- Email: [email protected]
0.59) mutants
107, 488-496
VIROLOGY
Biology
(1980)
of Simian
VII. Induction
SATVIR
Virus 40 (SV40) Transplantation
of SV40 TrAg in Nonpermissive SV40 Deletion (0.54/0.59)
S. TEVETHIA,*,’
* Department University
Antigen
(TrAg)
Mouse Cells by Early Viable Mutants
DAVID C. FLYER,* MARY WILLIAM C. TOPP’r
J. TEVETHIA,*,’
AND
of Microbiology, and Specialized Cancer Research Center, The Penxsyloania State College qf’Medicine. Hershey, Pennsylvarlia 17053, and tCold Spring Harbor Laboratory, Cold Spring Harbor. New York 11724 Accepted
August
1, 1980
Early viable deletion (0.54/0.59) mutants were tested for their ability to induce SV40 transplantation antigen (TrAg) in infected or transformed nonpermissive mouse cells in an attempt to determine the requirement for small t antigen in the expression of TrAg at the cell surface. The results indicate that dl(O.5410.59) mutants are as efficient as wild-type SV40 in generating specific cytotoxic lymphocytes in C57Bl/6 mice and in immunizing BALBic mice against an SV40 tumor cell challenge. Mouse (C57BV6) cells transformed by these mutants were also susceptible to lysis by the specifically sensitized lymphocytes. It can, therefore, be concluded that the synthesis of small t antigen is not an absolute requirement for expression of SV40 TrAg in SV40-infected or -transformed nonpermissive cells. INTRODUCTION
Cells transformed by Simian Virus 40 (SV40) express a specific transplantation rejection antigen (TrAg), also known as tumor-specific transplantation antigen (TSTA) at the cell surface. TrAg is specified by the early region of the SV40 genome (Tevethia and Tevethia, 1976; Girardi and Defendi, 1970; Anderson et al., 19’77a; Tevethia and Tevethia, 1977) and is involved in the immunological rejection of SV40 tumors by the immunized host (Tevethia, 1980). The early region of SV40 codes for two nonvirion proteins of molecular weight 94,000 (94K) and 17,000 (17K), also known as large T and small t antigens, respectively (Tegtmeyer et al., 1975; Prives et al., 1978; Paucha et al., 1978; Crawford et al., 1978; Sleigh et aZ., 1978). Efforts to relate these two proteins to SV40 TrAg have indicated that the product of the A gene, the large T antigen, is involved in the induction of SV40specific transplantation immunity in viva ’ Author dressed. 2 Scholar
to whom
reprint
of Leukemia
requests
Society
be ad-
of America.
0042-6822/80/160488-09.$02.00/O Copyright All rights
should
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
488
against a tumor cell challenge. The presence of TrAg activity in SV40-transformed cells correlates with the level of SV40 large T antigen (Chang et al., 1977) and preparations rich in SV40 T antigen from SV40transformed cells possess TrAg activity (Anderson et al., 197713;Rogers et al., 1977). Large T antigen purified to homogeneity can immunize mice against tumor cell challenge (Changet al., 1979) and can induce the generation of sensitized lymphocytes in vivo (Tevethia et al., 1980b). In addition, the SV40 A gene is involved in the expression of functional TrAg in SV40-infected permissive cells (Tevethia and Tevethia, 1977). It thus seems to be clear that the large T antigen possesses TrAg activity. The nature of the target antigen at the membranes of cells transformed by SV40 which reacts with specifically sensitized lymphocytes, however, remains unknown. Also, neither the role of small t antigen in the induction of an immune response in vivo nor its requirement at the cell surface for reactivity with specific lymphocytes have been established. In this report we have tested several viable mutants of SV40 with deletions in the
SIMIAN
VIRUS
40 TRANSPLANTATION
early region (0.54/0.59) for their ability to induce SV40 transplantation immunity in viva and to express TrAg at the surface of infected and transformed cells. The results indicate that, although the participation of small t antigen either as immunogen or as target antigen can not be ruled out, the presence of large T antigen alone in infected and transformed cells is sufficient for the expression of functional TrAg. MATERIALS
AND
METHODS
Cells. Primary mouse embryo fibroblast cultures (MEF) were derived from 13-to 15day-old embryos of C5’7BV6 mice as described previously (Pretell et al., 1979). BlGIPY is a transplantable cell line established from a tumor induced in newborn C57B116 mice by polyoma virus (Habel and Silverberg, 1960) and is susceptible to infection by SV40 (Pretell et al., 1979). B16/WT-3 is an SV40-transformed cell line derived from C57B116 MEF after infection with WT SV40 (Tevethia et al., 1980a). KCA is an adenovirus-5 transformed C57B116 kidney cell line (Knowles et al., 1979). CV-1 and TC-7 cells are continuous cell lines derived from African green monkey kidney cell cultures and were used to propagate WT SV40 and SV40 viable deletion mutants. All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 units/ml of penicillin, 100 pg/ml of streptomycin, 25 pg/ml of kanamycin, 0.03% glutamine, 0.075% NaHCO,, and 5% fetal calf serum (FCS). mKSA-ASC is an SV40-transformed cell line of BALBic origin (Kitet al., 1969) which grows as an ascites tumor in uivo (Rogers et al., 1977) and was passaged once weekly intraperitoneally in syngeneic mice. Viruses. SV40 (VA 45-54) was obtained from Dr. P. Tegtmeyer and was propagated in TC-7 cells as described previously (M. Tevethia et al., 1974). The viable deletion mutants used in this study have been described (Shenk et al., 1976; Sleigh et al., 1978). Transformation of C5?‘B1/6 MEF with WT SV40 and SV40 dl mutants. Actively
growing C57Bl/6 MEF cells at passage 4 were seeded at a density of 2 x lo4 per 25cm2 plastic flasks in DMEM containing 10%
ANTIGEN
INDUCTION
489
fetal bovine serum. After overnight incubation at 37” the medium was removed and virus was added to give a multiplicity of infection (m.o.i.> of 200. Control flasks received Tris-buffered saline (TBS) instead of virus. At the end of a 3-hr adsorption period at 37”, fresh medium was added and the incubation was continued for 3 weeks with weekly medium changes. Under these conditions normal C57Bl/6 MEF die and detach from the surface of the flask. In all virusinfected flasks between 10 and 50 dense clones of transformed cells developed. The cells in each flask were dispersed by treatment with trypsin and propagated in order to establish transformed cell lines. [“?S]Methionine labeling and extraction of SV40 WT and dl mutant-infected and -transformed cells. Confluent 75-cm’ flasks
of TC-7 cells grown in DMEM with 5% FCS were infected with SV40 WT and dl mutant virus at a m.o.i. of 5 PFUicell. Virus was allowed to adsorb for 90 min after which the flasks were refed with 15 ml DMEM containing 2% FCS. After 48 hr, infected TC-7 cells along with subconfluent monolayers of SV40 WT and dl mutant-transformed C57BV6 MEF were washed three times in TBS and incubated for 1 hr at 37” in methionine-free modified Eagle’s medium (MEM). The medium was removed and the cells radiolabeled for 1 hr at 37” by incubation in 3 ml of methionine-free MEM containing 50 &i/ml [?S]methionine (New England Nuclear, Boston, Mass). After. radiolabeling, each culture was washed three times in TBS and extraction of the cells was accomplished by adding 1 ml of extraction buffer containing 0.5%~ NP40, 0.1 M NaCl, 0.05 M TrisHCl, pH 8.0, 330 Fgiml phenylmethylsulfanyl fluoride, and 1% aprotinin. After incubation at 4” for 20 min, the extracts were centrifuged at 20,000 g and the supernatants used for immunoprecipitation of proteins. Zmmujnoprecipitation of SV4.0 turnor antigens from 3”S-labeled SV40 WT and dl mutant-infected and -transformed cell extracts by anti-T sera. ?S-Labeled SV40 WT
and dl mutant-infected and -transformed cell extracts were first reacted with 50 ~1 of normal hamster sera per milliliter of extract for 16 hr at 4”. Two hundred microliters of 10% (w/v> Staphylococcus aureus (Enzyme
490
TEVETHIA
Center, Boston, Mass.) was added per milliliter of extract and kept in suspension for 10 min at room temperature by using a rocking aparatus. The suspension was centrifuged at 1100 g for 10 min and the supernatant was divided into 600-~1 aliquots. Tumor antigens were immunoprecipitated by the addition of 20 ~1 of anti-T sera from hamsters bearing tumors induced by virus-free SV40-transformed hamster cells for 90 min at 4”. Staphylococcus aureus (200 ~1) was added, kept in suspension for 10 min at room temperature, and pelleted by centrifugation. The pellets were washed three times with TBS containing 1% NP-40 and 0.5 M LiCl. Immune complexes were eluted by resuspending the bacteria in 50 ~1 of SDSsample buffer which contained 1% SDS and 1% mercaptoethanol and heating for 3 min at 100”. The bacteria were pelleted and the supernatants were analyzed by SDS-polyacrylamide gel electrophoresis. SDS -polyacrylamide gel electrophoresis (SDS-PAGE). The immunoprecipitates
(2 x lo4 cpmwell) were analyzed by SDSPAGE on lo-15% acrylamide gradients using the Laemmli (19’70) buffer system. Gels were fixed for 1 hr in 5% trichloroacetic acid, prepared for fluorography (Bonner and Laskey, 1974), and exposed on Kodak X-Omat XR-5 film for 2-5 days at -76”. Generation
of
cytotoxic
lymphocytes.
