Journal of Virological Methods, 39 (1992) 185-195 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
185
VIRMET 01375
Generation of macaque B lymphoblastoid cell lines with simian Epstein-Barr-like viruses: transformation procedure, characterization of the cell lines and occurrence of simian foamy virus Gerald
Vo&,
Sigrid Nick”, Christiane Stahl-Henniga, and Gerhard HunsmanrP
Klaus Ritterb
aDeutsches Primatenzentrum. Abteilung Virologie und Immunologic, Giittingen (Germany) and bHygiene-Institut der Georg-August-Universitiit, Abteilung Medizinische Mikrobiologie, Gcttingen (Germany)
(Accepted
31 March 1992)
Summary Two simian Epstein-Barr-like viruses, a rhesus Epstein-Barr virus and Herpesvirus papio, were used to transform B cells from rhesus or cynomolgus macaques. The resulting cell lines exhibited predominantly a B lymphocyte phenotype and expressed Epstein-Barr virus antigens. The majority of B lymphoblastoid cell lines from macaques, which were seropositive for simian foamy virus, developed giant cells in culture. The cytopathic agent was identified as a foamy virus and was transmissible to human embryonal fibroblasts. Treatment of cell cultures with AZT abolished giant cell formation. B-lymphoblastoid cell line; Rhesus Epstein-Barr foamy virus; Syncytia formation; AZT
virus; Herpesvirus
papio; Simian
Introduction Transformed B lymphoblastoid cell lines (BLCL) have become important tools for immunological studies in human and non-human primates. Human BLCL, transformed with Epstein-Barr virus (EBV), are used widely for Correspondence to: G. Voss, Deutsches Kellnerweg 4, 3400 Giittingen, Germany.
Primatenzentrum,
Abteilung Virologle und Immunologie,
186
cytotoxicity assays. In particular, human immunodeliciency virus (HIV)specific cytotoxic T lymphocytes (CTL) were detected using BLCL infected with recombinant vaccinia viruses (for review see, Walker and Plata, 1990; Autran et al., 1991). Another important application for BLCL is the analysis of major histocompatibility antigens (MHC). In this experimental approach BLCL serve as a source for MHC antigens, which are radiolabeled and subsequently separated by isoelectric focusing (Yang et al., 1985; Neefjes et al., 1986). EBV B95-8 strain (Luka et al., 1978) is widely used for the transformation of human B lymphocytes. The method is well established and generates human BLCL (Sugden and Mark, 1977). Macaque BLCL for CTL-assays (Miller et al., 1990; Gotch et al., 1991) are obtained often by transformation with Herpesvirus papio (HVP) (Falk et al., 1976; Rabin et al., 1977). Although several other EBV-related herpesviruses were isolated from different species (Gerber et al., 1977; Rasheed et al., 1977; Neubauer et al., 1979; Backer et al., 1980; Heberling et al., 1981; Lapin et al, 1986; Rangan et al., 1986), these isolates are not used as often as HVP. In this study we intended to compare the transforming potential of different EBV-like simian viruses. In addition to HVP we used a second simian EBV-like Herpesvirus, rhesus Epstein-Barr virus (RhEBV) (Rangan et al., 1986), to transform macaque B lymphocytes. The resulting BLCL were compared and showed a B-cell phenotype. EBV-related antigens were detected in all BLCL. In some BLCL, originating from simian foamy virus (SFV)-seropositive macaques, giant cells containing SFV-antigens were observed. The infectious agent was transmitted to human embryonal fibroblasts by cocultivation. Giant cell formation was the main cause of cell death in outgrowing BLCL. To overcome this problem cultures were treated with 3’-azido-2’,3’-dideoxythymidine (AZT), which suppressed giant cell formation but shortened the lifetime of the BLCL cultures.
Materials and Methods Transforming
viruses
Two EBV-like simian viruses were used to transform macaque peripheral blood lymphocytes (PBLs). RhEBV was isolated from a lymphoma of a rhesus monkey (Macaca mulatta) (Rangan et al., 1986) and HVP originates from a lymphoma of a baboon (Papio hamadryas) (Falk et al., 1976; Rabin et al., 1977). LCL8664 cells (Rangan et al., 1986) were seeded at 2. lo5 per ml in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS) and maintained for 10 days at 37°C in a humidified 5% CO* atmosphere to produce infectious RhEBV for cell transformation. Thereafter, the cultures were incubated for another 10 days at 33°C. The culture supernatants were harvested, passed
187
through a 0.45 pm filter and aliquots were stored at -80°C. Infectious HVP was obtained by seeding 2.10’ cells per ml of the virusproducing S594 cell line (Falk et al., 1976; Rabin et al., 1977) in RPMI/lO% FCS and incubating the cultures subsequently for 10 days at 37°C in 5% CO*. The supernatant was treated as indicated for the LCL8664 cell line. Transformation
procedure
PBLs were isolated from fresh titrated blood of several rhesus monkeys of Indian or Chinese origin or cynomolgus monkeys kept at the DPZ. After separation on a Histopaque-1077 gradient (Sigma, Germany) the PBLs were seeded at a density of 2. IO6 cells per well in 1 ml RPM1 1640 supplemented with either 10% FCS or pooled human AB-serum and 1 lug per ml cyclosporin A (CsA) (Sandoz, Switzerland) in 12-well plates (Costar, USA). To transform these cultures, 1 ml of culture supernatant of LCL8664 or S594 cells was added. The culture medium was changed twice a week and the cell growth in the cultures was examined. Outgrowing cells were transferred to cell culture flasks and 2 weeks later, medium from all cultures was replaced stepwise by RPM1 1640 containing 10% FCS without CsA. Cocultivation
with HEL S37 cells
Human embryonal lung fibroblasts (HEL S37) were grown in Leighton tubes (Costar, USA) to a confluent monolayer. Subsequently the cells were cocultivated with 1 . lo5 transformed B-lymphoblastoid cells and examined for the expression of SFV antigens 48 h later. Detection of EBV-viral capsid antigen (VCA) antigen by indirect immunojluorescence
