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A fluorescence-based assay for quantitation of lymphokine-activated killer cell activity
Journal of Immunological Methods, 119 (1989) 45-51
45
Elsevier JIM05132
A fluorescence-based assay for quantitation of lymphokine-activated killer cell activity Thorsten Volgmann, Anette Klein-Struckmeier and Harald Mohr Blood Transfusion Service of Lower Saxony, Department of Research and Development, Eldagsener Str. 38, D-3257 Springe 1, F.R.G.
(Received24 August 1988, revisedreceived15 November 1988; accepted 25 November 1988)
A fluorescence assay for the quantitation of tumor cell lysis by activated and non-activated killer (LAK) cells is described. The target cells are labelled with a europium chelate (Eu-diethylenetriaminopentaacetate) and after cytolysis caused by the LAK cells the Eu 3+ complex is released into the culture supernatant. The addition of fl-naphthoyltrifluoroacetone to culture supematant aliquots leads to the formation of a highly fluorescent chelate which can be measured with a time-resolved fluorometer. The influence of various assay parameters has been evaluated including incubation time, effector-totarget cell ratio, the target cell line and different concentrations of interleukin-2 during cell culture. The optimized time-resolved fluorometric assay was found to be as simple and sensitive as the commonly used cytotoxicity assay in which the release of 51Cr from the labelled target cells is measured. In addition the assay is much faster and safer since the label is not radioactive. Key words: Cytotoxicityassay; Lymphokine-activatedkiller cell activity; Time-resolvedfluorescence
Introduction In cell-mediated cytolysis the cytotoxicity of effector cells (e.g., macrophages, cytotoxic T lymphocytes, natural killer (NK) cells) is usually determined by methods based on the release of different markers from the lysed cells. In general, these markers are compounds containing radioactive isotopes such as 5achromium, 75selenium or tritium, the 5aCr release assay being the one most commonly used. It is easy to perform, highly sensitive and the label is non-toxic to the cells
Correspondence to: Th. Volgmann, Blood Transfusion Service of Lower Saxony, Department of Research and Development, Eldagsener Str. 38, D-3257 Springe 1, F.R.G. Abbreviations: DTPA, diethylenetriaminopentaacetate;IL2, interleukin-2; LDH, lactate dehydrogenase; LAK cell, lymphokine-activatedkiller cell: NK cell, natural killer cell.
(Brunner et al., 1968). The use of SlCr, however, has some drawbacks, namely the health risk to personnel working with relatively high levels of the isotope which emits gamma rays. This situation had led to the development of non-radioactive cytotoxicity assays, e.g., the replacement of the radioactive label with a fluorescence dye which can be monitored with a fluorescence spectroscope when released (Brunning et al., 1980). Such an assay, however, lacks sensitivity because of the relatively high background fluorescence. In other test systems, the release of alkaline phosphatase or lactate dehydrogenase by the lysed cells is utilized for the determination of the cytolytic activity of effector cells (Szekeres et al., 1981; Korzeniewski et al., 1983). However, we have found it difficult to perform these assays within a normal working day, i.e., 8-9 h. Blomberg and co-workers (1986a,b) described an assay to mea-
0022-1759/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
46
sure the cytotoxicity of NK cells using target cells labelled with a europium complex. After release of the complex from lysed cells Eu 3+ is chelated with a fl-diketone. This chelate is highly fluorescent and can be quantified by time-resolved fluorometry. The fluorescence decay time of Eu chelates is in the range of 100-1000 ns compared to 10-20 ns decay time for fluorescent biological samples (Soini et al., 1979; Soini et al., 1983; Hemmila et al., 1984). This property of Eu chelates reduces the background fluorescence and considerably enhances the sensitivity of immunofluorescence assays in which Eu 3+ and other lanthanide ions are used. We have adopted the time-resolved fluorescence assay for the measurement of lymphokine-activated killer (LAK) cells. These are generated from their progenitors, cells belonging to the lymphocyte fraction within mononuclear blood cells, by exposure to interleukin-2 (IL-2) for 3-4 days (Grimm et al., 1984; Ortaldo et al., 1986). LAK cells are highly cytolytic towards autologous tumor cells and certain NK cell-resistant tumor cell lines; hence there is increasing interest in LAK cells for adoptive immune therapy (Rosenberg et al., 1986, 1987). We found that the time-resolved fluorescence assay with Eu 3+ as a label is as easy to perform, at least as sensitive as, and more rapid than either the 51Cr release or the lactate dehydrogenase assay. It thus represents a non-radioactive alternative for measuring the cytotoxicity of LAK and other cells.
Materials and methods
Reagents All chemicals were of analytical grade. DTPA and EuC13 were purchased from Aldrich-Chemie, Steinheim, F.R.G. Dextransulfate (MW 500000) was obtained from Pharmacia, Freiburg, F.R.G., iodophenylnitrotetrazolium chloride and NAD + were from Sigma, Deisenhofen, F.R.G. and diaphorase was purchased from Boehringer, Mannheim, F.R.G. All other chemicals were obtained from common suppliers (in general from Sigma). Microtiter strips and other plastic materials were
obtained from Flow, Meckenheim, F.R.G. and Nunc, Wiesbaden, F.R.G.
