 November 1993; revised received 14 February 1994; accepted 18 March 1994

Abstract

We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay. Key words: Cytolysis; Cytotoxicity assay; Natural killer cell; Time-resolved fluorometry

1. Introduction

T h e r e are many non-radioactive methods available for the m e a s u r e m e n t of natural cytotoxicity. In recent years methods using flow cytometry (Radosevic et al., 1990), p r o m p t fluorometry (Bruning et al., 1980; Kolber et al., 1987), MT'I'based colorimetry ( H e o et al., 1990), X T T - b a s e d

Abbreviations: cps, counts per second; CAPT, 6,6"-bis[N,N-

bis(carboxymethyl)-aminomethyl]-4'-phenyl-2,2';6'2"terpyridine; DTPA, diethylenetriaminopentaacetate; FBS, foetal bovine serum; Hepes, N-2-hydroxyethylpiperazineN'2-ethanesulphonic acid; NK, natural killer cell; PBS, phosphate-buffered saline. * Corresponding author.

colorimetry (Jost et al., 1992), enzyme release (Korzeniewski and Callewaert, 1983) and time-resolved fluorometry (Blomberg et al., 1986a,b,; Blomberg and Ulfstedt, 1993) have been introduced. Time-resolved fluorometry has also been applied in assays of lymphokine-activated killer cell activity (Volgmann et al., 1988), T lymphocyte activity (Granberg et al., 1988), complement mediated cytolysis (Cui and Bystryn, 1992) and in the determination of cytotoxic T lymphocyte precursors (Bouma et al., 1993). The method described here is an extension of the method introduced earlier for the m e a s u r e m e n t of N K cell cytotoxicity against one target cell line (Blomberg and Ulfstedt, 1993). Two different fluorescent lanthanide chelates are used. The amount of each

0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00094-D

120

J. L6vgren, K. Blomberg /Journal of lmmunological Methods 173 (1994) 119-125

chelate released from the cell lines during the cytotoxicity test can be detected simultaneously with great sensitivity from the same assay using time resolved fluorometry because the two lanthanides have different and distinct emission lines. The fluorescence decay time of the lanthanide chelates is much longer than that of the background. In time-resolved fluorometry the long decay time is utilized by measuring the fluorescence from the sample after a certain delay time, which makes it possible to avoid the short-lived background fluorescence (Soini and Kojola, 1983; Soini and L6vgren, 1987). This eliminates the need to correct for background which is necessary when using conventional fluorescent labels such as the carboxyfluorescein derivates. The method we have developed resembles the SlCr release assay. Two target cell-lines are labelled with different lanthanide chelates (Eu-CAPT and TbCAPT). The emission lines of these chelates are well separated from each other which permits the development of double-component assays in which the two components are measured simultaneously (Hemmil~i 1988, Hemmil~i et al. 1993). Cell death is quantified by measuring the amount of chelate released into the medium during the experiment. The labelling procedure of Blomberg et al. (1986a) has been modified in order to increase label incorporation with the goal of using two target cell lines in a cytotoxicity assay.

2. Materials and methods

2.1. Reagents Buffer A was a 50 mM Hepes buffer containing 93 mM NaCI, 5 mM KCI and 2 mM MgCI 2, pH 7.4. Buffer B contained, in addition to the contents of buffer A, 2 mM CaCI 2 and 10 mM glucose. Stock solutions of 40 mM fluorescent europium and terbium chelates were prepared in buffer A. The chelates used, europium-6,6"-bis [ N,N-bis(carboxymethyl)-aminomethyl]-4'-phenyl2,2';6',2"-terpyridine (Eu-CAPT) and terbium6,6"-bis[ N, N-bis(carboxymethyl)-aminomethyl]-4'phenyl-2,2';6'2"-terpyridine (Tb-CAPT) were

from Wallac (Turku, Finland). The dextran sulphate stock solution was prepared by dissolving 50 mg of dextran sulphate (MW = 500 000) in 10 ml of buffer A, The stock solutions could be stored for several months at 4°C.

2.2. Preparation of effector cells Peripheral blood mononuclear cells were isolated from buffy coats by Ficoll-Hypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden). The cells were then washed twice with PBS and resuspended in culture medium.

2.3. Target cells Three different cell lines were used as targets: K-562, a NK-sensitive human erythroleukemia cell line, Molt4, an NK-sensitive T lymphoblastoid cell line and Daudi, a relatively NK-resistant Burkitt's lymphoma cell line. The cells were cultured in RPMI 1640 medium (Gibco) containing 10% FBS, 100 ~ g / m l streptomycin and 100 I U / m l penicillin (Gibco).

