Establishment of an enzyme release assay for cytotoxic T lymphocyte activity

Journal of lnurnmological Methods, 147 (1992) 119-124 e 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00

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JIM 06199

Establishment of an enzyme release assay for cytotoxic T lymphocyte activity 1 Hitoshi Ohmori, Toshiyuki Takai, Takahiro Tanigawa and Yoichi Honma [)qJartment of Biotechnology, Faculty of Engineering, Okoyama University, Tsushima-Nalca, Okayama 700, Japan

(Received 30 August 1991, accepted 18 October 1991)

In the present report, we describe the establishment of a cell line that can be used as the target for measuring the activity of cytotoxic T lymphocytes (CfL) by an enzyme release assay. We transfected P3jNSI-Ag4-1 (NS-l), a myeloma cell line derived from BALBjc mice with Escherichia coli f3-galactosidase (I3-GaD gene, and isolated a stable transformant designated as NS-ljZ that expressed a high level of the enzyme activity intracellularly. The effector cells showing cytotoxicity against NS-ljZ were induced when the spleen cells of AKR or C3H mice were cultured with mitomycin C-treated BALBjc spleen cells for 4 days. When 2 X 10 4 NS-l jZ cells were incubated with varying numbers of effector cells, I3-Gal activity was released from the target cells depending on the number of effector cells and the time of incubation for up to 8 h. A highly sensitive enzyme assay was performed by using a fluorescent substrate, 4-methylumbelliferyl-l3-o-galactoside. The cytotoxicity was specific for H-2 haplotype of the stimulator cells, and was abolished by treating the effector cells with anti-Lyt 2 plus complement. The sensitivity of the enzyme release assay was comparable to that of 51Cr release assay. These results indicate that NS-1jZ can be used as a target cell line for the non-radioactive measurement of CTL activity. Key words: Cytotoxic T lymphocyte; Enzyme release assay; DNA transfection; /3-Galactosidase

Correspondence to: H. Ohmori. Department of BiotechnolDIY. Faculty of Engineering. Okayama University, TsushimaNaka. Okayama 700. Japan (Tel.: 81-862-52-1111. ext. 535; Fax: 81-862-53-7399). I This work was supported in part by a Grant-in-Aid for Scientific Research from The Ministry of Education, Science and Culture of Japan. AbbreviJltions: en.. cytotoxic T lymphocytes; E / T ratio. effeclor-to-target ratio; FCS. fetal calf serum; /3-Gal. /3,alactosidase; MMC, mitomycin C; 4-MU. 4-methylumbelliferone: 4-MUG. 4-methylumbelliferyl-/3-o-galactoside; XGal, 5-bromo-4-chloro-3-indolyl-/3-o-galactoside; NS-l, P3 /NSI-Ag4-1.

Introduction Cr release assay has been widely used for the measurement of cytotoxic T lymphocyte (CfL) activity. Although this is a very sensitive method, the following disadvantages have been pointed out. (1) The use of radioactive chromate is hazardous. (2) SlCr has a relatively short half-life, and is expensive. (3) One must prepare radiolabeled target cells just prior to every experiment. Therefore, several non-radioactive methods have been devised to overcome these problems. These 51 !

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include the assay of dye release from the target cells that had been loaded with fluorescein (Burning et aI., 1972, 1980) or neutral red (Parish and Mullbacher, 1983). Recently, Brenan and Parish (1988) have reported a sensitive fluorometric assay which assesses the release of DNA-binding dye H33342 from the target cells. Lactate dehydrogenase release assay has been reported in the measurement of natural killer cell activity (Korzeniewski and Callewaert, 1983). Because lactate dehydrogenase is present both in target cells and in effector cells, it is desirable to choose the indicator enzyme that is exclusively expressed in the target cells. For establishing an enzyme release assay of CfL, the target cells should satisfy the following conditions. (I) The target cells produce an intracellular enzyme that is not found in the effector cells. (2) A sensitive and simple assay of the enzyme is possible. To this end, we selected Escherichia coli f3-galactosidase (f3-GaO as an indicator enzyme that can be assayed very sensitively by using a fluorescent substrate, 4-methylumbelliferyl-f3-D-galactoside (4-MUG) (Ishikawa and Kato, 1978). A BALB/c mice-derived myeloma cell line, P3/NSI-Ag4-1 (NS-t) was transfected with the plasmid carrying E. coli f3-Gal gene, and the stable transformant expressing a high level of f3-Gal was isolated. In the present report, we describe the usefulness of this transformant as the target cells for the enzyme release assay of CfL activity.