Preparation of LAK cells Mononuclear cells were isolated from adeninesodium citrate-dextrose stabilized donor blood by the Ficoll-Hypaque-method as described by Boyum (B~yum et al., 1976). The cells were washed three times with isotonic sodium chloride solution. Then they were suspended in RPMI 1640 containing 10% heat-inactivated fetal calf serum, 50 g g / m l streptomycin, 100 g g / m l neomycin and human IL-2 (if not stated otherwise 1000 U/ml). IL-2 was produced from Ca ionophore A 23187/ phorbol ester induced donor lymphocytes (Mohr et al., 1986) and purified to apparent homogeneity. Briefly, IL-2 was first adsorbed to porous silicic acid (Amicon, Witten, F.R.G.) and, like interferon-~, (Yip et al., 1981), could be released with ethylene glycol containing phosphate buffer. This was followed by ion exchange chromatography using S-Sepharose Fast Flow (Pharmacia, Freiburg, F.R.G.). The IL-2-containing fractions were eluted at 100-200 mM NaC1. The last purification step was reverse phase HPLC using the octyl phase. IL-2 was eluted at approximately 40% propanol in phosphate buffer, pH 2.5. For LAK cell generation, the suspended cells were incubated for 3-4 days in a humidified 5% CO 2 atmosphere at 37 o C.
Cytotoxicity assays The cytolytic activity of the IL-2-activated cells was evaluated against the NK-sensitive cell line K562 and the LAK cell-sensitive cell line Raji at various effector-to-target ratios. Three different test systems were used (see below). Each assay was performed in triplicate and the cytotoxicity values were calculated by using the following formula:
% lysis =
test r e l e a s e - s p o n t a n e o u s r e l e a s e × 100 r e l e a s e - spontaneous release
maximum
The maximum release was the total release obtained from target cells treated with 0.5% Triton X-100, and spontaneous release was the normal release of Eu chelate during test time without effector cells.
47 Lytic units (LU) were defined as the number of cells/107 cells, which cause 20% (LU20) of the target cells and were calculated according to the method of Pross (Pross et al., 1981).
The reaction was measured after 20 min in an ELISA reader (ELISA Processor II from Behring, Marburg, F.R.G.) at a wavelength of 492 nm. The reference wavelength was 650 nm.
51Cr release
Europium release assay
assay
The 4 h standard 51Cr release assay was carried out as follows: effector cells were labelled with 200 ttCi Na2CrO4/107 cells and washed three times with isotonic NaC1 solution. 5 x 103 cells were pipetted into each cup of round-bottomed 96 well tissue culture plates. The effector cells were added to give a final volume of 200 #1. After incubation for 4 h in a humidified 5% CO 2 atmosphere at 37 ° C, the cells were centrifuged and 100 /tl of the supernatant were removed from each well and counted in a gamma-counter (Gammamatic I, Kontron Analytical, Basel, Switzerland). L D H assay
The LDH assay was carded out as described by Korzeniewski (Korzeniewski et al., 1983). The target ceils were washed three times with RPMI 1640 containing 3% FCS to remove adherent LDH derived from lysed cells. The number of viable cells was estimated using trypan blue exclusion. The cell suspension was diluted with RPMI 1640 plus 3% FCS to give final concentrations between 3 x 104 and 2 x 105 cells/ml. 100/L1 target cell suspension and 100/~1 of an effector cell suspension were pipetted together into the wells of a round-bottomed microtiter plate. Suspensions containing exclusively effector cells, target cells, or culture medium, respectively, served as controls to estimate the LDH background. The plates were incubated for 4-6 h in a humidified 5% CO 2 atmosphere at 37°C. After incubation they were centrifuged at 130 × g for 10 min. Then 150 #1 of the supernatant from each well were transferred to the corresponding well of a flat-bottomed microtiter plate. 20 /~1 of a 400 mM lactate solution ( p H = 6 . 8 in phosphatebuffered saline (PBS)) were added to each well, followed by 20/xl of a solution of 4 mM iodophenylnitrotetrazolium chloride in PBS and finally 20 #1 of a 'reaction mix' containing 2.7 U / m l diaphorase, 4.5 mM NAD +, 0.03% bovine serum albumin and 1.2% sucrose in PBS.
The target cells were labelled with Eu 3+ in labelling buffer (pH 7.4) containing 50 mM Hepes, 93 mM NaC1, 5 mM KC1, 2 mM MgC12 • 6H20 and either 50 /~M EuC13 plus 250 #M diethylenetriaminopentaacetate (DTPA) for labelling K562 cells or 100 #M EuC13 plus 500 /~M DTPA for labelling Raji cells. A stock solution of EuC13 (10 mM) was prepared by dissolving the salt at 60-70 °C in 0.1 N HC1. Immediately before use, 50 ~tl/ml of a solution containing 10 mg/ml dextran sulfate in destilled water were added to the labelling solution. This was followed by the addition of 5 x 106/ml washed target cells. The cells were labelled with Eu 3+ for 20 min in an icebath with occasional shaking. The labelling reaction was stopped by the addition of 30 /~l/ml of a 100 mM CaC12 solution. After 5 min the cells were washed 5-6 times with RPMI 1640 containing 2 mM CaCI 2. The cells were diluted to a concentration of 5 X 10 4 cells/ml in RPMI 1640 containing 10% heat-inactivated FCS, streptomycin (50 /xg/ml) and penicillin (50 U/ml). 100 #1 of this suspension and 100/xl of effector cells were pipetted into the cups of round-bottomed microtiter plates. The plates were incubated for 2.5 h at 37°C. After incubation the cells were centrifuged and 20 #1 of the supernatants were transferred to the cups of flat-bottomed microtiter strips containing 200 /tl of an enhancement solution (Delfia from LKBWallac, Turku, Finland). After thorough mixing for 5 min at room temperature fluorescence was measured in a time-resolved fluorometer (Arcus 1230 from LKB-Wallace, Turku, Finland).
Results and discussion Relation between fluorescence and target cell concentration
The initial experiments were carried out to determine the relationship between the maximal and spontaneous release of the Eua+-mediated
48
meximcll ~lease release
c-
o
c~
u
t~ J
la.