and simian foamy
virus (SFV)
Both, EBV-VCA and SFV antigens were detected by indirect immunofluorescence (Henle and Henle, 1966). 1 . lo6 suspension cells were removed from BLCL cultures and washed once in phosphate-buffered saline (PBS, 2 mM KH,PO,; 9 mM NazHP04; 180 mM NaCl, pH 7.3). Cells were resuspended in 100 ,ul PBS and aliquots of 10 ~1 were pipetted on to glass slides with 10 marked rings (Hiilzel, Germany). After 1 min at room temperature (RT), the cell suspension was removed and the slides were dried for 1 h at RT. Human embryonal tibroblasts were grown in Leighton tubes, cocultivated with BLCL and washed twice with PBS after 48 h. The suspension, as well as the monolayer cells, were fixed for 15 min at - 20°C in acetone and washed in PBS with 0.05% (v/v) Tween 20 (PBS-Tween). Sera were diluted 1:40 in PBS-Tween and incubated with the cells for 1 h at 37°C in a humidified atmosphere. After washing with PBS-Tween a FITC-labeled goat-anti-human IgG antiserum (Medac, Germany) diluted 1:40 was incubated with the cells for 45 min, as described above. The slides were washed once again and covered with 20% (v/
188
v) glycerol in PBS prior to examination with a fluorescence microscope (Zeiss, Germany). EBV-VCA was detected with human VCA-positive sera. SFV antigen was examined with positive reference macaque sera. Anti-complement
immunojluorescence
(ACIF)
EBV-nuclear antigen was detected according to the method described by Reedman and Klein (1973) with modifications developed by Henle et al. (1974). Briefly, cells were prepared and fixed on coverslips as described above. The cells were overlaid successively with the test specimen (inactivated antiEBNA positive human serum (1: 10 diluted); serum from guinea pig as source of complement (1:8); FITC-conjugated goat antibodies to guinea pig C3 (Nordic, NL) (1: 15)). All incubation steps were carried out for 1 h at 37°C. Following the last incubation step the coverslips were washed for 30 min in PBS containing 1% (w/v) Evans Blue and treated further, as described above. Screening of macaque sera for SFV antibodies and SFV reference sera Sera from rhesus or cynomolgus macaques were screened for the presence of antibodies against SFV by indirect immunofluorescence as described by Neumann-Haefelin et al. (1983). Briefly, Molt-4 cells infected with the SFV isolate LK-3 were suspended on teflon-coated glass slides, dried and then fixed for 15 min at -20°C in methanol. The samples were incubated first with monkey sera diluted 1:lO and subsequently with FITC-conjugated goat antihuman IgG antiserum diluted 1:20 (Medac, Germany). The specific fluorescence obtained with the monkey sera was compared to the fluorescence of SFV reference sera. Flow cytometry
analysis of cell surface antigens
Cell surface antigens were examined by flow cytometry analysis. 5. lo5 transformed cells from BLCL of rhesus or cynomolgus macaques were washed in phosphate-buffered saline (PBS) supplemented with 5% FCS and incubated in 40 ~1 PBS containing 5% FCS and 1 ~1 FITC-conjugated antibody for 1 h at 4°C. Monoclonal antibodies specific for Leu 5b (CD2), Leu 12 (CD19) and Leu 16 (CD20) were obtained from Becton-Dickinson, (USA) and goat-F(ab’)z anti-human IgG antiserum was purchase.d from Medac, Germany. Subsequently, the samples were washed with PBS/S% FCS and resuspended in 300 ~1 PBS containing 0.4% formaldehyde. At least 1 x lo4 cells were analyzed in each run using an EPICS profile flow cytometer (Coulter, Hialeah, USA).
189
Results Generation of BLCL
with RhEBV
or HVP
In total, 220 cultures of PBLs resulting in 94 growing BLCL from rhesus and cynomolgus monkeys, were initiated with supernatants of LCL8664 or S594 with different serum supplementation. Using RhEBV as transforming agent, 39 out of 83 initiated PBL cultures grew out to BLCL when supplemented with human AB-serum (Table 1). The first dividing cells were not visible before day 30 after the onset of the cultures. 55 transformed cell lines were established from 137 HVP-treated PBL cultures with human AB-serum supplementation (Table 1). The HVP-transformed cell lines grew out earlier, beginning from day 14. Successful transformation with RhEBV was achieved only when human AB-serum and CsA were supplemented, whereas HVP-transformed cells grew out also in FCS-containing medium without CsA (data not shown). Phenotypical
analysis of the transformed
BLCL
Thirteen RhEBV-transformed BLCL from rhesus or cynomolgus macaques and 2 HVP-transformed BLCL from cynomolgus monkeys were examined further for the expression of lymphocyte-specitic cell surface markers. Flow cytometry analysis showed that all cell lines expressed B-cell specific surface antigens in varying amounts. An average of 46.1% (6 20.1) of the cells were Leu12-positive. Leul2+ cells were observed in every cell line and the fractions of positive cells varied between 15.3 and 80.3%. Leul6+ (CD20) cells were detected in 3 of the 15 examined B-LCL and constituted 37.4% (6, 18.2) of the whole cells. The percentage of positive cells varied from 16.5 to 50.0%. Surface IgG was detected on 95.3% (6, 1.2) of the transformed cells. Each BLCL comprised an IgG-positive cell fraction constituting 94.3 to 96.3% of the whole cells. Only one BLCL exhibited a LeuSb+ (CD2) cell population (20.1%). Detection of EBV antigens VCA and EBNA
in the BLCL
In about 60% of the cells in the BLCL from both monkey species EBNA was induced by infection and transformation with EBV-like RhEBV or HVP (Fig. 1B). Two different fluorescence patterns were detected by ACIF. The majority TABLE 1 Survey of BLCL generated with RhEBV and HVP Transforming RhEBV HVP
virus
Total
MM=
MF=
39/83b 551137
14131 1l/45
25146 44192
a MM, Macaca mulatta; MF, Macaca fascicularis. b Number of successfully transformed/number of initiated cultures transformed
BLCL.