2.4. Labelling of target cells Around 10 × 106 cells were washed once with buffer A, the supernatant was removed and the cell pellet was resuspended in 900/zl of buffer A supplemented with 1-10 mM of Eu-CAPT or Tb-CAPT. Finally 100/~1 of the dextran sulphate stock solution was added to the cell suspension to make a total volume of 1 ml. The cell suspension was incubated at room temperature for 10 min and occasionally shaken. The addition of 3 ml of buffer B to the cell suspension stopped the labelling process. The incubation was continued for 5 more minutes to allow the membranes to seal. The labelled target cells were then washed three times with buffer B, twice with culture medium and resuspended to a concentration of 5 × 104 or 105 cells/ml.

2.5. Cytotoxicity assay When using only one target cell line 5000 target cells/100 /.d were added to each well of

J. Li~vgren, K. Blomberg /Journal of Immunological Methods 173 (1994) 119-125

U-bottomed microtitration plates (Nunc). If two different target cell lines were used 5000 target c e l l s / 5 0 ~I of each cell line were added to the wells. Triplicates of various effector cell concentrations were then pipetted into the wells. The volume used was 100 /zl and the number of effector cells ranged from 15 000 to 500000 per well. The final effector to target cell ratios ranged from 100:1 to 1.5:1. The effector to target cell ratios were always calculated in relation to both target cell lines. The spontaneous release was determined by incubating the targets with culture medium only and maximum release was obtained by incubating the target cells with 100 /zl 2% Triton X-100 in water. In order to determine the background fluorescence the remainder of the target cell suspension was centrifuged and 100/zl of the supernatant was incubated with 100 /zl medium. The plates were incubated for 2 and 4 h at 37°C in a humidified (95%) atmosphere with 5% CO 2. After the incubation the plates were centrifuged at 500 x g for 5 min and 150 /zl of the supernatant from each well was transferred to the wells of fiat-bottomed microtitration plates (Wallac). In addition 50/zl of medium were added to the maximum release wells and 50 /~1 of 3% Triton X-100 in water were added to the other wells. An equal amount of Triton X-100 was added to all wells because it is known to affect the signal level. The plates were measured in a time-resolved fluorometer (1234 Delfia, Wallac). The measuring conditions are given in Table 1. The percentage of cytolysis was determined by the following formula: experimental release - spontaneous release maximum release - spontaneous release x 100%

Table 1 Time-resolved fluorometric measuring conditions Chelate

Cycling time (ms)

Delay time (ms)

Counting time (ms)

Emission filter (nm)

Eu-CAPT Tb-CAPT

1 2

0.4 0.5

0.4 1.4

613 545

Excitation at 340 nm.

121

3. Results

3.1. Effect of temperature on target cell labelling The influence of the labelling time and temperature on label incorporation were studied. Labelling was carried out at 4 °, 20 ° and 37°C and the labelling time varied from 5 to 30 minutes. Label incorporation increased with the labelling time and temperature. Label incorporation at 20°C was usually twice that at 4°C and label incorporation at 37°C was approximately 1.5 times higher than at 20°C. The label incorporation between different labellings could vary by 50%. We standardized the labellings at room temperature since this was the most convenient and gave a satisfactory result.

3.2. Spontaneous release of the chelates The spontaneous release of the chelates varied with the cell line. The release rates of Eu-CAPT and Tb-CAPT were similar. After two hours the spontaneous release from K-562 ceils was 9%, from Molt4 cells 22% and from Daudi cells 21%. At 4 h the spontaneous release from the cells had increased to 11%, 27% and 28% respectively.

3.3. Specific release of the chelates The release rates of the two lanthanide chelates were studied by labelling the same pellet of K-562 cells with both chelates and running 2 and 4 h cytotoxicity assays. As can be seen from Fig. 1. the two chelates were released almost in identical fashion. The specific release of EuC A P T has earlier been compared to that of 51Cr and has been found to be somewhat faster (Blomberg and Ulfstedt, 1992). We also performed four assays with effector ceils from four donors in which K-562 cells labelled with Tb-CAPT and Molt4 cells labelled with Eu-CAPT were mixed and incubated with various numbers of lymphocytes. In the 2 h assays, at a 50:1 effector to target cell ratio, the specific release from K-562 cells was 48% (mean of four assays) and from

J. L6vgren, K. Blomberg/Journal of lmmunological Methods 173 (1994) 119-125

122

A.