1
i
2
i Ig~o of
cell
i
o
5
~
Fig. 1. Influence of various amounts of Eu-DTPA-labelled cells (e • Raji, • • K562) on fluorescence intensity and the relationship between background fluorescence and maximal fluorescence. The cells were labelled and incubated using the conditions described in the materials and methods section. Maximal release was obtained from cells which were treated with 0.5% Triton X100.
fluorescence and the number of target cells (Raji). Fig. 1 shows that the relationship was linear between 500 and 10000 cells per cup. The spontaneous release never exceeded 2-3% of the maximal fluorescence released from detergent lysed cells.
Since the commonly used cytotoxicity assays are performed with 5000 target cells/well, similar numbers were also used in our following experiments. Influence of incubation time on the release of Eu 3 ÷ To estimate the time dependency of the Eu 3+ release, labelled target cells were incubated with
Fig. 3. Dependence of the lytic activation of lymphocytes against two target cell lines (• •), Raji cells, o o, K562 cells) after treatment with various amounts of IL-2 (0, 10, 50, 100, 300, 600 and 1000 U / m l ) and after various times of incubation (closed symbols, 24 h; open symbols, 72 h).
LAK cells at an E / T ratio of 10 : 1 for different lengths of time. Fig. 2 shows a lag period of 30 rain before rapid release of the label from both K562 and Raji cells, with a linear increase of the fluorescence intensity up to 120 min. A plateau was reached after 150-180 min. Hence the assay can be stopped after 2-2.5 h, which is considerably shorter than in the classical 4 h 51Cr release assay and in the L D H assay, in which the release o f enzyme is still in its logarithmic phase after 4 h incubation (see Fig. 2b). We have found that the L D H assay requires at least 6 h to be adequately quantitative. This is in contrast to the observations of Korzeniewski and Callewaert (1983) whose data indicate that it requires an incubation time of 1(30
1OC
b
101 I
8(: n
6C
E~
D
3
~
] L - 2 concentrotion (Ig~o U / m l )
density
a
u
,
, ,
~ 60 •
$4c
,
~
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_'-2
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•
'~ 2C
•
?
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Time of europium- releose (h)
~
u3
240
2C
~
~
~
~
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7
Releasing t i m e (h)
Fig. 2. a: Dependence of specific lysis on time of Eu 3+ release during the cytotoxicity assay. Two target cell lines were tested m, Raji; [] D, K562) at an effector-to-target ratio of 10:1. E a c h point represents the mean of three separate experiments. (m
Fig. 2. b: Comparison of the kinetics of release times from K562 cells ( O O , L D H assay; [] D, SlCr-assay) using an effector-to-target ratio of 10 : 1. The shaded bars represent the specific lysis rates of K562 labelled w i t h E u - D T P A at the indicated effector-to-target ratios.
49
~2o~
not more than 2 - 4 h. The reason for this divergence remains obscure.
100~
? 8ow
LAK cell activation with IL-2 The use of the assay to measure L A K and N K cell activation by IL-2 is demonstrated in Fig. 3. Peripheral blood mononuclear cells were incubated with various amounts of IL-2 for 3 days. The cytotoxicity of the activated cells was tested on K562 as well as on Raji cells. As Fig. 3 indicates, the cytotoxic activity of the blood cells against K562 was detectable even in the absence of IL-2, which was obviously due to the presence of N K cells. This cytotoxic activity was drastically increased by IL-2 and reached its maximum at an IL-2 concentration of approximately 100 U / m l . NK-resistant Raji cells were not lysed by nonactivated mononuclear cells (see also Fig. 4). A threshold concentration of 30-50 U / m l IL-2 was needed to induce lytic activity. Maximal cytotoxicity was achieved at concentrations between 500-1000 U / m 1 IL-2. This is in agreement with other published results (Oshimi et al., 1988; Trenn et al., 1988).
Optimal effector-to-target ratio An E / T ratio of 3 0 : 1 to 5 0 : 1 was optimal when the Eu 3+ release assay was used to determine the cytotoxicity of unstimulated N K cells and this is similar to the classical 5]Cr release assay (Blomberg et al., 1986a,b). For measuring the cytotoxicity of lymphocytes activated with IL2, E / T ratios between 1 : 1 and 10 : 1 proved to be optimal (Fig. 4). This was independent of the target cell line used. The reason might be that IL-2 treatment leads to enhanced cytotoxicity of N K cells as well as L A K cells. This assumption is confirmed by the higher E / T ratios that were needed to lyse significant amounts of Raji and
•~-
60
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20
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~--q3-------13--O
0
20
,
[]
40
,
,
60 E / T - rQtio
,
80
,
D--
100
I
120
Fig. 4. Comparison of the lytic activity of activated (filled symbols) and non-activated (open symbols) lymphocytes against three different cell lines (O o, Raji; ,x ,x, K562; ~ - - ~ , Daudi) at various effector-to-target ratios. The Daudi cells were labelled in the same manner as the Raji cells. The lymphocytes were activated with 1000 U IL-2/ml. The activation time was 3 days.
Daudi targets with mononuclear cells not activated by IL-2. There was almost no cytolysis of Raji cells by effector cells not activated by IL-2.
