JOURNAL OF VIROLOGY, Dec. 1991, p. 6985-6988
Vol. 65, No. 12
0022-538X/91/126985-04$02.00/0 Copyright X 1991, American Society for Microbiology
NOTES Immunological Elimination of Infected Cells as the Candidate Mechanism for Tumor Protection in Polyomavirus-Infected Mice JULIA J. WIRTH* AND MICHELE M. FLUCK
Department of Microbiology, Interdepartmental Program in Molecular and Cellular Biology, Michigan State University, East Lansing, Michigan 48824-1101 Received 7 June 1991/Accepted 27 August 1991
The uniformly lethal development of mammary tumors in polyomavirus-infected adult female nude mice was prevented by adoptive cell transfer of polyomavirus-immune splenocytes or peritoneal cells. Transferred immune cells also lowered the growth rate of emerging tumors. The induction of other relatively less frequent tumors of the skin and bone was decreased as well. Using in situ hybridization of whole-body sections as well as hybridization of nucleic acids from the mammary glands, we show for the first time that transferred immune cells, but not normal cells, virtually eliminated virus signal in the whole mouse and in the mammary glands. Since infected and tumorous mammary glands produce very little infectious virus, it appears that a major mechanism mediating the prevention of polyomavirus oncogenesis involves the immunological elimination of nonproductively and persistently infected cells.
(at 6 weeks of age) with 107 PFU of wild-type strain A2. Six control mice received no cells. In repeat experiments, identical results were obtained with transfer of nonimmune splenocytes. The protocol is summarized in Table 1. Mice were monitored biweekly for the development of palpable tumors. At 10, 11, and 12 weeks postinfection, one or two mice per group were sacrificed after assessment of their tumor burden, to analyze viral replication. The experiment was terminated at 12 weeks postinfection because of the tumor load of the control mice. Palpable mammary tumors were detected beginning at 6 weeks postinfection for control mice and were observed for all control mice by 9 weeks postinfection (Fig. 1). The tumors, which were identified histologically as adenocarcinomas, appeared to be of polyclonal origin, since large tumor masses were usually composed of several smaller foci that coalesced and grew into the adjacent areas of the neck, back, and hindquarters. Of 24 mice receiving transferred immune cells, only 1 developed a tumor, first observed at 6 weeks postinfection, i.e., 1 week post-adoptive transfer. Ten mice receiving immune cells but not sacrificed for the replication study were tumor free at the end of the experiment. Of these, four remained tumor free for over 1 year, three developed mammary tumors with delays of 9 months to 1 year, two developed hind leg paralysis, suggesting osteosarcomas of the spine (11), and one died of undetermined causes. It must be noted that in adult female nude mice, mammary tumors develop with such rapidity and high frequencies as to mask the occurrence of other high-frequency tumors, i.e., those of the skin and the bone. The absence of nonmammary tumors in the long-term-surviving recipients of immune cells suggests that the tumors were also either prevented from developing or eliminated by the transferred cells. The total tumor burden of the mice is shown in Table 2. At 9 weeks postinfection, the six control mice bore a total of 20 tumors; by 10 weeks postinfection, the number had increased to 30; it increased to 35 by 11 weeks and finally to 46
The oncogenic potential of viruses depends on the ability of a virus to transform cells to the neoplastic state and on the ability of the transformed cells to evade the host's immune response. In the case of polyomavirus, infection of neonates and adult immunodeficient mice leads to the development of tumors in a wide spectrum of tissues (4, 16), whereas immunocompetent mice generate an immune response which eliminates the virus without any apparent pathology. How tumor development in these mice is prevented is unknown. We (12, 13) and others (2) have developed a system in which polyomavirus infection of adult female nude mice leads to the rapid induction of mammary tumors in almost 100% of the mice. In the present study, we have used this system to investigate the role of the immune response in controlling mammary neoplasia. Furthermore, we have used a new technique, direct in situ hybridization of whole mouse sections (recently modified in our laboratory [20]), to monitor the effect of the immune response on viral replication in the whole mouse, in the mammary glands, and in the tumors themselves. To investigate the relationship between the immune response and mammary neoplasia, a cell transfer experiment in which female immunocompetent (euthymic) BALB/c mice served as donors and female BALB/c nude mice were the recipients was designed. Donor mice were infected subcutaneously (at 6 weeks of age) with 2 x 106 PFU of wild-type polyomavirus strains that have been reported to induce different tumor profiles (6) or to stimulate different levels of antitumor immunity in tumor transplantation experiments (10). At 2 weeks postinfection, donor mice were sacrificed and their spleens and/or their peritoneal-cavity contents were removed. Single-cell suspensions (5 x 107 splenocytes or 5 x 106 peritoneal cells per mouse) were prepared and injected intravenously into 24 recipient female nude mice that had been infected subcutaneously 5 weeks previously *