Cytotoxic lymphocytes (CTL) capable of specifically killing syngeneic cells transformed or infected with WT SV40 were generated by immunizing C57Bl/6 mice (Jackson Laboratories, Bar Harbor, Maine) in the hind foot pad according to a procedure previously described (Knowles et al., 1979; Tevethia et al., 1980a). Mice were immunized with 2 x lo7 B16/WT-3 cells or with l-3 x lo7 plaque-forming units (PFU) of WT SV40 or dl mutants. The draining lymph nodes were excised 7 days postimmunization and lymphocyte suspensions were prepared by gently pressing the lymph nodes through a 60-gauge stainless-steel wire mesh. Viable cells were counted by trypan blue exclusion and resuspended at 4 x lo6 lymphocytes/ml
ET AL.
of RPM1 1640 media supplemented with 5 x lo-” M 2-mercaptoethanol, 20 mM HEPES buffer, 100 units/ml of penicillin, 100 pg/ml of streptomycin, 0.03% glutamine, 0.225% NaHCO,, and 10% heat-inactivated FCS. Lymphocytes (2 x 107) were incubated in 60-mm plastic tissue culture dishes at 37” in 5% CO, for 3 days to allow the sensitized lymphocytes to differentiate into CTL. Control lymphocytes from unimmunized mice were prepared similarly. 51Crrelease assay. Nonadherent lymphocytes were removed from tissue culture dishes by gentle pipetting and were washed once in RPM1 1640 medium. Subconfluent monolayers of target cells in 75-cm2 tissue culture flasks were labeled with 200-300 PCi of 51Cr (New England Nuclear Corp.) in DMEM with 5% FCS as described previously (Pretell et al., 1979; Tevethia et al., 1980a). At the time of testing, the labeled target cells were brought into a single cell suspension with 0.1% trypsin and washed three additional times with DMEM. Viable cells (2 x 10”) in 0.1 ml were added to 10 x 75mm glass culture tubes followed by an equal volume of effector lymphocytes in varying concentrations to provide effector: target cell ratios in the range of 4O:l and 1O:l. Culture tubes containing mixtures of j’Cr-labeled target cells and lymphocytes were centrifuged at 60 g for 5 min to increase their contact and then were incubated at 37 in 5% CO, for 8-10 hr. At the end of the incubation period, 0.8 ml of RPM1 1640 medium was added to each culture tube. The tubes were centrifuged at 250 g to pellet the cells and one-half of the supernatant fluid (0.5 ml) was withdrawn. Both the supernatant aliquot and the pellet containing the remainder of the supernatant and the cells were counted in a Beckman gamma counter. Spontaneous release of “‘Cr from the target cells was determined by incubating the labeled target cells alone and the maximum release of 51Cr was determined by lysing the target cells with 5% SDS. The percentage specific “‘Cr release was determined using the following formula:
% release (immune % specific j’Cr release =
lymphocytes) - % release (normal lymphocytes) % release (maximum) - % release (spontaneous)
’
SIMIAN
W T “7”
2001
‘y42005
VIRUS
40 TRANSPLANTATION
“i
FIG. 1. DNA of SV40 WT (776) and dl (0.5410.59) mutants cleaved with restriction endonuclease Hinf. Three micrograms of DNA of each virus were incubated for 2 hr at 37” with three units of Hinf and then electrophoresed overnight through a 4% polyacrylamide (2O:l Bis) gel cast in Tris-acetate buffer. The gel was incubated in ethidium bromide (10 pgiml) for 1 hr and photographed on Tri-X film under long-wave uv illumination.
ANTIGEN
INDUCTION
491
data in Fig. 1 show that each deletion is localized to the H&f-D fragment. These data are consistent with those reported earlier (Sleigh et al., 1978; Shenk et al., 1976). The early viable deletion mutants were also characterized with respect to their ability to induce tumor antigens in infected cells. Proteins were extracted for dl mutant-infected cells as described under Materials and Methods, immunoprecipitated with anti-T sera, and analyzed by SDS-PAGE. The results in Fig. 2 show that while the WT SV40 induced the synthesis of both large T and small t antigens, only large T antigen could be detected in monkey cells infected by dl884, dl890, dl2001, and dl2005 under the conditions used. Using different labeling conditions, the synthesis of a shortened small t polypeptide has been observed in monkey cells infected with dl884, 890, and 2001 but not dl 2005 (Sleigh et al., 19’78; Khoury et al., 1979).
--wt
NTNTNTNTNT
---
Induction of SV40-speci$c transplantation immunity. Adult BALB/c mice (kindly
provided by the Mammalian Genetics Branch, NCI; Frederick, Md.) were immunized with 1 x lo7 PFU of either WT SV40 or dl mutants intraperitoneally. Ten days later, mice were challenged with 1 x lo4 mKSAASC cells by the subcutaneous route. The animals were observed for tumor development. RESULTS
Characterization Mutants
of SV4.0 dl(0.5410.59)
The early viable deletion mutants were characterized for the size of deletion and the proteins they induced during permissive infection prior to testing for their ability to induce TrAg in vivo or in vitro in the nonpermissive host in order to insure that the virus stocks used were free of contaminating wild-type virus. DNA from WT SV40, dl 884, dl890, dl2001, and dl2005 was cleaved with the restriction enzyme Hinf and the fragments generated were separated by polyacrylamide gel electrophoresis as described previously (Sleigh et al., 1978). The
te FIG. 2. Synthesis of SV40 early proteins in SV40 wild-typeand dl mutant (0.54/0.59)-infected cells. TC7 cells were infected with either SV40 wild-type or dl mutant virus at an m.o.i. of 5 at 37”. Forty-eight hours postinfection, cells were starved for 1 hr in methioninefree media then radiolabeled with 150 &i of [YSmethionine in 3 ml of mediaicm* flask for 1 hr. Cell extracts were prepared with 0.5% NP40 buffer, pH 8.0, and immunoprecipitated with either normal hamster sera (N) or hamster anti-T sera (T). Immunoprecipitates were electrophoresed on lo- 15% gradient polya&amide gels as described under Materials and Methods.
492
TEVETHIA
Induction of SV40 Transplantation Im,munity in BALBIc Mice with SV40 dl Mutants
The mutants dl 884, dl 890, dl 2001, and dl 2005 were tested for their ability to immunize BALBic mice against a challenge of syngeneic SV40-transformed cells. The results in Table 1 show that all four dl mutants were able to immunize BALB/c against tumor cell challenge. The degree of immunity was comparable to that induced by WT SV40. These results indicate that a virus which induces and accumulates large T antigen alone (dl2005) is able to immunize mice against a tumor cell challenge, indicating that the synthesis of small t antigen is not necessary for immunization. Induction of Cytotoxic SV40 dl Mutants
Lymphocytes
by
Immunization of C57BV6 mice (responder strain) with SV40 results in the induction of CTL capable of killing syngeneic SV40 transformed cells in ,uitro (Knowles et al., 1979, Pretell et al., 1979). Since thymusderived lymphocytes are also involved in the rejection of SV40 tumors in mice (S. Tevethia et al., 1974) an experiment was performed to determine whether dl mutants would induce CTL in C57B116 mice. The results are shown in Table 2. Lymphocytes from C57Bl/6 mice immunized with WT SV40 specifically killed only the SV40transformed cells (B16/WT-3) and not cells transformed either by polyoma virus (B16/ PY) or by adenovirus-5 (KCA). Similarly, lymphocytes from mice immunized with dl 884, dl 2001, and dl 2005 were specifically cytotoxic only against B16/WT-3 cells. These results establish that early viable deletion mutants are capable of inducing CTL which specifically kill only the SV40transformed syngeneic cells. Expression qf TrAg at the Surface dl Mutant-Infected Cells
of SV40
The results presented thus far indicate that large T antigen alone is capable of inducing specific transplantation immunity in ,uivo and also induces the generation of CTL as the dl mutants used induce the synthesis
ET AL. TABLE
1
IMMUNIZATION OF BALB/c MICE BY SV40 dl (0.54/0.59) MUTANTS AGAINST TUMOR CELL (mKSA) CHALLENGE Mice immunized with” SV40 SV40 SV40 SV40 SV40 None
WT dl 884 dl890 dl2001 dl 2005
Tumor incidence” by mKSA cells: 2 x 10”
1 x IO’
O/5 015 O/5 015 O/5 515
015 015 O/5 O/5 O/5 616
(’ Adult BALBic mice were immunized twice at weekly interval with 1 x 10’ PFU of WT SV40 or SV40 dl mutants. Immunized and nonimmunized mice were challenged with mKSA cells subcutaneously. The animals were followed for tumor development. b Number of mice with tumors/number of mice inoculated.
