A novel method to analyze viral antigen-specific cytolytic activity in the chicken utilizing flow cytometry

Veterinary Immunology and Immunopathology 95 (2003) 1–9

A novel method to analyze viral antigen-specific cytolytic activity in the chicken utilizing flow cytometry$ Yongqiang Wang*, Mika Korkeama¨ki, Olli Vainio1 Department of Medical Microbiology, Turku Immunology Centre, Turku University, Kiinamyllynkatu 13, FIN-20520 Turku, Finland Received 10 April 2002; received in revised form 25 March 2003; accepted 11 April 2003

Abstract In order to overcome some of the main drawbacks that have emerged in the conventional assays for cytotoxic T-lymphocytes (CTLs) in the chicken, a novel approach to analyze viral antigen-specific cytolytic activity utilizing flow cytometry was developed. In this method, the target cells were distinguished from the effector cells by pre-labelling them with a fluorescent dye PKH67. Cell death was detected with propidium iodide which labels the DNA of damaged cells. Flow cytometric assay also enables phenotyping of the effector cells by direct or indirect immunofluorescence staining of lymphocyte surface molecules. The results showed that specific cytotoxic T cells were observed in the blood of chickens primed with fixed avian reticuloendotheliosis virus strain T transformed MHC-compatible B cells. Phenotypic analysis of the effector cells from blood demonstrated CTL activity both in CD8þ and CD4þ T cell populations and the majority CTLs were TCR2þ cells. # 2003 Elsevier B.V. All rights reserved. Keywords: Cytotoxicity assay; Phenotyping; Flow cytometry; Chicken; Reticuloendotheliosis virus

1. Introduction Cytotoxic T-lymphocyte (CTL) responses provide a major defence mechanism for elimination of virusinfected cells, and in some cases CTLs have been able to confer complete protection even in the absence of an antibody response (Luckacker et al., 1984; Bevan, $ This work was supported by the EU project FAIR3 PL961502, the Academy of Finland project No. 4293 and the Turku University Foundation. * Corresponding author. Present address: Zhengzhou College of Animal Engineering, 15 Beilin Road, 450011 Zhengzhou, Henan Province, PR China. Tel.: þ86-371-5720054; fax: þ86-371-5791315. E-mail addresses: [email protected] (Y. Wang), [email protected] (O. Vainio). 1 Tel.: þ358-2-3337428; fax: þ358-2-2330008.

1989; Seo et al., 1997). The conventional method used for the quantification of CTL activity is 51 chromium (51Cr)-release assay (Brunner et al., 1968). Although this method is the most common procedure used for analysing CTL activity in mammals, it has major disadvantages and limitations in analysing chicken cytotoxic T cells: (i) inefficient labelling of target cells with 51 Cr and (ii) high spontaneous release from the labelled target cells. The development of robust in vitro assays to measure cytotoxicity of cells derived from infected or immunized chickens has historically been very difficult, however, information concerning induction of CTL activity is of key importance in the design of improved vaccines, and the development of methods for CTL assays in chicken is an urgent task for avian immunologists.