Corresponding author. 6985
6986
NOTES
J. VIROL.
TABLE 1. Mouse treatment groups
1 2 3 4 5
TABLE 2. Cumulative number of mammary tumors
Groupa
Treatment (no. and type of cells received)
Strain'
(immune) (immune) (immune) (immune) (control)
5 x 107 splenocytes 5 x 10' splenocytes 5 x 107 splenocytes 5 x 106 peritoneal cells No cells
A2 3049 RA RA
Groups of six mice each were infected subcutaneously with 107 PFU of polyomavirus wild-type strain A2 at 6 weeks of age and were subjected to the indicated treatment 5 weeks later. b Strain of polyomavirus used to infect donor mice that were the source for the treatment cells. a
by 12 weeks. In contrast, a single tumor was observed in the immune-cell recipients through 12 weeks postinfection. Because of its early appearance, we assume that this tumor was already present at the time of cell transfer. The growth rate of this tumor was calculated to be 24.5 mm2/week, compared with growth rates of 53.5, 80, and 145 mm2/week for three randomly selected tumors from different control mice. Additionally, this tumor appeared to have stopped growing by 9 weeks postinfection. These and repeat experiments show that transfer of polyomavirus-immune cells can significantly decrease the incidence of mammary neoplasia and arrest the growth of established tumors. The immune cells may eliminate tumor cells present at the time of transfer as well as prevent the initiation of new tumors. Peritoneal cells, administered at 1/10 the concentration of splenocytes, were as efficient as the latter in conferring protection. Since peritoneal cells contain a high proportion of macrophages, this observation raises the possibility that macrophages play a role in controlling viral replication and tumor growth, perhaps through direct effects on virus-infected and/or virus-transformed cells. The different wild-type virus strains used to immunize the donor mice were chosen for their abilities to elicit different levels of antipolyomavirus immunity in other systems (6, 10). In the experiments reported here, however, no differences in the abilities of these viruses to stimulate either qualitative or quantitative differences in the protective capacities of the transferred cells could be detected. 6/6
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FIG. 1. Incidence of mammary tumors. Open bar, control mice which received no cells; solid bar, mice receiving transferred immune cells; ratios, number of mice with tumors/total number of mice in these two categories.
6
Cumulative no. of tumors detected (no. of tumor-bearing mice) at wk postinfectionb 7 8 9 10 11 12
0 0
4 (3) 1 (1)
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No cells Immune cells
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20 (6) 1 (1)
30 (6) 1 (1)
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a Mice received either no cells (6 mice) or polyomavirus-immune cells (24 mice [Table 1]). b Mice were palpated biweekly for the presence of tumors.
Clearance of the virus signal was analyzed by direct in situ hybridization (20) of whole-mouse sections and hybridization of total DNA extracted from mammary glands. In situ hybridization revealed extensive polyomavirus signal in control mice sacrificed at 10, 11, and 12 weeks postinfection (Fig. 2). As described in detail elsewhere (20) and as noted by others (5), most of the virus signal in these mice was in the mammary glands and mammary tumors. Significant levels of signal were also detected in the skin (Fig. 2) and bone, which also represent important target organs for tumorigenesis. Multiple tumors, most but not all of which contain high levels of viral nucleic acids, are visible in Fig. 2. (As described elsewhere, integration is not detected in most cases [18].) In contrast, very little signal could be detected in any of the mice receiving transferred polyomavirus-immune cells. Total DNA was extracted from mammary glands and analyzed by Southern transfer and hybridization. At 11 weeks postinfection, mammary glands from the mice not receiving cells displayed very high levels of viral genomes. This was observed both for the morphologically normal glands (Fig. 3, lanes 5, 7, and 9) and for the tumor-containing glands (Fig. 3, lanes 6, 8, and 10). In contrast, very low levels of viral DNA were detected in the mammary glands of the mice receiving transferred immune cells (Fig. 3, lanes 1 to 4). Identical results were obtained with mammary glands removed from the mice sacrificed at 12 weeks postinfection (data not shown). Thus, it appears that transfer of polyomavirus-immune cells greatly decreases the level of virus signal in the whole mouse as well as in the target tissues for tumor formation, in particular in the mammary gland. Since the infected mammary glands and the mammary gland tumors represent cases of essentially nonproductive infections with cells containing high levels of viral genomes but producing very low levels of viable virus (10-4 PFU per cell), it is likely that the killing of infected cells is a major mechanism involved in the clearance of the virus signal in infected adult animals. Thus, as has already been demonstrated for lymphocytic choriomeningitis virus (3, 22) and paramyxovirus (21) infections, transfer of immune cells can clear persistent polyomavirus infections. Within the polyomavirus tumor constellation, observed at least with wild-type strain A2, the induction of mammary tumors in adult female athymic mice represents the most oncogenic manifestation of polyomavirus infection (2, 12, 13); induction is rapid, synchronous, and polyclonal; within 9 weeks postinfection, tumors arise in every animal. Interestingly, these tumors, ductal adenocarcinomas, are very similar to human breast cancers. The results presented here illustrate the critical role that the immune response plays in preventing mammary tumor formation and in eliminating viral genomes, probably by eliminating infected cells. These findings build upon previous results obtained with immunosuppressed adult (1) or neonatally thymectomized (17) mice.