Materials and methods Mice Male BALB/c, AKR/J, C3H/HeN and C57BL/6 mice were obtained from The Animal Breeding Facility of Okayama University Medical School, Okayama, Japan. They were used at 8-15 weeks of age. Cel/lines NS-l, a BALB/c-derived myeloma cell line was obtained from Riken Cell Bank, Tsukuba, Japan.

Materials Chemicals, antibodies and other materials were purchased from the following sources: 4-MUG, 5-bromo-4-chloro-3-indolyl-f3-D-galactoside (XGaO and mycophenolic acid from Sigma, St. Louis, MO; 4-methylumbelliferone (4-MU) and polyethylene glycol 4000 from Nacalai tesque, Kyoto, Japan; DEAE-dextran from Pharmacia, Uppsala, Sweden; RPMI-1640 medium from Nissui Seiyaku, Tokyo, Japan; mitomycin C (MMC) from Kyowa Hakko, Tokyo, Japan; fetal calf serum (FCS) from Gibco, Grand Island, NY; monoclonal anti-Lyt 2.1 from Meiji Health Science Institute, Tokyo, Japan; low toxic rabbit complement from Cedarlane Laboratories, Ontario, Canada; Sodium [5l Cr]chromate from New England Nuclear, Boston, MA. Construction of plasmid vector The plasmid vector carrying both f3-Gal gene and a selection marker was constructed as follows. The plasmid pRSVlacZ was a generous gift from Dr. S. Tajima, Osaka University, Osaka, Japan. It harvours the pRSVcat sequence (Gorman et aI., 1982) in which the HindIII/BamHI fragment carrying chloramphenicol acetyltransferase gene is replaced by the HindIII/BamHI fragment encoding E. coli f3-Gal gene, lacZ derived from pCHllO (Pharmacia). This plasmid allows the expression of the lacZ gene in animal cells under the transcriptional control of Rous sarcoma virus long terminal repeat. The plasmid pSV2 gpt obtained from the Japanese Cancer Research Resources Bank allows the expression in animal cells of E. coli xanthine (guanine) phosphoribosyltransferase gene under the control of the SV40 early region promoter (Mulligan and Berg, 1980). It was digested with Pvu II, ligated with BamHI linker, and digested with BamHI. The resulting 2.2 kbase pair fragment was isolated and ligated with the Bam HI-digested pRSVlacZ to yield the plasmid pRSVlacZ-SVgpt. Establishment of stable transformants expressing f3-Gal !3-Gal-expressing target cell lines were established by the transfection of a parent cell line with pRSV/acZ-SVgpt in the following manner. The transfection was performed by the modified

121

DEAE-dextran method coupled with the osmotic shock procedure as reported from our laboratory (Takai and Ohmori, 199Oa, b). This procedure enables the efficient transfection of Iymphoblastoid cell lines. Briefly, 3 x 10 6 NS-l cells were suspended in 1.0 ml of RPMI 1640 medium containing 50 mM Tris-HCI buffer (pH 7.3), 20 J,Lg of DEAE-dextran and 10 J,Lg of pRSVlacZ-SVgpt. The cell suspension was incubated at 37'C for 30 min, and then mixed with an equal volume of a hypertonic solution containing 0.8 M sucrose, 16% (W IV) polyethyleneglycol 4000, 168 mM NaCI, 56 mM Tris-HCI (pH 7.3) and 20% (v Iv) dimethylsulfoxide. After incubation at 37'C for 10 min, the whole suspension was quickly diluted with 10 ml of serum-free RPMI 1640 medium. The cells were centrifuged, suspended in 10 ml of RPMI 1640 medium containing 10% FCS and plated into 96 well microtiter plates at 0.1 mIjwell. They were cultured at 37'C in the humidified atmosphere of 5% CO 2 and 95% air. 2 days later, the growth medium was changed to the selection medium containing 0.1 mM hypoxanthine, 16 J,LM thymidine, 1.6 mM xanthine, and 0.75 J,Lg/ml mycophenolic acid. 10-14 days later, growing colonies were split and examined for their expression of p-Gal activity by in situ staining with X-Gal, a chromogenic substrate as described by Sanes et al. (1986). The stable transformant of NS-l expressing the highest level of p-Gal was selected, and it was designated as NS-I/Z. The specific activity of I3-Gal in NS-1/Z was 105 nmoIjmin . mg protein. Growth of the target cell line, NS-J I Z NS-I/Z was maintained in RPMI 1640 medium containing 10% FCS without altering the level of I3-Gal expression for over several months, and was used at late logarithmic phase. Cells with less than 90% viability were discarded. Induction and assay of CTL activity All cultures were performed in RPMI 1640 medium supplemented with 10% FCS, 1 x 10.5 M 2-mercaptoethanol, 100 U Iml penicillin G and 50 J,Lg/ml streptomycin. Using 24 well multidish culture plates (Nunc, Roskilde, Denmark), unprimed 5 X 106 AKR (H-2 k) spleen cells were cultured with 2.5 x 106 BALB/c (H-2 d ) spleen