Comparison with other cytotoxicity assays In subsequent routine cyt0toxicity assays for the estimation of L A K cell activation by IL-2, effector and target cells were co-incubated for 2.5 h at E / T ratios of 2.5 : 1, 5 : 1 and 10 : 1 respectively. Table I shows that under these conditions cytotoxicity rates were comparable with those achieved by the 51Cr release assay which was incubated for 4 h at E / T ratios ranging from 1 0 : 1 and 4 0 : 1 . When the 51Cr release assay was performed at the same low E / T ratios as the Eu 3+ fluorescence assay, it proved to be much less sensitive. On the other hand there was a good correlation between the L D H release assay and the Eu 3+ assay (Fig. 5). It should be noted, however, that the latter
TABLE I COMPARISON OF SPECIFIC LYTIC RATES OF THE TWO CYTOTOXICITYASSAYS AT VARIOUS EFFECTOR TO TARGET RATIOS Specific lysis (%) Eu3+ assay
E/T ratio: K562 Raji
51Cr assay
2.5
5
10
10
20
40
24.2 8.5
48.0 20.6
61.0 31.6
37.2 18.4
56.2 29.1
90.7 38.8
50 10C
fluorometric assay can also be used to determine cytolytic activity of lymphoid cells activated by IL-2 against NK cell resistant target cells. In our laboratory the assay is routinely used to monitor the activation of in vitro generated LAK cells from tumor patients taking part in clinical trials evaluating the therapeutical potential of these cells.
8C 6C .u_
g
4C
e~
2C 0
5
10 E / T - ratio
15
20
Fig. 5. Comparison of the three cytotoxicity assays (A A, 51Cr-release; • • , LDH-release; " - - 0 , Eu3+ release). The filled symbols represents the specific rates of lysis of K562 cells produced using activated lymphocytes. The open symbols represents data obtained from K562 cells lysed by non-activated lymphocytes.
involved a 6 h incubation instead of the 2.5 h for the Eu 3+ release assay. The different velocities at which the two labels and the marker enzyme were released from the target cells may be explained as follows. The Eu-DTPA complex is chemically inert (Hemmil~i et al., 1984). It is relatively small and therefore able to leave the target cells just after they are rendered permeable by exposure to effector cells. 51Cr (used as 51CRO42- ion) is more reactive and will bind to cellular proteins by ionic or other forces, and LDH is a large molecule. Hence, 51Cr and LDH are not released into the cell culture supernatant until the target cells are completely lysed. As a result, the Eu 3+ fluorescence assay is the fastest of the three. The assay can easily be performed and because of the short labelling time, release period and counting time (1 s/sample) it can be evaluated during a normal working day. Compared to the commonly used 51Cr release assay, it has the additional advantage that no radioactive isotopes are used. Under appropriate conditions it is at least as sensitive and accurate as the two other assays tested. A disadvantage is the relatively high price of the time resolved fluorometer (approx. DM 35000). But this is not higher than that of a scintillation counter, and the fluorometer can be used for other assays than that described in this article (Soini et al., 1979; Jackson et al., 1986). Our data confirm and expand those of Blomberg et al. by demonstrating that the time-resolved
Acknowledgements This study was supported by the Bundesministerium of Forschung und Technologie (0318 707
A8). The authors would like to thank Mr. R. Rosinke for expert technical assistance and Mrs. J. Linneweber for preparing the manuscript.
References Blomberg, K., Granberg, C., Hemmil~i, I. and Ltivgren, T. (1986a) Europium-labelled target cells in an assay of natural killer cell activity. I. A novel non-radioactivity method based on time resolved fluorescence. J. Immunol. Methods 86, 225. Blomberg, K., Granberg, C., Hemmil~i, I. and LiSvgren, T. (1986b) Europium-labelled target cells in an assay of natural killer cell activity. II. Significance and specificity of the method. J. Immunol. Methods 92, 117. Boyum, A. (1976) Separation of leukocytes from blood and bone marrow. Scan& J. Chn. Lab. Invest. 21, 77. Brunner, K.T., Mavel, J., Cerottini, M.C. and Chapius, B. (1968) Qunatitative assay of the lytic action of immune lymphoid cells on 51Cr-labdled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14, 181. Brunning, J.W., Kardoi, M.J. and Arentzen, R. (1980) Carboxyfluorescein fluorochromasia assays. I. Non-radioactively labeled cell mediated lympholysis J. Immunol. Methods 33, 33. Grimm, E.A. and Rosenberg, S.A. (1984) The human lymphokine activated killer cell phenomenon. Lymphokines 9, 279. Hemmila, I., Dakubu, S., Mukkala, V.-M., Siitari, H. and L~Svgren, T. (1984) Europium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137, 335. Jackson, T.M. and Ekins, R.P. (1986) Theoretical limitations on immunoassay sensitivity: current practice and potential advantages of fluorescent Eu-chelates as non-radioisotopic tracers. J. Immunol. Methods 87, 13. Korzeniewski, C. and Callewaert, D.M. (1983) An enzyme-release assay for natural cytotoxicity. J. Immunol. Methods 64, 313.