VOL. 65, 1991
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FIG. 2. In situ hybridization of whole-mouse sections. Mice treated as described in the text and in Table 1, footnote a, were sacrificed at 10 (first column), 11 (second column), or 12 (third column) weeks postinfection (wpi). The procedure of Haase et al. (8), as modified by Wirth et al. (20), was used. Briefly, the frozen, embedded mice were sectioned sagitally by using an LKB cryomicrotome to expose brain, spleen, kidney, liver, salivary glands, lung, heart, skin, and bone. The sections (40 ,um) were collected on 3M tape, air dried, fixed in ethanol-acetic acid, dehydrated, and stored at room temperature. The DNA in the sections was denatured at 65°C in a 95% deionized formamide solution and hybridized in phosphate buffer at 37°C for 3 days with 106 cpm of denatured whole-polyomavirus-genome probe DNA per ml. The probe was column purified and had a specific activity greater than 109 cpm/,lg. The hybridized sections were washed, stained in Gill's hematoxylin, dried, and then autoradiographed. In order to compare data from different mice, all hybridizations and film exposures were carried out at the same time. Groups defined in Table 1 are numbered in the left margin.
According to the latter report, which represents the only previous in-depth study, mice thymectomized at birth and infected 7 to 9 days later primarily developed salivary gland tumors of longer latency (3 months on the average) and with a lower incidence (75%) than those observed in this study. Immune protection was induced at 6 weeks of age by transfer of spleen cells from donors hyperimmunized with tumor cells alone or tumor cells and virus. However, 44% of the thymectomized neonates were protected from tumor development by transfer of nonimmune spleen cells. The authors therefore concluded that most of the protection resulted from reconstituting recipient immunological competence. In the experiments described here, the immunization protocol was novel-a single infecting inoculation with a moderate dose of virus. As no protection was observed with nonimmune cells (resulting in a 100% incidence of tumors), we may infer that this system detects active participation of the transferred immune cells in the elimination of tumors. Although the effector mechanisms involved here are not known, it is clear from our results that reduction of the virus signal in the mammary glands (Fig. 3), most likely by the elimination of infected cells, is one, if not the major, mech-
anism required for tumor prevention. That an antitumor response is also operative is suggested by the reduced rate of tumor growth. However, the antitumor effector mechanism may not differ from that which eliminates infected cells, since tumor cells may represent a subset of these cells with similar antigenic profiles. Our results with viral expression in the infected mammary glands and in mammary gland tumors suggest that progression to the tumor stage is correlated with an increase in viral expression, since the level of viral transcripts in tumors is elevated compared with the level in nontumorous infected mammary glands (13). Thus, tumor cells in the mammary gland may in fact be easier to eliminate than infected cells are. Our studies of viral replication in the three major tissues which can sustain replication of the virus in adult animals (mammary gland, skin, and bone) (20) suggest that the elimination of infected cells may be a general mechanism for the clearance of the virus signal as well as the elimination of tumors in these tissues. The antigens recognized by this effector mechanism are unknown; however, the existence of polyomavirus tumor-specific transplantation antigens has been known for many years (9, 14, 19), and these are likely candidates. Recently, both middle (18) and
J. VIROL.
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FIG. 3. Analysis of mammary gland DNA. Mammary glands removed from mice sacrificed at 11 weeks postinfection were homogenized and treated with protease K. Total DNA was extracted and then digested (5 jig) with EcoRI, which linearizes the polyomavirus genome. DNA samples were then electrophoresed on a 0.8% agarose gel, blotted onto Hybond, probed with 32P-labeled polyomavirus whole-genome DNA, and autoradiographed. Arrowhead, linear polyomavirus DNA (5.3 kb). Lane 1, group 1 mouse (A2 donor [Table 1]); lane 2, group 2 mouse (3049 donor); lane 3, group 3 mouse (NG59RA donor, splenocytes); lane 4, (NG59RA donor, peritoneal cells); lanes 5 to 10, three control mice (infected mice which received no immune cells). In each case, a mammary tumor is shown (lanes 6, 8, and 10) juxtaposed to a macroscopically normal mammary gland from the same mouse (lanes 5, 7, and 9).
(15) T antigens have been shown to induce tumor rejection in rats, and the induction of a cytotoxic T-cell response in mice has been documented (7). Interestingly, both in the latter study (7) and in our work to date, the cytotoxic T-cell response tested in vitro was weak, in contrast to the powerful clearance demonstrated in the present experiments. In summary, the system described in this report identifies a major effector mechanism involved in preventing neoplasia and provides a unique opportunity to elucidate the antitumor immunological response.
large
This work was supported by grants from the Children's Leukemia Foundation and the American Cancer Society (grant MV-353).