0165-2427/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-2427(03)00109-0

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In order to improve the cytotoxic T cell assay in chicken, we applied a new method based on flow cytometry. This method avoids the danger and difficulties associated with the use of radioisotopes. With this method it is also possible to determine the phenotype of the effector cells, which has been impossible in the traditional 51 Cr-release assay. Flow cytometry has been used in many contexts to measure cell-mediated cytotoxicity. These studies use two-colour fluorescence staining and are restricted to the measurement of mouse or human natural killer (NK) cells (Papa et al., 1988; Vitale et al., 1989; Slezak and Horan, 1989; Radosˇevic´ et al., 1990; Chang et al., 1993; Hatam et al., 1994; Papadopoulos et al., 1994; Karawajew et al., 1994; Johann et al., 1995; Goldberg et al., 1999; Godoy-Ramirez et al., 2000), lymphokine-activated killer (LAK) cells (Papadopoulos et al., 1994; Godoy-Ramirez et al., 2000), antibody-dependent cell cytotoxicity (ADCC) (Flieger et al., 1995), cytotoxicity CD8þ cells in human peripheral blood induced by monoclonal antibody OKT3 (Karawajew et al., 1994), or cell-mediated lympholysis (CML) exerted by tumor-infiltration lymphocytes (TIL) against autologous and cancer tumor cells (Papadopoulos et al., 1994). Two-colour flow cytometry cytotoxicity assay has also been used to quantify CTL effector activity against allogeneic lymphoblasts in mouse (Mattis et al., 1997). The result shows an excellent correlation with data obtained using the standard 51 Cr-release assay. CTL assay based on 51 Cr-release has been described in chicken (Maccubbin and Schierman, 1986; Weinstock et al., 1989; Omar and Schat, 1996) but its use is generally hampered by a low specific 51 Cr-release. In this study we used avian reticuloendotheliosis virus immunization as a model system, to establish flow cytometrybased cytotoxicity assay in chicken, and to phenotype the effector cells through effector cell pre-staining. REV is an avian C-type retrovirus well suited for the study of anti-viral MHC-restricted cytotoxicity (Weinstock et al., 1989). MHC-restricted cellmediated cytotoxicity has been shown to be present in spleen cells harvested from chickens 7 days post inoculation with REV as measured by a 51 Cr-release assay (Maccubbin and Schierman, 1986; Weinstock and Schat, 1987; Weinstock et al., 1989). The present attempts have concentrated on trying to set-up a test assay in which specific cytotoxic cells are

induced by immunization of chickens with fixed REV transformed B cells, and then exposed to the REV transformed cells of that type in vitro. The results show that CTL were detected in peripheral blood lymphocytes (PBLs). Phenotyping assays show that the majority of the effective CTLs were CD8þ T cells and TCR2þ T cells. CD4þ cells may also play an important role in the cell-mediated immunity.

2. Materials and methods 2.1. Animals The MHC-homozygous chickens from H.B15 (MHC haplotype B15) and H.B21 (MHC haplotype B21) lines from the colonies at the Department of Medical Microbiology, Turku University (Turku, Finland) were used. 2.2. Reagents and antibodies PKH67 green fluorescent cell Linker Kit (Sigma, MO, USA) was used to label the target cells stably. Propidium iodide (Sigma), a red fluorescent DNA specific dye (Krishan, 1975; Ockleford et al., 1981; Yeh et al., 1981), was used to trace the dead cells. The following mouse anti-chicken mAb specific for chicken T cells made in our own laboratory were used in phenotype analysis: CD4: 2–6 (IgG1) (Luhtala et al., 1993), CD8a: 11–39 (IgG1) (Luhtala et al., 1995) and 3–298 (IgG2b) (Luhtala et al., 1997). TCR1 (IgG1) against gdTCR, TCR2 (IgG1) against ab1TCR, TCR3 (IgG1) against ab2TCR and the secondary antibodies, phycoerythrin (PE) conjugated goat anti-mouse IgG1 and IgG2b, were purchased from Southern Biotechnology Associates (Birmingham, AL). 2.3. Immunization H.B15 chickens were immunized with fixed REV transformed MHC-compatible cells in PBS. REV transformed MHC B15 B cell line was obtained from Dr. Thomas Go¨ bel. Cells were fixed in PBS/1% paraformaldehyde for 20 min at room temperature (RT) and washed thoroughly four times with 10 ml PBS. Chickens of 2-month-old were immunized three