45

_~40

i: ~z5

.;It -':::"

~20 ~ I0

15

.~::::'-

10

1.5:1

= 3:1

1,5:1

3:1

0

5

= 6:1 12:1 25:1 Effector :Target ratio

50:1

0 3:1

6:1

12:1 25:1 Effector : Target ratio

50:1

100:1 60

Fig. 1. Per cent specific release of E u - C A P T ( • ) and TbC A P T ( • ) from K-562 target cells when the same pellet was labelled with both chelates. Mean of three assays. The incubation times were 2 h ( - - - - - ) and 4 h ( ).

~ ,50 -~ 40 ~ 30

6:1

12:1

25:1

50:1

Effector :Target ratio

Molt4 cells 14%. In the 4 h assay the specific release from K-562 cells h a d increased to 55% and the specific release from Molt4 cells to 47% (Fig. 2). Cytotoxicity assays with K-562 cells labelled with T b - C A P T and Daudi cells labelled with E u - C A P T were carried out in the same manner. Parallel assays, in which the target cells were incubated separately with lymphocytes, were also performed. The total n u m b e r of target cells was 5000 per well when the cells were incubated separately and 10000 per well when the cells were mixed. When the cells were incubated separately (effector to target cell ratio 50 : 1) the specific release from K-562 cells after 2 h was 39% (mean of 3 assays) and the release from Daudi cells < 0%. The corresponding results from the

Fig. 2. Specific ma rke r release from K-562 cells ( • ) labelled with Tb-CAPT and Molt4 cells ( • ) labelled with E u - C A P T when incubated together for 2 h ( A ) and 4 h (B ) at different effector to target cell ratios. Mean of four assays.

assays in which the two target cells were mixed were 47% and 10%. After 4 h the specific release from the cells which were incubated separately had increased to 52% for K-562 and was < 0% for Daudi; the release from the cells which were incubated together had increased to 61% and 14% respectively (Table 2). In all assays the coefficient of variation for triplicate measurements was 5-10%.

Table 2 % specific release from two different cells lines (mean of three assays performed in triplicate) K-562 and Daudi in different wells

K-562 and Daudi in the same wells 2h

4h

2h

4h

E :T

K-562

Daudi

K-562

Daudi

K-562

Daudi

K-562

Daudi

3:1 6:1 12:'1 25:1 50:1

9 16 27 37 47

- 1 3 3 4 10

26 32 41 51 61

2 8 10 11 14

3 7 9 25 39

- 1 3 - 13 - 10 - 4

13 21 39 52

-6 - 17 - 17 - 27 - 7

J. L6vgren, I( Blomberg /Journal of Immunological Methods 173 (1994) 119-125

3.4. Sensitivity of the assay The sensitivity of the assay was estimated by the following formula: sensitivity 2 x SD (background of 5000 cells) maximum release (cps) - b a c k g r o u n d (cps) x 5000 cells The sensitivity when using different cell lines was calculated from at least ten 2 or 4 h assays. With K-562 cells the sensitivity was around 150 cells and with Molt4 cells it was 350 cells with both Eu-CAPT and Tb-CAPT when 5 mM chelate was used for the labelling. Daudi cells were only labelled with Eu-CAPT. The sensitivity with Daudi cells was 1000 cells. We also made three dilution series with K-562 cells that were labelled with different chelate concentrations (Table 3). When 10 mM chelate was used the maximum to background ratio remained at about 10 when the cell number was decreased. This suggests that less than 5000 cells can be used without any loss in assay sensitivity.

4. Discussion

In this paper we present a non-radioactive method which permits the simultaneous measurement of cytolysis using two different target cell types. The method is based on time-resolved fluo-

123

rometry and La-chelates with well separated emission lines. The use of the Eu-CAPT chelate as a target cell label in the assay of NK cytotoxicit3' has been previously described (Blomberg and Ulfstedt 1992) and here, in addition to the EuC A P T chelate, we have used a Tb-CAPT chelate. Even though two target cell lines are used the assay procedure is as simple as that of the 51Cr release assay. With the help of dextran sulphate the target cells can be labelled at room temperature with a lanthanide-CAPT chelate in only 10 min. The dextran sulphate treatment results in a nonspecific increase in the permeability of the cell membrane to small molecules, a process reversed by Ca 2+ and glucose (Kasahara 1977). The use of dextran sulphate does not make the cells more sensitive to lysis by NK cells (Blomberg et al., 1986b). The uptake of label is probably diffusion rate dependent and a prolonged labelling time results in cell death. This can be avoided by ensuring that the labelling time of sensitive cells is as short as possible. In order to be able to shorten the labelling time a higher chelate concentration or a higher labelling temperature can be used. Label incorporation varies depending on the cell line. Since the Eu-CAPT chelate has a somewhat higher signal to background ratio than the TbC A P T chelate we recommend its use with cell lines that are difficult to label. The amount of Eu- and Tb-CAPT chelate released from the cells are similar probably be-