REFERENCES 1. Allison, A. C. 1974. Interactions of antibodies, complement components and various cell types in immunity. Transplant. Rev. 19:3-55. 2. Berebbi, M. L., L. Dandolo, J. Hassoun, A. M. Bernard, and D. Blangy. 1988. Specific tissue targeting of polyomavirus oncogenicity in athymic nude mice. Oncogene 2:144-156. 3. Byrne, J. A., and M. B. A. Oldstone. 1984. Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo. J. Virol. 51:682-686. 4. Dawe, C. J., R. Freund, G. Mandel, K. Ballmer-Hofer, D. A. Talmage, and T. L. Benjamin. 1987. Variations in polyoma virus genotype in relation to tumor induction in mice. Am. J. Pathol. 127:243-261.
5. Demengeot, J., J. Jacquemier, M. Torrente, D. Blangy, and M. Berebbi. 1990. Pattern of polyomavirus replication from infection until tumor formation in the organs of athymic nulnu mice. J. Virol. 64:5633-5639. 6. Freund, R., G. Mandel, G. G. Carmichael, J. P. Barncastle, C. J. Dawe, and T. L. Benjamin. 1987. Polyomavirus tumor induction in mice: influences of viral coding and noncoding sequences on tumor profiles. J. Virol. 61:2232-2239. 7. Greene, M. I., L. L. Perry, E. Kinney-Thomas, and T. L. Benjamin. 1982. Specific thymus-derived (T) cell recognition of papova virus-transformed cells. J. Immunol. 128:732-736. 8. Haase, A., M. Brahic, L. Stowring, and H. Blum. 1984. Detection of viral nucleic acids by in situ hybridization. Methods Virol. 7:189-225. 9. Habel, K. 1961. Resistance of polyoma virus immune animals to transplanted polyoma tumors. Proc. Soc. Exp. Biol. Med. 106:722-729. 10. Hare, J. D. 1967. Transplant immunity to polyoma virusinduced tumor cells. IV. A polyoma strain defective in transplant antigen induction. Virology 31:625-632. 11. Harper, J. S., III, C. J. Dawe, B. D. Trapp, P. E. McKeever, M. Collins, J. L. Woyclechowska, D. L. Madden, and J. L. Sever. 1983. Paralysis in nude mice caused by polyomavirus-induced vertebral tumors, p. 359-367. In J. L. Sever and D. L. Madden (ed.), Polyomaviruses and human neurological diseases. Alan R. Liss, Inc., New York. 12. Haslam, S. A., L. J. Counterman, J. J. Wirth, and M. M. Fluck. 1989. Histopathogenesis and characterization of mammary tumors induced by polyomavirus in athymic nu/nu mice. Proc. Am. Assoc. Cancer Res. 30:455. 13. Haslam, S. Z., J. J. Wirth, L. J. Counterman, and M. M. Fluck. Characterization of the mammary hyperplasia, dysplasia and synchronous polyclonal neoplasia induced in athymic female adult mice by polyomavirus. Submitted for publication. 14. Ito, Y., J. R. Brockelhurst, and R. Dulbecco. 1977. Virus specific proteins in the plasma membrane of cells lytically infected or transformed by polyoma virus. Proc. Natl. Acad. Sci. USA 74:4666-4670. 15. Lathe, R., M. P. Kieny, P. Gerlinger, P. Clertant, I. Guizani, F. Cuzin, and P. Chambon. 1987. Tumour prevention and rejection with recombinant vaccinia. Nature (London) 326:878-880. 16. Law, L. W., and C. J. Dawe. 1960. Influence of total body X-irradiation on tumor induction by parotid tumor agent in adult mice. Proc. Soc. Exp. Biol. Med. 105:414-419. 17. Law, L. W., R. C. Ting, and E. Leckbard. 1967. Prevention of virus-induced neoplasms in mice through passive transfer of immunity by sensitized syngeneic lymphoid cells. Proc. Natl. Acad. Sci. USA 57:1068-1075. 18. Reinholdsson, G., T. Ramquist, J. Brandberg, and T. Dalianis. 1988. A polyomavirus tumor-specific transplantation antigen (TSTA) epitope is situated within the N-terminal amino acid sequence common to middle and small T antigens. Virology 166:616-619. 19. Sjogren, H. O., I. Hellstrom, and G. Klein. 1961. Transplantation of polyoma virus-induced tumors in mice. Cancer Res. 21:324-337. 20. Wirth, J. J., A. Amalfitano, R. Gross, M. B. A. Oldstone, and M. M. Fluck. Organ, age and sex specific replication of polyomavirus in the mouse. Submitted for publication. 21. Young, D. F., R. E. Randall, J. A. Hoyle, and B. E. Souberbielle. 1990. Clearance of a persistent paramyxovirus infection is mediated by cellular immune responses but not by serumneutralizing antibody. J. Virol. 64:5403-5411. 22. Zinkernagel, R. M., and R. W. Welsh. 1976. H-2 compatibility requirement for virus-specific T cell mediated effector functions in vivo. I. Specificity of T cells conferring anti-viral protection against lymphocytic choriomeningitis virus is associated with H-2K and H-2D. J. Immunol. 117:1495-1502.