190
191
of cells showing fluorescence presented a granular nuclear staining. A small fraction had a brilliant nuclear fluorescence. These cells were larger than the other cells showing fluorescence and the negative cells (Fig. 1B). The examination of VCA-expressing cells from the BLCL transformed with RhEBV or HVP revealed that up to 35% of the cells expressed VCA (Fig. 1B). Giant cell formation in transformed BLCL Giant cells were observed in 39 BLCL from 25 animals, beginning from 4 weeks after the start of the cultures. Giant cells increased in 31 cultures, and the cells died within a month. The other cultures continued to grow, but still contained giant cells. Comparison to serological data of the macaques from which the transformed PBLs were derived revealed that all cultures with giant cells originated from monkeys seropositive for SFV. PBLs from 36 seropositive macaques were transformed and giant cells were found in cultures originating from 25 animals, whereas the BLCL from 11 seropositive monkeys did not demonstrate giant cell formation. The giant cells were examined for the presence of SFV antigens by indirect immunofluorescence to prove this correlation between giant cell formation and SFV-seropositivity. The giant cells showed a staining only with SFV-positive reference sera, whereas the cells with normal size showed frequent staining with SFV-positive and negative sera (Fig. IC). This staining was probably due to EBV-related antigens, since the SFV reference sera contained EBV-VCA antibodies (data not shown). In order to transmit the infectious agent, fetal human embryonal tibroblasts were cocultivated with BLCL positive or negative for SFV-induced giant cells. After 48 h, cultures cocultivated with SFV-positive BLCL exhibited giant multinucleated fibroblasts. These cells were stained with the SFV-positive control serum (Fig. 1D). The control cultures did not exhibit any cytopathic effect. These observations indicated that infectious SFV was present in the infected BLCL. In an attempt to suppress SFV replication in HVP-transformed BLCL showing giant cell formation, cultures were split and one sample each was maintained in medium containing 4 pmol per ml AZT. In five of the six cultures the giant cells disappeared (Table 2). One culture (from Mf760) was not affected by AZT treatment and still contained giant cells. Except the BLCL from Mm 1707, which could be maintained for a longer period with AZT supplementation, the other AZT-treated BLCL cultures died earlier than the untreated parallel samples (Table 2). + Fig. 1. EBV-related and SFV antigens detected by indirect immunofluorescence. BLCL of rhesus or cynomolgus macaques were examined for the expression of EBV-related antigens, using positive reference sera. Staining with a VCA-specific serum is shown in (A) and ACIF staining for EBNA in (B). Giant cellcontaming BLCL cultures (C) and syncytia of HEL S37 tibroblasts after cocultivatton with giant cellpositive BLCL (D) were examined for SFV antigen expression using a SFV reference serum.
192 TABLE 2 AZT treatment
of giant cell-positive
BLCL
Animal
Occurrence of giant cellsa
AZT treatmentb
Giant cellsC
Death of cellsd
MF723e
8
+ _
_ +
20 -
MF760
8
+ _
+ +
28 -
MF778
8
+ -
_ +
20 -
MF5357
8
+ _
_ +
-
MM1663e
8
+ -
_ +
20 -
MM1707
4
+ -
+
22 8
a Week after transformation when first giant cells were observed. b Cultures were not treated with AZT (-) or with 4 pm01 per mi AZT (+). ’ Occurrence of giant cells 4 weeks after initial AZT treatment. d Cell lines dies at the indicated week after transformation or could not be cultivated further. e MM, Macaca mulatta; MF, Macaca fascicularis.
Discussion We investigated the capacity of two EBV-like simian viruses to transform macaque B-cells. The outgrowing BLCL were characterized phenotypically by flow cytometry and showed predominantly B-cell characteristics. VCA, as well as EBNA, was detected in the transformed cells. Occurrence of giant cells in the BLCL was correlated to SFV-seropositivity of the animals from which the cell lines were derived. The infectious virus was transmitted successfully in vitro to human embryonal Iibroblasts. Giant cell formation was suppressed by addition of AZT to the cultures. The two viruses RhEBV and HVP did not display characteristic differences in the induction of the virus-specific antigens VCA and EBNA in the BLCL from cynomolgus or rhesus macaques. In contrast to observations of human EBV-transformed BLCL, when 100% of the infected cells were EBNA-positive (Fresen and zur Hausen, 1976) only up to 60% of the monkey BLCL expressed EBNA. This may be due either to a reduced expression of EBNA genes in the cells without fluorescence or may be related to an incomplete replication cycle of EBV in some of the cells. Furthermore it cannot be excluded that some cells were transformed without being infected (Klein et al., 1974). The fact that EBNA was only expressed in a fraction of all the cells indicates that the BLCL are of polyclonal origin.