of large T antigen only. The results do not, however, indicate whether dl mutant-infected cells synthesize TrAg at the cell surface. To demonstrate this point, BlG/PY cells were infected with dl mutants and used as target cells in the cytotoxicity assay with lymphocytes generated against virus-free B16/WT-3 cells. The results in Fig. 3 show that BlG/PY cells infected with WT SV40 were specifically lysed by lymphocytes generated against B16/WT-3 cells. The specific “‘Cr release from WT-infected cells was approximately 18% as compared to 28% 3’Cr release from B16/WT-3 cells. The specific siCr release from dl mutant (dl884, dl 890, dl 2001, dl 2005)-infected cells was lower than that of WT SV40-infected cells. These results indicate that SV40 TrAg is expressed at the surface of dl mutant-infected cells since cellular immune reactions leading to cell death take place at the membrane level (Cerottini and Brunner, 1974). Expression of TrAg at the Surface of SV40 dl Mutant-Transformed Cells
C57Bl/6 MEF cells were transformed with either mutants dl884 or dl2005 or with WT SV40. All transformants were 100%
SIMIAN
VIRUS 40 TRANSPLANTATION TABLE
GENERATION
OF LYMPHOCYTES
ANTIGEN
INDUCTION
493
2
CYTOTOXIC TO SV40-TRANSFORMED BY SV40 dl (0.5410.59) MUTANTS
CELLS
IN C57Bll6
MICE
Target cells Bl6/WT-3 Lymph node effector cells” SV40 SV40 SV40 SV40
Percentage “‘Cr release
WT immunized d1884 immunized dl 2001 immunized dl 2005 immunized
79 76 74 79
Nonimmunized None (spontaneous) None (maximum)
BIGIPY
Percentage specific “‘Cr releaseb
KCA
Percentage “I&. release
Percentage specific “lCr releaseb
Percentage “‘Cr release
26 26 26 31
3 3 3 9
20 20 21 23
75 ‘71 69 75
21 23 100
24 23 98
Percentage specific 51Cr release” 0 0 2 4 20 18 100
(I Adult C57BV6 mice were immunized with 2 x 10’ PFU of WT SV40 or dl mutants by inoculation in the hind footpad. Seven days later lymphocytes from immunized and nonimmunized mice were cultured in vitro for 3 days and reacted with “‘Cr-labeled B16/WT-3 cells (SV40 transformed), B16IPY cells (polyoma virus transformed), and KCA (adenovirus-5 transformed) as described under Materials and Methods. * Lymphocyte/target cell ratio of 10~1.
positive for SV4Q T antigen by the immunofluorescence test. The absence of 17K protein was confirmed in one line each of dl2005 and dl884-transformed C57BU6 cells by the immunoprecipitation test using anti-T sera. The results in Fig. 4 show that while WT SVBO-transformed cells contain the 17K protein (small t antigen), it is not present in detectable quantities in the dl mutantBIG/PY
Cells
Infected
by:
sv40
percent 0
transformed cells. The large T antigen was present in WT SV40- and dl mutant-transformed cells. Two WT SV40 and three dl 884 and dl 2005 transformed cell lines were used as targets in the lymphocyte-mediated cytotoxicity assay to determine if these cells are susceptible to lysis by lymphocytes sensitized to SV40 TrAg. The results in Table 3 Speclflc
10 I
51 Cr
Release 20 1
30 I
wt
SV 40
dl884
SV40
dl690
SV40
dl2001
SV 40
dI2005
616/Wt-3 umnfecfed
m
FIG. 3. Expression of SV4OTrAg at the surface of dl mutant (0.54/0.59)-infected cells. Polyoma-transformed mouse cells (BlG/Py) were infected with either SV40 wild-type or dl mutant at an m.o.i. of 50 and used as targets in a “‘Cr release assay 72 hr after infection as described under Materials and Methods. SV40-transformed cells (B16/WT-3) were also used as targets. Target cells were incubated with syngenie lymphocytes from mice sensitized to SV40 TrAg at an effector:target cell ratio of approximately 4O:l.
494
TEVETHIA
demonstrate that dl mutant-transformed cells which synthesize only the large T antigen can act as a target for lymphocytes generated in response to immunization of syngeneic mice with SV40. The specific “‘Cr release from dl mutant-transformed cells was comparable to the “‘Cr release from WT-transformed cells. Cells transformed by adenovirus-5 (KCA) or polyoma virus (BIGIPY) were not susceptible to lysis by lymphocytes sensitized to SV40 TrAg. DISCUSSION
The findings of this study show that cells transformed by SV40 early viable deletion (0.54/0.59) mutants which synthesize large T antigen alone can express SV40-specific 616
B16
wt 2005 NTNTNT
B16 884
t-
FIG. 4. Synthesis of SV40 early proteins in SV40 wild-typeand dl mutant (0.54/0.59)-transformed cells. C57B116 mouse embryo fibroblasts transformed by either wild-type SV40 (BlGIWT cells), dl 2005 (B16/ 2005 cells), or dl 884 (B16/884 cells) were starved for methionine for 1 hr in methionine-free media then radiolabeled with 150 &i of [35S]methionine in 3 ml of mediaR&m* flask for 1 hr. Cell extracts were prepared in 0.5% NP40 buffer, pH 8.0, and immunoprecip itated with either normal hamster sera (N) or hamster anti-T sera (T). Immunoprecipitates were electrophoresed on IO- 15% gradient polyacrylamide gels as described under Materials and Methods.
ET AL. TABLE
Y
EXPRESSION OF SV40 TrAg AT THE SURFACE OF SV40 dl (0.5410.59) MUTANT-TRANSFORMED CELI.S Lymphocyte donor immunized with”
Target cella
Transformed by
sv40 SV4O
BlG/WT-17 BIUWT-18
SV40 SV40
WT WT
74 HO
sv40 sv40 sv40
Bl6/884-1 B16/884-2 Bl6/884-3
sv40 SV40 SV40
dl 884 dl 884 dl 884
48 82 78
sv40 sv40 sv40
B16/2005-1 B16i2005-2 B16i2005-3
SV40 SV40 SV40
dl2005 dl2005 dl2005
79 80 80
SV4O sv40
KCA BlGIPY
AdenoviruG Polyoma virus
6 9
a Adult C57BV6 mice were immunized with 2 x IO’ PFU of SV40 via hind footpad inoculation. Seven days later draining lymph nodes were excised and the lymphocytes cultured in vitro for 3 days prior to use in the “Cr release assay against the indicated target cells. li Lymphocywtarget cell ratios of 4O:l.