Vol. 65, No. 12
0022-538X/91/126985-04$02.00/0 Copyright X 1991, American Society for Microbiology
NOTES Immunological Elimination of Infected Cells as the Candidate Mechanism for Tumor Protection in Polyomavirus-Infected Mice JULIA J. WIRTH* AND MICHELE M. FLUCK
Department of Microbiology, Interdepartmental Program in Molecular and Cellular Biology, Michigan State University, East Lansing, Michigan 48824-1101 Received 7 June 1991/Accepted 27 August 1991
The uniformly lethal development of mammary tumors in polyomavirus-infected adult female nude mice was prevented by adoptive cell transfer of polyomavirus-immune splenocytes or peritoneal cells. Transferred immune cells also lowered the growth rate of emerging tumors. The induction of other relatively less frequent tumors of the skin and bone was decreased as well. Using in situ hybridization of whole-body sections as well as hybridization of nucleic acids from the mammary glands, we show for the first time that transferred immune cells, but not normal cells, virtually eliminated virus signal in the whole mouse and in the mammary glands. Since infected and tumorous mammary glands produce very little infectious virus, it appears that a major mechanism mediating the prevention of polyomavirus oncogenesis involves the immunological elimination of nonproductively and persistently infected cells.
(at 6 weeks of age) with 107 PFU of wild-type strain A2. Six control mice received no cells. In repeat experiments, identical results were obtained with transfer of nonimmune splenocytes. The protocol is summarized in Table 1. Mice were monitored biweekly for the development of palpable tumors. At 10, 11, and 12 weeks postinfection, one or two mice per group were sacrificed after assessment of their tumor burden, to analyze viral replication. The experiment was terminated at 12 weeks postinfection because of the tumor load of the control mice. Palpable mammary tumors were detected beginning at 6 weeks postinfection for control mice and were observed for all control mice by 9 weeks postinfection (Fig. 1). The tumors, which were identified histologically as adenocarcinomas, appeared to be of polyclonal origin, since large tumor masses were usually composed of several smaller foci that coalesced and grew into the adjacent areas of the neck, back, and hindquarters. Of 24 mice receiving transferred immune cells, only 1 developed a tumor, first observed at 6 weeks postinfection, i.e., 1 week post-adoptive transfer. Ten mice receiving immune cells but not sacrificed for the replication study were tumor free at the end of the experiment. Of these, four remained tumor free for over 1 year, three developed mammary tumors with delays of 9 months to 1 year, two developed hind leg paralysis, suggesting osteosarcomas of the spine (11), and one died of undetermined causes. It must be noted that in adult female nude mice, mammary tumors develop with such rapidity and high frequencies as to mask the occurrence of other high-frequency tumors, i.e., those of the skin and the bone. The absence of nonmammary tumors in the long-term-surviving recipients of immune cells suggests that the tumors were also either prevented from developing or eliminated by the transferred cells. The total tumor burden of the mice is shown in Table 2. At 9 weeks postinfection, the six control mice bore a total of 20 tumors; by 10 weeks postinfection, the number had increased to 30; it increased to 35 by 11 weeks and finally to 46
The oncogenic potential of viruses depends on the ability of a virus to transform cells to the neoplastic state and on the ability of the transformed cells to evade the host's immune response. In the case of polyomavirus, infection of neonates and adult immunodeficient mice leads to the development of tumors in a wide spectrum of tissues (4, 16), whereas immunocompetent mice generate an immune response which eliminates the virus without any apparent pathology. How tumor development in these mice is prevented is unknown. We (12, 13) and others (2) have developed a system in which polyomavirus infection of adult female nude mice leads to the rapid induction of mammary tumors in almost 100% of the mice. In the present study, we have used this system to investigate the role of the immune response in controlling mammary neoplasia. Furthermore, we have used a new technique, direct in situ hybridization of whole mouse sections (recently modified in our laboratory [20]), to monitor the effect of the immune response on viral replication in the whole mouse, in the mammary glands, and in the tumors themselves. To investigate the relationship between the immune response and mammary neoplasia, a cell transfer experiment in which female immunocompetent (euthymic) BALB/c mice served as donors and female BALB/c nude mice were the recipients was designed. Donor mice were infected subcutaneously (at 6 weeks of age) with 2 x 106 PFU of wild-type polyomavirus strains that have been reported to induce different tumor profiles (6) or to stimulate different levels of antitumor immunity in tumor transplantation experiments (10). At 2 weeks postinfection, donor mice were sacrificed and their spleens and/or their peritoneal-cavity contents were removed. Single-cell suspensions (5 x 107 splenocytes or 5 x 106 peritoneal cells per mouse) were prepared and injected intravenously into 24 recipient female nude mice that had been infected subcutaneously 5 weeks previously *