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times by intravenous injection (i.v.) with 30  106 cells at 3–5 day intervals. 2.4. Effector cell preparation PBL from immunized chickens were used as effector cells. Blood was drawn from the jugular vein into heparinized syringes and PBL were prepared by a low speed centrifugation technique (Vainio and Ratcliffe, 1984). Briefly, the blood was mixed with 2 vol. of pre-warmed (þ40 8C) HBSS and centrifuged at RT for 20 min at 62  g. Lymphocytes were harvested from the plasma, washed once with serum-free chicken IMDM (Vainio et al., 1990). Lymphocytes were resuspended at 2:5  107 ml1 in chicken IMDM containing 5% fetal calf serum (FCS) and 1% normal chicken serum (NCS). They were used as the effector cells directly or after immunofluorescence staining for the purpose of killer cell phenotyping. Cells from the unimmunized hatchmates were used as the control effector cells. 2.5. Target cell preparation REV transformed B cells which expressing both high level of MHC class I and class II molecules, were used as the target cells. For keeping the proportion of live cells, they were centrifuged 1 day before the assay over a Ficoll-Paque gradient and seeded at 1  106 cells per millilitre in culture medium. On the day of the assay, 5  106 cells were washed once in serum-free chicken IMDM in order to remove serum proteins and lipids that may interfere with staining. Target cells were labeled with PKH67 using the Linker Kit (Sigma). Briefly, the pellet was resuspended in 1 ml of Diluent C from the kit. An amount of 1 ml of 2 mM PKH67 was made into a polypropylene tube with Diluent C. PKH67 dilution was quickly pipetted into the tube with cells, swirled and mixed carefully but thoroughly. Cells were incubated for 3 min at RT with gentle mixing. The labeling was stopped by adding an equal volume of heat-inactivated FCS, and incubated for 1 min. The sample was diluted with equal volume of chicken IMDM containing 5% FCS and 1% NCS. After centrifugation at 400  g for 7 min at RT, the cells were transferred into a new tube. The wash was repeated three times with 10 ml chicken IMDM containing 5% FCS and 1% NCS and the cells were

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counted before the last wash. The cells were resuspended into chicken IMDM containing 5% FCS and 1% NCS to the concentration of 2:5  105 cells per millilitre. 2.6. Cytotoxicity assay An amount of 25,000 pre-labelled target cells (100 ml of the target cell suspension) and graded numbers of effector cells (effector:target (E:T) ratio 100:1, 50:1, 25:1 and 12.5:1) were added in a volume of 200 ml in chicken IMDM in triplicate into the wells of 96-well round-bottom microtiter plate (Nunclon@, Roskilde, Denmark). Two micrograms of propidium iodide (1 mg/ml, in PBS) was added and the cells were centrifugated briefly to promote contact between effector cells and target cells. Cell mixture was incubated for 50 min at 39 8C and transferred into flow cytometry tubes with 300 ml of FACS solution (PBS with 2% FCS and 0.01% Na3N). Fluorescence analysis was done immediately with a FACScan instrument (Becton-Dickinson, Mountain View, CA). In the killer cell phenotyping experiment, the effector cells were pre-incubated with various of chicken T cell specific antibodies for 30 min, followed by PE-conjugated anti-Ig isotype-specific antibody for another 30 min, or simply incubated with PE-conjugated antibodies for 30 min, on ice. In the flow cytometry data acquisition, the PKH67 labeled REV transformed B target cells were detected at FL1 channel and PI labeled dead target cells were detected at FL2 (in two-colour assays) or FL3 (in three-colour assays). An electronic gate was made at FL1 on PKH67-positive target cells, in order to discard the unlabeled effector and target cells during counting. In three-colour effector cell phenotyping assays, the gated FL1-positive cells were analyzed at FL2 and FL3 channels, the PI labeled dead target cells were detected at FL3 and the PE-labeled effector-target cell conjugates were detected at FL2 channel. 2.7. Data processing The percentage of specific target cells killed was considered as the total dead target cells at each E:T ratio excluding the cell death in controls in which target cells were alone with PI.

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3. Results 3.1. Two-colour CTL assays H.B15 chickens of 8-week old were immunized three times with fixed histocompatible REV-B cells to induce the generation of cytotoxic effector cells. Killing was analysed by PBL. Almost all (99.0%) of PBL were considered as the live cells by the examination of 0.4% Tryphan Blue. The percentage of cytotoxicity at various E:T cell ratios was determined using flow cytometric analysis after 50 min of incubation of the effector cells and target cells, with the presence of PI. Target cells alone incubated with PI were used as the control. The result shows that the background target cell death, normally was 2–8%, much less than that in 51 Cr-release assay, which in our experience was 16–25% in chicken (Vainio et al., unpublished data). These two-colour CTL assays were repeated at least 10 times at the E:T ratio 100:1. The dot plots from one representative experiment are shown in Fig. 1. The target cells were 80.6% stained with PKH67. The PKH67-positive target cells were gated and the percentage of PI staining was measured. There were very few dead cells (3.2%) without the participation of effector cells during the target cell incubation (Fig. 1A). The cell death is just the cell damaged by pipeting and/or labelling processes. Analyses from effector-target co-incubation samples show that the pathogenic specific killing was 20% more, at E:T 100:1 (Fig. 1B). The mean percentage of killing at different E:T rations in three immunized chickens was shown in