transplantation antigen at the cell surface. It appears that the synthesis of small t antigen is not an absolute requirement for the expression of immunoreactive TrAg. These results support earlier findings that purified large T antigen can immunize syngeneic mice against SV40 tumor cells and can induce a cell-mediated immune response in the inoculated host (Chang et nl., 1979; Tevethia et al., 1980b). The studies carried out so far, however, have not ruled out the participation of small t antigen either as an immunogen or as a target antigen at the cell surface. Studies with nondefective adenoSV40 hybrid viruses indicate that TrAg sites which are involved in the sensitization of host against tumor cell challenge are located in the carboxy region of T antigen (Jay et al., 1978; 1979; Lewis and Rowe, 1973). These studies, however, have not excluded the existence of TrAg sited on the amino terminus of T antigen. Both large T and small t antigens share amino acid sequences at the amino terminus and also contain common antigenic sites in this region as demonstrated by the ability of antibody to denatured large T antigen to immunoprecipitate small t antigen (Crawford et aZ., 1980; Lane
SIMIAN
VIRUS 40 TRANSPLANTATION
and Robbins, 1978; Greenfield et al., 1980). It is quite possible that these common antigeneic sites shared between the large T and small t proteins may also be involved in the induction of cellular immune response of the host against tumor cell challenge. In addition, antigenic sites specified by amino acids in the unique carboxy terminal portion of small t antigen (Greenfield et al., 1980) may be involved in the cell-mediated immune reactions. By using mouse cells infected or transformed by viable deletion (0.54.10.59) mutants which induce the synthesis of large T antigen only, we hoped to define the role of small t antigen in the expression of TrAg at the cell surface by determining whether differences in the susceptibility of cell-mediated lysis of WT or dl mutant-infect.ed or -transformed cells could be demonstrated. Although mouse cells infected by dl mutants were not as susceptible to lymphocytemediated cytolysis as the WT-infected cells, this difference was not noticed when WT and dl mutant-transformed cells were used as target, cells. The lower release of “‘Cr from dl mutant-infected cells upon interaction with lymphocytes sensitized to SV40 TrAg could be explained by variation in the efficiency of infection of nonpermissive cells by dl mutants. The question regarding the nature of TrAg at the surface of SV40-transformed cells and its relationship to SV40-coded proteins still requires clarification, although the available evidence strongly suggests that large T antigen possesses TrAg sites which are immunogenic in the syngeneic host. Whether small t antigen also possesses TrAg sites can most convincingly be demonstrated by using purified small t antigen as immunogen. ACKNOWLEDGMENTS The studies reported here were supported by Research Grant CA 25000, CA 24694. CA 18450, CA 13106, and CA 24803 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. The excellent technical assistance of C. Paul is gratefully acknowledged. REFERENCES ANDERSON, J. L., MARTIN, R. G., CHANG, C., and MORA, P. T. (1977a). Tumor specific transplantation
ANTIGEN
INDUCTION
495
antigen is expressed during SV40 lytic infection with wild-type and tsA mutant viruses. Virology 76, 254-262. ANDERSON, J. L., MARTIN, R. G. CHANG, C., MORA, P. T., and LIVINGSTON, D. M. (1977b). Nuclear preparations of SV40 transformed cells containing tumor specific transplantation rejection activity. Virology 76, 420-425. BONNER. W. M., and LASKEY, R. A. (1974). A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biothem. 46,84-88. CEROTTINI, J. C., and BRUNNER, K. T. (1974). Cell mediated cytotoxicity: Allograft rejection and tumor immunity. Advan. I?nmur~o/. 18, 67-132. CHANG, C., ANDERSON, J. L.. MARTIN, R. G., and MORA, P. T. (1977). Expression of tumor-specific transplantation antigen in cell lines transformed by wild-type or tsA mutant simian virus 40. J. Viral. 22,281.-289. CHANG, C., MARTIN, R. G., LIVINGSTON, D. M., LUBORSKY, S. W., Hu, C., and MORA, P. T. (1979). Relationship between T-antigen and tumor specific transplantation antigen in simian virus 40 transformed cells. J. Viral. 29, 69-75. CRAWFORD, L. V., COLE, C. N., SMITH, A. E., PAUCH& E., TEGTMEYER, P., RUNDELL, K., and BERG, P. (1978). Organization and expression of early genes of simian virus 40. Proc. Nat. Acad. Sci. USA 75, 117-121. CRAWFORD, L. V., PIM, D. C., and LANE, D. P. (1980). An immunochemical investigation of SV40 T-antigens. 2. Quantitation of antigen and antibody activities. Virology 100, 314-325. GREENFIELD, R. S., FLYER, D. C., and TEVETHIA, S. S. (1980). Demonstration of unique and common antigenic sites located on the SV40 large T and small t antigens. Virology 104, 312-322. GIRARDI, A. J., and DEFENDI, V. (1970). Induction of SV40 transplantation antigen (TrAg) during the lytic cycle. Virology 42, 688-698. HABEL, K., and SILVERBERG, R. J. (1960). Relationship of polyoma virus and tumor in viva. Vimlogy 12 463-476. JAY,‘G., JAY, F. T., CHANG, C., FRIEDMAN, R. M., and LEXINE, A. S. (1978). Tumor specific transplantation: Use of the Ad2+ND,, hybrid virus to identify the protein responsible for simian virus 40 tumor rejection and its genetic origin. Proc. Naf. Acad. Sci.
USA
75, 3055-3059.
JAY, G., JAY, F. T., CHANG, C., LEVINE, A. S., and FRIEDMAN, R. M. (1979). Induction of simian virus 40.specific tumor rejection by the AD2+ND, hybrid virus. J. Gen. Viral. 44, 287-296. KIT, S., KURIMURA, T., and DUBBS, D. R. (1969). Transplantable mouse tumor line induced by injection of SV40-transformed mouse kidney cells. Znt. J. Center 4,384-392.
TEVETHIA
496
KNOWLES, B. B., KONCAR, M., PFIZENMAIER, K., SOLTER, D., ADEN, D. P., and TRINCHIERI, G. (1979). Genetic control of the cytotoxic T cell response to SV40 tumor-associated specific antigen. J. Immunol. 122, 17981806. KHOURY, G., GRUSS. P., DHAR, R., and LAI, C. J. (1979). Processing and expression of early SV40 mRNA: A role for RNA conformation in splicing. Cell
18, 85-92.
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LANE, D. P., and ROBBINS, A. K. (1978). An immunochemical investigation of SV40 T antigens. 1. Production, properties and specificity of a rabbit antibody to purified simian virus 40 large T antigen. Virology 87, 182-193. LEWIS, A. M., JR., and ROWE, W. P. (1973). Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. VIII. Association of simian virus 40 transplantation antigen with a specific region of the early viral genome. J. Viral. 12, 836-840. PAUCHA, E., HARVEY, R., and SMITH, A. E. (1978). Cell-free synthesis of simian virus 40 T antigens. J. Viral. 28, 154-170. PRETELL, J., GREENFIELD, R. S., and TEVETHIA, S. S. (1979). Biology of simian virus 40 (SV40) transplantation antigen (TrAg). V. In vitro demonstration of SV40 TrAg in SV40 infected nonpermissive mouse cells by the lymphocyte mediated cytotoxicity assay. Virology 97, 32-41. PRIVES, C., GLUZMAN, Y., and WINOCUR, E. (1978). Cellular and cell-free synthesis of simian virus 40 T antigens in permissive and transformed cells. J. Viral.
25, 587-595.
ROGERS, M. J., LAW, L. W., and APPELLA, E. (1977). Subcellular distribution of the tumor specific transplantation antigen of SV40 transformed cells. J. Nat. Cancer Inst. 59, 1291-1295. SHENK, T. E., CARBON, J., and BERG, P. (1976). Construction and analysis of viable deletion mutants of simian virus 40. J. Viral. 18, 664-671.
ET AL.
SLEIGH, M. J., TOPP, W. C., HLJNICH, R., and SAMBROOK, J. F. (1978). Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell 14, 79-88. TEGTMEYER. P., SCHWARTZ, M., COLLINS, J. K., and RUNDELL, K. (1975). Regulation of tumor antigen synthesis by simian virus 40 gene A. J. Viral. 16, 168-178.
TEVETHIA, M. J., and TEVETHIA, S. S. (1976). Biology of SV40 transplantation antigen (TrAg). I. Demonstration of SV40 TrAg on glutaraldehyde fixed SV40infected African green monkey kidney cells. Viralogy 69, 474-489.
TEVETHIA, M. J., and TEVETHIA, S. S. (1977). Biology of simian virus 40 (SV40) transplantation antigen (TrAg). III. Involvement of SV40 gene A in the expression of TrAg in permissive cells. Virology 81, 212-223.
TEVETHIA, M. J., RIPPER, L. W., ~~~TEVETHIA, S. S. (1974). A simple qualitative spot complementation test for temperature sensitive mutants of SV40. Intervirology
3, 245-255.
TEVETHIA, S. S. (1980). Immunology of simian virus 40. In “Virus Oncology” (G. Klein, ed.), pp. 581-601. Raven Press, New York, TEVETHIA, S. S., BLASECKI, J. W., WANECK, G., and GOLDSTEIN, A. (1974). Requirement of thymus-derived 0 positive lymphocytes for rejection of DNA virus (SV40) tumors in mice. J. Zmmunol. 113,14171423. TEVETHIA, S. S., GREENFIELD, R. S., FLYER, D., and TEVETHIA, M. J. (1980a). Simian virus 40 transplantation rejection antigen: Relationship to SV40 specific proteins. Cold Spring Harbor Symp. 44, 235242. TEVETHIA, S. S., FLYER, D. C., and TJIAN, R. (1980b). Biology of simian virus 40 (SV40) transplantation antigen (TrAg) VI. Mechanism of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein). Virology 107, 13-23.