Corresponding author. 6985
6986
NOTES
J. VIROL.
TABLE 1. Mouse treatment groups
1 2 3 4 5
TABLE 2. Cumulative number of mammary tumors
Groupa
Treatment (no. and type of cells received)
Strain'
(immune) (immune) (immune) (immune) (control)
5 x 107 splenocytes 5 x 10' splenocytes 5 x 107 splenocytes 5 x 106 peritoneal cells No cells
A2 3049 RA RA
Groups of six mice each were infected subcutaneously with 107 PFU of polyomavirus wild-type strain A2 at 6 weeks of age and were subjected to the indicated treatment 5 weeks later. b Strain of polyomavirus used to infect donor mice that were the source for the treatment cells. a
by 12 weeks. In contrast, a single tumor was observed in the immune-cell recipients through 12 weeks postinfection. Because of its early appearance, we assume that this tumor was already present at the time of cell transfer. The growth rate of this tumor was calculated to be 24.5 mm2/week, compared with growth rates of 53.5, 80, and 145 mm2/week for three randomly selected tumors from different control mice. Additionally, this tumor appeared to have stopped growing by 9 weeks postinfection. These and repeat experiments show that transfer of polyomavirus-immune cells can significantly decrease the incidence of mammary neoplasia and arrest the growth of established tumors. The immune cells may eliminate tumor cells present at the time of transfer as well as prevent the initiation of new tumors. Peritoneal cells, administered at 1/10 the concentration of splenocytes, were as efficient as the latter in conferring protection. Since peritoneal cells contain a high proportion of macrophages, this observation raises the possibility that macrophages play a role in controlling viral replication and tumor growth, perhaps through direct effects on virus-infected and/or virus-transformed cells. The different wild-type virus strains used to immunize the donor mice were chosen for their abilities to elicit different levels of antipolyomavirus immunity in other systems (6, 10). In the experiments reported here, however, no differences in the abilities of these viruses to stimulate either qualitative or quantitative differences in the protective capacities of the transferred cells could be detected. 6/6
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TIME POST INFECTION (WEEKS)
FIG. 1. Incidence of mammary tumors. Open bar, control mice which received no cells; solid bar, mice receiving transferred immune cells; ratios, number of mice with tumors/total number of mice in these two categories.
6
Cumulative no. of tumors detected (no. of tumor-bearing mice) at wk postinfectionb 7 8 9 10 11 12
0 0
4 (3) 1 (1)
Treatmenta
No cells Immune cells
6 (5) 1 (1)
20 (6) 1 (1)
30 (6) 1 (1)
35 (6) 1 (1)
46 (6) 1 (1)
a Mice received either no cells (6 mice) or polyomavirus-immune cells (24 mice [Table 1]). b Mice were palpated biweekly for the presence of tumors.
Clearance of the virus signal was analyzed by direct in situ hybridization (20) of whole-mouse sections and hybridization of total DNA extracted from mammary glands. In situ hybridization revealed extensive polyomavirus signal in control mice sacrificed at 10, 11, and 12 weeks postinfection (Fig. 2). As described in detail elsewhere (20) and as noted by others (5), most of the virus signal in these mice was in the mammary glands and mammary tumors. Significant levels of signal were also detected in the skin (Fig. 2) and bone, which also represent important target organs for tumorigenesis. Multiple tumors, most but not all of which contain high levels of viral nucleic acids, are visible in Fig. 2. (As described elsewhere, integration is not detected in most cases [18].) In contrast, very little signal could be detected in any of the mice receiving transferred polyomavirus-immune cells. Total DNA was extracted from mammary glands and analyzed by Southern transfer and hybridization. At 11 weeks postinfection, mammary glands from the mice not receiving cells displayed very high levels of viral genomes. This was observed both for the morphologically normal glands (Fig. 3, lanes 5, 7, and 9) and for the tumor-containing glands (Fig. 3, lanes 6, 8, and 10). In contrast, very low levels of viral DNA were detected in the mammary glands of the mice receiving transferred immune cells (Fig. 3, lanes 1 to 4). Identical results were obtained with mammary glands removed from the mice sacrificed at 12 weeks postinfection (data not shown). Thus, it appears that transfer of polyomavirus-immune cells greatly decreases the level of virus signal in the whole mouse as well as in the target tissues for tumor formation, in particular in the mammary gland. Since the infected mammary glands and the mammary gland tumors represent cases of essentially nonproductive infections with cells containing high levels of viral genomes but producing very low levels of viable virus (10-4 PFU per cell), it is likely that the killing of infected cells is a major mechanism involved in the clearance of the virus signal in infected adult animals. Thus, as has already been demonstrated for lymphocytic choriomeningitis virus (3, 22) and paramyxovirus (21) infections, transfer of immune cells can clear persistent polyomavirus infections. Within the polyomavirus tumor constellation, observed at least with wild-type strain A2, the induction of mammary tumors in adult female athymic mice represents the most oncogenic manifestation of polyomavirus infection (2, 12, 13); induction is rapid, synchronous, and polyclonal; within 9 weeks postinfection, tumors arise in every animal. Interestingly, these tumors, ductal adenocarcinomas, are very similar to human breast cancers. The results presented here illustrate the critical role that the immune response plays in preventing mammary tumor formation and in eliminating viral genomes, probably by eliminating infected cells. These findings build upon previous results obtained with immunosuppressed adult (1) or neonatally thymectomized (17) mice.