193
Up to 35% of the cells expressed VCA after transformation. This indicates a high production of new virus particles. The level of virus production may vary, depending on the infected cell line, since Miller and Lipman (1973) have found considerable differences in the release of infectious virus from human cell lines transformed with EBV. The majority of BLCL from SFV-positive macaques exhibited giant cells beginning 4 weeks after initiation of the cultures. Many of these cultures could not be maintained for more than a month. This phenomenon was the main cause for the failure to establish permanently growing BLCL. The giant cells were shown to contain SFV antigen and infectious virus was transmitted to human embryonal fibroblasts. Thus, it is very likely that the cytopathic agent was SFV. Furthermore, SFV was isolated frequently from lymphoblastoid cells of several primate species (Feldman et al., 1975; Rabin et al., 1976; Barahona et al., 1976; Schnitzer, 1981; Neumann-Haefelin et al., 1983). Since it is difficult to obtain SFV-seronegative macaques we attempted to suppress giant cell formation with AZT. This drug was shown to inhibit HIV-I replication in vitro (Mitsuya et al., 1985) by selective interaction with the reverse transcriptase (Furman et al., 1986). We observed an inhibition of giant cell formation in the treated cultures. This observation suggests that AZT can also inhibit SFV replication. Nevertheless, the approach of treating BLCL cultures with AZT is not yet well established, since most of the treated cultures died earlier than untreated ones. This may be due to the development of AZTresistant SFV in the cultures or AZT is cytotoxic over a long culture period. Further evaluation of a more sophisticated cell culture technique using AZT may lead to an efficient method of suppressing SFV without a reduction in the life of the cultures.
Acknowledgements We would like to thank Dr. G. Nelke, Staatliches Kontrollinstitut fur immunbiologische Arzneimittel, Berlin, Germany, for providing us with HEL S37 cells and Dr. D. Neumann-Haefelin, Institut fur Virologie, Universitat Freiburg, for fixed Molt-4/LK-3 cells and SFV reference sera. CsA was kindly supplied by Sandoz, Basel, Switzerland. The excellent technical assistance of S. Aurisch and S. Noch is greatfully acknowledged. This manuscript contains parts of the doctoral thesis of G.V.
References Autran, B., Plata, F. and Debre, P. (1991) MHC-restricted cytotoxicity against HIV. J. AIDS 4, 361-367. Barahona, H., Garcia, F.G., Melendez, L.V., King, N.W., Ingalls, J.K. and Daniel, M.D. (1976) Isolation and characterization of a lymphocyte associated foamy virus from a red uakari monkey (Cucajuo rubicundus). J. Med. Primatol. 5, 253-265.
194 Backer, J.F., Tiedemann, K.H., Bornkamm, G.W. and zur Hausen, H. (1980) Characterization of the EBV-like virus from African green monkey lymphoblasts. Virology 101, 291-295. Falk, L., Deinhardt, F., Nonoyama, M., Wolfe, L.G., Bergholz, C., Lapin, B., Yakovleva, L. and Agrba, V. (1976)Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus. Int. J. Cancer 18, 798-807. Feldman, M.D., Dunnick, N.R., Barry, D.W. and Parkman, P.D. (1975) Isolation of foamy virus from rhesus, African green and cynomolgus monkey leukocytes. J. Med. Primatol. 4, 287-295. Fresen, K.O. and zur Hausen, H. (1976) Establishment of EBNA-expressing cell lines by infection of Epstein-Barr virus (EBV)-genome-negative human lymphoma cells with different EBV strains. Int. J. Cancer 17, 161-166. Furman, P.A., Fyfe, J.A., St.Clair, M.H., Weinhold, K., Rideout, J.L., Freeman, G.A., NusinoffLehrman, S., Bolognesi, D.P., Broder, S., Mitsuya, H. and Barry, D.W. (1986) Phosphorylation of 3’-azido-3’-deoxythymidine and selective interaction of the S-triphosphate with human immunodeticiency virus reverse transcriptase. Proc. Nat]. Acad. Sci. USA 83, 8333-8337. Gerber, P., Kaher, S.S., Schidlovsky, G. Peterson, W.D. and Daniel, M.D. (1977) Biologic and antigenic characteristics of Epstein-Barr virus-related herpesvirus of chimpanzees and baboons. Int. J. Cancer 20, 448459. Gotch, F.M., Hove& R., Delchambre, M., Silvera, P. and McMichael, A.J. (1991) Cytotoxic T-cell response to simian immunodeficiency virus by cynomolgus macaque monkeys immunized with recombinant vaccinia virus. AIDS 5, 317-320. Heberling, R.L., Bieber, C.P. and Kaher, S.S. (1981) Establishment of a lymphoblastoid cell line from a lymphomous cynomolgus monkey. In: D.S. Yohn and J.R. Blakeslee (Eds), Advances in comparative leukemia research. Elsevier, Amsterdam, pp. 385-386. Henle, G. and Henle, W. (1966) Immunofluorescence in cells derived from Burkitt’s lymphoma. J. Bacterial. 