VIROLOGY
Biology
(1980)
of Simian
VII. Induction
SATVIR
Virus 40 (SV40) Transplantation
of SV40 TrAg in Nonpermissive SV40 Deletion (0.54/0.59)
S. TEVETHIA,*,’
* Department University
Antigen
(TrAg)
Mouse Cells by Early Viable Mutants
DAVID C. FLYER,* MARY WILLIAM C. TOPP’r
J. TEVETHIA,*,’
AND
of Microbiology, and Specialized Cancer Research Center, The Penxsyloania State College qf’Medicine. Hershey, Pennsylvarlia 17053, and tCold Spring Harbor Laboratory, Cold Spring Harbor. New York 11724 Accepted
August
1, 1980
Early viable deletion (0.54/0.59) mutants were tested for their ability to induce SV40 transplantation antigen (TrAg) in infected or transformed nonpermissive mouse cells in an attempt to determine the requirement for small t antigen in the expression of TrAg at the cell surface. The results indicate that dl(O.5410.59) mutants are as efficient as wild-type SV40 in generating specific cytotoxic lymphocytes in C57Bl/6 mice and in immunizing BALBic mice against an SV40 tumor cell challenge. Mouse (C57BV6) cells transformed by these mutants were also susceptible to lysis by the specifically sensitized lymphocytes. It can, therefore, be concluded that the synthesis of small t antigen is not an absolute requirement for expression of SV40 TrAg in SV40-infected or -transformed nonpermissive cells. INTRODUCTION
Cells transformed by Simian Virus 40 (SV40) express a specific transplantation rejection antigen (TrAg), also known as tumor-specific transplantation antigen (TSTA) at the cell surface. TrAg is specified by the early region of the SV40 genome (Tevethia and Tevethia, 1976; Girardi and Defendi, 1970; Anderson et al., 19’77a; Tevethia and Tevethia, 1977) and is involved in the immunological rejection of SV40 tumors by the immunized host (Tevethia, 1980). The early region of SV40 codes for two nonvirion proteins of molecular weight 94,000 (94K) and 17,000 (17K), also known as large T and small t antigens, respectively (Tegtmeyer et al., 1975; Prives et al., 1978; Paucha et al., 1978; Crawford et al., 1978; Sleigh et aZ., 1978). Efforts to relate these two proteins to SV40 TrAg have indicated that the product of the A gene, the large T antigen, is involved in the induction of SV40specific transplantation immunity in viva ’ Author dressed. 2 Scholar
to whom
reprint
of Leukemia
requests
Society
be ad-
of America.
0042-6822/80/160488-09.$02.00/O Copyright All rights
should
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
488
against a tumor cell challenge. The presence of TrAg activity in SV40-transformed cells correlates with the level of SV40 large T antigen (Chang et al., 1977) and preparations rich in SV40 T antigen from SV40transformed cells possess TrAg activity (Anderson et al., 197713;Rogers et al., 1977). Large T antigen purified to homogeneity can immunize mice against tumor cell challenge (Changet al., 1979) and can induce the generation of sensitized lymphocytes in vivo (Tevethia et al., 1980b). In addition, the SV40 A gene is involved in the expression of functional TrAg in SV40-infected permissive cells (Tevethia and Tevethia, 1977). It thus seems to be clear that the large T antigen possesses TrAg activity. The nature of the target antigen at the membranes of cells transformed by SV40 which reacts with specifically sensitized lymphocytes, however, remains unknown. Also, neither the role of small t antigen in the induction of an immune response in vivo nor its requirement at the cell surface for reactivity with specific lymphocytes have been established. In this report we have tested several viable mutants of SV40 with deletions in the
SIMIAN
VIRUS
40 TRANSPLANTATION
early region (0.54/0.59) for their ability to induce SV40 transplantation immunity in viva and to express TrAg at the surface of infected and transformed cells. The results indicate that, although the participation of small t antigen either as immunogen or as target antigen can not be ruled out, the presence of large T antigen alone in infected and transformed cells is sufficient for the expression of functional TrAg. MATERIALS
AND
METHODS
Cells. Primary mouse embryo fibroblast cultures (MEF) were derived from 13-to 15day-old embryos of C5’7BV6 mice as described previously (Pretell et al., 1979). BlGIPY is a transplantable cell line established from a tumor induced in newborn C57B116 mice by polyoma virus (Habel and Silverberg, 1960) and is susceptible to infection by SV40 (Pretell et al., 1979). B16/WT-3 is an SV40-transformed cell line derived from C57B116 MEF after infection with WT SV40 (Tevethia et al., 1980a). KCA is an adenovirus-5 transformed C57B116 kidney cell line (Knowles et al., 1979). CV-1 and TC-7 cells are continuous cell lines derived from African green monkey kidney cell cultures and were used to propagate WT SV40 and SV40 viable deletion mutants. All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 units/ml of penicillin, 100 pg/ml of streptomycin, 25 pg/ml of kanamycin, 0.03% glutamine, 0.075% NaHCO,, and 5% fetal calf serum (FCS). mKSA-ASC is an SV40-transformed cell line of BALBic origin (Kitet al., 1969) which grows as an ascites tumor in uivo (Rogers et al., 1977) and was passaged once weekly intraperitoneally in syngeneic mice. Viruses. SV40 (VA 45-54) was obtained from Dr. P. Tegtmeyer and was propagated in TC-7 cells as described previously (M. Tevethia et al., 1974). The viable deletion mutants used in this study have been described (Shenk et al., 1976; Sleigh et al., 1978). Transformation of C5?‘B1/6 MEF with WT SV40 and SV40 dl mutants. Actively
growing C57Bl/6 MEF cells at passage 4 were seeded at a density of 2 x lo4 per 25cm2 plastic flasks in DMEM containing 10%
ANTIGEN
INDUCTION
489
fetal bovine serum. After overnight incubation at 37” the medium was removed and virus was added to give a multiplicity of infection (m.o.i.> of 200. Control flasks received Tris-buffered saline (TBS) instead of virus. At the end of a 3-hr adsorption period at 37”, fresh medium was added and the incubation was continued for 3 weeks with weekly medium changes. Under these conditions normal C57Bl/6 MEF die and detach from the surface of the flask. In all virusinfected flasks between 10 and 50 dense clones of transformed cells developed. The cells in each flask were dispersed by treatment with trypsin and propagated in order to establish transformed cell lines. [“?S]Methionine labeling and extraction of SV40 WT and dl mutant-infected and -transformed cells. Confluent 75-cm’ flasks
of TC-7 cells grown in DMEM with 5% FCS were infected with SV40 WT and dl mutant virus at a m.o.i. of 5 PFUicell. Virus was allowed to adsorb for 90 min after which the flasks were refed with 15 ml DMEM containing 2% FCS. After 48 hr, infected TC-7 cells along with subconfluent monolayers of SV40 WT and dl mutant-transformed C57BV6 MEF were washed three times in TBS and incubated for 1 hr at 37” in methionine-free modified Eagle’s medium (MEM). The medium was removed and the cells radiolabeled for 1 hr at 37” by incubation in 3 ml of methionine-free MEM containing 50 &i/ml [?S]methionine (New England Nuclear, Boston, Mass). After. radiolabeling, each culture was washed three times in TBS and extraction of the cells was accomplished by adding 1 ml of extraction buffer containing 0.5%~ NP40, 0.1 M NaCl, 0.05 M TrisHCl, pH 8.0, 330 Fgiml phenylmethylsulfanyl fluoride, and 1% aprotinin. After incubation at 4” for 20 min, the extracts were centrifuged at 20,000 g and the supernatants used for immunoprecipitation of proteins. Zmmujnoprecipitation of SV4.0 turnor antigens from 3”S-labeled SV40 WT and dl mutant-infected and -transformed cell extracts by anti-T sera. ?S-Labeled SV40 WT
and dl mutant-infected and -transformed cell extracts were first reacted with 50 ~1 of normal hamster sera per milliliter of extract for 16 hr at 4”. Two hundred microliters of 10% (w/v> Staphylococcus aureus (Enzyme
490
TEVETHIA
Center, Boston, Mass.) was added per milliliter of extract and kept in suspension for 10 min at room temperature by using a rocking aparatus. The suspension was centrifuged at 1100 g for 10 min and the supernatant was divided into 600-~1 aliquots. Tumor antigens were immunoprecipitated by the addition of 20 ~1 of anti-T sera from hamsters bearing tumors induced by virus-free SV40-transformed hamster cells for 90 min at 4”. Staphylococcus aureus (200 ~1) was added, kept in suspension for 10 min at room temperature, and pelleted by centrifugation. The pellets were washed three times with TBS containing 1% NP-40 and 0.5 M LiCl. Immune complexes were eluted by resuspending the bacteria in 50 ~1 of SDSsample buffer which contained 1% SDS and 1% mercaptoethanol and heating for 3 min at 100”. The bacteria were pelleted and the supernatants were analyzed by SDS-polyacrylamide gel electrophoresis. SDS -polyacrylamide gel electrophoresis (SDS-PAGE). The immunoprecipitates
(2 x lo4 cpmwell) were analyzed by SDSPAGE on lo-15% acrylamide gradients using the Laemmli (19’70) buffer system. Gels were fixed for 1 hr in 5% trichloroacetic acid, prepared for fluorography (Bonner and Laskey, 1974), and exposed on Kodak X-Omat XR-5 film for 2-5 days at -76”. Generation
of
cytotoxic
lymphocytes.