VOL. 65, 1991
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FIG. 2. In situ hybridization of whole-mouse sections. Mice treated as described in the text and in Table 1, footnote a, were sacrificed at 10 (first column), 11 (second column), or 12 (third column) weeks postinfection (wpi). The procedure of Haase et al. (8), as modified by Wirth et al. (20), was used. Briefly, the frozen, embedded mice were sectioned sagitally by using an LKB cryomicrotome to expose brain, spleen, kidney, liver, salivary glands, lung, heart, skin, and bone. The sections (40 ,um) were collected on 3M tape, air dried, fixed in ethanol-acetic acid, dehydrated, and stored at room temperature. The DNA in the sections was denatured at 65°C in a 95% deionized formamide solution and hybridized in phosphate buffer at 37°C for 3 days with 106 cpm of denatured whole-polyomavirus-genome probe DNA per ml. The probe was column purified and had a specific activity greater than 109 cpm/,lg. The hybridized sections were washed, stained in Gill's hematoxylin, dried, and then autoradiographed. In order to compare data from different mice, all hybridizations and film exposures were carried out at the same time. Groups defined in Table 1 are numbered in the left margin.
According to the latter report, which represents the only previous in-depth study, mice thymectomized at birth and infected 7 to 9 days later primarily developed salivary gland tumors of longer latency (3 months on the average) and with a lower incidence (75%) than those observed in this study. Immune protection was induced at 6 weeks of age by transfer of spleen cells from donors hyperimmunized with tumor cells alone or tumor cells and virus. However, 44% of the thymectomized neonates were protected from tumor development by transfer of nonimmune spleen cells. The authors therefore concluded that most of the protection resulted from reconstituting recipient immunological competence. In the experiments described here, the immunization protocol was novel-a single infecting inoculation with a moderate dose of virus. As no protection was observed with nonimmune cells (resulting in a 100% incidence of tumors), we may infer that this system detects active participation of the transferred immune cells in the elimination of tumors. Although the effector mechanisms involved here are not known, it is clear from our results that reduction of the virus signal in the mammary glands (Fig. 3), most likely by the elimination of infected cells, is one, if not the major, mech-
anism required for tumor prevention. That an antitumor response is also operative is suggested by the reduced rate of tumor growth. However, the antitumor effector mechanism may not differ from that which eliminates infected cells, since tumor cells may represent a subset of these cells with similar antigenic profiles. Our results with viral expression in the infected mammary glands and in mammary gland tumors suggest that progression to the tumor stage is correlated with an increase in viral expression, since the level of viral transcripts in tumors is elevated compared with the level in nontumorous infected mammary glands (13). Thus, tumor cells in the mammary gland may in fact be easier to eliminate than infected cells are. Our studies of viral replication in the three major tissues which can sustain replication of the virus in adult animals (mammary gland, skin, and bone) (20) suggest that the elimination of infected cells may be a general mechanism for the clearance of the virus signal as well as the elimination of tumors in these tissues. The antigens recognized by this effector mechanism are unknown; however, the existence of polyomavirus tumor-specific transplantation antigens has been known for many years (9, 14, 19), and these are likely candidates. Recently, both middle (18) and
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FIG. 3. Analysis of mammary gland DNA. Mammary glands removed from mice sacrificed at 11 weeks postinfection were homogenized and treated with protease K. Total DNA was extracted and then digested (5 jig) with EcoRI, which linearizes the polyomavirus genome. DNA samples were then electrophoresed on a 0.8% agarose gel, blotted onto Hybond, probed with 32P-labeled polyomavirus whole-genome DNA, and autoradiographed. Arrowhead, linear polyomavirus DNA (5.3 kb). Lane 1, group 1 mouse (A2 donor [Table 1]); lane 2, group 2 mouse (3049 donor); lane 3, group 3 mouse (NG59RA donor, splenocytes); lane 4, (NG59RA donor, peritoneal cells); lanes 5 to 10, three control mice (infected mice which received no immune cells). In each case, a mammary tumor is shown (lanes 6, 8, and 10) juxtaposed to a macroscopically normal mammary gland from the same mouse (lanes 5, 7, and 9).
(15) T antigens have been shown to induce tumor rejection in rats, and the induction of a cytotoxic T-cell response in mice has been documented (7). Interestingly, both in the latter study (7) and in our work to date, the cytotoxic T-cell response tested in vitro was weak, in contrast to the powerful clearance demonstrated in the present experiments. In summary, the system described in this report identifies a major effector mechanism involved in preventing neoplasia and provides a unique opportunity to elucidate the antitumor immunological response.
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This work was supported by grants from the Children's Leukemia Foundation and the American Cancer Society (grant MV-353).