91, 1248-1256. Henle, W., Guerra, A. and Henle, G. (1974) False negative and prozone reactions in tests for antibodies to Epstein-Barr virus-associated nuclear antigen. Int. J. Cancer 13, 751-754. Klein, G., Sugden, B., Leibold, W. and Menezes, J. (1974) Infection of EBV-genome-negative and positive human lymphoblastoid cell lines with biologically different preparations of EBV. Intervirology 3, 232-244. Lapin, B.A., Timanovskaya, V.V. and Yakovleva, L.A. (1985) Herpesvirus HVMA: a new representative in the group of the EBV-like B-lymphotropic herpesviruses of primates. Haematol. Blood Transf. 29, 312-313. Luka, J., Lindahl, T. and Klein, G. (1978) Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines. J. Viral. 27, 604611. Miller, G. and Lipman, M. (1973) Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc. Natl. Acad. Sci USA 70, 19&194. Miller, M.D., Lord, C.I., Stallard, V., Mazzara, G.P. and Letvin, N.L. (1990) The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques. J. Immunol. 144, 122-128. Mitsuya, H., Weinhold, K.J., Furman, P.A., St.Clair, M.H., Nusinoff-Lehrman, S., Gallo, R.C., Bolognesi, D., Barry, D.W. and Broder, S. (1985) 3’-azido-3’-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc. Natl. Acad. Sci. USA 82, 70967100. Neefjes, J.J., Breur-Vriesendorp, B.S., van Seventer, G.A., Ivanyi, P. and Ploegh, H.L. (1986) An improved biochemical method for the analysis of HLA-class I antigens. Definition of new HLAclass I subtypes. Hum. Immunol. 16, 169-181. Neubauer, R.H., Rabin, H., Strnad, B.C., Nonoyama, M. and Nelson-Rees, W.A. (1979) Establishment of a lymphoblastoid cell line and isolation of an Epstein-Barr (EBV)-related virus of gorilla origin. J. Virol. 31, 845-848. Neumann-Haefehn, D., Rethwilm, A., Bauer, G., Gudat, F. und zur Hausen, H. (1983) Characterization of a foamy virus isolated from Cercopithecus aethiops lymphoblastoid cells. Med. Microbial. Immunol. 172, 75-86. Rabin, H., Neubauer, R.H., Woodside, N.J., Cicmanec, J.L., Wallen, W.C., Lapin, B.A., Agrba,
195 V.A., Yakoleva, L.A. and Chuvirov, G.N. (1976) Virological studies of baboon (Pupio hamadryas) lymphoma: isolation and characterization of foamy viruses. J. Med. Primatol. 5, 1322. Rabin, H., Neubauer, R., Hopkins, R., Dzhikidze, E., Shevtsova, Z. and Lapin, B. (1977) Transforming activity and antigenicity of an Epstein-Barr-like virus from lymphoblastoid cell lines of baboons with lymphoid disease. Intervirology 8, 24&249. Rangan, S.R.S., Martin, L.N., Bozelka, B.E., Wang, N. and Gormus, B.J. (1986) Epstein-Barr virus-related herpesvirus from a rhesus monkey (Mucacu muluttu) with malignant lymphoma. Int. J. Cancer 38, 4255432. Rasheed, S., Rongey, R.W., Bruszweski, J., Nelson-Rees, W.A., Rabin, H., Neubauer, R.H., Esra, G. and Gardner, M.B. (1977) Establishment of a cell line with associated Epstein-Barr-like virus from a leukemic orangutan. Science 198, 407409. Reedman, B.M. and Klein, G. (1973) Cellular localization of an Epstein-Barr virus (EBV)associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11, 4999520. Schnitzer, T.J. (1981) Characterization of a simian foamy virus isolated from a spontaneous primate lymphoma. J. Med. Primatol. 10, 312-328. Sugden, B. and Mark, W. (1977) Clonal transformation of adult human leukocytes by Epstein-Barr virus. J. Virol. 23, 503-508. Walker, B.D. and Plats, F. (1990) Cytotoxic T-lymphocytes against HIV. AIDS 4, 177-184. Yang, S.Y., Chang, A., Ohvero, R., Rehas, V. and Yunis, E.J. (1985) IEF pattern of HLA-B13 antigens from orientals and Caucasians. Immunogenetics 21, 125-134.
185
VIRMET 01375
Generation of macaque B lymphoblastoid cell lines with simian Epstein-Barr-like viruses: transformation procedure, characterization of the cell lines and occurrence of simian foamy virus Gerald
Vo&,
Sigrid Nick”, Christiane Stahl-Henniga, and Gerhard HunsmanrP
Klaus Ritterb
aDeutsches Primatenzentrum. Abteilung Virologie und Immunologic, Giittingen (Germany) and bHygiene-Institut der Georg-August-Universitiit, Abteilung Medizinische Mikrobiologie, Gcttingen (Germany)
(Accepted
31 March 1992)
Summary Two simian Epstein-Barr-like viruses, a rhesus Epstein-Barr virus and Herpesvirus papio, were used to transform B cells from rhesus or cynomolgus macaques. The resulting cell lines exhibited predominantly a B lymphocyte phenotype and expressed Epstein-Barr virus antigens. The majority of B lymphoblastoid cell lines from macaques, which were seropositive for simian foamy virus, developed giant cells in culture. The cytopathic agent was identified as a foamy virus and was transmissible to human embryonal fibroblasts. Treatment of cell cultures with AZT abolished giant cell formation. B-lymphoblastoid cell line; Rhesus Epstein-Barr foamy virus; Syncytia formation; AZT
virus; Herpesvirus
papio; Simian
Introduction Transformed B lymphoblastoid cell lines (BLCL) have become important tools for immunological studies in human and non-human primates. Human BLCL, transformed with Epstein-Barr virus (EBV), are used widely for Correspondence to: G. Voss, Deutsches Kellnerweg 4, 3400 Giittingen, Germany.