Cytotoxic lymphocytes (CTL) capable of specifically killing syngeneic cells transformed or infected with WT SV40 were generated by immunizing C57Bl/6 mice (Jackson Laboratories, Bar Harbor, Maine) in the hind foot pad according to a procedure previously described (Knowles et al., 1979; Tevethia et al., 1980a). Mice were immunized with 2 x lo7 B16/WT-3 cells or with l-3 x lo7 plaque-forming units (PFU) of WT SV40 or dl mutants. The draining lymph nodes were excised 7 days postimmunization and lymphocyte suspensions were prepared by gently pressing the lymph nodes through a 60-gauge stainless-steel wire mesh. Viable cells were counted by trypan blue exclusion and resuspended at 4 x lo6 lymphocytes/ml
ET AL.
of RPM1 1640 media supplemented with 5 x lo-” M 2-mercaptoethanol, 20 mM HEPES buffer, 100 units/ml of penicillin, 100 pg/ml of streptomycin, 0.03% glutamine, 0.225% NaHCO,, and 10% heat-inactivated FCS. Lymphocytes (2 x 107) were incubated in 60-mm plastic tissue culture dishes at 37” in 5% CO, for 3 days to allow the sensitized lymphocytes to differentiate into CTL. Control lymphocytes from unimmunized mice were prepared similarly. 51Crrelease assay. Nonadherent lymphocytes were removed from tissue culture dishes by gentle pipetting and were washed once in RPM1 1640 medium. Subconfluent monolayers of target cells in 75-cm2 tissue culture flasks were labeled with 200-300 PCi of 51Cr (New England Nuclear Corp.) in DMEM with 5% FCS as described previously (Pretell et al., 1979; Tevethia et al., 1980a). At the time of testing, the labeled target cells were brought into a single cell suspension with 0.1% trypsin and washed three additional times with DMEM. Viable cells (2 x 10”) in 0.1 ml were added to 10 x 75mm glass culture tubes followed by an equal volume of effector lymphocytes in varying concentrations to provide effector: target cell ratios in the range of 4O:l and 1O:l. Culture tubes containing mixtures of j’Cr-labeled target cells and lymphocytes were centrifuged at 60 g for 5 min to increase their contact and then were incubated at 37 in 5% CO, for 8-10 hr. At the end of the incubation period, 0.8 ml of RPM1 1640 medium was added to each culture tube. The tubes were centrifuged at 250 g to pellet the cells and one-half of the supernatant fluid (0.5 ml) was withdrawn. Both the supernatant aliquot and the pellet containing the remainder of the supernatant and the cells were counted in a Beckman gamma counter. Spontaneous release of “‘Cr from the target cells was determined by incubating the labeled target cells alone and the maximum release of 51Cr was determined by lysing the target cells with 5% SDS. The percentage specific “‘Cr release was determined using the following formula:
% release (immune % specific j’Cr release =
lymphocytes) - % release (normal lymphocytes) % release (maximum) - % release (spontaneous)
’
SIMIAN
W T “7”
2001
‘y42005
VIRUS
40 TRANSPLANTATION
“i
FIG. 1. DNA of SV40 WT (776) and dl (0.5410.59) mutants cleaved with restriction endonuclease Hinf. Three micrograms of DNA of each virus were incubated for 2 hr at 37” with three units of Hinf and then electrophoresed overnight through a 4% polyacrylamide (2O:l Bis) gel cast in Tris-acetate buffer. The gel was incubated in ethidium bromide (10 pgiml) for 1 hr and photographed on Tri-X film under long-wave uv illumination.
ANTIGEN
INDUCTION
491
data in Fig. 1 show that each deletion is localized to the H&f-D fragment. These data are consistent with those reported earlier (Sleigh et al., 1978; Shenk et al., 1976). The early viable deletion mutants were also characterized with respect to their ability to induce tumor antigens in infected cells. Proteins were extracted for dl mutant-infected cells as described under Materials and Methods, immunoprecipitated with anti-T sera, and analyzed by SDS-PAGE. The results in Fig. 2 show that while the WT SV40 induced the synthesis of both large T and small t antigens, only large T antigen could be detected in monkey cells infected by dl884, dl890, dl2001, and dl2005 under the conditions used. Using different labeling conditions, the synthesis of a shortened small t polypeptide has been observed in monkey cells infected with dl884, 890, and 2001 but not dl 2005 (Sleigh et al., 19’78; Khoury et al., 1979).
--wt
NTNTNTNTNT
---
Induction of SV40-speci$c transplantation immunity. Adult BALB/c mice (kindly
provided by the Mammalian Genetics Branch, NCI; Frederick, Md.) were immunized with 1 x lo7 PFU of either WT SV40 or dl mutants intraperitoneally. Ten days later, mice were challenged with 1 x lo4 mKSAASC cells by the subcutaneous route. The animals were observed for tumor development. RESULTS
Characterization Mutants
of SV4.0 dl(0.5410.59)
The early viable deletion mutants were characterized for the size of deletion and the proteins they induced during permissive infection prior to testing for their ability to induce TrAg in vivo or in vitro in the nonpermissive host in order to insure that the virus stocks used were free of contaminating wild-type virus. DNA from WT SV40, dl 884, dl890, dl2001, and dl2005 was cleaved with the restriction enzyme Hinf and the fragments generated were separated by polyacrylamide gel electrophoresis as described previously (Sleigh et al., 1978). The
te FIG. 2. Synthesis of SV40 early proteins in SV40 wild-typeand dl mutant (0.54/0.59)-infected cells. TC7 cells were infected with either SV40 wild-type or dl mutant virus at an m.o.i. of 5 at 37”. Forty-eight hours postinfection, cells were starved for 1 hr in methioninefree media then radiolabeled with 150 &i of [YSmethionine in 3 ml of mediaicm* flask for 1 hr. Cell extracts were prepared with 0.5% NP40 buffer, pH 8.0, and immunoprecipitated with either normal hamster sera (N) or hamster anti-T sera (T). Immunoprecipitates were electrophoresed on lo- 15% gradient polya&amide gels as described under Materials and Methods.
492
TEVETHIA
Induction of SV40 Transplantation Im,munity in BALBIc Mice with SV40 dl Mutants
The mutants dl 884, dl 890, dl 2001, and dl 2005 were tested for their ability to immunize BALBic mice against a challenge of syngeneic SV40-transformed cells. The results in Table 1 show that all four dl mutants were able to immunize BALB/c against tumor cell challenge. The degree of immunity was comparable to that induced by WT SV40. These results indicate that a virus which induces and accumulates large T antigen alone (dl2005) is able to immunize mice against a tumor cell challenge, indicating that the synthesis of small t antigen is not necessary for immunization. Induction of Cytotoxic SV40 dl Mutants
Lymphocytes
by
Immunization of C57BV6 mice (responder strain) with SV40 results in the induction of CTL capable of killing syngeneic SV40 transformed cells in ,uitro (Knowles et al., 1979, Pretell et al., 1979). Since thymusderived lymphocytes are also involved in the rejection of SV40 tumors in mice (S. Tevethia et al., 1974) an experiment was performed to determine whether dl mutants would induce CTL in C57B116 mice. The results are shown in Table 2. Lymphocytes from C57Bl/6 mice immunized with WT SV40 specifically killed only the SV40transformed cells (B16/WT-3) and not cells transformed either by polyoma virus (B16/ PY) or by adenovirus-5 (KCA). Similarly, lymphocytes from mice immunized with dl 884, dl 2001, and dl 2005 were specifically cytotoxic only against B16/WT-3 cells. These results establish that early viable deletion mutants are capable of inducing CTL which specifically kill only the SV40transformed syngeneic cells. Expression qf TrAg at the Surface dl Mutant-Infected Cells
of SV40
The results presented thus far indicate that large T antigen alone is capable of inducing specific transplantation immunity in ,uivo and also induces the generation of CTL as the dl mutants used induce the synthesis
ET AL. TABLE
1
IMMUNIZATION OF BALB/c MICE BY SV40 dl (0.54/0.59) MUTANTS AGAINST TUMOR CELL (mKSA) CHALLENGE Mice immunized with” SV40 SV40 SV40 SV40 SV40 None
WT dl 884 dl890 dl2001 dl 2005
Tumor incidence” by mKSA cells: 2 x 10”
1 x IO’
O/5 015 O/5 015 O/5 515
015 015 O/5 O/5 O/5 616
(’ Adult BALBic mice were immunized twice at weekly interval with 1 x 10’ PFU of WT SV40 or SV40 dl mutants. Immunized and nonimmunized mice were challenged with mKSA cells subcutaneously. The animals were followed for tumor development. b Number of mice with tumors/number of mice inoculated.