REFERENCES 1. Allison, A. C. 1974. Interactions of antibodies, complement components and various cell types in immunity. Transplant. Rev. 19:3-55. 2. Berebbi, M. L., L. Dandolo, J. Hassoun, A. M. Bernard, and D. Blangy. 1988. Specific tissue targeting of polyomavirus oncogenicity in athymic nude mice. Oncogene 2:144-156. 3. Byrne, J. A., and M. B. A. Oldstone. 1984. Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo. J. Virol. 51:682-686. 4. Dawe, C. J., R. Freund, G. Mandel, K. Ballmer-Hofer, D. A. Talmage, and T. L. Benjamin. 1987. Variations in polyoma virus genotype in relation to tumor induction in mice. Am. J. Pathol. 127:243-261.
5. Demengeot, J., J. Jacquemier, M. Torrente, D. Blangy, and M. Berebbi. 1990. Pattern of polyomavirus replication from infection until tumor formation in the organs of athymic nulnu mice. J. Virol. 64:5633-5639. 6. Freund, R., G. Mandel, G. G. Carmichael, J. P. Barncastle, C. J. Dawe, and T. L. Benjamin. 1987. Polyomavirus tumor induction in mice: influences of viral coding and noncoding sequences on tumor profiles. J. Virol. 61:2232-2239. 7. Greene, M. I., L. L. Perry, E. Kinney-Thomas, and T. L. Benjamin. 1982. Specific thymus-derived (T) cell recognition of papova virus-transformed cells. J. Immunol. 128:732-736. 8. Haase, A., M. Brahic, L. Stowring, and H. Blum. 1984. Detection of viral nucleic acids by in situ hybridization. Methods Virol. 7:189-225. 9. Habel, K. 1961. Resistance of polyoma virus immune animals to transplanted polyoma tumors. Proc. Soc. Exp. Biol. Med. 106:722-729. 10. Hare, J. D. 1967. Transplant immunity to polyoma virusinduced tumor cells. IV. A polyoma strain defective in transplant antigen induction. Virology 31:625-632. 11. Harper, J. S., III, C. J. Dawe, B. D. Trapp, P. E. McKeever, M. Collins, J. L. Woyclechowska, D. L. Madden, and J. L. Sever. 1983. Paralysis in nude mice caused by polyomavirus-induced vertebral tumors, p. 359-367. In J. L. Sever and D. L. Madden (ed.), Polyomaviruses and human neurological diseases. Alan R. Liss, Inc., New York. 12. Haslam, S. A., L. J. Counterman, J. J. Wirth, and M. M. Fluck. 1989. Histopathogenesis and characterization of mammary tumors induced by polyomavirus in athymic nu/nu mice. Proc. Am. Assoc. Cancer Res. 30:455. 13. Haslam, S. Z., J. J. Wirth, L. J. Counterman, and M. M. Fluck. Characterization of the mammary hyperplasia, dysplasia and synchronous polyclonal neoplasia induced in athymic female adult mice by polyomavirus. Submitted for publication. 14. Ito, Y., J. R. Brockelhurst, and R. Dulbecco. 1977. Virus specific proteins in the plasma membrane of cells lytically infected or transformed by polyoma virus. Proc. Natl. Acad. Sci. USA 74:4666-4670. 15. Lathe, R., M. P. Kieny, P. Gerlinger, P. Clertant, I. Guizani, F. Cuzin, and P. Chambon. 1987. Tumour prevention and rejection with recombinant vaccinia. Nature (London) 326:878-880. 16. Law, L. W., and C. J. Dawe. 1960. Influence of total body X-irradiation on tumor induction by parotid tumor agent in adult mice. Proc. Soc. Exp. Biol. Med. 105:414-419. 17. Law, L. W., R. C. Ting, and E. Leckbard. 1967. Prevention of virus-induced neoplasms in mice through passive transfer of immunity by sensitized syngeneic lymphoid cells. Proc. Natl. Acad. Sci. USA 57:1068-1075. 18. Reinholdsson, G., T. Ramquist, J. Brandberg, and T. Dalianis. 1988. A polyomavirus tumor-specific transplantation antigen (TSTA) epitope is situated within the N-terminal amino acid sequence common to middle and small T antigens. Virology 166:616-619. 19. Sjogren, H. O., I. Hellstrom, and G. Klein. 1961. Transplantation of polyoma virus-induced tumors in mice. Cancer Res. 21:324-337. 20. Wirth, J. J., A. Amalfitano, R. Gross, M. B. A. Oldstone, and M. M. Fluck. Organ, age and sex specific replication of polyomavirus in the mouse. Submitted for publication. 21. Young, D. F., R. E. Randall, J. A. Hoyle, and B. E. Souberbielle. 1990. Clearance of a persistent paramyxovirus infection is mediated by cellular immune responses but not by serumneutralizing antibody. J. Virol. 64:5403-5411. 22. Zinkernagel, R. M., and R. W. Welsh. 1976. H-2 compatibility requirement for virus-specific T cell mediated effector functions in vivo. I. Specificity of T cells conferring anti-viral protection against lymphocytic choriomeningitis virus is associated with H-2K and H-2D. J. Immunol. 117:1495-1502.