Fig. 2. Cytotoxicity induced by PBL from immunized and unimmunized chickens.

Fig. 2. The percentage of target cells killed by effector cells increased dramatically from E:T 12.5:1 to 50:1, and then slightly decreasing, but no significant difference between 50:1 and 100:1. In the experiments of the effector cell phenotyping assays, E:T ratio 100:1 was taken. PBL from chickens immunized with fixed REV transformed B cells kill target cells in an MHCdependent manner. REV transformed MHC-haplotype B2 cells have no responses to the effector cells from the immunized B15 chickens. The percentage of the killing was 3.27% (n ¼ 3). The mean cytotoxicity from three un-immunized hatchmates was shown also in Fig. 2. The killing was little bit enhanced from the E:T ratio 12.5:1–100:1, but the percentage of killing was far away behind the cytotoxicity derived from the immunized chickens. This demonstrates that the cytotoxicity from the immunized chickens is REV specific. 3.2. Three-colour phenotypic analysis of the effector cells

Fig. 1. The basic set-up of a cytotoxicity assay based on flow cytometry. Target cell distribution in various cases is shown after a 50 min assay: (A) PKH67-labeled target cells with PI but without effector cells; (B) PKH67-labeled target cells after a 50 min coincubation with PI and effector cells from an immunized chicken.

Multicolour flow cytometric analysis gives the advantage to determine the phenotype of cytotoxic killer cells, which attached to the dying target cells. In the phenotyping assays three colours (PKH67, PI and PE) were used. The effector cells were interacted with anti-CD (CD4 or CD8), or anti-TCR (TCR1, TCR2,

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when the effector cells were stained (Fig. 3B–F). Events in the lower right quadrant represent live effector cell–target cell conjugates. The phenotyping assays show that the majority of the killer cells were CD8þ or TCR2þ T cells, and some CD4þ and TCR3þ cells were also involved. The flow cytometric analysis observed that it is not only CD8þ T cells contacted with the dying target cells, CD4þ T cells do as well. Double staining of PBL from these chickens with mAb 2–6 (IgG1, FITC) and 3–298 (IgG2b, PE) showing that there were no CD8þ/ CD4þ T cells in the blood from chickens that used in these studies, this exclude the possibility that double positive cells were recognized as CD4þ cells. CD4þ cells conjugated with the dying target cells does not imply that this cell type acts as the killer cells, but at least there was some kind of important help from them in the killing action.

4. Discussion

Fig. 3. The basis of a method to analyze the phenotype of cytotoxic effector cells: (A) gated PKH67-labelled target cells after co-incubation with anti-IgG1-PE stained effector cells and PI, used as control for effector cell phenotyping; (B) gated PKH67-labelled target cells after co-incubation with anti-CD4 ðmAb 26Þ þ antiIgG1-PE stained effector cells and PI; (C) gated PKH67-labelled target cells after co-incubation with anti-CD8 ðmAb 1139Þ þ antiIgG1-PE stained effector cells and PI; (D) gated PKH67-labelled target cells after co-incubation with anti-TCR1 þ anti-IgG1-PE stained effector cells and PI; (E) gated PKH67-labelled target cells after co-incubation with anti-TCR2 þ anti-IgG1-PE stained effector cells and PI; (F) gated PKH67-labelled target cells after coincubation with anti-TCR3 þ anti-IgG1-PE stained effector cells and PI.