of large T antigen only. The results do not, however, indicate whether dl mutant-infected cells synthesize TrAg at the cell surface. To demonstrate this point, BlG/PY cells were infected with dl mutants and used as target cells in the cytotoxicity assay with lymphocytes generated against virus-free B16/WT-3 cells. The results in Fig. 3 show that BlG/PY cells infected with WT SV40 were specifically lysed by lymphocytes generated against B16/WT-3 cells. The specific “‘Cr release from WT-infected cells was approximately 18% as compared to 28% 3’Cr release from B16/WT-3 cells. The specific siCr release from dl mutant (dl884, dl 890, dl 2001, dl 2005)-infected cells was lower than that of WT SV40-infected cells. These results indicate that SV40 TrAg is expressed at the surface of dl mutant-infected cells since cellular immune reactions leading to cell death take place at the membrane level (Cerottini and Brunner, 1974). Expression of TrAg at the Surface of SV40 dl Mutant-Transformed Cells
C57Bl/6 MEF cells were transformed with either mutants dl884 or dl2005 or with WT SV40. All transformants were 100%
SIMIAN
VIRUS 40 TRANSPLANTATION TABLE
GENERATION
OF LYMPHOCYTES
ANTIGEN
INDUCTION
493
2
CYTOTOXIC TO SV40-TRANSFORMED BY SV40 dl (0.5410.59) MUTANTS
CELLS
IN C57Bll6
MICE
Target cells Bl6/WT-3 Lymph node effector cells” SV40 SV40 SV40 SV40
Percentage “‘Cr release
WT immunized d1884 immunized dl 2001 immunized dl 2005 immunized
79 76 74 79
Nonimmunized None (spontaneous) None (maximum)
BIGIPY
Percentage specific “‘Cr releaseb
KCA
Percentage “I&. release
Percentage specific “lCr releaseb
Percentage “‘Cr release
26 26 26 31
3 3 3 9
20 20 21 23
75 ‘71 69 75
21 23 100
24 23 98
Percentage specific 51Cr release” 0 0 2 4 20 18 100
(I Adult C57BV6 mice were immunized with 2 x 10’ PFU of WT SV40 or dl mutants by inoculation in the hind footpad. Seven days later lymphocytes from immunized and nonimmunized mice were cultured in vitro for 3 days and reacted with “‘Cr-labeled B16/WT-3 cells (SV40 transformed), B16IPY cells (polyoma virus transformed), and KCA (adenovirus-5 transformed) as described under Materials and Methods. * Lymphocyte/target cell ratio of 10~1.
positive for SV4Q T antigen by the immunofluorescence test. The absence of 17K protein was confirmed in one line each of dl2005 and dl884-transformed C57BU6 cells by the immunoprecipitation test using anti-T sera. The results in Fig. 4 show that while WT SVBO-transformed cells contain the 17K protein (small t antigen), it is not present in detectable quantities in the dl mutantBIG/PY
Cells
Infected
by:
sv40
percent 0
transformed cells. The large T antigen was present in WT SV40- and dl mutant-transformed cells. Two WT SV40 and three dl 884 and dl 2005 transformed cell lines were used as targets in the lymphocyte-mediated cytotoxicity assay to determine if these cells are susceptible to lysis by lymphocytes sensitized to SV40 TrAg. The results in Table 3 Speclflc
10 I
51 Cr
Release 20 1
30 I
wt
SV 40
dl884
SV40
dl690
SV40
dl2001
SV 40
dI2005
616/Wt-3 umnfecfed
m
FIG. 3. Expression of SV4OTrAg at the surface of dl mutant (0.54/0.59)-infected cells. Polyoma-transformed mouse cells (BlG/Py) were infected with either SV40 wild-type or dl mutant at an m.o.i. of 50 and used as targets in a “‘Cr release assay 72 hr after infection as described under Materials and Methods. SV40-transformed cells (B16/WT-3) were also used as targets. Target cells were incubated with syngenie lymphocytes from mice sensitized to SV40 TrAg at an effector:target cell ratio of approximately 4O:l.
494
TEVETHIA
demonstrate that dl mutant-transformed cells which synthesize only the large T antigen can act as a target for lymphocytes generated in response to immunization of syngeneic mice with SV40. The specific “‘Cr release from dl mutant-transformed cells was comparable to the “‘Cr release from WT-transformed cells. Cells transformed by adenovirus-5 (KCA) or polyoma virus (BIGIPY) were not susceptible to lysis by lymphocytes sensitized to SV40 TrAg. DISCUSSION
The findings of this study show that cells transformed by SV40 early viable deletion (0.54/0.59) mutants which synthesize large T antigen alone can express SV40-specific 616
B16
wt 2005 NTNTNT
B16 884
t-
FIG. 4. Synthesis of SV40 early proteins in SV40 wild-typeand dl mutant (0.54/0.59)-transformed cells. C57B116 mouse embryo fibroblasts transformed by either wild-type SV40 (BlGIWT cells), dl 2005 (B16/ 2005 cells), or dl 884 (B16/884 cells) were starved for methionine for 1 hr in methionine-free media then radiolabeled with 150 &i of [35S]methionine in 3 ml of mediaR&m* flask for 1 hr. Cell extracts were prepared in 0.5% NP40 buffer, pH 8.0, and immunoprecip itated with either normal hamster sera (N) or hamster anti-T sera (T). Immunoprecipitates were electrophoresed on IO- 15% gradient polyacrylamide gels as described under Materials and Methods.
ET AL. TABLE
Y
EXPRESSION OF SV40 TrAg AT THE SURFACE OF SV40 dl (0.5410.59) MUTANT-TRANSFORMED CELI.S Lymphocyte donor immunized with”
Target cella
Transformed by
sv40 SV4O
BlG/WT-17 BIUWT-18
SV40 SV40
WT WT
74 HO
sv40 sv40 sv40
Bl6/884-1 B16/884-2 Bl6/884-3
sv40 SV40 SV40
dl 884 dl 884 dl 884
48 82 78
sv40 sv40 sv40
B16/2005-1 B16i2005-2 B16i2005-3
SV40 SV40 SV40
dl2005 dl2005 dl2005
79 80 80
SV4O sv40
KCA BlGIPY
AdenoviruG Polyoma virus
6 9
a Adult C57BV6 mice were immunized with 2 x IO’ PFU of SV40 via hind footpad inoculation. Seven days later draining lymph nodes were excised and the lymphocytes cultured in vitro for 3 days prior to use in the “Cr release assay against the indicated target cells. li Lymphocywtarget cell ratios of 4O:l.