Primatenzentrum,
Abteilung Virologle und Immunologie,
186
cytotoxicity assays. In particular, human immunodeliciency virus (HIV)specific cytotoxic T lymphocytes (CTL) were detected using BLCL infected with recombinant vaccinia viruses (for review see, Walker and Plata, 1990; Autran et al., 1991). Another important application for BLCL is the analysis of major histocompatibility antigens (MHC). In this experimental approach BLCL serve as a source for MHC antigens, which are radiolabeled and subsequently separated by isoelectric focusing (Yang et al., 1985; Neefjes et al., 1986). EBV B95-8 strain (Luka et al., 1978) is widely used for the transformation of human B lymphocytes. The method is well established and generates human BLCL (Sugden and Mark, 1977). Macaque BLCL for CTL-assays (Miller et al., 1990; Gotch et al., 1991) are obtained often by transformation with Herpesvirus papio (HVP) (Falk et al., 1976; Rabin et al., 1977). Although several other EBV-related herpesviruses were isolated from different species (Gerber et al., 1977; Rasheed et al., 1977; Neubauer et al., 1979; Backer et al., 1980; Heberling et al., 1981; Lapin et al, 1986; Rangan et al., 1986), these isolates are not used as often as HVP. In this study we intended to compare the transforming potential of different EBV-like simian viruses. In addition to HVP we used a second simian EBV-like Herpesvirus, rhesus Epstein-Barr virus (RhEBV) (Rangan et al., 1986), to transform macaque B lymphocytes. The resulting BLCL were compared and showed a B-cell phenotype. EBV-related antigens were detected in all BLCL. In some BLCL, originating from simian foamy virus (SFV)-seropositive macaques, giant cells containing SFV-antigens were observed. The infectious agent was transmitted to human embryonal fibroblasts by cocultivation. Giant cell formation was the main cause of cell death in outgrowing BLCL. To overcome this problem cultures were treated with 3’-azido-2’,3’-dideoxythymidine (AZT), which suppressed giant cell formation but shortened the lifetime of the BLCL cultures.
Materials and Methods Transforming
viruses
Two EBV-like simian viruses were used to transform macaque peripheral blood lymphocytes (PBLs). RhEBV was isolated from a lymphoma of a rhesus monkey (Macaca mulatta) (Rangan et al., 1986) and HVP originates from a lymphoma of a baboon (Papio hamadryas) (Falk et al., 1976; Rabin et al., 1977). LCL8664 cells (Rangan et al., 1986) were seeded at 2. lo5 per ml in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS) and maintained for 10 days at 37°C in a humidified 5% CO* atmosphere to produce infectious RhEBV for cell transformation. Thereafter, the cultures were incubated for another 10 days at 33°C. The culture supernatants were harvested, passed
187
through a 0.45 pm filter and aliquots were stored at -80°C. Infectious HVP was obtained by seeding 2.10’ cells per ml of the virusproducing S594 cell line (Falk et al., 1976; Rabin et al., 1977) in RPMI/lO% FCS and incubating the cultures subsequently for 10 days at 37°C in 5% CO*. The supernatant was treated as indicated for the LCL8664 cell line. Transformation
procedure
PBLs were isolated from fresh titrated blood of several rhesus monkeys of Indian or Chinese origin or cynomolgus monkeys kept at the DPZ. After separation on a Histopaque-1077 gradient (Sigma, Germany) the PBLs were seeded at a density of 2. IO6 cells per well in 1 ml RPM1 1640 supplemented with either 10% FCS or pooled human AB-serum and 1 lug per ml cyclosporin A (CsA) (Sandoz, Switzerland) in 12-well plates (Costar, USA). To transform these cultures, 1 ml of culture supernatant of LCL8664 or S594 cells was added. The culture medium was changed twice a week and the cell growth in the cultures was examined. Outgrowing cells were transferred to cell culture flasks and 2 weeks later, medium from all cultures was replaced stepwise by RPM1 1640 containing 10% FCS without CsA. Cocultivation
with HEL S37 cells
Human embryonal lung fibroblasts (HEL S37) were grown in Leighton tubes (Costar, USA) to a confluent monolayer. Subsequently the cells were cocultivated with 1 . lo5 transformed B-lymphoblastoid cells and examined for the expression of SFV antigens 48 h later. Detection of EBV-viral capsid antigen (VCA) antigen by indirect immunojluorescence
and simian foamy
virus (SFV)
Both, EBV-VCA and SFV antigens were detected by indirect immunofluorescence (Henle and Henle, 1966). 1 . lo6 suspension cells were removed from BLCL cultures and washed once in phosphate-buffered saline (PBS, 2 mM KH,PO,; 9 mM NazHP04; 180 mM NaCl, pH 7.3). Cells were resuspended in 100 ,ul PBS and aliquots of 10 ~1 were pipetted on to glass slides with 10 marked rings (Hiilzel, Germany). After 1 min at room temperature (RT), the cell suspension was removed and the slides were dried for 1 h at RT. Human embryonal tibroblasts were grown in Leighton tubes, cocultivated with BLCL and washed twice with PBS after 48 h. The suspension, as well as the monolayer cells, were fixed for 15 min at - 20°C in acetone and washed in PBS with 0.05% (v/v) Tween 20 (PBS-Tween). Sera were diluted 1:40 in PBS-Tween and incubated with the cells for 1 h at 37°C in a humidified atmosphere. After washing with PBS-Tween a FITC-labeled goat-anti-human IgG antiserum (Medac, Germany) diluted 1:40 was incubated with the cells for 45 min, as described above. The slides were washed once again and covered with 20% (v/