51 Molar, H., Monner, D. and Plessing, A. (1986) Calcium ionophore A23187 in the presence of phorbol ester PMA: a potent inducer of interleukin-2 and interferon-gammasynthesis by human blood cells. Immunobiology 171, 159. Ortaldo, J.O., Mason, A. and Oveston, R. (1986) Lymphokine activated killer cells. Analysis of progenitors and effectors. J. Exp. Med. 164, 1193. Oshimi, K., Oshimi, Y., Saito, H. and Mizoguchi, H. (1988) Cytotoxicity of interleukin-2-activated lymphocytes for autologous normal blood mononuclear cells. J. Immunol. Methods 109, 161. Pross, H.F., Baines, M.G., Rubin, P., Shragge, P. and Patterson, M.S. (1981) Spontaneous human lymphocyte mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity. J. Clin. Immunol. 1, 51. Rosenberg, S.A. and Lotze, M.T. (1986) Cancer immunotherapy using interleukin-2 and interleukin-2 activated lymphocytes. Ann. Rev. Immunol. 4, 681. Rosenberg, S.A., Lotze, M.T., Muul. L.M., Chang, A.E., Avis, A.P., Leitman, S., et al. (1987) A progress report on the
treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or highdose interleukin-2 alone. New Engl. J. Med. 316, 889. Soini, E. and Hemmil~i, I. (1979) Fluoroimmunoassay: Present status and key problems. Chn. Chem. 25, 353. Soini, E. and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates - a new generation of non-isotopic immunoassays. Clin. Chem. 29, 65. Szekeres, I., Pasca, A.S. and Peytsik, B. (1981) Measurement of lymphocyte cytotoxicity by assessing endogeneous alkaline phosphatase activity of the target cells. J. Immunol. Methods 40, 151. Trenn, G., Pettit, G.R., Takayama, H., Hu-Li, J. and Sitkovsky, M.V. (1988) Immunomodulating properties of a novel series of protein kinase C activators. J. Immunol. 140, 433. Yip, Y.K., Pang, R.H.L., Urban, C. and Vilcek, J. (1981) Partial purification and characterization of human gamma interferon (immune interferon). Proc. Natl. Acad. Sci. U.S.A. 78, 1601.
45
Elsevier JIM05132
A fluorescence-based assay for quantitation of lymphokine-activated killer cell activity Thorsten Volgmann, Anette Klein-Struckmeier and Harald Mohr Blood Transfusion Service of Lower Saxony, Department of Research and Development, Eldagsener Str. 38, D-3257 Springe 1, F.R.G.
(Received24 August 1988, revisedreceived15 November 1988; accepted 25 November 1988)
A fluorescence assay for the quantitation of tumor cell lysis by activated and non-activated killer (LAK) cells is described. The target cells are labelled with a europium chelate (Eu-diethylenetriaminopentaacetate) and after cytolysis caused by the LAK cells the Eu 3+ complex is released into the culture supernatant. The addition of fl-naphthoyltrifluoroacetone to culture supematant aliquots leads to the formation of a highly fluorescent chelate which can be measured with a time-resolved fluorometer. The influence of various assay parameters has been evaluated including incubation time, effector-totarget cell ratio, the target cell line and different concentrations of interleukin-2 during cell culture. The optimized time-resolved fluorometric assay was found to be as simple and sensitive as the commonly used cytotoxicity assay in which the release of 51Cr from the labelled target cells is measured. In addition the assay is much faster and safer since the label is not radioactive. Key words: Cytotoxicityassay; Lymphokine-activatedkiller cell activity; Time-resolvedfluorescence
Introduction In cell-mediated cytolysis the cytotoxicity of effector cells (e.g., macrophages, cytotoxic T lymphocytes, natural killer (NK) cells) is usually determined by methods based on the release of different markers from the lysed cells. In general, these markers are compounds containing radioactive isotopes such as 5achromium, 75selenium or tritium, the 5aCr release assay being the one most commonly used. It is easy to perform, highly sensitive and the label is non-toxic to the cells
Correspondence to: Th. Volgmann, Blood Transfusion Service of Lower Saxony, Department of Research and Development, Eldagsener Str. 38, D-3257 Springe 1, F.R.G. Abbreviations: DTPA, diethylenetriaminopentaacetate;IL2, interleukin-2; LDH, lactate dehydrogenase; LAK cell, lymphokine-activatedkiller cell: NK cell, natural killer cell.
(Brunner et al., 1968). The use of SlCr, however, has some drawbacks, namely the health risk to personnel working with relatively high levels of the isotope which emits gamma rays. This situation had led to the development of non-radioactive cytotoxicity assays, e.g., the replacement of the radioactive label with a fluorescence dye which can be monitored with a fluorescence spectroscope when released (Brunning et al., 1980). Such an assay, however, lacks sensitivity because of the relatively high background fluorescence. In other test systems, the release of alkaline phosphatase or lactate dehydrogenase by the lysed cells is utilized for the determination of the cytolytic activity of effector cells (Szekeres et al., 1981; Korzeniewski et al., 1983). However, we have found it difficult to perform these assays within a normal working day, i.e., 8-9 h. Blomberg and co-workers (1986a,b) described an assay to mea-
0022-1759/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)
46
sure the cytotoxicity of NK cells using target cells labelled with a europium complex. After release of the complex from lysed cells Eu 3+ is chelated with a fl-diketone. This chelate is highly fluorescent and can be quantified by time-resolved fluorometry. The fluorescence decay time of Eu chelates is in the range of 100-1000 ns compared to 10-20 ns decay time for fluorescent biological samples (Soini et al., 1979; Soini et al., 1983; Hemmila et al., 1984). This property of Eu chelates reduces the background fluorescence and considerably enhances the sensitivity of immunofluorescence assays in which Eu 3+ and other lanthanide ions are used. We have adopted the time-resolved fluorescence assay for the measurement of lymphokine-activated killer (LAK) cells. These are generated from their progenitors, cells belonging to the lymphocyte fraction within mononuclear blood cells, by exposure to interleukin-2 (IL-2) for 3-4 days (Grimm et al., 1984; Ortaldo et al., 1986). LAK cells are highly cytolytic towards autologous tumor cells and certain NK cell-resistant tumor cell lines; hence there is increasing interest in LAK cells for adoptive immune therapy (Rosenberg et al., 1986, 1987). We found that the time-resolved fluorescence assay with Eu 3+ as a label is as easy to perform, at least as sensitive as, and more rapid than either the 51Cr release or the lactate dehydrogenase assay. It thus represents a non-radioactive alternative for measuring the cytotoxicity of LAK and other cells.