transplantation antigen at the cell surface. It appears that the synthesis of small t antigen is not an absolute requirement for the expression of immunoreactive TrAg. These results support earlier findings that purified large T antigen can immunize syngeneic mice against SV40 tumor cells and can induce a cell-mediated immune response in the inoculated host (Chang et nl., 1979; Tevethia et al., 1980b). The studies carried out so far, however, have not ruled out the participation of small t antigen either as an immunogen or as a target antigen at the cell surface. Studies with nondefective adenoSV40 hybrid viruses indicate that TrAg sites which are involved in the sensitization of host against tumor cell challenge are located in the carboxy region of T antigen (Jay et al., 1978; 1979; Lewis and Rowe, 1973). These studies, however, have not excluded the existence of TrAg sited on the amino terminus of T antigen. Both large T and small t antigens share amino acid sequences at the amino terminus and also contain common antigenic sites in this region as demonstrated by the ability of antibody to denatured large T antigen to immunoprecipitate small t antigen (Crawford et aZ., 1980; Lane
SIMIAN
VIRUS 40 TRANSPLANTATION
and Robbins, 1978; Greenfield et al., 1980). It is quite possible that these common antigeneic sites shared between the large T and small t proteins may also be involved in the induction of cellular immune response of the host against tumor cell challenge. In addition, antigenic sites specified by amino acids in the unique carboxy terminal portion of small t antigen (Greenfield et al., 1980) may be involved in the cell-mediated immune reactions. By using mouse cells infected or transformed by viable deletion (0.54.10.59) mutants which induce the synthesis of large T antigen only, we hoped to define the role of small t antigen in the expression of TrAg at the cell surface by determining whether differences in the susceptibility of cell-mediated lysis of WT or dl mutant-infect.ed or -transformed cells could be demonstrated. Although mouse cells infected by dl mutants were not as susceptible to lymphocytemediated cytolysis as the WT-infected cells, this difference was not noticed when WT and dl mutant-transformed cells were used as target, cells. The lower release of “‘Cr from dl mutant-infected cells upon interaction with lymphocytes sensitized to SV40 TrAg could be explained by variation in the efficiency of infection of nonpermissive cells by dl mutants. The question regarding the nature of TrAg at the surface of SV40-transformed cells and its relationship to SV40-coded proteins still requires clarification, although the available evidence strongly suggests that large T antigen possesses TrAg sites which are immunogenic in the syngeneic host. Whether small t antigen also possesses TrAg sites can most convincingly be demonstrated by using purified small t antigen as immunogen. ACKNOWLEDGMENTS The studies reported here were supported by Research Grant CA 25000, CA 24694. CA 18450, CA 13106, and CA 24803 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. The excellent technical assistance of C. Paul is gratefully acknowledged. REFERENCES ANDERSON, J. L., MARTIN, R. G., CHANG, C., and MORA, P. T. (1977a). Tumor specific transplantation
ANTIGEN
INDUCTION
495
antigen is expressed during SV40 lytic infection with wild-type and tsA mutant viruses. Virology 76, 254-262. ANDERSON, J. L., MARTIN, R. G. CHANG, C., MORA, P. T., and LIVINGSTON, D. M. (1977b). Nuclear preparations of SV40 transformed cells containing tumor specific transplantation rejection activity. Virology 76, 420-425. BONNER. W. M., and LASKEY, R. A. (1974). A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biothem. 46,84-88. CEROTTINI, J. C., and BRUNNER, K. T. (1974). Cell mediated cytotoxicity: Allograft rejection and tumor immunity. Advan. I?nmur~o/. 18, 67-132. CHANG, C., ANDERSON, J. L.. MARTIN, R. G., and MORA, P. T. (1977). Expression of tumor-specific transplantation antigen in cell lines transformed by wild-type or tsA mutant simian virus 40. J. Viral. 22,281.-289. CHANG, C., MARTIN, R. G., LIVINGSTON, D. M., LUBORSKY, S. W., Hu, C., and MORA, P. T. (1979). Relationship between T-antigen and tumor specific transplantation antigen in simian virus 40 transformed cells. J. Viral. 29, 69-75. CRAWFORD, L. V., COLE, C. N., SMITH, A. E., PAUCH& E., TEGTMEYER, P., RUNDELL, K., and BERG, P. (1978). Organization and expression of early genes of simian virus 40. Proc. Nat. Acad. Sci. USA 75, 117-121. CRAWFORD, L. V., PIM, D. C., and LANE, D. P. (1980). An immunochemical investigation of SV40 T-antigens. 2. Quantitation of antigen and antibody activities. Virology 100, 314-325. GREENFIELD, R. S., FLYER, D. C., and TEVETHIA, S. S. (1980). Demonstration of unique and common antigenic sites located on the SV40 large T and small t antigens. Virology 104, 312-322. GIRARDI, A. J., and DEFENDI, V. (1970). Induction of SV40 transplantation antigen (TrAg) during the lytic cycle. Virology 42, 688-698. HABEL, K., and SILVERBERG, R. J. (1960). Relationship of polyoma virus and tumor in viva. Vimlogy 12 463-476. JAY,‘G., JAY, F. T., CHANG, C., FRIEDMAN, R. M., and LEXINE, A. S. (1978). Tumor specific transplantation: Use of the Ad2+ND,, hybrid virus to identify the protein responsible for simian virus 40 tumor rejection and its genetic origin. Proc. Naf. Acad. Sci.
USA
75, 3055-3059.
JAY, G., JAY, F. T., CHANG, C., LEVINE, A. S., and FRIEDMAN, R. M. (1979). Induction of simian virus 40.specific tumor rejection by the AD2+ND, hybrid virus. J. Gen. Viral. 44, 287-296. KIT, S., KURIMURA, T., and DUBBS, D. R. (1969). Transplantable mouse tumor line induced by injection of SV40-transformed mouse kidney cells. Znt. J. Center 4,384-392.
TEVETHIA
496
KNOWLES, B. B., KONCAR, M., PFIZENMAIER, K., SOLTER, D., ADEN, D. P., and TRINCHIERI, G. (1979). Genetic control of the cytotoxic T cell response to SV40 tumor-associated specific antigen. J. Immunol. 122, 17981806. KHOURY, G., GRUSS. P., DHAR, R., and LAI, C. J. (1979). Processing and expression of early SV40 mRNA: A role for RNA conformation in splicing. Cell
18, 85-92.
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LANE, D. P., and ROBBINS, A. K. (1978). An immunochemical investigation of SV40 T antigens. 1. Production, properties and specificity of a rabbit antibody to purified simian virus 40 large T antigen. Virology 87, 182-193. LEWIS, A. M., JR., and ROWE, W. P. (1973). Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. VIII. Association of simian virus 40 transplantation antigen with a specific region of the early viral genome. J. Viral. 12, 836-840. PAUCHA, E., HARVEY, R., and SMITH, A. E. (1978). Cell-free synthesis of simian virus 40 T antigens. J. Viral. 28, 154-170. PRETELL, J., GREENFIELD, R. S., and TEVETHIA, S. S. (1979). Biology of simian virus 40 (SV40) transplantation antigen (TrAg). V. In vitro demonstration of SV40 TrAg in SV40 infected nonpermissive mouse cells by the lymphocyte mediated cytotoxicity assay. Virology 97, 32-41. PRIVES, C., GLUZMAN, Y., and WINOCUR, E. (1978). Cellular and cell-free synthesis of simian virus 40 T antigens in permissive and transformed cells. J. Viral.
25, 587-595.
ROGERS, M. J., LAW, L. W., and APPELLA, E. (1977). Subcellular distribution of the tumor specific transplantation antigen of SV40 transformed cells. J. Nat. Cancer Inst. 59, 1291-1295. SHENK, T. E., CARBON, J., and BERG, P. (1976). Construction and analysis of viable deletion mutants of simian virus 40. J. Viral. 18, 664-671.
ET AL.
SLEIGH, M. J., TOPP, W. C., HLJNICH, R., and SAMBROOK, J. F. (1978). Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell 14, 79-88. TEGTMEYER. P., SCHWARTZ, M., COLLINS, J. K., and RUNDELL, K. (1975). Regulation of tumor antigen synthesis by simian virus 40 gene A. J. Viral. 16, 168-178.
TEVETHIA, M. J., and TEVETHIA, S. S. (1976). Biology of SV40 transplantation antigen (TrAg). I. Demonstration of SV40 TrAg on glutaraldehyde fixed SV40infected African green monkey kidney cells. Viralogy 69, 474-489.
TEVETHIA, M. J., and TEVETHIA, S. S. (1977). Biology of simian virus 40 (SV40) transplantation antigen (TrAg). III. Involvement of SV40 gene A in the expression of TrAg in permissive cells. Virology 81, 212-223.
TEVETHIA, M. J., RIPPER, L. W., ~~~TEVETHIA, S. S. (1974). A simple qualitative spot complementation test for temperature sensitive mutants of SV40. Intervirology
3, 245-255.
TEVETHIA, S. S. (1980). Immunology of simian virus 40. In “Virus Oncology” (G. Klein, ed.), pp. 581-601. Raven Press, New York, TEVETHIA, S. S., BLASECKI, J. W., WANECK, G., and GOLDSTEIN, A. (1974). Requirement of thymus-derived 0 positive lymphocytes for rejection of DNA virus (SV40) tumors in mice. J. Zmmunol. 113,14171423. TEVETHIA, S. S., GREENFIELD, R. S., FLYER, D., and TEVETHIA, M. J. (1980a). Simian virus 40 transplantation rejection antigen: Relationship to SV40 specific proteins. Cold Spring Harbor Symp. 44, 235242. TEVETHIA, S. S., FLYER, D. C., and TJIAN, R. (1980b). Biology of simian virus 40 (SV40) transplantation antigen (TrAg) VI. Mechanism of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein). Virology 107, 13-23.