cells, that had been pretreated with 25 J,Lg/ml MMC, in 1.6 ml culture medium for 4 days at 37°C in the humidified atmosphere of 5% CO 2 and 95% air. Nonadherent cells were harvested, and their CTL activities were measured by the enzyme release assay in the following manner. Varying numbers of effector cells 0-4 X 10 5 ) were cultured in triplicate with 2 X 10 4 NS-I/Z in 0.2 ml of RPMI 1640 containing 2% FCS in Falcon microtiter plates. Culture plates were centrifuged for 2 min at 800 rpm, and incubated at 37°C for 2-8 h in a humidified atmosphere of 5% CO 2 and 95% air. After incubation, microtiter plates were centrifuged at 800 rpm for 2 min, and the supernatants were collected. For assaying the released f3-Gal activity, 0.1 ml of the supernatant was incubated for 30 min at 37°C with 0.3 ml of 10 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaCi, 1 mM MgCI 2 , 0.1% bovine serum albumin and 10 J,Lg of 4-MUG. The reactions were stopped by the addition of 2 ml of 0.1 M glycine buffer (pH 10.3). Released 4-MU were determined fluorometrically with the excitation at 360 nm and the emission at 450 nm. Relative fluorescent intensities were read using Fluororead fluorescence spectrophotometer (Ajinomoto, Tokyo, Japan). The fluorescence intensity of 10- 6 M 4-MU was usually defined as 10. This equipment allows the determination of 10- 9 M 4-MU. The enzyme release in the absence of effector cells was designated as spontaneous release (S). Total enzyme activity (T) of the target cells was measured after disrupting the cells with 0.0425% Triton X-loo. This concentration of the detergent had no significant effects on the assay of f3-Gal. Percent enzyme release was defined as follows,

R-S % enzyme release = --S- X 100

T-

where R is the observed enzyme release by CTL. Data were usually presented as the mean ± standard deviation (SD) from triplicate experiments.

Cr release assay Labeling of NS-I/Z cells with sodium [SICr]chromate was done as described by Grab-

51

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stein and Chen (1980). Briefly, 1 X 10 6 NS-I/Z cells were incubated with 100 /LCi of sodium 51 [ Cr]chromate in 0.5 ml of RPM I 1640 medium containing 5% FCS at 37°C for 1 h. They were thoroughly washed with the same medium to remove the free radioactivity, and resuspended in RPMI 1640 medium containing 2% FCS at 2 X 10 5 /ml. Labeled target cells (2 X 10 4 ) were incubated with varying numbers of effector cells, and the release of the radioactivity from the target cells was measured under the same experimental conditions as those in the enzyme release assay. Results We attempted to establish a procedure to estimate the activity of CTL by measuring the release of enzyme activity from the target cells. To this end, we transfected BALB/c-derived myeloma cell line NS-l with E. coli I3-Gal gene, and obtained a stable transformant designated as NSliZ that expressed a high level of I3-Gal activity intracellularly. The advantage of the use of I3-Gal as the indicator enzyme is that one can measure the enzyme activity sensitively by employing a fluorescent substrate, 4-MUG. As illustrated in Fig. 1A, I3-Gal activity could be assayed using a small number of cells as few as 2 X 10 3 • Fig. 18 demonstrates that the enzyme activity was stable when the cell-free extracts were kept at 37°C for up to 8 h in the medium for CTL assay. Neither the total enzyme activity nor the spontaneous release of the enzyme did significantly increase or decrease when NS-I/Z cells were incubated for 8 h in the assay medium as shown in Fig. Ie. The spontaneous release was kept below 10% in each experiment. Cytotoxic cells were induced by stimulating AKR spleen cells with MMC-treated allogeneic BALB/c spleen cells for 4 days. Then, we tested whether the stimulated AKR spleen cells could cause the release of I3-Gal from NS-1/Z cells. When AKR effector cells were incubated for varying hours with 2 x 10 4 NS-ljZ at the effector to target (E IT) ratio of 20: 1, I3-Gal activity was released into the medium depending on the incubation period for up to 8 h as shown in Fig. 2A. In the following experiments, incubations