Materials and methods
Reagents All chemicals were of analytical grade. DTPA and EuC13 were purchased from Aldrich-Chemie, Steinheim, F.R.G. Dextransulfate (MW 500000) was obtained from Pharmacia, Freiburg, F.R.G., iodophenylnitrotetrazolium chloride and NAD + were from Sigma, Deisenhofen, F.R.G. and diaphorase was purchased from Boehringer, Mannheim, F.R.G. All other chemicals were obtained from common suppliers (in general from Sigma). Microtiter strips and other plastic materials were
obtained from Flow, Meckenheim, F.R.G. and Nunc, Wiesbaden, F.R.G.
Preparation of LAK cells Mononuclear cells were isolated from adeninesodium citrate-dextrose stabilized donor blood by the Ficoll-Hypaque-method as described by Boyum (B~yum et al., 1976). The cells were washed three times with isotonic sodium chloride solution. Then they were suspended in RPMI 1640 containing 10% heat-inactivated fetal calf serum, 50 g g / m l streptomycin, 100 g g / m l neomycin and human IL-2 (if not stated otherwise 1000 U/ml). IL-2 was produced from Ca ionophore A 23187/ phorbol ester induced donor lymphocytes (Mohr et al., 1986) and purified to apparent homogeneity. Briefly, IL-2 was first adsorbed to porous silicic acid (Amicon, Witten, F.R.G.) and, like interferon-~, (Yip et al., 1981), could be released with ethylene glycol containing phosphate buffer. This was followed by ion exchange chromatography using S-Sepharose Fast Flow (Pharmacia, Freiburg, F.R.G.). The IL-2-containing fractions were eluted at 100-200 mM NaC1. The last purification step was reverse phase HPLC using the octyl phase. IL-2 was eluted at approximately 40% propanol in phosphate buffer, pH 2.5. For LAK cell generation, the suspended cells were incubated for 3-4 days in a humidified 5% CO 2 atmosphere at 37 o C.
Cytotoxicity assays The cytolytic activity of the IL-2-activated cells was evaluated against the NK-sensitive cell line K562 and the LAK cell-sensitive cell line Raji at various effector-to-target ratios. Three different test systems were used (see below). Each assay was performed in triplicate and the cytotoxicity values were calculated by using the following formula:
% lysis =
test r e l e a s e - s p o n t a n e o u s r e l e a s e × 100 r e l e a s e - spontaneous release
maximum
The maximum release was the total release obtained from target cells treated with 0.5% Triton X-100, and spontaneous release was the normal release of Eu chelate during test time without effector cells.
47 Lytic units (LU) were defined as the number of cells/107 cells, which cause 20% (LU20) of the target cells and were calculated according to the method of Pross (Pross et al., 1981).
The reaction was measured after 20 min in an ELISA reader (ELISA Processor II from Behring, Marburg, F.R.G.) at a wavelength of 492 nm. The reference wavelength was 650 nm.
51Cr release
Europium release assay
assay
The 4 h standard 51Cr release assay was carried out as follows: effector cells were labelled with 200 ttCi Na2CrO4/107 cells and washed three times with isotonic NaC1 solution. 5 x 103 cells were pipetted into each cup of round-bottomed 96 well tissue culture plates. The effector cells were added to give a final volume of 200 #1. After incubation for 4 h in a humidified 5% CO 2 atmosphere at 37 ° C, the cells were centrifuged and 100 /tl of the supernatant were removed from each well and counted in a gamma-counter (Gammamatic I, Kontron Analytical, Basel, Switzerland). L D H assay
The LDH assay was carded out as described by Korzeniewski (Korzeniewski et al., 1983). The target ceils were washed three times with RPMI 1640 containing 3% FCS to remove adherent LDH derived from lysed cells. The number of viable cells was estimated using trypan blue exclusion. The cell suspension was diluted with RPMI 1640 plus 3% FCS to give final concentrations between 3 x 104 and 2 x 105 cells/ml. 100/L1 target cell suspension and 100/~1 of an effector cell suspension were pipetted together into the wells of a round-bottomed microtiter plate. Suspensions containing exclusively effector cells, target cells, or culture medium, respectively, served as controls to estimate the LDH background. The plates were incubated for 4-6 h in a humidified 5% CO 2 atmosphere at 37°C. After incubation they were centrifuged at 130 × g for 10 min. Then 150 #1 of the supernatant from each well were transferred to the corresponding well of a flat-bottomed microtiter plate. 20 /~1 of a 400 mM lactate solution ( p H = 6 . 8 in phosphatebuffered saline (PBS)) were added to each well, followed by 20/xl of a solution of 4 mM iodophenylnitrotetrazolium chloride in PBS and finally 20 #1 of a 'reaction mix' containing 2.7 U / m l diaphorase, 4.5 mM NAD +, 0.03% bovine serum albumin and 1.2% sucrose in PBS.
The target cells were labelled with Eu 3+ in labelling buffer (pH 7.4) containing 50 mM Hepes, 93 mM NaC1, 5 mM KC1, 2 mM MgC12 • 6H20 and either 50 /~M EuC13 plus 250 #M diethylenetriaminopentaacetate (DTPA) for labelling K562 cells or 100 #M EuC13 plus 500 /~M DTPA for labelling Raji cells. A stock solution of EuC13 (10 mM) was prepared by dissolving the salt at 60-70 °C in 0.1 N HC1. Immediately before use, 50 ~tl/ml of a solution containing 10 mg/ml dextran sulfate in destilled water were added to the labelling solution. This was followed by the addition of 5 x 106/ml washed target cells. The cells were labelled with Eu 3+ for 20 min in an icebath with occasional shaking. The labelling reaction was stopped by the addition of 30 /~l/ml of a 100 mM CaC12 solution. After 5 min the cells were washed 5-6 times with RPMI 1640 containing 2 mM CaCI 2. The cells were diluted to a concentration of 5 X 10 4 cells/ml in RPMI 1640 containing 10% heat-inactivated FCS, streptomycin (50 /xg/ml) and penicillin (50 U/ml). 100 #1 of this suspension and 100/xl of effector cells were pipetted into the cups of round-bottomed microtiter plates. The plates were incubated for 2.5 h at 37°C. After incubation the cells were centrifuged and 20 #1 of the supernatants were transferred to the cups of flat-bottomed microtiter strips containing 200 /tl of an enhancement solution (Delfia from LKBWallac, Turku, Finland). After thorough mixing for 5 min at room temperature fluorescence was measured in a time-resolved fluorometer (Arcus 1230 from LKB-Wallace, Turku, Finland).