or TCR3) antibodies, followed by labelling with PE-conjugated anti-IgG1. The pre-labelled cells were used as the effector cells at E:T ratio of 100:1. Fig. 3 shows the basic set-up of the phenotype analysis. In the analysis PKH67-labeled target cells were gated at FL1 channel. The dead target cells (PI-positive) were detected at FL3 channel and target-effector conjugated cells were detected at FL2 (stained with mAb). The results show that spontaneous cell death was around 4%. Specific cell lysis was around 20% (Fig. 3). No events emerged at FL2 (Fig. 3A) unless in some cases

Most current assays for measuring cytotoxicity are based on alterations of plasma membrane permeability and the consequent release (leakage) of self- or preloaded-components into the supernatant or the uptake of dyes, normally excluded by viable cells. Among these, 51 Cr-release assay is the well-known and most widely practiced one. However, it has many apparent disadvantages: (i) 51 Cr-release assay is a radioactive method with potential health and environmental hazards; (ii) a relatively long effector-target cell cocultivation time is needed; (iii) the inefficient labelling of the target cells, which is mostly chicken cell-specific disadvantage; (iv) a high spontaneous release and (v) it does not allow one to analyze the phenotype of the killer cells. Almost all of these shortcomings also exist in other similar methods based on the release of other radioactive markers, e.g. ½3 Hproline or 5-½125 I-2-deoxyuridine (Oldham et al., 1977), ½3 H-thymidine (Matzinger, 1991), 14 C-leucine (Kubo et al., 1999), 111 InOx- (Shortman and Wilson, 1981; Wiltrout et al., 1981; Jakubek et al., 1983; Russell et al., 1986), ½35 S- (Andoins et al., 1996) or ½75 Se-methionine (Leibold and Bridge, 1979), or fluorescent dyes such as carboxyfluorescein diacetate (Bruning et al., 1980), europium (Blomberg et al., 1986a,b), bis-carboxyethyl-carboxyfluorescein

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(BCECF) (Kolber et al., 1988) or Calcein-acetoxymethyl (Calcein-AM) (Roden et al., 1999), from the damaged target cells. Another group of assays is based on the measurement of cytoplasmic enzymes released into the supernatant by damaged cells. The amount of enzyme activity detected in the culture supernatant corresponds to the proportion of lysed cells. Enzyme release assays have been described for lactatic dehydrogenase (LDH) (Korzeniewski and Callewaert, 1983; Decker and Lohmann-Matthes, 1988; Kubo et al., 1999), for alkaline (Szekeres et al., 1981) and acid (Martin and Clynes, 1991) phosphatase, for hemoglobin-enzyme (Hou and Zheng, 1985). However, their use has been hampered by the low amount of those enzymes present in many cells and by the elaborate kinetic assays required to quantify most enzyme activities. Although LDH is a stable cytoplasmic enzyme present in all cells, and it is released into the cell culture supernatant, rapidly when the plasma membrane is damaged, it is an elaborate work to measure the enzyme activity, and it is impossible to distinguish between enzyme produced from effector cells and target cells. Alternately, to the release of cell-self enzyme, target cell line has been transfected with the plasmid carrying an E. Coli b-galactosidase gene (Ohmori et al., 1992; Scha¨ fer et al., 1997) or a luciferase reporter gene (Scha¨ fer et al., 1997). Although those enzymes are exclusively and highly expressed in the target cells, the effector cell phenotyping remained as a problem. The third group assays is based on determination of cytotoxicity by dye staining, e.g. trypan blue (Renoux et al., 1980) or Propidium iodide/CellTrackerTM Green (CTTMG) 5-chloromethyl fluorescein diacetate double staining (Kubo et al., 1999). Instead of microscopic observation and cell counts in this type of assays, the tool of flow cytometry now is used as a modification. The use of flow cytometry is now established as an important tool in the laboratory studies of immunology. Through labelling the effector cells, flow cytometry-based method in the cell lysis assay enables to follow the behaviour of the cytotoxic T cells. The flow cytometry-based method avoids the use of radioactivity, overcoming many of the limitations encountered with radioactive labelling of chicken target cells, such as poor incorporation, high spontaneous release and low antigen-specific release.