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Fig. 1. A: f3-Gal activity in NS-l/Z. Varying numbers of NS-l/Z cells (I x 10 3 to 2 X 10 4 ) were lysed in 0.2 ml of RPM I 1640 medium containing 2% FCS and 0.0425% Triton X-100. 0.1 ml of the cell-free extracts was used for the measurement of the enzyme activity. B: stability of f3-Gal activity in the assay medium. The cell-free extracts of NS-1/Z prepared by sonication were incubated at 37"C in RPM I 1640 medium containing 2% FCS for the indicated time. C: the level of the total enzyme activity and the spontaneous release of the enzyme in NS-t/Z cells during the incubation in the assay medium. NS-t/Z cells (2 x 10 4 ) were incubated in 0.2 ml of the assay medium for 2-8 h. At the indicated time, supernatants were collected for the assay of the spontaneous release (0). Total enzyme activities (e) were measured after lysing the cells in 0.0425% Triton X-tOO. The enzyme activity was expressed as a relative fluorescence intensity where 10- 6 M 4-MU was defined as 10. Data were presented as the mean value of duplicate experiments.

were carried out for 6 h. Fig. 28 illustrates that the release of the enzyme activity was also dependent on E IT ratio. No significant release was A)

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Fig. 2. Effects of incubation time or E / T ratio on the cytolysis of NS-t/Z. A: NS-1/Z cells (2 x 104 ) were incubated with BALB/c-stimulated AKR effector cells at a E/T ratio of 20: 1 for the indicated time. B: AKR spleen cells that had been unstimulated (0) or stimulated by BALB/c cells (e) were incubated at varying E/T ratio with 2xI0 4 NS-l/Z cells for 6 h. The cytolysis was estimated as percent f3-Gal release. Each point represents the mean ± SO from triplicate experiments.

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Fig. 3. Specificity of stimulator cells for the induction of the cytotoxicity against NS-I/Z cells. AKR spleen cells were stimulated in vitro with MMC-treated BALB/c or C57BL/6 spleen cells for 4 days as described in the materials and methods section. The effector cells were incubated with the target cells at the indicated E / T ratio for 6 h. Each point represents the mean ± SO from triplicate experiments. (0) unstimulated, (e) stimulated with BALB/c, (.) stimulated with C57BL/6.

observed with unstimulated AKR cells. Although data were not presented, the cytotoxicity against NS-I/Z was similarly induced in C57BL/6 (H-2b) spleen cells that had been cultured with BALB/c stimulator cells. We then carried out the following experiments to confirm whether the cytotoxicity observed by using NS-I/Z is mediated by CfL. As illustrated in Fig. 3, the cytotoxicity against NS-I/Z (H_2d) was induced when AKR (H_2k) spleen cells were stimulated with BALB/c (H-2d) spleen cells, but not with those of C57BL/6 (H-2 b). Thus, the cytotoxicity was specific for the haplotype of the stimulator cells. Fig. 4 shows that the cytotoxic activity was abolished when the effector cells were treated with anti-Lyt 2.1 plus complement prior to the assay. These results indicate that the cytotoxicity against NS-I/Z is mediated by CfL that are activated by allogeneic H-2 antigen. Fig. 5 illustrates the comparison of the enzyme release assay with StCr release assay. In this case, C3H spleen cells were stimulated for 4 days with MMC-treated BALB/c spleen cells, and incubated with unlabeled or StCr labeled NS-l/Z cells for the-enzyme release assay or for StCr release assay, respectively. The percent release in the enzyme release assay was approximately 50% lower than that in StCr release assay. However,