Results and discussion Relation between fluorescence and target cell concentration
The initial experiments were carried out to determine the relationship between the maximal and spontaneous release of the Eua+-mediated
48
meximcll ~lease release
c-
o
c~
u
t~ J
la.
1
i
2
i Ig~o of
cell
i
o
5
~
Fig. 1. Influence of various amounts of Eu-DTPA-labelled cells (e • Raji, • • K562) on fluorescence intensity and the relationship between background fluorescence and maximal fluorescence. The cells were labelled and incubated using the conditions described in the materials and methods section. Maximal release was obtained from cells which were treated with 0.5% Triton X100.
fluorescence and the number of target cells (Raji). Fig. 1 shows that the relationship was linear between 500 and 10000 cells per cup. The spontaneous release never exceeded 2-3% of the maximal fluorescence released from detergent lysed cells.
Since the commonly used cytotoxicity assays are performed with 5000 target cells/well, similar numbers were also used in our following experiments. Influence of incubation time on the release of Eu 3 ÷ To estimate the time dependency of the Eu 3+ release, labelled target cells were incubated with
Fig. 3. Dependence of the lytic activation of lymphocytes against two target cell lines (• •), Raji cells, o o, K562 cells) after treatment with various amounts of IL-2 (0, 10, 50, 100, 300, 600 and 1000 U / m l ) and after various times of incubation (closed symbols, 24 h; open symbols, 72 h).
LAK cells at an E / T ratio of 10 : 1 for different lengths of time. Fig. 2 shows a lag period of 30 rain before rapid release of the label from both K562 and Raji cells, with a linear increase of the fluorescence intensity up to 120 min. A plateau was reached after 150-180 min. Hence the assay can be stopped after 2-2.5 h, which is considerably shorter than in the classical 4 h 51Cr release assay and in the L D H assay, in which the release o f enzyme is still in its logarithmic phase after 4 h incubation (see Fig. 2b). We have found that the L D H assay requires at least 6 h to be adequately quantitative. This is in contrast to the observations of Korzeniewski and Callewaert (1983) whose data indicate that it requires an incubation time of 1(30
1OC
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density
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Time of europium- releose (h)
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Releasing t i m e (h)
Fig. 2. a: Dependence of specific lysis on time of Eu 3+ release during the cytotoxicity assay. Two target cell lines were tested m, Raji; [] D, K562) at an effector-to-target ratio of 10:1. E a c h point represents the mean of three separate experiments. (m
Fig. 2. b: Comparison of the kinetics of release times from K562 cells ( O O , L D H assay; [] D, SlCr-assay) using an effector-to-target ratio of 10 : 1. The shaded bars represent the specific lysis rates of K562 labelled w i t h E u - D T P A at the indicated effector-to-target ratios.
49
~2o~
not more than 2 - 4 h. The reason for this divergence remains obscure.
100~
? 8ow
LAK cell activation with IL-2 The use of the assay to measure L A K and N K cell activation by IL-2 is demonstrated in Fig. 3. Peripheral blood mononuclear cells were incubated with various amounts of IL-2 for 3 days. The cytotoxicity of the activated cells was tested on K562 as well as on Raji cells. As Fig. 3 indicates, the cytotoxic activity of the blood cells against K562 was detectable even in the absence of IL-2, which was obviously due to the presence of N K cells. This cytotoxic activity was drastically increased by IL-2 and reached its maximum at an IL-2 concentration of approximately 100 U / m l . NK-resistant Raji cells were not lysed by nonactivated mononuclear cells (see also Fig. 4). A threshold concentration of 30-50 U / m l IL-2 was needed to induce lytic activity. Maximal cytotoxicity was achieved at concentrations between 500-1000 U / m 1 IL-2. This is in agreement with other published results (Oshimi et al., 1988; Trenn et al., 1988).
Optimal effector-to-target ratio An E / T ratio of 3 0 : 1 to 5 0 : 1 was optimal when the Eu 3+ release assay was used to determine the cytotoxicity of unstimulated N K cells and this is similar to the classical 5]Cr release assay (Blomberg et al., 1986a,b). For measuring the cytotoxicity of lymphocytes activated with IL2, E / T ratios between 1 : 1 and 10 : 1 proved to be optimal (Fig. 4). This was independent of the target cell line used. The reason might be that IL-2 treatment leads to enhanced cytotoxicity of N K cells as well as L A K cells. This assumption is confirmed by the higher E / T ratios that were needed to lyse significant amounts of Raji and
•~-
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,
,
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,
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Fig. 4. Comparison of the lytic activity of activated (filled symbols) and non-activated (open symbols) lymphocytes against three different cell lines (O o, Raji; ,x ,x, K562; ~ - - ~ , Daudi) at various effector-to-target ratios. The Daudi cells were labelled in the same manner as the Raji cells. The lymphocytes were activated with 1000 U IL-2/ml. The activation time was 3 days.