188
v) glycerol in PBS prior to examination with a fluorescence microscope (Zeiss, Germany). EBV-VCA was detected with human VCA-positive sera. SFV antigen was examined with positive reference macaque sera. Anti-complement
immunojluorescence
(ACIF)
EBV-nuclear antigen was detected according to the method described by Reedman and Klein (1973) with modifications developed by Henle et al. (1974). Briefly, cells were prepared and fixed on coverslips as described above. The cells were overlaid successively with the test specimen (inactivated antiEBNA positive human serum (1: 10 diluted); serum from guinea pig as source of complement (1:8); FITC-conjugated goat antibodies to guinea pig C3 (Nordic, NL) (1: 15)). All incubation steps were carried out for 1 h at 37°C. Following the last incubation step the coverslips were washed for 30 min in PBS containing 1% (w/v) Evans Blue and treated further, as described above. Screening of macaque sera for SFV antibodies and SFV reference sera Sera from rhesus or cynomolgus macaques were screened for the presence of antibodies against SFV by indirect immunofluorescence as described by Neumann-Haefelin et al. (1983). Briefly, Molt-4 cells infected with the SFV isolate LK-3 were suspended on teflon-coated glass slides, dried and then fixed for 15 min at -20°C in methanol. The samples were incubated first with monkey sera diluted 1:lO and subsequently with FITC-conjugated goat antihuman IgG antiserum diluted 1:20 (Medac, Germany). The specific fluorescence obtained with the monkey sera was compared to the fluorescence of SFV reference sera. Flow cytometry
analysis of cell surface antigens
Cell surface antigens were examined by flow cytometry analysis. 5. lo5 transformed cells from BLCL of rhesus or cynomolgus macaques were washed in phosphate-buffered saline (PBS) supplemented with 5% FCS and incubated in 40 ~1 PBS containing 5% FCS and 1 ~1 FITC-conjugated antibody for 1 h at 4°C. Monoclonal antibodies specific for Leu 5b (CD2), Leu 12 (CD19) and Leu 16 (CD20) were obtained from Becton-Dickinson, (USA) and goat-F(ab’)z anti-human IgG antiserum was purchase.d from Medac, Germany. Subsequently, the samples were washed with PBS/S% FCS and resuspended in 300 ~1 PBS containing 0.4% formaldehyde. At least 1 x lo4 cells were analyzed in each run using an EPICS profile flow cytometer (Coulter, Hialeah, USA).
189
Results Generation of BLCL
with RhEBV
or HVP
In total, 220 cultures of PBLs resulting in 94 growing BLCL from rhesus and cynomolgus monkeys, were initiated with supernatants of LCL8664 or S594 with different serum supplementation. Using RhEBV as transforming agent, 39 out of 83 initiated PBL cultures grew out to BLCL when supplemented with human AB-serum (Table 1). The first dividing cells were not visible before day 30 after the onset of the cultures. 55 transformed cell lines were established from 137 HVP-treated PBL cultures with human AB-serum supplementation (Table 1). The HVP-transformed cell lines grew out earlier, beginning from day 14. Successful transformation with RhEBV was achieved only when human AB-serum and CsA were supplemented, whereas HVP-transformed cells grew out also in FCS-containing medium without CsA (data not shown). Phenotypical
analysis of the transformed
BLCL
Thirteen RhEBV-transformed BLCL from rhesus or cynomolgus macaques and 2 HVP-transformed BLCL from cynomolgus monkeys were examined further for the expression of lymphocyte-specitic cell surface markers. Flow cytometry analysis showed that all cell lines expressed B-cell specific surface antigens in varying amounts. An average of 46.1% (6 20.1) of the cells were Leu12-positive. Leul2+ cells were observed in every cell line and the fractions of positive cells varied between 15.3 and 80.3%. Leul6+ (CD20) cells were detected in 3 of the 15 examined B-LCL and constituted 37.4% (6, 18.2) of the whole cells. The percentage of positive cells varied from 16.5 to 50.0%. Surface IgG was detected on 95.3% (6, 1.2) of the transformed cells. Each BLCL comprised an IgG-positive cell fraction constituting 94.3 to 96.3% of the whole cells. Only one BLCL exhibited a LeuSb+ (CD2) cell population (20.1%). Detection of EBV antigens VCA and EBNA
in the BLCL
In about 60% of the cells in the BLCL from both monkey species EBNA was induced by infection and transformation with EBV-like RhEBV or HVP (Fig. 1B). Two different fluorescence patterns were detected by ACIF. The majority TABLE 1 Survey of BLCL generated with RhEBV and HVP Transforming RhEBV HVP
virus
Total
MM=
MF=
39/83b 551137
14131 1l/45
25146 44192
a MM, Macaca mulatta; MF, Macaca fascicularis. b Number of successfully transformed/number of initiated cultures transformed
BLCL.