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Fig. 4 ..~he cytotoxicity against NS-l/Z is mediated by Lyt2.1-posltlve cells. BALB/c-stimulated AKR spleen cells were treated with complement (e) or with anti-Lyt-2.l plus complement (0) prior to the enzyme release assay. The effector cells were incubated with the target cells for 6 h. Each point represents the mean ± SO from triplicate experiments.

the spontaneous release of the enzyme was always 10% or less. These values were approximately a half of those observed in StCr release assay. Therefore, it is likely that these two procedures show comparable sensitivity. The experimental variation among triplicate experiments of the enzyme release assay was usually less than ± 10% of the average (Figs. 2-5), thus indicating the reliability of this assay method. A) Enzyme

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Fig. S. Comparison between the enzyme release assay and SICr release assay. For SICr release assay, NS-l/Z were labeled with sodium [SICr] chromate as indicated in the materials and methods section. C3H spleen cells were stimulated with MMC-treated BALB/c spleen cells for 4 days. The effector cells were incubated with unlabeled or slCr-labeled NS-l/Z cells for 6 h. A: enzyme release assay; B: SICr release assay. The spontaneous release in the enzyme release assay or in SICr release assay was 10% or 21 %, respectively. Each point represents the mean ± SO from triplicate experiments.

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Discussion

In the present report, we described the establishment of a novel tJ-Gal release assay for measuring the activities of CfL. This procedure has several advantages as follows. (I) One does not have to prepare labeled target cells at every experiment. (2) Non-radioactive experiments are more economical and not hazardous. (3) This enzyme release assay shows enough sensitivity. Therefore, various types of CfL activities can be measured if appropriate tJ-Gal-expressing target cell lines are established. For obtaining stable transformants expressing tJ-Gal, we transfected cell lines with the lacZ-carrying plasmid by modified DEAE-dextran method coupled with the osmotic shock procedure developed in our laboratory (Takai and Ohmori, 1990a,b). This procedure is effective particularly in the transfection of Iymphoblastoid cell lines. In our experiences, it was necessary to use a large number of cells (3-6 x 10 7 ) to obtain transformants expressing a high level of the enzyme stably because the levels of the enzyme expression were highly variable among isolated transformants. The use of tJ-Gal as the indicator of enzyme release has the following advantages. (1) tJ-Gal-expressing transform ants can be easily detected by in situ staining of the cells using choromogenic substrate, X-Gal (Takai and Ohmori, 1990a). (2) Released enzyme activity can be sensitively measured by a fluorescent substrate, 4MUG. (3) The enzyme activity is stable under the experimental conditions for CfL assay. Percent release of the enzyme was usually 4050% lower than that of Ster. However, the enzyme release assay is likely to give sensitivity comparable to that of Ster release assay due to its lower spontaneous release. It has been demonstrated that the cytolysis of the target cells is due to the exocytosis of the pore-forming proteins from CfL (Henkart, 1985). The pore-forming proteins integrated into the cell membrane would enable the leakage of small molecules like Ster, whereas macromolecular tJ-Gal might be released only after larger pores are formed or the osmotic lysis of the target cells occurs. The similar assay method can be applied to other types of cytotoxic cells. We are currently

trying to establish various target cell lines expressing tJ-Gal.

Acknowledgment

We thank Ms. Sayako Ariyoshi for supplying us experimental animals.

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225. Sanes, J.R., Rubenstein, J.L.R. and Nicolas, J.-F. (1986) Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 5, 3133. Takai, T. and Ohmori, H. (1990a) DNA transfection of mouse lymphoid cells by the combination of DEAE-dextranmediated DNA uptake and osmotic shock procedure. Biochim. Biophys. Acta 1048, 105. Takai, T. and Ohmori, H. (1990b) Hiahly efficient DNA transfection of hematopoietic cell lines by an improved DEAE-dextran method: A synergistic enhancement by hypertonic treatment and dimethylsulfoxide. Methods Mol. Cell. BioI. 2, 82.