Daudi targets with mononuclear cells not activated by IL-2. There was almost no cytolysis of Raji cells by effector cells not activated by IL-2.
Comparison with other cytotoxicity assays In subsequent routine cyt0toxicity assays for the estimation of L A K cell activation by IL-2, effector and target cells were co-incubated for 2.5 h at E / T ratios of 2.5 : 1, 5 : 1 and 10 : 1 respectively. Table I shows that under these conditions cytotoxicity rates were comparable with those achieved by the 51Cr release assay which was incubated for 4 h at E / T ratios ranging from 1 0 : 1 and 4 0 : 1 . When the 51Cr release assay was performed at the same low E / T ratios as the Eu 3+ fluorescence assay, it proved to be much less sensitive. On the other hand there was a good correlation between the L D H release assay and the Eu 3+ assay (Fig. 5). It should be noted, however, that the latter
TABLE I COMPARISON OF SPECIFIC LYTIC RATES OF THE TWO CYTOTOXICITYASSAYS AT VARIOUS EFFECTOR TO TARGET RATIOS Specific lysis (%) Eu3+ assay
E/T ratio: K562 Raji
51Cr assay
2.5
5
10
10
20
40
24.2 8.5
48.0 20.6
61.0 31.6
37.2 18.4
56.2 29.1
90.7 38.8
50 10C
fluorometric assay can also be used to determine cytolytic activity of lymphoid cells activated by IL-2 against NK cell resistant target cells. In our laboratory the assay is routinely used to monitor the activation of in vitro generated LAK cells from tumor patients taking part in clinical trials evaluating the therapeutical potential of these cells.
8C 6C .u_
g
4C
e~
2C 0
5
10 E / T - ratio
15
20
Fig. 5. Comparison of the three cytotoxicity assays (A A, 51Cr-release; • • , LDH-release; " - - 0 , Eu3+ release). The filled symbols represents the specific rates of lysis of K562 cells produced using activated lymphocytes. The open symbols represents data obtained from K562 cells lysed by non-activated lymphocytes.
involved a 6 h incubation instead of the 2.5 h for the Eu 3+ release assay. The different velocities at which the two labels and the marker enzyme were released from the target cells may be explained as follows. The Eu-DTPA complex is chemically inert (Hemmil~i et al., 1984). It is relatively small and therefore able to leave the target cells just after they are rendered permeable by exposure to effector cells. 51Cr (used as 51CRO42- ion) is more reactive and will bind to cellular proteins by ionic or other forces, and LDH is a large molecule. Hence, 51Cr and LDH are not released into the cell culture supernatant until the target cells are completely lysed. As a result, the Eu 3+ fluorescence assay is the fastest of the three. The assay can easily be performed and because of the short labelling time, release period and counting time (1 s/sample) it can be evaluated during a normal working day. Compared to the commonly used 51Cr release assay, it has the additional advantage that no radioactive isotopes are used. Under appropriate conditions it is at least as sensitive and accurate as the two other assays tested. A disadvantage is the relatively high price of the time resolved fluorometer (approx. DM 35000). But this is not higher than that of a scintillation counter, and the fluorometer can be used for other assays than that described in this article (Soini et al., 1979; Jackson et al., 1986). Our data confirm and expand those of Blomberg et al. by demonstrating that the time-resolved
Acknowledgements This study was supported by the Bundesministerium of Forschung und Technologie (0318 707
A8). The authors would like to thank Mr. R. Rosinke for expert technical assistance and Mrs. J. Linneweber for preparing the manuscript.
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51 Molar, H., Monner, D. and Plessing, A. (1986) Calcium ionophore A23187 in the presence of phorbol ester PMA: a potent inducer of interleukin-2 and interferon-gammasynthesis by human blood cells. Immunobiology 171, 159. Ortaldo, J.O., Mason, A. and Oveston, R. (1986) Lymphokine activated killer cells. Analysis of progenitors and effectors. J. Exp. Med. 164, 1193. Oshimi, K., Oshimi, Y., Saito, H. and Mizoguchi, H. (1988) Cytotoxicity of interleukin-2-activated lymphocytes for autologous normal blood mononuclear cells. J. Immunol. Methods 109, 161. Pross, H.F., Baines, M.G., Rubin, P., Shragge, P. and Patterson, M.S. (1981) Spontaneous human lymphocyte mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity. J. Clin. Immunol. 1, 51. Rosenberg, S.A. and Lotze, M.T. (1986) Cancer immunotherapy using interleukin-2 and interleukin-2 activated lymphocytes. Ann. Rev. Immunol. 4, 681. Rosenberg, S.A., Lotze, M.T., Muul. L.M., Chang, A.E., Avis, A.P., Leitman, S., et al. (1987) A progress report on the
treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or highdose interleukin-2 alone. New Engl. J. Med. 316, 889. Soini, E. and Hemmil~i, I. (1979) Fluoroimmunoassay: Present status and key problems. Chn. Chem. 25, 353. Soini, E. and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates - a new generation of non-isotopic immunoassays. Clin. Chem. 29, 65. Szekeres, I., Pasca, A.S. and Peytsik, B. (1981) Measurement of lymphocyte cytotoxicity by assessing endogeneous alkaline phosphatase activity of the target cells. J. Immunol. Methods 40, 151. Trenn, G., Pettit, G.R., Takayama, H., Hu-Li, J. and Sitkovsky, M.V. (1988) Immunomodulating properties of a novel series of protein kinase C activators. J. Immunol. 140, 433. Yip, Y.K., Pang, R.H.L., Urban, C. and Vilcek, J. (1981) Partial purification and characterization of human gamma interferon (immune interferon). Proc. Natl. Acad. Sci. U.S.A. 78, 1601.