In this study, REV specific cytotoxicity was used as a model system. Flow cytometry-based assays show that the cytotoxicity was haplotype-specific and effector cell dose-dependent. Our data also show that certainly CD8þ T cells were involved in the killing, but also CD4þ T cells, may be involved to some extent. This is because CD4þ T cell/ killed target cell conjugates were observed in the effector phenotyping assay. Effector cells from unimmunized chickens can induce target cells death to some extent which may indicate that NK cells also kill REV transformed B cells. It has been shown that there are NK positive cells in the PBL (2–6%) from chickens by NK cell specific antibodies 14H12, 15E7 and 20E5 staining. The phenomenon of CTL with CD4 marker has been reported in infectious bronchitis virus in mice (Zajac et al., 1996; Williams and Engelhard, 1996), in equine (Hammond et al., 1998), in poultry (Collisson et al., 2000), although most of the studies employed the CD4þ or CD8þ cell depletion method. In contrast to CD8þ CTLs, however, the biological role of CD4þ T cells in the cell-mediated immunity is poorly understood, especially in vivo. So far, even the definition of CTL is not very clear in chicken and they should be further characterized. This studies show that the major CTLs were TCR2þ cells, and some are TCR3þ cells. This is consistent with the research concerning with the characterization of the avian T cell receptor. Lahti et al. (1988) have shown that in the peripheral blood the vast majority of the TCR1þ cells are CD4/CD8. The TCR3þ cells co-express either the CD4 or CD8 antigen, but the CD4/CD8 ratio for the TCR3þ cells differs from that of the TCR2þ cells. Approximately 88% of the TCR3þ cells are CD4þ and 12% are CD8þ, while 74% of the TCR2þ cells are CD4þ and 26% are CD8þ. In the effector cell phenotyping assays, the effector cells were pre-stained with a number of chicken T cell specific antibodies. Reasoningly, such process might block CTL. In these experiments, the CD4 antibody is blocking the killing just by 1.6% (Fig. 3A UL þ UR ¼ 25:2%, Fig. 3B UL þ UR ¼ 23:6%, 25:23:6% ¼ 1:6%), CD8 by 3.7%, TCR1 by 3%, TCR2 by 5.5%, TCR3 by 2.6%. All of the tested antibodies are blocking the killing but the effect is weak. The relative bigger effect is seen with TCR2 and CD8 antibodies. There are several putative reasons of the weak blocking effect of anti-CD8 antibody. Firstly,

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the CD8 (or other antibodies TCR1, 2, 3) antibody might bind to an epitope that is not interacting with MHC and thus not interfering with the specific recognition and cytotoxicity. Secondly, as shown in Fig. 3C, there are 11.4% CD8þ cells and only 3.3% of them are involved in cytotoxic killing, 8.1% of CD8þ cells are associated with PI negative cells. So even if the antibody would block the killing there are other CD8 ‘‘negative’’ cells which could still kill target cells. Thirdly, the amount of T cells (UR þ LR) in Fig. 3D–F is 29.5%. Reduced CD4þ cells 5.5% (Fig. 3B UR þ LR), results in 24% T cells. However, there are 11.4% CD8þ cells were caught by staining (Fig. 3C UR þ LR). Might be that the CD8 antibody used here is staining only approximately 50% of the CD8þ cells. Fourthly, CD8, CD4, TCRs and background altogether gives 14.7% cells associated with the killed cells. And the mean percentage of dead cells is 22.5%. There is 7.8% of killed cells could also be killed by some other cell population such as chicken NK cells. The flow cytometry assay to measure cytotoxicity introduced here can be used as an alternative to the standard 51 Cr-release, especially in chicken. Knowledge derived from this study will be useful for the analysis of cytotoxicity induced by other chicken infectious disease related viral or parasitic antigens. By using recombinant virus techniques to express antigens and the flow cytometry-based cytotoxicity assay, it is possible to determine which pathogen encoded proteins or peptides are actually immunologically important.

Acknowledgements We thank Dr. Thomas Go¨ bel (Institute for Animal Physiology, Munchen, Germany) for kindly providing the REV transformed B cell lines, and chicken NK antibodies. We are grateful to Mrs. Taina Niitynpera¨ with her expert technical helps.

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