190
191
of cells showing fluorescence presented a granular nuclear staining. A small fraction had a brilliant nuclear fluorescence. These cells were larger than the other cells showing fluorescence and the negative cells (Fig. 1B). The examination of VCA-expressing cells from the BLCL transformed with RhEBV or HVP revealed that up to 35% of the cells expressed VCA (Fig. 1B). Giant cell formation in transformed BLCL Giant cells were observed in 39 BLCL from 25 animals, beginning from 4 weeks after the start of the cultures. Giant cells increased in 31 cultures, and the cells died within a month. The other cultures continued to grow, but still contained giant cells. Comparison to serological data of the macaques from which the transformed PBLs were derived revealed that all cultures with giant cells originated from monkeys seropositive for SFV. PBLs from 36 seropositive macaques were transformed and giant cells were found in cultures originating from 25 animals, whereas the BLCL from 11 seropositive monkeys did not demonstrate giant cell formation. The giant cells were examined for the presence of SFV antigens by indirect immunofluorescence to prove this correlation between giant cell formation and SFV-seropositivity. The giant cells showed a staining only with SFV-positive reference sera, whereas the cells with normal size showed frequent staining with SFV-positive and negative sera (Fig. IC). This staining was probably due to EBV-related antigens, since the SFV reference sera contained EBV-VCA antibodies (data not shown). In order to transmit the infectious agent, fetal human embryonal tibroblasts were cocultivated with BLCL positive or negative for SFV-induced giant cells. After 48 h, cultures cocultivated with SFV-positive BLCL exhibited giant multinucleated fibroblasts. These cells were stained with the SFV-positive control serum (Fig. 1D). The control cultures did not exhibit any cytopathic effect. These observations indicated that infectious SFV was present in the infected BLCL. In an attempt to suppress SFV replication in HVP-transformed BLCL showing giant cell formation, cultures were split and one sample each was maintained in medium containing 4 pmol per ml AZT. In five of the six cultures the giant cells disappeared (Table 2). One culture (from Mf760) was not affected by AZT treatment and still contained giant cells. Except the BLCL from Mm 1707, which could be maintained for a longer period with AZT supplementation, the other AZT-treated BLCL cultures died earlier than the untreated parallel samples (Table 2). + Fig. 1. EBV-related and SFV antigens detected by indirect immunofluorescence. BLCL of rhesus or cynomolgus macaques were examined for the expression of EBV-related antigens, using positive reference sera. Staining with a VCA-specific serum is shown in (A) and ACIF staining for EBNA in (B). Giant cellcontaming BLCL cultures (C) and syncytia of HEL S37 tibroblasts after cocultivatton with giant cellpositive BLCL (D) were examined for SFV antigen expression using a SFV reference serum.
192 TABLE 2 AZT treatment
of giant cell-positive
BLCL
Animal
Occurrence of giant cellsa
AZT treatmentb
Giant cellsC
Death of cellsd
MF723e
8
+ _
_ +
20 -
MF760
8
+ _
+ +
28 -
MF778
8
+ -
_ +
20 -
MF5357
8
+ _
_ +
-
MM1663e
8
+ -
_ +
20 -
MM1707
4
+ -
+
22 8
a Week after transformation when first giant cells were observed. b Cultures were not treated with AZT (-) or with 4 pm01 per mi AZT (+). ’ Occurrence of giant cells 4 weeks after initial AZT treatment. d Cell lines dies at the indicated week after transformation or could not be cultivated further. e MM, Macaca mulatta; MF, Macaca fascicularis.
Discussion We investigated the capacity of two EBV-like simian viruses to transform macaque B-cells. The outgrowing BLCL were characterized phenotypically by flow cytometry and showed predominantly B-cell characteristics. VCA, as well as EBNA, was detected in the transformed cells. Occurrence of giant cells in the BLCL was correlated to SFV-seropositivity of the animals from which the cell lines were derived. The infectious virus was transmitted successfully in vitro to human embryonal Iibroblasts. Giant cell formation was suppressed by addition of AZT to the cultures. The two viruses RhEBV and HVP did not display characteristic differences in the induction of the virus-specific antigens VCA and EBNA in the BLCL from cynomolgus or rhesus macaques. In contrast to observations of human EBV-transformed BLCL, when 100% of the infected cells were EBNA-positive (Fresen and zur Hausen, 1976) only up to 60% of the monkey BLCL expressed EBNA. This may be due either to a reduced expression of EBNA genes in the cells without fluorescence or may be related to an incomplete replication cycle of EBV in some of the cells. Furthermore it cannot be excluded that some cells were transformed without being infected (Klein et al., 1974). The fact that EBNA was only expressed in a fraction of all the cells indicates that the BLCL are of polyclonal origin.
193
Up to 35% of the cells expressed VCA after transformation. This indicates a high production of new virus particles. The level of virus production may vary, depending on the infected cell line, since Miller and Lipman (1973) have found considerable differences in the release of infectious virus from human cell lines transformed with EBV. The majority of BLCL from SFV-positive macaques exhibited giant cells beginning 4 weeks after initiation of the cultures. Many of these cultures could not be maintained for more than a month. This phenomenon was the main cause for the failure to establish permanently growing BLCL. The giant cells were shown to contain SFV antigen and infectious virus was transmitted to human embryonal fibroblasts. Thus, it is very likely that the cytopathic agent was SFV. Furthermore, SFV was isolated frequently from lymphoblastoid cells of several primate species (Feldman et al., 1975; Rabin et al., 1976; Barahona et al., 1976; Schnitzer, 1981; Neumann-Haefelin et al., 1983). Since it is difficult to obtain SFV-seronegative macaques we attempted to suppress giant cell formation with AZT. This drug was shown to inhibit HIV-I replication in vitro (Mitsuya et al., 1985) by selective interaction with the reverse transcriptase (Furman et al., 1986). We observed an inhibition of giant cell formation in the treated cultures. This observation suggests that AZT can also inhibit SFV replication. Nevertheless, the approach of treating BLCL cultures with AZT is not yet well established, since most of the treated cultures died earlier than untreated ones. This may be due to the development of AZTresistant SFV in the cultures or AZT is cytotoxic over a long culture period. Further evaluation of a more sophisticated cell culture technique using AZT may lead to an efficient method of suppressing SFV without a reduction in the life of the cultures.
Acknowledgements We would like to thank Dr. G. Nelke, Staatliches Kontrollinstitut fur immunbiologische Arzneimittel, Berlin, Germany, for providing us with HEL S37 cells and Dr. D. Neumann-Haefelin, Institut fur Virologie, Universitat Freiburg, for fixed Molt-4/LK-3 cells and SFV reference sera. CsA was kindly supplied by Sandoz, Basel, Switzerland. The excellent technical assistance of S. Aurisch and S. Noch is greatfully acknowledged. This manuscript contains parts of the doctoral thesis of G.V.
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