Table 3 Dilution series of cells labelled in 10 rain at room temperature Number of Chelate concentration K-562 cells 0.1 mM Eu-CAPT

1 mM Eu-CAPT

10 mM Eu-CAPT

Maximum Back-ground Maximum/ Maximum Back-ground Maximum/ Maximum Back-ground Maximum/ release (cps) background release (cps) background release (cps) background

(cps) 50 100 500 1000 5000 25000 50000

390 487 1038 1804 7624 32454 72598

(cps) 299 330 442 384 712 2198 4071

1 1 2 5 11 15 18

496 536 1793 3 500 15706 76018 142738

(cps) 531 368 395 473 1305 5495 10457

1 1 5 7 12 14 14

2155 5490 25 127 51909 257502 1322777 2467153

580 902 3 324 5 647 23 680 108199 200872

4 6 8 9 11 12 12

124

J. L6vgren, K. BIomberg /Journal o flmmunological Methods 173 (1994) 119-125

c a u s e t h e labels a r e i d e n t i c a l a p a r t f r o m t h e c h e l a t e d l a n t h a n i d e s . W h e n using 5000 t a r g e t cells t h e d e a t h o f as few as 150 cells c o u l d b e d e t e c t e d above the background. Because the background d e c r e a s e s with cell n u m b e r s t h e d e a t h o f an even s m a l l e r n u m b e r o f cells c a n b e d e t e c t e d w h e n f e w e r t a r g e t cells a r e used. In o r d e r to achieve this k i n d o f sensitivity t h e m a x i m u m signal to background ratio after the labelling should be a b o u t 1 0 : 1 . D a u d i cells a r e h a r d e r to label t h a n K-562 o r M o l t 4 cells a n d t h e s p o n t a n e o u s r e l e a s e f r o m t h e m is also higher. This l e a d s to lower sensitivity a n d p o o r e r r e p r o d u c i b i l i t y which aga.in can l e a d to n e g a t i v e results w h e n t h e specific r e l e a s e o f label is low. T h e m e t h o d d e s c r i b e d has b e e n u s e d in two a n d f o u r h o u r N K cytotoxicity assays. T h e lant h a n i d e C A P T assay c a n b e p e r f o r m e d within o n e w o r k i n g d a y d u e to t h e s h o r t l a b e l l i n g time, t h e s i m p l e assay p r o c e d u r e a n d t h e fast r e l e a s e of label. Since t h e m e a s u r e m e n t o f 96 s a m p l e s t a k e s less t h a n 10 m i n u t e s t h e results a r e available i m m e d i a t e l y . T h e m e t h o d is less s u i t a b l e for long t e r m cytotoxicity assays d u e to t h e relatively high s p o n t a n e o u s r e l e a s e . In o u r h a n d s t h e p r e c i s i o n o f t h e assay is similar to t h a t o f t h e 5~Cr r e l e a s e assay. I n o r d e r to d e m o n s t r a t e t h e c a p a b i l i t i e s of t h e assay we have p r e s e n t e d results f r o m N K cytotoxicity assays using two N K - s e n s i t i v e cell lines (K-562 a n d M o l t 4 ) a n d N K - s e n s i t i v e a n d N K - r e s i s t a n t cell lines (K-562 a n d D a u d i ) as targets. A s can b e s e e n f r o m T a b l e 2 t h e m e a n o f t h e results f r o m t h e assays d i f f e r e d d e p e n d i n g u p o n w h e t h e r K-562 a n d D a u d i cells w e r e incub a t e d in t h e s a m e wells o r in s e p a r a t e wells. B e c a u s e t h e results o b t a i n e d with t h e D a u d i cells w e r e b e l o w t h e e s t i m a t e d sensitivity o f t h e assay a n d t h e v a r i a t i o n was high an analysis o f v a r i a n c e was d o n e . T h e d i f f e r e n c e s b e t w e e n t h e results f r o m t h e p a r a l l e l tests w e r e f o u n d to b e signific a n t with b o t h cell lines (results n o t shown). T h e h i g h e r c o n c e n t r a t i o n in t h e wells o f b o t h K-562 cell a n d D a u d i cells p r o b a b l y r e s u l t e d in t h e h i g h e r cytolysis. Thus, t h e results f r o m tests in which d i f f e r e n t cell lines a r e i n c u b a t e d e i t h e r s e p a r a t e l y o r t o g e t h e r can n o t be d i r e c t l y c o m p a r e d . H o w e v e r , t h e use o f two l a b e l l e d t a r g e t cell lines p r o v i d e s a b e t t e r o p p o r t u n i t y to gain

i n f o r m a t i o n a b o u t t h e specificity o f t h e e f f e c t o r cells t h a n t h e use o f only o n e l a b e l l e d t a r g e t cell line. It also p r o v i d e s a novel a p p r o a c h to studying t h e effects o f cytotoxic events on b y s t a n d e r cells.

References Blomberg, K. and Ulfstedt, A.-C. (1993) Fluorescent europium chelates as target cell markers in the assessment of natural killer cell cytotoxicity. J. Immunol. Methods 160, 24. Blomberg, K., Granberg, C., Hemmil~i, I. and L6vgren, T. (1986) Europium-labelled target cells in assay of natural killer cell activity. I. A novel non-radioactive method based on time-resolved fluorescence. J. Immunol. Methods 86, 225. Blomberg, K., Granberg, C., Hemmilh, I. and L6vgren, T. (1986) Europium-labelled target cells in assay of natural killer cell activity. II. A novel non-radioactive method based on time-resolved fluorescence, Significance and specificity of the method. J. Immunol. Methods 92, 117. Bouma, G.J., Van der Meer-Prins, P.M.W., Van Bree, F.P.M.J., Van Rood, J.J. and Claas, F.H.J. (1992) Determination of cytotoxic T-lymphocyte precursor frequencies using europium labelling as nonradioactive alternative to labelling with chromium-51. Hum. Immunol. 35, 85. Brunning, J.W., Kardol, M.J. and Arentzen, R. (1980) Carboxyfluorescein fluorochromasia assays. I. Non-radioactively labelled cell mediated lympholysis J. Immunol. Methods 33, 33. Cui, J. and Bystryn, J.-C. (1992) An improved europium release assay for complement-mediated cytolysis. J. Immunol. Methods 147, 13. Granberg, C., Blomberg, K., Hemmil~i, I. and L6vgren T. (1988) Determination of cytotoxic T lymphocyte activity by time-resolved fluorometry using europium-labelled concanavalin-A stimulated cells as targets. J. Immunol. Methods 114, 191. Hemmil[i, I. (1988) Lanthanides as probes for time-resolved fluorometric immunoassays. Scand. J. Clin. Lab. Invest 48, 389. Hemmilh, I., Mukkala, V.-M., Latva, M. and Kiilholma, P. (1993) Di- and tetracarboxylate derivatives of pyridines, bipyridines and terpyridines as lumonigenic reagents for time-resolved fluorometric determination of terbium and dysprosium. J. Biochem. Biophys. Methods 26, 283. Heo, D.S., Park, J.-G., Hata, K., Day, R., Herberman, R.B. and Whiteside, T.L. (1990) Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity. Cancer Res. 50, 3681. Kasahara, M. (1977) A permeability change in erlich ascites tumor cells caused by dextran sulfate and its repair by ascites fluid or Ca 2+ ions. Arch. Biochem. Biophys. 184, 400.

J. Liivgren, K. Blomberg / Journal of Immunological Methods 173 (1994) 119- 125 Koiber, M.A., Quinones, R.R., Gress, R.E. and Henkart, P.A. (1988) Measurement of cytotoxicity by target cell release and retention of the fluorescent dye bis-carboxyethylcarboyfluorescein (BCECF) J. Immunol. Methods 108, 255. Korzeniewski, C. and Callewaert, D.M. (1983) An enzyme-release assay for natural cytotoxicity. J. Immunol. Methods 64, 313. Jost, L.M., Kirkwood, J.M. and Whiteside, T.L. (1992) Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J. Immunol. Methods 147, 153. Radosevic, K., Garritsen, H.S.P., Van Graft, M., De Grooth,

125

B. and Greve, J, (1990) A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells. J Immunol. Methods 135, 81. Soini, E. and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates - A new generation of non-isotopic immunoassays. Clin. Chem. 29, 65. Soini, E. and Lfvgren T. (1987) Time-resolved fluorescence of lanthanide probes and applications in biotechnology. CRC Crit. Rev. Anal. Chem. 18, 105. Volgmann, T., Klein-Struckmeier, A. and Mohr, H. (1989) A fluorescence-based assay for quantization of lymfokineactivated killer cell activity. J. Immunol. Methods 119, 45.